Download Regulation of Ocular Growth in Wild

Regulation of Ocular Growth in Wild-Type and Retinopathy,
Globe Enlarged (RGE) Chickens
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Eric R. Ritchey, OD, MS
Graduate Program in Vision Science
The Ohio State University
2011
Dissertation Committee:
Andy J. Fischer, PhD, Advisor
Donald O. Mutti, OD, PhD
Andrew Hartwick, OD, PhD
Peter Reiser, PhD
Copyright by
Eric R. Ritchey, OD, MS
2011
Abstract
According to the Centers for Disease Control, the most frequent ocular
condition afflicting the United States population is refractive error. In most
instances, refractive error occurs due to an improper match between the axial
length of the eye and objects at optical infinity (i.e., myopia, hyperopia or
astigmatism) or due to the loss of ability to focus on near objects with age
(presbyopia). The ability of the eye to coordinate growth of the organ while the
organism physically matures is of particular interest to vision scientists. In
humans, this relationship appears to be changing as the prevalence of refractive
errors, particularly myopia, increases. This alteration in the regulation on ocular
growth can be detrimental, as myopia is associated with potentially blinding
diseases such as glaucoma, retinal detachment, retinoschesis and myopic retinal
degeneration. A number of potential mechanisms have been proposed for why
the prevalence of myopia is on the rise; however, our knowledge of ocular growth
regulation is incomplete. Further insight into how ocular growth is regulated may
help scientists and clinicians develop effective therapeutic interventions to slow,
or possibly prevent, myopia development.
Chapter 1 of the dissertation addresses the significant literature regarding
the development of myopia in experimental animals, with an emphasis on the
role of unobstructed vision in normal refractive development and potential
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mechanisms involved in regulating ocular growth. Chapter 2 of the dissertation
examines the role of Insulin-like Growth Factor 1 (IGF1) and Fibroblast Growth
Factor 2 (FGF2) in the regulation of ocular growth. Recent research implicates
insulin as a potent stimulator of ocular growth. In chickens, a combination of
IGF1 & FGF2 radically alters ocular growth, leading to increased intraocular
pressure, axial length and anterior chamber changes leading to the development
of extreme myopia. Chapter 3 examines the expression of guanine-nucleotide
binding protein β3 (GNB3), also known as cone β-transducin, in multiple species.
We demonstrate that a mutant chicken model with congenital reduction in vision,
the Retinopathy, Globe Enlarged (RGE), lacks GNB3 in retinal neurons critical for
normal visual signal transduction. Chapter 4 of the dissertation examines
whether the RGE chicken can respond to experimental visual manipulations
known to cause ocular growth and myopia in multiple animal species. The
dissertation concludes with Chapter 5, a discussion of the pertinence of the
finding of this research and proposed future avenues of investigation to further
our understanding of regulation of ocular growth.
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Dedication
This dissertation is dedicated to my family: my wife Moriah, my son Evan and my
parents Earl and Mary, for whom I have tried to do my very best. Their love and
support have made my pursuit of the PhD in Vision Science possible.
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Acknowledgments
In a letter to Robert Hook, Sir Isaac Newton stated “If I have seen a little
further it is by standing on the shoulders of Giants”. There have been many
giants in my professional life that I would like to acknowledge. Foremost, I would
like to thank Dr. Andy Fischer for everything he has done in helping me reach
this milestone in my life. Andy has been a terrific mentor as he has guided my
transition from clinical to basic science. Under his tutelage, I have learned that
there is so much about the eye left to discover. Andy has given me the tools to
continue my development as a scientist and his support, encouragement and
critique have been invaluable during my time in the Fischer Lab. I have had the
opportunity to work with talented individuals who made coming to lab enjoyable
every day and I would be remiss not to thank them for their support and
encouragement. I would like to thank Dr. Kanika Ghai, Dr. Jennifer Stanke,
Ghezel Omar, Melissa Scott, Rachel Bongini, Pat Sherwood and Kim Code for
their patient and tolerance. I would especially like to thank our lab manager,
Chris Zelinka, whose assistance has been invaluable. From Optometry, I would
like to acknowledge Drs. Joseph Barr, Karla Zadnik, Mark Bullimore and Donald
Mutti. They have been my ardent supporters throughout the PhD and have
provided me with invaluable advice in times of need. Without your support I
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would not have flourished at Ohio State. Finally, I would like to thank my fellow
Vision Science graduate students, particularly Drs. David Berntsen, Jeff Schafer,
Kathryn Richdale and Tracy Bildstein. You have always been there when I have
needed encouragement and support. There have been countless other
individuals that I have not mentioned here that have significantly contributed to
my growth as a person and a scientist. I am sorry that I cannot acknowledge
every individual by name.
As a final thought, it is said that the measure of a man can been seen in
the company that he keeps. If that is true, I think that I am a very good man. I
have had the good fortune to be part of a tremendous community while at Ohio
State. Thank you one and all for having helped make my success in graduate
school possible.
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Vita
1973 ....................................................... Akron, Ohio
1997 ....................................................... BA, Anthropology,
The Ohio State University
2001 ....................................................... OD, Doctor of Optometry,
The Ohio State University
2003 ....................................................... MS, Vision Science,
The Ohio State University
2004 ....................................................... Graduate Teaching Associate,
The Ohio State University
2005 ....................................................... Postdoctoral Fellow,
The Ohio State University
2006-Present .......................................... Senior Research Associate
The Ohio State University
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Publications
Ritchey ER, Bongini RE, Code KA, Zelinka C, Petersen-Jones S, Fischer AJ. The
pattern of expression of guanine nucleotide-binding protein beta3 in the retina is
conserved across vertebrate species. Neuroscience (2010). Sep 1; 169(3): 1376-1391.
Fischer AJ, Scott MA, Ritchey ER, Sherwood P. Mitogen activated protein kinasesignaling regulates the ability of Müller glia to proliferate and protect retinal neurons
against excitotoxicity. Glia (2009). Nov 1; 57(14): 1538-52.
Fischer AJ, Ritchey ER, Scott MA, Wynne A. Bullwhip Neurons in the Retina Regulate
the Size and Shape of the Eye. Developmental Biology (2008). May 1; 317(1): 196-212.
Ritchey ER, Barr JT, Mitchell GL. The Comparison of Overnight Lens Modalities (COLM)
Study. Eye and Contact Lens (2005). 31(2): 70-75.
Fields of Study
Major Field: Vision Science
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Table of Contents
Abstract .................................................................................................................ii
Dedication ............................................................................................................iv
Acknowledgments ................................................................................................ v
Vita ...................................................................................................................... vii
Publications ........................................................................................................ viii
Fields of Study .................................................................................................... viii
Table of Contents .................................................................................................ix
List of Tables ........................................................................................................xi
List of Figures ...................................................................................................... xii
Chapter 1: Introduction ......................................................................................... 1
Myopia: A growing problem ........................................................................................ 1
Unobstructed vision is required for proper ocular growth ...................................... 5
The peripheral retina verses the central retina in refractive development ........ 12
Retinal neurons and their role in ocular growth ..................................................... 16
The identification of Egr1 and Glucagon as potential eye growth modulators.. 21
The role of insulin as a stimulator of ocular growth ............................................... 28
Retinoic Acid: A sign-sensitive modulator of ocular growth? ............................... 30
Post-retinal tissue and ocular growth ...................................................................... 32
Potential pharmacological modulators of ocular growth? .................................... 36
Summary ...................................................................................................................... 41
Chapter 2: The combination of IGF1 and FGF2 and the induction of excessive
ocular growth and extreme myopia......................................................................43
Abstract ............................................................................................................43
Introduction ......................................................................................................44
Results.......................................................................................................................... 55
Discussion.................................................................................................................... 75
Conclusions ................................................................................................................. 82
Acknowledgements .................................................................................................... 82
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Chapter 3: The pattern of expression of guanine nucleotide-binding protein β3
(GNB3) in the retina is conserved across vertebrate species ..............................84
Abstract ............................................................................................................84
Introduction ......................................................................................................85
Methods and Materials .............................................................................................. 87
Results.......................................................................................................................... 93
Discussion.................................................................................................................. 123
Conclusions ............................................................................................................... 130
Acknowledgements .................................................................................................. 130
Chapter 4: Vision-guided ocular growth in a mutant chicken model with
diminished visual acuity ..................................................................................... 132
Abstract ...................................................................................................................... 132
Introduction ................................................................................................................ 133
Methods and Materials ............................................................................................ 136
Results........................................................................................................................ 142
Discussion.................................................................................................................. 177
Conclusions ............................................................................................................... 177
Acknowledgements .................................................................................................. 178
Chapter 5: Summary and Future Directions ...................................................... 179
Summary of Findings ............................................................................................... 179
Future directions ....................................................................................................... 182
Future directions for the role of insulin and IGF in ocular growth. .................... 182
Data from RGE chickens suggest that normal photoreceptor and bipolar cell
function are not critical for vision-guided ocular growth ..................................... 183
Can visually-guided ocular growth stimuli (plus lenses) slow the progressive
globe enlargement of the RGE chick? .................................................................. 186
Future directions for the Egr1/glucagon pathway in ocular growth .................. 188
Concluding Remarks ................................................................................................ 190
References ....................................................................................................... 191
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List of Tables
Table 2.1: PCR primers (5’ to 3’), target and predicted product sizes .................50
Table 2.2: Antibodies, sources and working dilutions ..........................................51
Table 3.1: Table of Antibodies used in immunohistochemistry and Western
Blotting…………………………………………………………………………….........91
Table 3.2: GNB3 protein sequence homology across specied referenced to
Homo Sapiens ................................................................................................... 121
Table 4.1: PCR primers (5’ to 3’), target and predicted product sizes ............... 141
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List of Figures
Figure 1.1: Application of form-deprivation goggles prevents presentation of clear
images to the retina, causing excessive ocular growth ......................................... 8
Figure 1.2: Egr1 expression is up-regulated in eyes recovering from formdeprivation compared to untreated control eyes ..................................................23
Figure 1.3: Application of lenses alters retinal Egr1 expression, glucagon
synthesis and subsequent ocular growth .............................................................26
Figure 2.1: Receptors to insulin, IGF and FGF are widely expressed in different
ocular tissues.......................................................................................................56
Figure 2.2: Three consecutive daily intraocular injections of 800ng IGF1 and
200ng FGF2 stimulate excessive ocular growth …. ............................................58
Figure 2.3: Treatment with 3 consecutive daily injections of 800ng IGF1 and
200ng FGF2 ........................................................................................................60
Figure 2.4: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 stimulate excessive ocular growth ..................................................63
Figure 2.5: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 do not have short-term effects upon cell death, glial reactivity or the
integrity of ganglion cells, whereas the long-term survival of ganglion cells in the
peripheral retina is compromised.........................................................................67
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Figure 2.6: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 dramatically change the anterior segment of the eye .....................71
Figure 2.7: Intraocular injections of 200ng IGF1 and 200ng FGF2 stimulate the
proliferation of cells in far peripheral regions of the retina, progenitors in the CMZ,
equatorial regions of lens capsule, and non-pigmented epithelial cells in the
zonules ................................................................................................................73
Figure 2.8: Intraocular injections of the combination of IGF1 and FGF2 increase
intraocular pressure (IOP) in a dose-dependent manner.....................................75
Figure 3.1: Expression of GNB3 mRNA and protein in wild-type and RGE -/chickens ..............................................................................................................94
Figure 3.2: GNB3 expression in the developing chicken retina ...........................98
Figure 3.3: GNB3 is expressed by photoreceptors and bipolar cells in the retinas
of wildtype chicks............................................................................................... 102
Figure 3.4 GNB3 is expressed by all photoreceptors in retinas of wildtype chicken
.......................................................................................................................... 104
Figure 3.5 GNB3 is expressed by cone photoreceptors and bipolar cells in the
goldfish retina…………………………………………………………………….........107
Figure 3.6 GNB3 is expressed by photoreceptors and bipolar cells in the
Xenopus retina .................................................................................................. 108
Figure 3.7: GNB3 is expressed by cone photoreceptors and bipolar cells in the
mouse retina ...................................................................................................... 112
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Figure 3.8: GNB3 is expressed by cone photoreceptors and bipolar cells in the
guinea pig retina ................................................................................................ 115
Figure 3.9: GNB3 is expressed by cone photoreceptors and bipolar cells in the
dog retina .......................................................................................................... 118
Figure 3.10: GNB3 is expressed by cone photoreceptors and bipolar cells in the
primate retina (Macaca fascicularis) .................................................................. 120
Figure 4.1: Biometric measurements obtained from pictographs of enucleated
eyes ................................................................................................................... 138
Figure 4.2: Retinoscopy indicates that RGE chickens are emmetropic at age P12
.......................................................................................................................... 142
Figure 4.3: Percentage change in ocular growth observed in WT and RGE chicks
with form-deprivation: ........................................................................................ 145
Figure 4.4: Percentage change in ocular growth observed in WT and RGE chicks
with lens treatment ............................................................................................ 149
Figure 4.5: Percentage change in circumferential ocular growth and corneal
curvature observed in WT and RGE chicks with lens treatment using ImagePro
.......................................................................................................................... 153
Figure 4.6: Non-cycloplegic retinoscopy demonstrates that RGE chickens
develop myopia with 4.5 days of –7 lens wear and hyperopia with 4.5 days of +7
lens wear ........................................................................................................... 156
xiv
Figure 4.7: Recovery from form-deprivation (FD) slows ocular enlargement and
prevents the development of myopia in RGE chicks ......................................... 161
Figure 4.8: The expression of Egr1 in glucagoneric amacrine cells changes with
visual manipulation in WT and RGE chickens ................................................... 167
Figure 4.9: Levels of pro-glucagon mRNA are altered with visual manipulation in
RGE chickens .................................................................................................... 169
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Chapter 1: Introduction
Myopia: A growing problem
Myopia is defined as the condition of the eye where parallel rays of
incoming light focus in front of the retina in the absence of accommodation
(Hofstetter, Griffin et al. 2000). Myopic individuals are commonly referred to as
“near-sighted”, due to the blurring of objects at optical infinity given that the far
point of the optical system lies in front of the retina (Edwards 1998). Humans
have been aware of the concept of myopia for centuries, with historical records
revealing that the Greek philosopher Aristotle was aware of the condition
(reviewed in Grosvenor 2007). As a field of scientific inquiry, the study of myopic
refractive error has been an area of interest for almost 200 years, with
observations correlating myopia with education and near work dating from 1813
(Ware 1813). However, rigorous scientific study on the subject did not begin until
the 1900s.
The dilemma in the study of ocular growth is that the phenomenon can be
divided into two distinct categories. The first category can best be described as
the growth of the eye associated with physical maturation. From birth, the human
eye must grow in proportion to the rest of the body as the child matures
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(reviewed in Wallman and Winawer 2004). The second category of ocular growth
corresponds to the eye’s function as the major sensory organ of the body. As an
organism matures, ocular growth must coordinate the changes in the major
refractive components of the eye, the cornea and crystalline lens, with the overall
length of the eye, known as axial length. The interplay between the cornea and
crystalline lens with axial length must be properly regulated to obtain the desired
refractive outcome. The significance of both categories of growth can be
displayed by examining the axial length of full-term newborn children versus
adults. The average axial length in full-term newborn children has been reported
to be 16.8mm. In adults, the average axial length increases to 23.6mm (Gordon
and Donzis 1985). Thus, the eye must increase in axial length approximately
40% from birth to adulthood to reach maturity. During this time, the eye must
maintain an axial length commensurate to the refractive power of the cornea and
crystalline lens at that particular point in time to allow for clear distance vision for
the child to be emmetropic. This can be demonstrated by examining the change
in corneal curvature from birth to adulthood. At birth, humans primarily show
hyperopic, or far-sighted, refractive errors (Graham and Gray 1963; Mehra,
Khare et al. 1965; Kuo, Sinatra et al. 2003). This corresponds with the eye being
shorter in the axial dimension, despite the increased corneal curvature observed
at birth, where the mean corneal power is 51.2 diopters in newborns, which
decreases to 43.5 diopters at adulthood (Gordon and Donzis 1985). Therefore,
as humans approach adulthood the cornea flattens significantly, reducing the
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dioptric power and subsequent convergence of incoming light while the eye
elongates. The coordinated flattening of the cornea works in concordance with
the increase in axial length to allow for emmetropization as children mature and
become less hyperopic. Failure to maintain the balance between the refractive
elements of the eye and the axial length can have significant effects on the final
refractive error, given that a change of 1mm can generate approximately 2.5
diopters of refractive error, dependent upon age (Hart 1999).
Given this, most animals at birth are not emmetropic and have some
significant refractive error. For humans and other primates, the trend is to be
born hyperopic; however, it should be noted that some species begin life myopic
(reviewed in Wallman and Winawer 2004). For most species, the final refractive
outcome achieved at maturity approaches mild hyperopia or emmetropia, the
condition of having no refractive error. In emmetropia, incoming light refracted by
the cornea and cystalline lens in a non-accommodating eye is focused on the
retina, providing clear distance vision. If the axial length of the eye is too short or
the dioptric power of the eye insufficient, incoming light will focus behind the
retina, also known as hyperopia. Conversely, if axial length is too long for the
refractive components of the eye or the dioptric power of the eye excessive, light
focuses in front of the retina causing myopia. If the goal of ocular development is
to have clear distance vision at maturity, the refractive endpoint should be
emmetropia, or alternatively, hyperopia. Hyperopic refractive errors can be
overcome through accommodation, which releases tension on the crystalline lens
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zonules, allowing the curvature of the crystalline lens to steepen and add plus
power to the optical system to clear objects at optical infinity (Helmholtz and
Southall 1962). The least desirable outcome would be myopia, where
accommodation would simply increase the blur of distance objects.
The fact that humans begin life hyperopic and move towards emmetropia
is not unusual (Saunders, Woodhouse et al. 1995; Mutti, Mitchell et al. 2005)
(reviewed in Mutti 2007). What makes humans unusual is that they develop
myopia at a significant rate when the prevalence of myopia in other animals is
relative rare (reviewed in Smith 1998). The prevalence of myopia in humans
varies with ethnicity and region of the world. In the United States, current
estimates indicate that approximately 33% of the population between ages 12-54
years old is myopic (Vitale, Sperduto et al. 2009). In Asian countries such as
Taiwan and Singapore, myopia has become pandemic, with the prevalence of
myopia approaching 80% (Lin, Shih et al. 2001; Edwards and Lam 2004; Lin,
Shih et al. 2004). More concerning to eye care practitioners, the prevalence of
myopia appears to be increasing (Lin, Shih et al. 2001; Lin, Shih et al. 2004;
Vitale, Sperduto et al. 2009) (reviewed in Morgan and Rose 2005). This increase
in the number of myopic individuals has become a significant public health
concern. Not only is myopia associated with visually debilitating conditions such
as glaucoma, retinoschesis, retinal detachment, staphyloma, and maculopathy; it
is also associated with significant expense associated with visual correction
(Saw, Gazzard et al. 2005; Kuzin, Varma et al. 2010; Liu, Xu et al. 2010; Perera,
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Wong et al. 2010). Current estimates indicate that the treatment and correction of
myopia is approximately 4.6 billion dollars per annum (Javitt and Chiang 1994).
Given the significant public health costs and the potential impact of myopia
related visual disorders; it is of tremendous interest how ocular growth is
regulated and how this growth may be modulated to achieve a desired refractive
state.
Unobstructed vision is required for proper ocular growth
It is well established that proper ocular growth requires that the eye is
exposed to normal, unobstructed vision. There are multiple reports of abnormal
ocular growth in children who have ocular pathology or surgical interventions that
prevent clear images from reaching the retina. Examples of children who develop
myopia in an eye that is partially or completely occluded due to the presence of
hemangiomas of the eyelid, neonatal lid closure, ptosis or cataract have been
reported (Robb 1977; O'Leary and Millodot 1979; Hoyt, Stone et al. 1981; Rabin,
Van Sluyters et al. 1981; Nathan, Kiely et al. 1985).
While these correlations between observed ophthalmic disease and
refractive error are interesting to clinicians and scientists, animal experiments
have provided far more insight into the process of vision-guided ocular growth.
The origins of these experiments can be traced to the work of Levinsohn at the
turn of the century, who hypothesized that gravity would passively stretch the eye
with near work and lead to increased axial length. Levinsohn placed monkeys in
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boxes that restricted their head movements and forced the face and eyes into a
downward position to simulate the downward position of the eyes with chronic
near work. These animals became myopic compared to control animals placed in
boxes that held the face in a typical up-right posture and held the eyes in a
normal, forward position (reviewed in Smith 1998). Although it is not certain if the
myopia observed was due to the positional location induced by the apparatus or
by a restriction of the field of vision, the work of Levisohn proved that ocular
growth could be manipulated experimentally in animals.
Given the historical observations that myopic individuals tended to have
more formal education and performed more near vision tasks, researchers
looked to use animal models to explore the relationship between near work and
myopia. Francis Young, a psychologist who established a primate research
center at Washington State University in 1957, published a series of reports
examining the role of environmental factors in myopia development (Eye on Psi
Chi 2006). Young hypothesized that near work and accommodation were
myopigenic. Using semi-transparent hoods to create a near vision-restricted
environment, Young was able to generate myopia in macaques (Macaca
nemestrina) (Young 1961). Young was also able to prevent myopia in these
animals using topical atropine (Young 1965). Given that atropine prevented
myopia in these animals, Young proposed that near convergence and
accommodation in these animals were the cause of the generated myopia,
similar to the anecdotal reports of myopia individuals in near-work intensive
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activities. Moreover, Young believed that the cycloplegic effects of atropine were
responsible for the lack of myopia in those animals (reviewed in Smith 1998).
The work of Young and previous investigators appeared to support the
longstanding hypothesis that excessive near work and the accommodative
system was the primary factor involved in the development of myopia. In the
1970’s, a chance observation would radically change the way scientists thought
about myopia development. Hubel and Wiesel noted that macaque monkeys that
underwent monocular lid suturing developed myopia in the occluded eye (Hubel,
Wiesel et al. 1976). This was further quantified by the work of Wiesel and Raviola
in 1977, which showed that the amount of myopia produced by lid fusion
increased with longer periods of deprivation and that the effect was diminished
as animals approached maturity. The result of the observations of Hubel and
Wiesel was the inspiration for a number of studies from various researchers that
would examine the phenomenon of form-deprivation and myopia. Thus, ocular
growth in the absence of form-vision has been studied in numerous animal
models either through suturing of the eyelids or by application of diffuser goggles.
Among the animal models that have been examined are chickens, marmosets,
tree shrews, mice, monkeys, cats, tree squirrels and rabbits (Sherman, Norton et
al. 1977; Wiesel and Raviola 1977; Wallman, Turkel et al. 1978; Kirby, Sutton et
al. 1982; Norton 1990; McBrien, Moghaddam et al. 1993; Troilo and Judge 1993;
Tejedor and de la Villa 2003; Gao, Liu et al. 2006).
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Figure1.1: Application of form-deprivation goggles prevents presentation of clear
images to the retina, causing excessive ocular growth
Regardless of the species, the requirement of clear retinal images for
proper ocular growth has been shown to be highly conserved. When deprived of
clear vision, increases in axial length are observed compared to untreated control
eyes (Wallman, Turkel et al. 1978) (reviewed in Morgan 2003). The importance
of clear retinal images is emphasized by the fact that the effects of formdeprivation myopia can be reduced or eliminated by short periods of clear,
unobstructed vision. In chickens, 30 minutes of unrestricted vision reduced formdeprivation myopia by 50% and a 95% reduction was obtained with 130 minutes
of unobstructed vision (Napper, Brennan et al. 1995; Napper, Brennan et al.
1997). Another observation that demonstrates the importance of clear retinal
images is that animals in which form-deprivation myopia has been induced show
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the ability to recover from the induced refractive error if form-deprivation is
discontinued before the animal reached maturity and the induced refractive error
is left uncorrected (Wallman and Adams 1987; McBrien, Gentle et al. 1999).
These studies clearly demonstrate that the retina is tuned to respond rapidly to
image quality and properly adjust the rate of ocular growth.
Form-deprivation is not the only visual manipulation that has been shown
to modulate rates of ocular growth. Application of spectacle or contact lenses has
been shown to dramatically affect the rate of growth of the eye. When hyperopic
defocus is induced through the use of divergent (a.k.a. minus) lenses, the focal
point of the optical system is placed behind the retina leading to ocular growth.
Conversely, when myopic defocus is induced through the use of convergent
(a.k.a. plus) lenses, the focal point of the eye is placed in front of the retina and
ocular growth slows. These experiments clearly show that ocular growth can be
modified not only by a form-deprivation paradigm; rates of ocular growth can be
manipulated by changing the relationship between the retina and the point-focus
of the optical system. Furthermore, the retina has the intrinsic ability to determine
not only that the point-focus of light is not conjugate with the retina, but can also
determine the direction of the sign of defocus. This ability of the retina to
determine the sign of imposed defocus through application of lenses has been
shown to be well conserved across multiple species (Wildsoet and Wallman
1995; Howlett and McFadden 2009; Troilo, Totonelly et al. 2009; Smith, Hung et
al. 2010).
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It is important to note that the ability of the eye to alter rates of growth in
response to visual clues is not dependent on higher visual centers. There have
been a number of experiments that have examined how the eye responds to
visually-guided ocular growth clues in the absence of input from the central
nervous system. It is possible to isolate the eye from the CNS by severing the
optic nerve, damaging the optic nerve by pinching or crushing the nerve, or by
blocking neurological impulses via pharmacological agents. In chickens, eyes
that have undergone a complete optic nerve section still show the ability to grow
in response to form-deprivation or negative lens wear (Wildsoet 2003) (Troilo,
Gottlieb et al. 1987). In tree shrews and chickens, intact optic nerves where
synaptic transmission of ganglion cells has been blocked using teterodotoxin still
respond to form-deprivation (Norton, Essinger et al. 1994; McBrien, Moghaddam
et al. 1995). These experiments indicate that ganglion cell function is not required
to generate vision-guided ocular growth; however, they do not completely
preclude that the CNS plays a role in ocular growth. Tetrodotoxin-treated eyes
without form-deprivation in chickens had significantly flatter corneas than
contralateral control eyes, revealing that ganglion cell function may play a role in
regulating the shape of the anterior chamber (McBrien, Moghaddam et al. 1995).
While chickens with a complete optic nerve section respond to form-deprivation
in a normal manner, non form-deprived eyes undergoing optic nerve section
were significantly smaller and more hyperopic than controls (Troilo, Gottlieb et al.
1987; Wildsoet 2003). Moreover, while optic nerve-sectioned eyes in chickens
10
respond to visual manipulation, the recovery responses to the generated
refractive error are altered, with the eye over-compensating for the error (Troilo
and Wallman 1991). These results indicate that the retina is able to alter the local
rate of ocular growth independently of higher visual centers; however, an intact
connection between the eye and the CNS may have some influence over the
ocular growth associated with a maturing animal.
The response of the eye to growth-modulating stimuli in the absence of
communication with the higher visual centers of the brain clearly demonstrates
that visually-guided ocular growth is a locally-mediated phenomenon; meaning
that ocular growth is modulated by cells intrinsic to the eye. More importantly,
these cells act in a regional manner, with different retinal hemispheres capable of
growing at different rates. This was clearly demonstrated in the chicken, where
occlusion of one half of the visual field caused excessive ocular growth in the
corresponding retinal hemisphere (Hodos and Kuenzel 1984; Wallman, Gottlieb
et al. 1987). At the time of these experiments, it was unclear if this response was
limited to avian species or if this type of growth could be elicited across species.
Importantly, this observation of localized retinal growth with hemispherical formdeprivation appears to be conserved, with similar results observed in mammalian
models. Guinea pigs and macaques (Macaca mulatta) respond in a similar
manner to chickens under hemifield occlusion (McFadden 2002; Smith, Huang et
al. 2009).
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The peripheral retina verses the central retina in refractive development
The observation that different regions of the retina can grow independently
of one another calls into question the importance of the central retina in the
regulation of vision-guided ocular growth. The central retina in most species is a
specialized region designed for high spatial frequency vision accomplished by an
increased density of cone photoreceptors. This area, referred to as a visual
streak, area centralis or fovea centralis depending upon the species, provides the
best visual acuity. In the correction of refractive error, the point-focus of light is
theoretically placed on the fovea to maximize visual acuity. Given the role the
fovea plays in visual acuity, it seems inherent that the fovea would play a critical
role in vision-guided ocular growth; however, the fovea does not seem to be a
primary regulator of vision-guided ocular growth. This has been clearly
demonstrated in rhesus monkeys, where laser photoablation of the fovea does
not prevent emmetropization nor does it inhibit the development of refractive
error in response to form-deprivation or lens-induced ametropia (Smith,
Ramamirtham et al. 2007; Smith, Hung et al. 2009). Rhesus monkeys treated
with annular diffusing goggles that form-deprived the peripheral retina without
depriving foveal vision developed myopia as well. After goggle removal, laser
photoablation of the fovea did not prevent recovery from the induced refractive
error (Smith, Kee et al. 2005). In humans, diseases and surgical procedures that
affect the peripheral retina but spare the fovea have been associated with
myopia development (Sieving and Fishman 1978; Knight-Nanan and O'Keefe
12
1996; Connolly, Ng et al. 2002; Al-Ghamdi, Albiani et al. 2004). Combined with
the evidence from animal models, the association of peripheral retinal damage in
humans with myopia suggests that proper peripheral retinal function is essential
for proper ocular growth.
In the development of myopic refractive error, the peripheral retina
appears to play a significant role while the fovea is relatively inconsequential. In
particular, hyperopic refractive errors in the peripheral retina appear to be a
myopigenic stimulus. In rhesus monkeys undergoing form-deprivation, relative
hyperopic peripheral refractions and more prolate ocular shapes are observed
(Huang, Hung et al. 2009). When rhesus monkeys are treated with annulardesign divergent lenses that generate relative peripheral hyperopia without
affecting central vision, myopic refractive errors are generated; moreover, rhesus
monkeys that have undergone foveal photoablation became myopic when
divergent lenses were applied (Smith, Hung et al. 2009). Furthermore, if relative
peripheral hyperopia is selectively generated in one hemisphere, the myopia
produced by the procedure occurs in the treated hemisphere (Smith, Hung et al.
2010).
Clinical observations support the assertion that relative peripheral
hyperopia is associated with myopia. Hoogerheide observed that young adults
(18 years old or over) in pilot training who demonstrated peripheral hyperopic
refractive errors were more likely to become myopic than those with peripheral
myopic refractive errors (Hoogerheide, Rempt et al. 1971) (reviewed in Charman
13
and Radhakrishnan 2010). Furthermore, myopic children with relative peripheral
hyperopia have been shown to have deeper vitreous chambers, thinner
crystalline lenses and steeper corneas than emmetropic and hyperopic children
who displayed relative peripheral myopic refractions (Mutti, Sholtz et al. 2000).
The observation of peripheral hyperopic refraction with myopia has been
observed in individuals of different ethnicities; however, the amount of relative
peripheral hyperopia may vary by ethnicity (Kang, Gifford et al. 2010; Sng, Lin et
al. 2011).
Given the association of relative peripheral hyperopia with myopic
refractive error, it is reasonable to question if conventional treatments for myopic
refractive error could contribute to myopic progression. It is postulated that
correction of myopia with corrective lenses, while focusing light on the fovea to
maximize acuity, would generate hyperopia in the peripheral retina and thus be
myopigenic. Indeed, relative peripheral hyperopia has been shown to increase
patients with moderate myopia (–3.25 DS to –6.00DS) who were treated with
spectacle lenses (Lin, Martinez et al. 2010). Therefore, if the hyperopic refractive
error in the retinal periphery were corrected, could myopic progression be
retarded or eliminated? There is evidence in chickens that lenses with peripheral
plus power can neutralize peripheral hyperopia and inhibit axial growth (Liu and
Wildsoet 2011). In humans, patients who have undergone corneal reshaping
using orthokeratology contact lenses to treat myopia show relative peripheral
myopia after lens wear compared to relative peripheral hyperopia before
14
treatment (Queiros, Gonzalez-Meijome et al. 2010; Kang and Swarbrick 2011).
Moreover, orthokeratology has been shown to retard myopia progression (Cho,
Cheung et al. 2005; Walline, Jones et al. 2009). Per clinical reports,
discontinuation of orthokeratology lenses lead to increased rates of axial growth
compared to those observed during lens wear (Lee and Cho 2010).
Although there is a body of evidence that implicates relative peripheral
hyperopia with myopia progression, questions remain on the exact role of
peripheral refractive error and ocular growth. At least one investigator has
reported relative peripheral astigmatism, with peripheral hyperopia in the
horizontal meridian and relative peripheral myopia in the vertical meridian. Given
the evidence from animals models that indicates stop-grow signals are far more
potent than growth signals, the relative peripheral myopia observed by Berntsen
et al. should act as a “stop” signal for ocular growth (Berntsen, Mutti et al. 2010).
Moreover, a large longitudinal study of non-myopic school aged children reported
that relative peripheral hyperopia had minimal influence on the development of
myopia or on the rate of myopia progression. This group also showed that there
was no significant association between hyperopic relative peripheral refraction
and the risk of myopia onset in Hispanic, Native American or white subjects.
Furthermore, axial elongation was not related to the average relative peripheral
refractive error for any ethnicity (Mutti, Sinnott et al. 2011). The results of these
studies show that the peripheral retina plays a major role in regulating ocular
growth and may promote myopia progression; however, relative peripheral
15
hyperopic refraction may not be the stimulus to generate myopia in an
emmetropic individual. Thus, the correction of relative peripheral hyperopia may
inhibit further myopic progression; however, it may not be an effective preventive
treatment for myopia development.
Retinal neurons and their role in ocular growth
The establishment of local retinal control of eye growth has led
researchers to investigate which retinal cells are responsible for the regulation of
vision-guided ocular growth. Among the candidates are:
Photoreceptors and Bipolar Cells: Photoreceptors are the primary light
detecting neuron of the eye. Two toxins with purported specificity for damaging
photoreceptors, formoguanamine and tunicamycin, have been used to examine
the role of photoreceptors in ocular growth. In newly hatched chickens,
formoguanamine has been shown to damage the retinal pigment epithelium and
photoreceptor layer by inhibiting mitochondrial ornithine amino transferase,
causing subsequent blindness (Obara, Matsuzawa et al. 1985) (reviewed in
Crewther 2000). The eyes of formoguanamine-treated chickens do not develop
lid suture myopia, indicating that phototransduction is required for the eye to
develop deprivational myopia; however, loss of photoreceptor function does not
lead to inhibited ocular growth in itself as formoguanamine-treated eye become
no larger than non-sutured control eyes (Oishi and Lauber 1988; Lauber and
Oishi 1990). Tunicamycin, an inhibitor of dolichylphosphate-mediated protein
glycosylation, has been reported to be a toxin that is selective for the
16
photoreceptor layer of the retina (Fliesler, Rapp et al. 1984; Anderson, Williams
et al. 1988). Experiments examining ocular growth after intraocular administration
of tunicamycin display similar results when compared to formoguanamine. As
with formoguanamine, eyes treated with tunicamycin do not respond to formdeprivation; however, eyes that were treated with tunicamycin without formdeprivation did not show changes in vitreous chamber depth when compared to
control animals (Ehrlich, Sattayasai et al. 1990). The results of these studies that
attempt to selectively ablate photoreceptors can be difficult to interpret given that
these agents may not be truly specific to the photoreceptor layer. While
formoguanamine has been shown to be neurotoxic to photoreceptors, it is not
truly photoreceptor specific as it also damages the retinal pigment epithelium.
The effects of formoguanamine may have dramatic effects on vision-guided
ocular growth. In his tunicamycin study, Ehrlich reported that the treatment
showed significant damage to cell types across the retina, making his
interpretation that photoreceptors may play a critical role in form-deprivation
myopia tenuous. Given the results of these experiments, photoreceptors do not
appear to play a significant role in regulating ocular growth overall; however, it
appears that some photoreceptor function is required for the eye to detect the
visual clues that drive vision-guided ocular growth.
Bipolar cells have also been suggested as potential modulators of ocular
growth given their role in the ON/OFF center-surround receptive field
organization of the retina. Beyond their function in the vertical transmission of
17
visual signal from photoreceptor to ganglion cells, bipolar cells play a critical role
in contrast detection (reviewed in Lawrence and Azar 2002). In humans,
abnormal ON- and OFF-bipolar cell function has been detected in myopic eyes
with multifocal electroretinogram (mfERG) (Chen, Brown et al. 2006).
Furthermore, alteration of bipolar cell function and development of refractive error
has been reported in animal models. In cats, inhibiting ON-bipolar cell response
via intravitreal injections of D, L-2-amino-4-phosphonobutyric acid (a.k.a. L-AP4
or APB) caused reduced axial lengths and hyperopic refractive errors; indicating
a potential role for ON-bipolar cells and emmetropization (Smith, Fox et al. 1991).
L-AP4 has also been shown to cause reduced axial length in chickens and to
suppress the ERG ON-response; as well as render the eye insensitive to formdeprivation (Crewther, Crewther et al. 1996; Fujikado, Hosohata et al. 1996).
Alteration of ON- and OFF-bipolar cell function through the use of the glial toxins
levo-aminoadipic acid (ON-bipolar cells) and dextro-aminoadipic acid (OFFbipolar cells) has also been examined. L-aminoadipic acid reduced ocular growth
in unoccluded eyes compared to controls; however, it enhanced form-deprivation
myopia. Conversely, D- aminoadipic acid caused enhanced ocular growth in
unoccluded eyes but inhibited form-deprivation myopia (Crewther and Crewther
1990). In macaques, PKCα-positive ON-bipolar cells alter regulation of Erg1, an
immediate-early gene associated with ocular growth in animals, in response to
plus lenses or form-deprivation (Zhong, Ge et al. 2004).
18
Amacrine cells: The retinal neurons that are the most likely modulators of
ocular growth are amacrine cells. While photoreceptors and bipolar cells cannot
be dismissed outright due to their fundamental role in visual signal transduction,
the role of amacrine cells in temporal visual signal processing appears to be
particularly well suited for regulating ocular growth. Furthermore, amacrine cells
are the most diverse population of neurons present in the retina, with over 20
different types identified in the primate and human retina (Mariani 1990; Kolb,
Linberg et al. 1992). Experiments that focus on the ablation of sub-classes of
amacrine cells through the use of exogenous neurotoxins have demonstrated the
profound effect these cells have in visually-guided ocular growth. Among the
early toxins used in an attempt to selectively ablate target populations of retinal
neurons was kainic acid. Kainic acid is an excitatory neurotoxin that acts at
kainate ionotropic glutamate receptors, which was reported in early experiments
to selectively ablate amacrine cells without damaging bipolar cells, ganglion cells
and photoreceptors (Ehrlich and Morgan 1980; Morgan and Ingham 1981;
Ingham and Morgan 1983; Wildsoet and Pettigrew 1988; Huettner 2003).
However, kainic acid in excessive doses has been shown to be neurotoxic to
biopolar cells and horizontal cells (Wildsoet and Pettigrew 1988). Kainic acid
when applied in low doses (2 nanomoles) in chickens without form-deprivation
did not affect ocular growth compared to control eyes; however, in larger doses
(20 and 200 nanomoles), kainic acid-treated eyes became significantly larger
(Wildsoet and Pettigrew 1988). Kainic acid has also been reported restrict form19
deprivation induced growth in chickens (Ehrlich, Sattayasai et al. 1990).
However, given the widespread damage reported with the use of kainic acid in
the retinal neurons which were ablated, these studies become difficult to
interpret. These early studies did demonstrate that ocular growth could be
altered through the use of exogenously-applied toxins and the overall ocular
growth could be separated from vision-guided ocular growth
Further attempts were made to selectively ablate populations of retinal
neurons with a variety of toxins, primarily in the chicken. Among the toxins
investigated have been quisqualic acid, N-methyl-D-aspartate (NMDA),
ethylcholine mustard aziridinium (ECMA) and colchicine. Examination of these
studies revealed that some toxins could cause significant enlargement of the eye
while making the eye unresponsive to form-deprivation; however, other toxins did
not affect the ability of the eye to respond to vision-guided ocular growth stimuli.
For example, quisqualic acid has been reported to lesion amacrine cells,
horizontal cells and disrupt the outer segments of photoreceptors; however, it
does not affect the development of form-deprivation myopia (Barrington,
Sattayasai et al. 1989; Ehrlich, Sattayasai et al. 1990). Another toxin,
ethylcholine mustard aziridinium ion (ECMA), performed in a similar manner to
quisqualic acid in that eyes treated with ECMA did not show abnormal ocular
growth and form-deprivation myopia was not affected (Fischer, Miethke et al.
1998). In contrast, treatment with NMDA caused ocular enlargement; however,
the eyes became unresponsive to form-deprivation (Fischer, Seltner et al. 1997;
20
Fischer, Seltner et al. 1998). Moreover, treatment with colchicine in early
postnatal chicks leads to excessive eye growth and myopia; however, colchicinetreated eyes did not increase further in eye size or in myopia when placed under
form-deprivation (Fischer, Morgan et al. 1999). This would seem to indicate that
each toxin damaged subtly different populations of retinal neurons; moreover,
some toxins affected neurons that are required to induce vision-guided ocular
growth.
The identification of Egr1 and Glucagon as potential eye growth
modulators
Based on the results of chemical ablation studies, retinal targets that
classically were associated with ocular growth, such as cholinergic and
dopaminergic amacrine cells were eliminated as the primary modulators of
vision-guided ocular growth. Despite significant damage to type I, II and III
cholinergic amacrine cells in retinas treated with quisqualic acid, quisqualatetreated eye continue to respond to form-deprivation (Fischer, Miethke et al.
1998). Conversely, colchicine treatment spares most cholinergic amacrine cells,
yet colchicine-treated eyes show excessive growth and no longer respond to
form-deprivation (Fischer, Seltner et al. 1998). Dopaminergic amacrine cells have
also been suggested as the primary modulators of ocular growth. Treatment with
colchicine has been shown to cause a loss of 90% of the dopaminergic amacrine
cells, as identified via tyrosine hydroxylase immunoreactivity (Fischer, Morgan et
al. 1999). Given that colchicine-treatment causes excessive ocular growth, the
21
loss of dopaminergic amacrine cells could play a significant role in regulating the
growth of the eye. This theory does not explain the varying effects that quisqualic
acid and NMDA have on ocular growth. Neither quisqualic acid nor NMDA
treatment damages dopaminergic amacrine cells; however, quisqualate treated
eyes continue to respond to form-deprivation while NMDA-treated eyes lose this
ability. If dopaminergic amacrine cells were the primary modulators of visionguided ocular growth, NMDA treated eyes should continue to respond to formdeprivation. The results of these various chemical ablation studies ruled out
various populations of amacrine cell that had been proposed as regulators of eye
growth. If a resident population of amacrine cells were major modulators of
ocular growth, they should be relatively insensitive to quisqualic acid and ECMA;
however, they should be sensitive to NMDA- and colchicine-induced damage
(Fischer, Seltner et al. 1998).
Two observations regarding the effect of NMDA on ocular growth led to a
discovery that demonstrated that amacrine cells were indeed the major
modulators of ocular growth. The first observation, discussed previously, was the
effect of NMDA on ocular growth and the subsequent insensitivity to formdeprivation. The second observation was that NMDA receptor antagonists
dextromethorphan, MK801, and AP5 inhibit form-deprivation myopia (Fischer,
Seltner et al. 1998). Noting that the activation of NMDA receptors often lead to
activation of intermediate-early genes, Fischer examined the regulation of the
immediate-early genes ZENK (a.k.a. Zif268, Egr1, NGFI-A, Krox-24) and Fos in
22
amacrine cells under growth-stimulating paradigms. This led to the discovery that
in chickens a specific class of amacrine cells was responsible for regulating
ocular growth. Glucagon-positive amacrine cells, representing 1% of the total
amacrine cell population in chickens, have been shown to respond to visual
signals by up- or down- regulating ZENK, the avian ortholog of early growth
response 1 (Egr1) gene (Fischer, McGuire et al. 1999). Egr1 is an inducible
nuclear protein of the immediate- early gene family with zinc-finger DNA-binding
domains (Fischer, McGuire et al. 1999). These genes, which include Egr1, Fos
and Jun, show quick, short-term transient responses to stimuli. Activation of
immediate-early genes leads to the production of transcriptional products that
activate late response genes, which induce longer lasting effects (reviewed in
Hughes and Dragunow 1995)).
Figure 1.2: Egr1 expression is up-regulated in chicken retinas recovering from
form-deprivation (panel b, large arrows) compared to untreated control eyes
(panel a, small arrows) (Ritchey, Code et al. 2009)
23
Egr1 appears to play a critical role in the control of ocular growth in
chickens regardless of whether the eyes have undergone experimental
manipulation. The expression of Egr1 has been observed in untreated eyes, with
approximately 40% of glucagon-positive amacrine cells co-expressing Egr1. The
active expression of Egr1 in untreated eyes indicates that Egr1 potentially plays
an active role in normal eye growth and emmetropization. Of significant
importance, the work of Fischer et al. was the first demonstration that a specific
retinal neuron was sensitive to form-deprivation stimuli and directional lensinduced retinal blur. Specifically, the percentage of glucagon-positive amacrine
cells expressing Egr1 in the chick retina changes dramatically with visual
manipulation. Egr1 synthesis was down-regulated by growth-promoting stimuli;
both hyperopic defocus and form deprivation. Conversely, Egr1 synthesis was
up-regulated by growth-suppressing stimuli; both myopic defocus and the
presentation of unobstructed vision with the discontinuation of form-deprivation
(Figure 1.2). This up-regulation of Egr1 in glucagon-positive amacrine cells is
consistent with the observed action of intermediate-early genes, with Egr1
expression levels increasing in as little as 30 minutes after goggle removal. This
is not to say, however, that Egr1 expression is limited to glucagon-positive
amacrine cells. Egr1 has been shown to be present in presumptive cone ONbipolar cells under control conditions; moreover, Egr1 expression levels in
presumptive cone ON-bipolar cells change with levels of retinal illumination.
Müller glia also express Egr1 in response to NMDA treatment. However, the
24
changes in the expression of Egr1 in bipolar cells or in Müller glia were not
associated with detection of vision-guided ocular growth stimuli. Combining the
work of Fischer et al. with observations of Egr1 in myopia development in other
mice and monkeys helps to build a compelling argument for the role of Egr1 in
the regulation of ocular growth. Egr1 knockout mice have been shown to develop
myopic eyes compared to wild-type animals. Moreover, form-deprivation has
been shown to down-regulate Egr1 expression in mice (Brand, Schaeffel et al.
2007; Schippert, Burkhardt et al. 2007). Focus dependent activation of retinal
neurons has also been detected in primates. In macaques, Egr1 expression was
shown to be down-regulated by form-deprivation in GABAergic amacrine cells
and some bipolar cells, while plus lens treatment increased Egr1 expression
(Zhong, Ge et al. 2004). Despite these findings, further studies are required to
fully elucidate the role of Egr1 gene regulation in ocular growth.
25
Figure 1.3: Application of lenses alters retinal Egr1 expression, glucagon
synthesis and subsequent ocular growth
Changes in Egr1 regulation in glucagon-positive amacrine cells with visual
manipulation have been associated with changes in glucagon peptide production
in chickens. This observed up- or down-regulation in peptide production in
coordination with Egr1 regulation has led to investigation of glucagon as a
potential modulator of eye growth. Glucagon is a highly-conserved peptide
consisting of 29 amino acids and is produced by multiple organ systems,
including the pancreas, intestines and nervous system by cleavage of the
immediate polypeptide precursor proglucagon (Drucker and Asa 1988)
(reviewed in Kieffer and Habener 1999). Visual manipulation studies have shown
that glucagon production can be altered, with decreased retinal glucagon levels
26
after minus lens wear. Eyes treated with plus lenses show increased levels of
glucagon mRNA in the retina (Feldkaemper and Schaeffel 2002; Buck, Schaeffel
et al. 2004). When treated with a glucagon antagonist, des-His1-Glu1-glucagonamide, the effect of plus lenses on eye growth was diminished; however, the
antagonist did not alter the effect of minus lens wear (Feldkaemper and Schaeffel
2002). Conversely, the ocular growth observed with minus lenses treatment can
be prevented by concurrent application of a glucagon-receptor agonist,
Lys17,18,Glu21-glucagon, in a dose-dependent manner (Feldkaemper and
Schaeffel 2002). Attempts to supplement retinal glucagon levels through
injections of exogenous glucagon have been shown to disrupt ocular growth from
form-deprivation. Moreover, application of the glucagon antagonist [desHis(1),des- Phe(6),Glu(9)]-glucagon-NH(2) inhibited the growth-modulating effect
of plus lenses (Vessey, Lencses et al. 2005; Vessey, Rushforth et al. 2005).
Despite the strong evidence in the chick model for glucagon being a
significant modulator of ocular growth, the relationship between Egr1 and
glucagon in mammals continues to be poorly understood. Glucagon expression
in mammals has been examined primarily in mouse models. The link between
Egr1 and glucagon in mice has been shown to be tenuous at best. While Egr1
expression has been identified in the mouse retina, glucagon peptide was not
detected. Several peptides related to glucagon were identified; however, the
expression of these peptides did not change with visual manipulation (Mathis and
Schaeffel 2007). Conversely, myopia and increased axial length has been
27
detected in Egr1-knockout mice when compared to wild-type animals (Schippert,
Burkhardt et al. 2007). In primates, the evidence for glucagon is far less
compelling. Although Egr1 gene expression has been shown to change in
response to visual manipulation; these changes were associated with GABAergic
amacrine cells and presumptive ON-bipolar cells in macaques (Zhong, Ge et al.
2004). Furthermore, retinal glucagon has not been detected in macaques,
suggesting a potential evolutionary split between avian and primates (Andy J.
Fischer, personal communication). This evidence indicates that although Egr1
expression in mammals may have a role in the regulation of eye growth; the
glucagon peptide is most likely not the peptide modulating ocular growth in these
animals.
The role of insulin as a stimulator of ocular growth
Insulin has also been proposed as a regulator of ocular growth. It is well
established that glucagon and insulin play opposite roles in regulating blood
glucose levels; with insulin decreasing blood glucose levels and glucagon acting
in opposition (reviewed in Brockman 1978). Given that glucagon levels are
altered by different visually-guided ocular growth paradigms; combined with
glucagon’s ability to prevent form-deprivation and minus lens-induced ocular
growth, there has been speculation that insulin may play a role in promoting
excessive ocular growth. Specifically, it has been hypothesized that the observed
increase in the prevalence of diabetes due to changing dietary habits, particularly
28
to a Westernized diet from a hunter-gatherer diet, may alter insulin signaling and
be a significant risk factor for myopia development (Cordain, Eaton et al. 2002;
Prada, Zecchin et al. 2005). Animal models have provided some preliminary
evidence that may support a role for insulin in the regulation of ocular growth.
Exogenously-delivered insulin has been shown to stimulate the proliferation of
retinal progenitors in the circumferential marginal zone of chickens (Fischer and
Reh 2000). Conversely, exogenous glucagon and glucagon-like peptide 1
(GLP1) suppress proliferation of peripheral retinal progenitors (Fischer, Omar et
al. 2005). When combined with lenses in chickens, insulin blocked the
development of hyperopia with plus lens wear and exacerbated negative lensinduced myopia (Feldkaemper, Neacsu et al. 2009); as well as caused increased
axial length without visual manipulation (Zhu and Wallman 2009). In guinea pigs,
exogenous recombinant human IGF-2 did not promote increased axial length in
eyes without visual manipulation; however, it did enhance form-deprivation
myopia (Deng, Tan et al. 2010).
Despite the preliminary evidence for insulin promoting ocular growth, the
exact role insulin plays in ocular growth is yet to be determined as various
discrepancies must be addressed. For example, patients with diabetes without
retinopathy have been shown to have no difference in axial length when
compared to matched, non-diabetic patients (Pierro, Brancato et al. 1999). This
contrasts reports linking insulin and insulin-like growth factor (IGF) in humans
with myopia. Patients with type-I diabetes and elevated HbA1C levels have been
29
shown to have a statistically higher risk of being myopic (Jacobsen, Jensen et al.
2008); furthermore, short-nucleotide IGF-1 polymorphisms have been associated
with myopia in humans (Metlapally, Ki et al. 2010). In animal models,
Feldkaemper and Deng report that exogenous insulin and IGF did not promote
ocular growth in eyes without visual manipulation; however, Zhu reported ocular
growth. Moreover, the observed changes in axial length reported by both
Feldkaemper and Zhu were due to increases in anterior chamber depth and not
vitreous chamber depth (Feldkaemper, Neacsu et al. 2009; Zhu and Wallman
2009). Given the differences observed between studies and the apparent affect
of insulin and IGF on the anterior chamber of the eye, more investigation into
how insulin fits as a potential mechanism for myopia development is warranted.
Retinoic Acid: A sign-sensitive modulator of ocular growth?
Retinoic acid has also been examined as a modulator of ocular growth,
due to work that indicates that retinoic acid levels alter in response to the
direction of visual manipulation. Examination of chickens demonstrated that
stimulating ocular growth through form-deprivation increased levels of retinoic
acid in the retina (Seko, Shimizu et al. 1998) and levels of retinoic acid receptor
in the sclera (Seko, Shimokawa et al. 1996). Moreover, inhibition of retinoic acid
synthesis using disulfiram inhibits form-deprivation myopia (Bitzer, Feldkaemper
et al. 2000). Chickens also showed directional shifts in retinoic acid in the
choroid. Choroidal retinoic acid levels decreased in form-deprivation and minus
30
lens treated eyes, whereas choroidal retinoic acid levels increase with removal of
form-deprivation goggles or plus lens treatment (Mertz and Wallman 2000). In
guinea pigs, form-deprivation and minus lens wear caused increases in retinal
retinoic acid levels and decreases with plus lens wear or discontinuation of formdeprivation, similar to that observed in chickens (McFadden, Howlett et al. 2004).
However, choroidal levels of retinoic acid in guinea pigs were opposite those
observed in the chicken, with form-deprivation increasing retinoic acid levels and
recovery from form-deprivation decreasing retinoic acid levels (McFadden,
Howlett et al. 2004).
Although the regulation of retinoic acid appears to be a potential player in
the regulation of ocular growth, the role of retinoic acid in human myopia remains
uncertain. Examination of the response of retinoic acid regulation to formdeprivation in marmosets suggests that retinoic acid may have a significant role
in myopia development. In a subset of marmosets that developed myopia with
form-deprivation, increases in retinoic acid levels were observed in the retina and
the choroid, along with decreased scleral glycosaminoglycan synthesis. In a
subset of marmosets that did not become myopic with form-deprivation, no
changes in retinoic acid levels were observed in the retina or choroid (Troilo,
Nickla et al. 2006). Retinoic acid has also been shown to regulate levels of
Fibulin-1, a protein associated in tissue remodeling, in cultured human scleral
fibroblasts (Li, McFadden et al. 2010). Conversely, investigations into genes
associated with retinoic acid in humans have failed to find an association with
31
myopia. Examination of single nucleotide polymorphisms (SNPs) in the gene for
All-trans-retinol dehydrogenase (RDH8), an enzyme in retinoic acid metabolism,
failed to find an association with high myopia in a set of Chinese subjects (Yu,
Wang et al. 2010). Moreover, SNPs variants in the genes for retinoic acid
receptor alpha or beta were not associated with myopia (Veerappan, Schache et
al. 2009; Ding, Chen et al. 2010). Although there is evidence for retinoic acid
playing a role in regulation of ocular growth in animal models, more research into
the role of retinoic acid and human myopia is required.
Post-retinal tissue and ocular growth
While the retina has been shown to alter gene regulation in response to
form-deprivation and lens wear, changes in post-retinal ocular tissue are required
to alter axial length and develop refractive error. Both the choroid and the sclera
have both been shown to change in response to visual modulation of ocular
growth. It has been postulated that the role of the observed changes in the
choroid are to compensate for the lag in the response time in remodeling of the
sclera to visually-guided ocular growth clues (reviewed in Wallman and Winawer
2004). It has been clearly demonstrated that the thickness of the choroid
changes in response to vision-guided ocular growth clues. Moreover, the
observed changes occur in a manner directional to the induced defocus. When
presented with a growth-promoting stimulus that would move the focus of
incoming light behind the retina, such as hyperopic defocus, or in situations that
prevent a clear foci of light on the retina, such as in form-deprivation, choroidal
32
thickness decreases in an attempt to place the image on the retina. Conversely,
when presented with a growth-slowing stimulus that would move the foci of light
in front of the retina, choroidal thickness increases. As the vascular tunic of the
eye, rapid changes in choroidal thickness are possible by altering tissue
hydration levels when compared to the sclera (Junghans, Crewther et al. 1999;
Kee, Marzani et al. 2001). Indeed, choroidal blood flow has been shown to
significantly decrease in eyes that undergo form-deprivation (Shih, Fitzgerald et
al. 1993). When form-deprivation goggles are removed, choroidal thickness
increases rapidly to partially compensate for the induced myopia (Irving,
Callender et al. 1995; Wallman, Wildsoet et al. 1995). This response has been
shown to be a local phenomenon, as the choroidal will thicken in the
corresponding area placed under partial form-deprivation with a hemi-occlusion
goggle (Wallman, Wildsoet et al. 1995). Choroidal thickness changes have also
been demonstrated with plus and minus lens wear (Wallman, Wildsoet et al.
1995; Wildsoet and Wallman 1995). The observed response of the choroid to
brief periods of visual stimuli occur rapidly, in as short as a few minutes to hours
(Zhu and Wallman 2009) (Zhu, Park et al. 2005). This response has been
demonstrated to be highly conserved across many species, including chickens,
guinea pigs, marmosets and macaques (Hung, Wallman et al. 2000; Troilo,
Nickla et al. 2000; Howlett and McFadden 2009; Lu, Zhou et al. 2009) (reviewed
in Wallman and Winawer 2004). These observed changes in choroidal thickness
clearly indicate that retinal detection of the visual stimuli that signal changes in
33
ocular growth are transmitted to post-retinal ocular tissue; however, the exact
mechanism of transmission remains poorly understood.
Although the choroid has been shown to alter its thickness in response to
visual stimuli, the development of irreversible axial myopia ultimately requires
changes to the sclera. This is reflected in human myopia, where excessive axial
length is associated with the vast majority of myopic patients (Zadnik 1997).
Given the necessary changes in the ocular dimensions of the eye in response to
the maturation of the animal and the matching of axial length to the refractive
elements of the eye, the sclera cannot be a rigid tissue. This is observed in
humans with excessive amounts of myopia, which are associated with posterior
staphyloma, a thinning and distension of the sclera (Curtin 1977). In these
extremely myopic patients, the development of posterior staphylomas does not
appear to be simply a matter of passively stretching the sclera. Importantly,
morphological changes in scleral composition have been observed in these
patients (Curtin, Iwamoto et al. 1979; reviewed in McBrien and Gentle 2003). In
myopic patients, thinning of collagen fibrils and fiber bundles has been observed
(reviewed in Rada, Shelton et al. 2006).
It is important to note that the observed changes in scleral composition do
not appear to be a simple secondary effect of myopia. The sclera has been
shown to be a dynamic, elastic tissue that undergoes remodeling in response to
visual stimuli, assuming the stimuli are provided for a significant amount of time
(Wang, Shih et al. 1997). This observation comes from examination of multiple
34
animal models; however, when examining these models, one must acknowledge
the differences in scleral composition among species. A division exists in the
structure of the sclera between placental mammals and other vertebrate animals.
In placental mammals, such as humans, the sclera is a single fibrous structure
(a.k.a. the fibrous sclera); whereas in other vertebrates it is composed of two
distinct layers, one fibrous and one cartilaginous (reviewed in Rada, Shelton et
al. 2006). The difference in scleral composition leads to differences in the
response to growth-inducing stimuli across species. Most notably, chickens show
growth in the cartilaginous layer with myopia development (Gottlieb, Joshi et al.
1990; Rada, Thoft et al. 1991; Rada and Matthews 1994; Rada, Matthews et al.
1994; reviewed in Rada, Shelton et al. 2006). However, the fibrous layer of the
sclera responds in a manner similar to that observed in placental mammals
(Gottlieb, Joshi et al. 1990). Although certain animal models have a cartilaginous
layer, such as the chicken, which differs from humans, this does not mean that
these animal models can be dismissed when considering the scleral response to
ocular growth. For example, evidence suggests that evolutionary remnants of the
cartilaginous sclera remain in placental animals and may play an important role
in the regulation of scleral function (Rada, Shelton et al. 2006). Importantly,
thinning of the posterior fibrous sclera with induced myopia is observed in
multiple species. The thinning of the sclera appears to be bimodal, with the first
phase demonstrating rapid early thinning and a second, less rapid phase
demonstrating changes in collagen fiber diameter (McBrien, Cornell et al. 2001).
35
Glycosaminoglycan and collagen production decreases, while matrix
metalloproteinase production increases to actively degrade collagen, leading to
extension of the sclera and myopia development (reviewed in McBrien and
Gentle 2003).
The signaling mechanisms involved in modulating sclera changes are
unclear; however, it is generally accepted that the retina & choroid are involved in
the pathway. Visual signals have been shown to promote substantial changes in
the sclera (Christensen and Wallman 1991; Marzani and Wallman 1997; Gentle
and McBrien 1999). In vitro studies have suggested that the RPE can alter
scleral chondrocyte proliferation in chicks (Seko, Tanaka et al. 1994). Several
molecules have been suggested as potential chemical messengers from the
retina & choroid to the scleral, such as fibroblast growth factor-2 (FGF-2),
transforming growth factor beta (TGF-β) and retinoic acid; however, no definitely
chemical messenger/s have been identified as the primary agents for sclera
change in ocular growth (Rohrer and Stell 1994; Seko, Tanaka et al. 1995; Seko,
Shimokawa et al. 1996; Mertz and Wallman 2000; reviewed in McBrien and
Gentle 2003).
Potential pharmacological modulators of ocular growth?
Atropine and Pirenzepine: Pharmacological agents targeting muscarinic
acetylcholine receptors have been the subject of intense investigation due to the
early theories postulating that the accommodative system played a significant
36
role in myopia development. The non-specific muscarinic antagonist atropine
was one of the first potential pharmacological interventions identified, due to its
well established cycloplegic effects. Early primate research indicated that
atropine could indeed be an effective treatment for induced myopia. The work of
Francis Young showed that myopic progression in macaques raised in a
simulated near-vision only environment was effectively retarded through the use
of topical atropine (Young 1965; reviewed in Edwards 1996) (reviewed in Smith
1998). This work, combined with reports that atropine reduced myopic
progression or may prevent myopia development in pre-myopic children
suggests that the cholinergic system is a key modulator of ocular growth (Gimbel
1973; Bedrossian 1979; Dyer 1979; Brodstein, Brodstein et al. 1984; Chou, Shih
et al. 1997; Shih, Chen et al. 1999; Fang, Chung et al. 2010). Although atropine
is effective at slowing myopic progression, this ability appears to act via nonaccommodative mechanisms. Studies examining the use of bifocal or
progressive addition lenses (PALs) to relax accommodative effort to slow myopic
progression, as well as the deliberate under-correction of myopic patients, have
shown variable results. Some studies indicate that the use of bifocal lenses or
PALs may provide some protective effect from myopic progression; however
other studies show no protective effect (Grosvenor, Perrigin et al. 1987; Leung
and Brown 1999) (reviewed in Saw, Gazzard et al. 2002). More troubling was the
finding that the treatment effects of these interventions were minimal and
transient in nature (Fulk, Cyert et al. 2002; Gwiazda, Hyman et al. 2003).
37
Furthermore pirenzepine, an M1-preferential cholinergic antagonist, has also
been reported to slow myopia progression in humans, despite having a minimal
response on accommodation due to mediation of accommodation through the
M3-muscarinic receptor (Pang, Matsumoto et al. 1994; Siatkowski, Cotter et al.
2004; Tan, Lam et al. 2005; Siatkowski, Cotter et al. 2008). Animal models
further suggest that the effect of muscarinic antagonists is separate from
accommodation. For example, atropine and pirenzepine inhibit form-deprivation
myopia in chickens; despite the fact that chickens lack a homologue to the M-1
receptor in mammals and nicotinic acetylcholine receptors control their
accommodative response (McBrien, Moghaddam et al. 1993; Luft, Ming et al.
2003; Yin, Gentle et al. 2004; McBrien, Arumugam et al. 2011). This evidence
provides a compelling argument that muscarinic antagonists modulate ocular
growth through a non-accommodative mechanism.
Given that muscarinic antagonists alter ocular growth through a nonaccommodative mechanism, animal models offers potential insights into the
potential targets of action. Evidence for retinal sites of action has been observed
in chickens. In chickens subjected to form-deprivation or minus lens wear,
intravitreal injection of atropine prevented down-regulation of mRNA transcript
levels in the retina for Egr1, suggesting a possible interaction of muscarinic
antagonists with the glucagoneric system (Ashby, McCarthy et al. 2007; Ashby,
Kozulin et al. 2010). Furthermore, application of the M-4 selective muscarinic
antagonist MT-3 in chicks reduced form-deprivation myopia in a dose dependent
38
manner, while the M-1 selective antagonist MT-7 had no effect (McBrien,
Arumugam et al. 2011). It has also been suggested that muscarinic antagonsists
act directly on extra-retinal tissue. In scleral cultures, atropine was shown to
inhibit scleral cell proliferation. Atropine, and to a lesser extent pirenzepine, were
also shown to inhibit chondrocyte matrix production in these cultures (Lind, Chew
et al. 1998). Deither also proposes that atropine acts via a scleral mechanism
rather than through retinal receptors (Diether and Schaeffel 1999). In mouse
eyes fitted with –10DS lenses, gene expression levels for M-1, M-3 and M-4
receptors in the sclera were upregulated with atropine treatment, whereas
expression levels for M-2 and M-5 were decreased (Barathi and Beuerman
2011). Despite this evidence, the site of action for muscarinic antagonists
remains unknown and further research is warranted to determine if these agents
work through retinal or extra-retinal targets.
Dopamine: Dopamine has been suggested as a potential modulator of
ocular growth. Early experiments in the chicken and rhesus monkeys revealed
that form-deprivation decreased retinal levels of dopamine and dopamine
synthesis (Stone, Lin et al. 1989; Stone, Lin et al. 1990). After removal of formdeprivation goggles, retinal dopamine levels increase (Pendrak, Nguyen et al.
1997). A similar trend has been observed with lens-induced ametropias, where
retinal dopamine levels increase with myopic defocus and decrease with
hyperopic defocus, suggesting that ocular growth corresponds to reductions in
dopamine (Guo, Sivak et al. 1995). Injection of dopamine into form-deprived eyes
39
has been shown to prevent myopia and attempts to negate ocular growth from
form-deprivation or minus lens wear through the use of apomorphine, a nonselective dopamine receptor agonist, have been successful in animal models
(Iuvone, Tigges et al. 1991; Schmid and Wildsoet 2004; Gao, Liu et al. 2006).
Furthermore, intraperitoneal injection of the dopamine precursor L-DOPA in a
guinea pig has been shown to reduce form-deprivation myopia and offset the
reduction of retinal dopamine levels typically observed with form-deprivation
(Mao, Liu et al. 2010). This work suggests that retinal dopamine levels are
intimately associated with regulation of ocular growth.
The proposed site of action for dopaminergic control of eye growth acts is
the D2 dopamine receptor, as determined primarily through the use of D-2
receptor selective antagonists. Concurrent application of spiperone, a D2receptor selective antagonist, was shown to reduce the ability of apomorphine to
prevent form-deprivation myopia (Rohrer, Spira et al. 1993). Sulpiride, another
D2-selective dopamine antagonist, enhances form-deprivation myopia in
chickens (Schaeffel, Bartmann et al. 1995). Despite evidence pointing to the D-2
receptor as a potential modulator of ocular growth, depletion of retinal dopamine
through the use of 6-hydroxydopamine (6-OHDA), a dopamine-depleting
neurotoxin, casts some doubt on the importance of dopamine in regulating eye
growth. In chickens, myopia did not develop under form-deprivation conditions
after depletion of retinal dopamine with 6-OHDA. Conversely, 6-OHDA treatment
did not alter spectacle lens induced ocular growth (Li, Schaeffel et al. 1992;
40
Schaeffel, Hagel et al. 1994). Furthermore, altering retinal dopamine levels
through illumination provide disparate results. Chickens exposed to constant light
showed a reduction in retinal dopamine levels; however, they were unresponsive
to form-deprivation (Bartmann, Schaeffel et al. 1994). When treated with plus or
minus lenses under high illuminace levels, the rate growth with minus lens wear
was slowed and the effects of plus wear enhanced; however, despite alterations
in growth rate, the animals fully compensated for the induced ametropia (Ashby
and Schaeffel 2010). Ultimately, these results suggest that the retinal
dopaminergic system may be a regulator of ocular growth; however, the
mechanism is poorly understood and more research is required to clarify its role
in myopia development.
Summary
Our understanding of the mechanisms that regulate vision-guided ocular
growth has improved greatly over the past 40 years. The discovery that the
regulation of ocular growth is intrinsic to the eye itself had led to discovery that
ocular growth is most likely regulated by retinal amacrine cells. In at least one
animal model, a specific sub-type of amacrine cell has been shown to alter gene
regulation in response to induced ametropias. Furthermore, the peripheral retina
appears to play a significant role in detecting the sign of defocus in animals and
in humans. The detection of these visual signals leads to subsequent transient
changes in the choroid and permanent changes in the sclera. The application of
41
receptor agonists and antagonists have helped to determine the potential role the
retinal cholinergic and dopaminergic systems play in the regulation of eye
growth. Despite these discoveries, the factors that lead humans to fail to
emmetropize are poorly understood. Moreover, questions remain as to the site of
action for many growth modulating factors. Continued research into the
mechanisms that regulate ocular growth is required before an effective
intervention to retard the development or progression of myopia in humans can
be discovered.
42
Chapter 2: The combination of IGF1 and FGF2 and the induction of
excessive ocular growth and extreme myopia
Abstract
Recent studies have indicated that insulin and IGF1 exacerbate the
excessive ocular growth that results from form deprivation- and lens-induced
myopia (Feldkaemper, Neacsu et al. 2009; Zhu and Wallman 2009). Here we
investigate whether the combination of insulin-like growth factor 1 (IGF1) and
fibroblast growth factor 2 (FGF2) influence rates of ocular growth in eyes with
unrestricted vision. FGF2 combined with different doses of IGF1 were injected
into the vitreous chamber of postnatal chicks. Measurements of ocular
dimensions and intraocular pressure (IOP) were made during and at the
completion of different treatment paradigms. In addition, histological and
immunocytochemical analyses were performed to assess cell death, cellular
proliferation and the integrity of the retina, ciliary body, lens and cornea. Treated
eyes had significant increases equatorial diameter, vitreous chamber depth, and
lens thickness, whereas anterior chamber depth was decreased. Treated eyes
developed extreme myopia, in excess of 20 diopters of refractive error. Lower
doses of IGF1 and FGF2, that increased eye size, did not result in acute retinal
damage, whereas higher doses of IGF1 and FGF2 resulted in widespread
43
degeneration of ganglion cells and glial reactivity. Shortly after treatment, eyes
had angle closure accompanied by increased (IOP). The combination of IGF1
and FGF2 increased IOP in a dose-dependent manner, whereas IGF1 alone,
FGF2 alone and the combination of insulin and FGF2 had no effect on IOP.
Seven days after treatment with IGF1 and FGF2 changes to anterior chamber
depth and elevated IOP were no longer present, whereas increases in the
vitreous chamber were persistent. We conclude that the extreme myopia in
IGF1/FGF2-treated eyes results from increased vitreous chamber depth,
decreased anterior chamber depth, and changes in the lens and cornea. We
propose that factor-induced ocular enlargement result, in part, from long-lasting
changes to the sclera.
Introduction
Eye growth is a complex, well-coordinated process with an endpoint of
minimal to no refractive error; a process known as emmetropization. In most, if
not all, vertebrate species, the eyes have significant refractive error at the time of
birth (reviewed in Wallman and Winawer 2004). During adolescence, the eye
grows so that the length of the vitreous chamber precisely matches the combined
refractive power of the lens and cornea to eliminate refractive error. Humans
undergo active emmetropization; however we differ from other vertebrate species
in that a significant number of individuals fail to emmetropize and, consequently,
develop myopia (nearsightedness). Myopia commonly results from elongation of
44
the vitreous chamber which is determined by the growth of the sclera; the
connective tissue sheath of the eye.
In animal models, attenuated clear vision has been shown to cause
excessive ocular growth and myopic refractive errors. This model is known as
form-deprivation myopia (FDM). The requirement for clear vision is wellconserved across species including chicks, marmosets, tree shrews, mice,
monkeys, and rabbits (Wiesel and Raviola 1977; Wallman, Turkel et al. 1978;
Norton 1990; Troilo and Judge 1993; Guo, Sivak et al. 1995; Tejedor and de la
Villa 2003) (reviewed in Wallman and Winawer 2004). It is currently believed that
the retina detects defocus cues and liberates factors that accelerate or
decelerate the growth of the sclera (reviewed in Wallman and Winawer 2004)).
The retina-derived factors that regulate eye growth remain poorly understood.
Several peptide and amino acid-derived neurotransmitters in the retina have
been implicated as growth-regulators in animal models of myopia (reviewed in
Wallman and Winawer 2004). In the chick model system, the most promising
candidate for a retina-derived signal that regulates ocular growth is glucagon
peptide. We have reported that the glucagon-expressing retinal amacrine cells
(GACs) respond to growth-slowing visual stimuli (plus-defocus and recovery from
FDM) by up-regulating the immediate early gene Egr1 (Fischer, McGuire et al.
1999). By contrast, the GACs respond to growth-accelerating visual cues by
down-regulating Egr1 (Fischer, McGuire et al. 1999). Additional reports have
indicated that growth-guiding visual cues influence retinal levels of glucagon
45
mRNA (Feldkaemper and Schaeffel 2002), and that agonists and antagonists to
the glucagon receptor influence rates of ocular growth in form-deprived and lenstreated eyes (Feldkaemper and Schaeffel 2002; Vessey, Lencses et al. 2005;
Fischer, Ritchey et al. 2008). Recent studies have indicated that exogenous
insulin and Insulin-like growth factor 1 (IGF1) can exacerbate the excessive
ocular growth that results from form-deprivation, and can counter-act the growthslowing effects of glucagon (Feldkaemper, Neacsu et al. 2009; Zhu and Wallman
2009). However, injections of insulin and IGF1 into eyes with unrestricted vision
have little effect upon ocular growth (Feldkaemper, Neacsu et al. 2009; Zhu and
Wallman 2009). In addition, a recent study has identified genetic associations
between IGF1 polymorphisms and high-grade myopia in humans (Metlapally, Ki
et al. 2010), consistent with the hypothesis that insulin and IGF1 have important
roles in regulating rates of ocular growth and the pathogenesis of myopia.
The mechanisms underlying insulin/IGF1-induced changes in ocular growth
remain uncertain. The identity of the cells that express receptors, signaling
pathways and the types of cells that directly respond to insulin and IGF1 have
recently been studied. Feldkaemper and colleagues (2009) reported that the
expression of the immediate early gene Egr1 near the middle of the INL suggests
that the Müller glia may respond to insulin. We have reported that intraocular
injections of insulin selectively stimulate the Müller glia to accumulate Egr1, cFos
and low levels of pERK, whereas retinal neurons do not appear to respond
(Fischer, Scott et al. 2010). By comparison, IGF1 stimulates the Müller glia to
46
accumulate p38 MAPK and cFos, whereas retinal neurons do not appear to
respond (Fischer, Scott et al. 2010). In addition, we have reported that insulin
and IGF1 stimulate the reactivity of microglia and a novel type of glial cell, termed
Non-astrocytic Inner Retinal Glial (NIRG) cells (Fischer, Scott et al. 2009;
Fischer, Scott et al. 2010), and stimulate the proliferation of retinal progenitors in
the circumferential marginal zone (CMZ) and non-pigmented epithelium of the
pars plana (Fischer and Reh 2000; Fischer, Dierks et al. 2002; Fischer and Reh
2003). Collectively, these findings indicate that the effects of insulin and IGF1 are
subtly different, but are centered on the retinal glia. The possibility remains that
insulin and IGF1 act at extra-retinal tissues to influence rates of ocular growth.
Another growth factor that may be involved in regulating ocular growth is
fibroblast growth factor (FGF). Rohrer and Stell (1994) reported that low doses
(<5ng) of FGF1 inhibit FDM, whereas high doses (~300ng) cause retinal
degeneration that includes the loss of photoreceptors and detachment from the
RPE (Rohrer and Stell 1994). However, it has been our experience, and that of
others (Hicks 1998; LaVail, Yasumura et al. 1998; Valter, Maslim et al. 1998;
Chaum 2003), that FGF’s is potently neuroprotective for different types of retinal
cells including photoreceptors and that these neuroprotective actions may be
elicited through the Müller glia (Fischer, Scott et al. 2009). Through the course of
studies into effects of IGF1 and FGF2 on retinal glia and neuronal survival, we
noticed that treated eyes appeared significantly larger than contra lateral control
eyes. Thus, the purpose of the current study was to investigate how the
47
combination of IGF1 and FGF2 influence ocular growth. Some of this work has
been presented at the 13th International Myopia Conference (Tuebingen,
Germany) (Tarutta, Chua et al. 2011).
Animals:
The use of animals in these experiments was in accordance with the
guidelines established by the National Institutes of Health, the ARVO Statement
for the Use of Animals in Ophthalmic and Vision Research, and the Ohio State
University. Fertilized eggs and newly hatched wild type leghorn chickens (Gallus
gallus domesticus) were obtained from the Department of Animal Sciences at the
Ohio State University. The stage of the chick embryos was determined according
to the guidelines established by Hamburger and Hamilton (Hamburger 1951).
Postnatal chicks were kept on a cycle of 12 hours light, 12 hours dark (lights on
at 8:00 AM). Chicks were housed in a stainless steel brooder at about 25 oC and
received water and chick starter (Purina, St. Louis, MO) ad libitum.
Intraocular injections:
Intraocular injections were made as described in previous reports
(Fischer, Seltner et al. 1998; Fischer, Morgan et al. 1999). In short, chicks were
anesthetized by inhalation of 2.5% isoflurane in O2 at a flow rate of 1.5 L/min.
Injections were made with a 25 l Hamilton syringe and 26G needle with a
cutting tip. The needle was consistently inserted into the dorsal quadrant of the
vitreous chamber thought the pars plana and dorsal eye lid, which was sterilized
48
with iodine solution. Daily injections were made between 1 and 2 pm. For all
experiments, the left eyes of chicks were injected with the “test” compound and
the contra-lateral eyes were injected with vehicle as a control. Compounds were
injected in 20 l sterile saline with 0.05 mg/ml bovine serum albumin added as
carrier. Compounds included insulin (1 ug per dose; Sigma-Aldrich, St. Louis,
MO), IGF1 (50 to 800 ng per dose; Cell Signaling Technology, Danvers, MA),
FGF2 (200ng per dose; Cell Signaling Technology). Two g of BrdU was
included with each injection to label proliferating cells.
Reverse transcriptase PCR:
Retinas from two P7 chicks were pooled and placed in 1.5 ml of Trizol
Reagent (Invitrogen; Carlsbad, CA) and total RNA was isolated according to the
Trizol protocol and resuspended in 50 μl RNAse free water. Genomic DNA was
removed by using the DNA FREE kit provided by Ambion (Austin, TX). cDNA
was synthesized from mRNA by using Superscripttm III First Strand Synthesis
System (Invitrogen) and oligo dT primers according to the manufacturer’s
protocol. PCR primer sequences are listed in table 2.1. Primers were designed
by using the Primer-BLAST primer design tool at NCBI
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR reactions were performed
by using standard protocols, Platinumtm Taq (Invitrogen) or TITANIUMtm Taq
(Clontech; Mountain View, CA) and an Eppendorf thermal cycler. PCR products
were run on a 1.2% agarose gel to verify the predicted product sizes. PCR
49
products were excised from the gels, extracted, purified (Qiaex II kit, Qiagen;
Valencia, CA), and sequenced to verify the identity of the products. Control
reactions were performed using all components with the exception of the reverse
transcriptase to exclude the possibility that primers were amplifying genomic
DNA.
Target mRNA
Sequence
Insulin receptor
forward TTG CCA TAA TCA TTG CTG GA
reverse CCT CTG TTT CCA GAG GCT TG
forward GGC AAA GCT GAC ACA TCT GA
reverse TCC AGG TCA AGC TCC TCT GT
forward GCT GGA TGT GAA GCA GAC AA
reverse GCC TCC CAG TTC TCT CTG TG
forward ATA ACA CCA AGC CGA ACC AG
reverse GGC AGC TCA TAC TCG GAG AC
forward GCT GTC CAC AAG CTG ACA AA
reverse GGT GCA GTT GGC AGG TTT AT
forward TTG GCC TTG CTA GAG ACG TT
reverse AGG GCA GTA CCC TCA GGT TT
forward GGA ACA CTA TAA AGG CGA
GAT
reverse TCA CAA GTT TCC CGT TCT CA
IGF1 receptor
IGF2 receptor
FGFR1
FGFR2
FGFR3
GAPDH
Predicted product
size
1065
1221
1199
970
948
854
218
Table 2.1: PCR primers (5’ to 3’), target and predicted product sizes
Fixation, sectioning and immunocytochemistry
Tissues were fixed, sectioned and immunolabeled as described previously
(Fischer, Omar et al. 2005; Fischer, Foster et al. 2008). Working dilutions and
sources of antibodies used in this study included are listed in table 2.2. Antigen
retrieval was used to permit immunolabeling for BrdU and PCNA, sections were
washed for 7 minutes in 4M HCl. Secondary antibodies included donkey-antigoat-Alexa488, goat-anti-rabbit-Alexa488, goat-anti-mouse-Alexa488/568, rabbit
50
anti-goat Alexa488 and goat-anti-mouse-IgM-Alexa568 (Invitrogen) diluted to
1:1000 in PBS plus 0.2% Triton X-100.
We evaluated the specificity of primary antibodies by comparison with
published examples of results and assays for specificity. None of the observed
labeling was due to non-specific labeling of secondary antibodies or
autofluorescence because sections labeled with secondary antibodies alone
were devoid of fluorescence. Secondary antibodies included goat-anti-rabbitAlexa488/568/647 and goat-anti-mouse-Alexa488/568/647 (Invitrogen) diluted to
1:1000 in PBS plus 0.2% Triton X-100.
Antigen
Working
dilution
1:2000
Host
rabbit
Clone or
catalog number
AB5535
Brn3a
(Pou4f2)
transitin
1:200
mouse
mab1585
1:80
mouse
EAP3
TopAP
1:100
mouse
2M6
Cleaved
caspase 3
Proliferating
cell nuclear
antigen
BrdU
1:500
rabbit
AF583
1:1000
mouse
M0879
1:200
rat
OBT00030S
BrdU
1:100
mouse
G3G4
Sox9
Table 2.2: Antibodies, sources and working dilutions
TUNEL:
51
Source
Millipore
Billerica, MA
Millipore
Billerica, MA
Developmental Studies
Hybridoma Bank (DSHB)
Iowa City, IA
Dr. P. Linser
University of Florida (Fischer,
Miethke et al. 1998; Fischer,
Seltner et al. 1998)
R&D Systems
Minneapolis, MN
Dako Immunochemicals
Carpinteria, CA
AbD Serotec
Raleigh, NC
DSHB
To identify dying cells that contained fragmented DNA we used the
TUNEL method. We used an In Situ Cell Death Kit (TMR red; Roche Applied
Science; Indianapolis, IN), as per the manufacturer’s instructions.
Retinoscopy and refraction:
We used trial lenses and streak retinoscopy to measure refractive error in
control and treated eyes, similar to previous reports (Fischer, Miethke et al. 1998;
Fischer, Seltner et al. 1998). Retinoscopy was performed by one individual to
prevent inter-individual variability.
Measurement of Intraocular Pressure (IOP):
A TonoLabtm tonometer was used to measure IOP. The device was used
with the factory-set calibration for rat eyes, similar to recent reports (Morrison, Jia
et al. 2009; Pease, Cone et al. 2010)
Microscopy, measurements, cell counts and statistics:
Photomicrographs were obtained using a Leica DM5000B microscope
equipped with epifluorescence and a Leica DC500 digital camera. Confocal
images were obtained using a Zeiss LSM 510 imaging system at the Hunt-Curtis
Imaging Facility at the Ohio State University. Images were optimized for color,
brightness and contrast, multiple channels overlaid and figures constructed by
using Adobe Photoshop™6.0. Cell counts were performed on representative
52
images. To avoid the possibility of region-specific differences within the retina,
cell counts were consistently made from the same region of retina for each data
set. Central retina was as assessed within 20o of the posterior pole of the eye,
with a radius of approximately 1.3 mm. Peripheral retina was considered at
approximately 2.5 mm from the peripheral retinal margin, or about 65 o eccentric
to the posterior pole of the eye. Postnatal chick retina is approximately 13 mm
across.
To account for inter-individual variability of eye size, statistical analyses
were performed for the differences between measurements of treated and control
eyes for each individual. Thus, significance of difference (p-values) for the
magnitude of interocular change was calculated for the mean differences from
zero by using a one-tailed student’s t-test.
Measurements of eye size:
Photographs of enucleated eyes were taken using a 6.1 megapixel Nikon
D100 SLR camera. High resolution digital images (>50 pixels/mm) of enucleated
eyes were measured by using Image Pro Plus 6.2 (Media Cybernetics;
Bethesda, MD). Measurements obtained using Image Pro Plus 6.2 were highly
reproducible and had low levels of sampling error (± 0.22%) at a resolution of 20
pixels/µm or greater, similar to prior descriptions (Fischer, Ritchey et al. 2008).
Corneal arc was measured as the linear distance, on profile, across the cornea
from ventral limbus to dorsal limbus.
53
High-resolution A-scan ultrasonography was used to measure corneal
thickness, anterior chamber depth, lens thickness and vitreous chamber depth
along the optical axis. Corneal anesthesia was achieved using one drop of
topical 0.5% proparacaine hydrochloride ophthalmic solution. After insertion of a
4mm-barraquer pediatric lid speculum, a 20 MHz Panametrics-NDT (Waltham,
MA) transducer with a polystyrene delay line offset (V208-RM) driven by a
Panametrics-NDT 5072 pulser-receiver was coupled to the corneal apex using
ultrasound coupling gel (Medline Industries, Inc.; Mundelein, IL). The acoustic
reflections were collected and digitized using a PicoScope® 5203 USB-PC
oscilloscope and the PicoScope® 6 PC Oscilloscope software, version 6.3.43.0.
Ultrasonic radio frequency (RF) signals were first filtered with a low-pass filter at
the cutoff frequency of 80MHz to exclude high frequency noise. The envelope of
the signals was then extracted using the analytic signal magnitude (Gammell
1981). Peaks corresponding to anterior cornea surface and retina were selected
for axial length measurement. Times-of-flight were determined and converted to
distance assuming the constant speed of sound of 1540m/s. It is noted that the
speed of sound in cornea and lens may be slightly higher that the assumed
value. Since the goal of the present study was to compare the thickness and
depths between control and treated tissues, the assumption of a uniform speed
of sound should have minimal influence on the outcome measures.
54
Results
Expression of receptors in different ocular tissues:
The expression of receptors for insulin and IGF1 in different ocular tissues
remains uncertain. In a previous study, using RT-PCR, we failed to detect a
splice variant of the insulin receptor in retinal cells, whereas this variant was
readily detected in the liver (Fischer, Scott et al. 2009). By comparison, using a
different set of primers for the insulin receptor, we detect mRNA for the insulin
receptor in the choroid+RPE and sclera, whereas faint bands for insulin receptor
were detected in the retina, ciliary body and lens (Figure 2.1). Similar to the
insulin receptor, we detected IGF1R and IGF2R in many different ocular tissues
(Figure 2.1). Transcripts for IGF1R and IGF2R were detected in the retina,
choroid+RPE, lens fiber cells and sclera (Fig. 1). IGF1R was not detected in the
ciliary body+lens capsule, whereas IGF2R was detected (Figure 2.1). All
isoforms of the FGF receptor were detected in all tissues of the eye; FGFR1,
FGFR2 and FGFR3 were detected in the retina, choroid+RPE, ciliary body, lens
and sclera (Figure 2.1). Currently, there is no known homologue to FGFR4 in the
chick.
55
Figure 2.1: Receptors to insulin, IGF and FGF are widely expressed in different
ocular tissues. RT-PCR was used to detect transcripts to the insulin receptor,
IGF1 receptor, IGF2 receptor, FGFR1, FGFR2 and FGFR3. PCR was performed
on cDNA pools obtained from retina, choroid+RPE, ciliary body+lens capsule,
lens fiber cells and sclera. Abbreviations: FGFR – fibroblast growth factor
receptor, IGF – insulin-like growth factor, GAPDH - Glyceraldehyde 3-phosphate
dehydrogenase, RPE – retinal pigmented epithelium.
Effects of high doses of IGF1 and FGF2 on eye size and retinal integrity:
In previous studies, we applied consecutive daily injections of 800ng IGF1
to stimulate retinal glia, including Müller glia, microglia and NIRG cells (Fischer,
Scott et al. 2010). Thus, we began by applying 800ng-doses of IGF1. Consistent
with prior reports (Feldkaemper, Neacsu et al. 2009; Zhu and Wallman 2009), we
56
found that 3 consecutive daily intraocular injections of 800ng IGF1 had no
significant effect upon ocular growth (difference (treated-control) in axial length
0.04 ± 0.41 mm; p=0.94). By contrast, when 800ng IGF1 was injected with 200ng
FGF2 there were significant increases in eye size; including increases in
equatorial diameter and axial length, which were accompanied by increases in
corneal circumference and arc (Figure 2.2a-c). These eyes were extremely
myopic, with more than 20 diopters of refractive error; significant myopic
refractions persisted in treated eyes after correction with 20 diopter lenses.
57
Figure 2.2: Three consecutive daily intraocular injections of 800ng IGF1 and
200ng FGF2 stimulate excessive ocular growth. Injections began at P5, ended
at P7, and measurements were performed on enucleated eyes at P8.
Measurements of enucleated eyes were made from digital images using
ImagePro6.2. Measurements of eyes (n=6) were made on axis (a) and on profile
(b). The histogram illustrates the mean (±SD) difference in ocular dimension
(mm; c). Measurements included corneal circumference (corneal circ), corneal
arc, equatorial circumference (eq circ), dorsal-ventral equatorial diameter (D-V eq
diameter), and axial length. Statistical significance (*p<0.05, **p<0.001, n=6)
was determined by using a paired, one-tailed student’s t-test.
We found that retinas were damaged in eyes treated with 800ng IGF1 and
200ng FGF2. In situ labeling of fragmented DNA revealed numerous dying cells
in the proximal INL and in the GCL in treated eyes (Figure 2.3a and 2.3b). In
addition, we found that Sox9-expressing Müller glia underwent nuclear migration
and up-regulated transitin, the avian homologue of mammalian nestin, in treated
58
retinas (Figure 2.3c-2.3f). In normal retinas, the nuclei of Sox9-positive Müller
glia were tightly stratified in the middle of the INL, and levels of transitin in Müller
glia were low (Figure 2.3c and 2.3e). Reactive and proliferating Müller gliaderived progenitor cells are known to undergo interkinetic nuclear migration
(Fischer and Reh 2001; Fischer 2005; Fischer and Bongini 2010) and transiently
up-regulate transitin (Fischer and Omar 2005). Consistent with the hypothesis
that treatment with 800ng IGF1 and 200ng FGF2 stimulates proliferation, we
observed many cells that expressed proliferating cell nuclear antigen (PCNA) in
the INL (Figure 2.3g and h); in Sox9-positive Müller glia-derived progenitors (not
shown). In addition, we observed PCNA-positive cells scattered across the IPL
and GCL (Figure 2.3g and 2.3h); these cells expressed Nkx2.2 (not shown)
indicating that these cells were non-astrocytic inner retinal glial (NIRG) cells
(Fischer, Scott et al. 2010). Since significant numbers of dying cells were
detected in the GCL we probed for the integrity of ganglion cells by
immunolabeling for Pou4f2 (Brn3a); a member of the POU-family of transcription
factors that are known to be expressed by the vast majority of ganglion
cells(Xiang, Zhou et al. 1993). Immunolabeling for Pou4f2 indicated that
treatment with 800ng IGF1 and 200ng FGF2 caused damage and loss of
ganglion cells; the remaining ganglion cells appeared atrophic with shrunken
nuclei (Figure 2.3i and 2.3j).
59
Figure 2.3: Treatment with 3 consecutive daily injections of 800ng IGF1 and
200ng FGF2 causes cell death, glial migration and proliferation within the retina.
Retinas were harvested 1 day after the last of 3 consecutive daily injections of
factors. Vertical sections of central regions of the retina were labeled for
fragmented DNA using the TUNEL method (a and b) or with antibodies to Sox9
(c and d), transitin (e and f), PCNA (g and h) or Pou4f2 (I and j). Arrows indicate
dying cells (b), reactive Müller glia (d), proliferating Müller glia (h) or abnormal
ganglion cell nuclei (j). Arrow-heads indicate putative NIRG cells that are PCNApositive (h). The insets (i’ and j’) in panel j are 2-fold enlargements of the boxedout regions in i and j. The calibration bar (50 µm) in panel j applies to all panels.
Abbreviations: INL – inner nuclear layer, IPL – inner plexiform layer, GCL –
ganglion cell layer, PCNA – proliferating cell nuclear antigen.
60
Effects of low doses of IGF1 and FGF2 on eye size and retinal integrity:
The widespread cell death observed with 3 consecutive daily injections of
800ng IGF1 and 200ng FGF2 complicates the interpretation of mechanisms
stimulating excessive eye growth. Different types of toxins that destroy retinal
neurons are known to cause ocular enlargement (Wildsoet and Pettigrew 1988;
Ehrlich, Sattayasai et al. 1990; Fischer, Seltner et al. 1997; Fischer, Morgan et al.
1999). Thus, we sought to determine whether lower doses of IGF1 in
combination with FGF2 influenced ocular growth. Accordingly, we tested whether
4 consecutive daily intraocular injections of 200ng IGF1 and 200ng FGF2
influenced the size and shape of the eye, and the “health” of retinal cells. Eyes
treated with 200ng IGF1 and 200ng FGF2 were significantly larger than contralateral control eyes (Figure 2.4a and 2.4b). Measurements from digital images
and ultrasonography indicated significant increases in equatorial diameter, axial
length, and vitreous chamber depth (Figure 2.4c and 2.4g). In addition, we
observed increases in corneal circumference and corneal arc, but not corneal
radius of curvature (Figure 2.4d). Measurements using A-scan ultrasound
indicated significant increases in axial length, vitreous chamber depth and lens
thickness in eyes treated with 200ng IGF1 and 200ng FGF2 (Figure 2.4g). By
contrast, ultrasound measurements indicated significant decreases in anterior
chamber depth (Figure 2.4g).
61
Figure 2.4: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 stimulate excessive ocular growth. Injections began at P5, ended
at P8, and ocular measurements were performed at P9 (a-d and g) or P15 (e,f
and h). Measurements were made by using ImagePro6.2 on digital images of
enucleated eyes (a-f) and by using A-scan ultrasonography (g and h).
Histograms represent the mean and standard deviation for data-sets.
Measurements using ImagePro6.2 included nasal-temporal equatorial diameter
(N-T diameter), dorsal-ventral equatorial diameter (D-V diameter), equatorial
circumference, axial length, corneal circumference, corneal arc, and corneal
radius. Measurements using A-scan ultrasound included axial length, vitreous
chamber depth, anterior chamber depth and lens thickness. Statistical
significance was determined by using a paired, one-tailed student’s t-test (n= 5 to
10).
62
Figure 2.4
63
We next tested whether the effects of IGF1 and FGF2 on changes in
ocular dimensions were transient or long-lasting. IGF1/FGF2-mediated changes
in axial length, namely vitreous chamber depth, and equatorial circumference
were maintained at 7 days after treatment (Figure 2.4e, f and g). These increases
in axial length, vitreous chamber depth and equatorial circumference were not
significantly different from those seen at 1 day after treatment (compare Figures
2.4c,d,g to 2.4e,f,h). In addition, we found persistent increases in corneal
circumference and arc (Figure 2.4f), however these increases were diminished in
amplitude compared to those seen at 1 day after treatment (Figure 2.4d). By
contrast, decreases in anterior chamber depth and increases in lens thickness
had disappeared by 7 days after treatment (Figure 2.4h). We found that 4
consecutive daily injections of 200ng FGF2 alone have no effects upon ocular
growth (difference in axial length (treated-control) 0.08 ± 0.29 mm; p=0.71),
consistent with previous reports (Rohrer and Stell 1994). Further, 4 consecutive
daily injections of 200ng of IGF1 alone have no effects upon ocular growth
(difference in axial length (treated-control) 0.08 ± 0.42 mm; p=0.91), consistent
with previous reports (Feldkaemper, Neacsu et al. 2009; Zhu and Wallman
2009). Similarly, the combination of 1000ng insulin and 200ng FGF2 did not
significantly influence ocular size (difference in axial length (treated-control) 0.05
± 0.37 mm; p=0.78). Collectively, these findings indicate that the IGF1 and FGF2
act synergistically to stimulate ocular growth.
64
One day after the last injection of 200ng IGF1 and 200ng FGF2, we
examined the retina and found no indication of cell death (Figure 2.5a and 2.5b),
reactivity of Müller glia (Figure 2.5c and 2.5d), or pyknotic ganglion cell nuclei
(Figure 2.5e and 2.5f). There was no difference in cell death or glial reactivity in
central or peripheral regions of the retina (not shown). We next tested whether
injections of 200ng IGF1 and 200ng FGF2 had any long-lasting effects on the
retina. At 7 days after the injection, we detected a few scattered TUNEL-positive
cells in peripheral regions of the GCL (Figure 2.5g and 2.5h). TUNEL-positive
cells were not detected in central regions of treated retinas (data not shown).
Consistent with the distribution of TUNEL-positive cells at 7 days after treatment,
most (8/10) of the IGF1/FGF2-treated eyes contained regions of peripheral retina
that had reduced numbers of Pou4f2-positive ganglion cells (Figure 2.5i and
2.5j). By comparison, there was no detectable depletion of Pou4f2-positive
ganglion cells in central regions of treated retinas (not shown). Seven days after
treatment with IGF1 and FGF2, the nuclei of Müller glia were delaminated away
from the center of the INL (Figure 2.5l-n). In peripheral regions of treated retinas,
Sox9-positive Müller glia-derived cells were found scattered across the INL and
in the ONL (Figure 2.5n). The de-lamination of somata of Müller glia occurs with
the reactivity and/or proliferation that can occur with retinal damage or growth
factor treatment (reviewed in Fischer 2005; Fischer and Bongini 2010). Retinas
treated with IGF1 alone or FGF2 alone showed no indications of ganglion cell
loss or proliferating Müller glia (data not shown), consistent with previous reports
65
(Fischer, McGuire et al. 2002; Fischer, Scott et al. 2009; Fischer, Scott et al.
2010).
66
Figure 2.5: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 do not have short-term effects upon cell death, glial reactivity or the
integrity of ganglion cells, whereas the long-term survival of ganglion cells in the
peripheral retina is compromised. Injections of IGF1 and FGF2 began at P5,
ended at P8, and tissues were harvested at P9 (a-f) or P15 (g-n). Vertical
sections of control (a,c,e,g,i and k) and treated retinas (b,d,f,h,j and l-n) were
labeled for TUNEL (a,b,g and h), Sox9 (c,d and k-n), transitin (c and d), and
Pou4f2 (e,f,i and j). Arrows indicate labeled nuclei. The calibration bar (50 µm) in
panel n applies to all panels. Abbreviations: INL – inner nuclear layer, IPL – inner
plexiform layer, GCL – ganglion cell layer, IOP – intraocular pressure.
67
Effects of IGF1 and FGF2 on the lens, ciliary body and anterior chamber:
Eyes treated with IGF1 and FGF2 developed extreme myopic shifts with
refractive error in excess of 20 diopters over only 4 days of treatment. These
measurements were made using trial lenses and a slit-lamp retinoscope; strong
myopic reflexes remained for all treated animals corrected with 20 diopter lenses.
This extreme myopia likely resulted from both increases in vitreous chamber
depth and increased refractive power of the lens and/or cornea. To better assess
factor-induced changes in the anterior segment, we examined anterior ocular
structures in transverse sections at the level of the pupil. Treatment with IGF1
and FGF2 significantly reduced anterior chamber depth and caused angle
closure (Figure 2.6a-d). These changes were apparent in all animals (n=7).
Measurements from histological sections revealed that at 7 days after treatment
with IGF1 and FGF2 the anterior chamber depth was not significantly different
from control eyes (Figure 2.6e-i), consistent with ultrasound measurements of
intact eyes (Fig. 4). By comparison, measurements from histological sections
revealed that at 7 days after treatment with IGF1 and FGF2 the angle remained
significantly narrower compared to the angle of control eyes (Figure 2.6i).
However, the mean narrowing of the angle at 7 days after treatment was not as
significant as the narrowing at 1 day after treatment (Figure 2.6d and 2.6i).
Histological observations revealed persistent changes in the lens capsule (Figure
2.6e-g). The lens capsule of treated eyes appeared abnormal, filled with
vacuoles (Figure 2.6e-g). In most (8/10) of the IGF1/FGF2-treated eyes, an over68
growth of non-pigmented epithelial (NPE) cells persisted within the zonules that
attached to the lens (indicated by arrows in Figure 2.6e-g). There was variability
in the long-lasting effects of IGF1 and FGF2 on anterior chamber collapse and
hypertrophy of NPE cells in the pars plicata (compare Figure 2.6g and 2.6h).
Consistent with ultrasound measurements (see Figure 2.4h), histological
measurements at 7 days after treatment indicated that there was no significant
difference in anterior chamber depths between control and treated eyes (Figure
2.6h). At 7 days after treatment, the angle remained significantly narrower than in
eyes treated with IGF1 and FGF2 compared to the angle of control eyes (Figure
2.6i). The variation in measurements of anterior chamber depth and the angle
was large in the treated group (Figure 2.6h and 2.6i).
69
Figure 2.6: Four consecutive daily intraocular injections of 200ng IGF1 and
200ng FGF2 dramatically change the anterior segment of the eye. Sections were
obtained from control eyes (a, a’ and e) and from eyes treated with IGF1 and
FGF2 (b, b’, f and g) and were stained with hematoxylin and eosin. The arrows
indicate the zonules that attach to the lens, straight lines indicate the angle,
double-ended arrows indicate the anterior chamber depth. Histograms indicate
the mean and standard deviation (n=6) for anterior chamber depth (mm; c and h)
and angle (degrees; d and i). Significance of difference (p-value; n=5) was
determined using a two-tailed student t-test.
70
Figure 2.6
71
To better understand IGF1/FGF2-induced changes in the retina, lens and
zonules we tested whether proliferation occurred. Proliferating cells were
identified by probing for the expression of PCNA and accumulation of BrdU.
Consistent with previous reports (Fischer and Reh 2000; Fischer, Dierks et al.
2002; Fischer and Reh 2003), we found that the combination of IGF1 and FGF2
stimulated the proliferation of progenitors in the CMZ at the peripheral edge of
the retina (Figure 2.7a and 2.7b). In eyes treated with IGF1 and FGF2, we did not
find a significant induction of proliferation of cells within the iris or anterior lens
epithelium (Figure 2.7c and 2.7d). By contrast, we found numerous proliferating
cells within equatorial regions of the lens epithelium and within NPE of the ciliary
folds that are adjacent to equatorial regions of the lens (Figure 2.7e and 2.7f).
These findings suggest that at least some of the changes seen in the anterior
segment of eyes treated with IGF1 and FGF2 result from the proliferation of cells.
72
Figure 2.7: Intraocular injections of 200ng IGF1 and 200ng FGF2 stimulate the
proliferation of cells in far peripheral regions of the retina, progenitors in the CMZ,
equatorial regions of lens capsule, and non-pigmented epithelial cells in the
zonules. Sections were labeled with DRAQ5 (nuclei; blue), PCNA (red) and BrdU
(green). Sections were obtained from control eyes (a, c and e) and eyes 1 day
after the last of 4 consecutive daily injections of IGF1 and FGF2 (b, d and f). The
calibration bar (50 µm) in panel b applies to panels a and b, the bar in d applies
to c and d, and the bar in f applies to e and f. Abbreviations: CMZ –
circumferential marginal zone, PCNA – proliferating cell nuclear antigen.
73
Effects of IGF1 and FGF2 on intraocular pressure (IOP):
The angle closure observed in IGF1/FGF2-treated eyes (Figure 2.6b and
2.6d) suggested that IOP might be elevated. Accordingly we used a TonoLabtm
rebound tonometer to measure IOP in control and treated eyes. In control eyes
(n=20) the IOP was 19.4 ± 3.1 mm Hg. The IOP was not significantly elevated in
eyes treated with saline, 200ng IGF1 alone, 200ng FGF2, the combination of
insulin and FGF2, or 50ng IGF1 + 200ng FGF2 (Figure 2.8). By comparison, the
IOP was significantly elevated by approximately 7 mm Hg in eyes treated with
200ng IGF1 + 200ng FGF2 and by approximately 11 mm Hg in eyes treated with
300ng IGF1 + 200ng FGF2 (Figure 2.8). All IOP measurements were made at 1
day after the last injection. Interestingly, the IOP of treated eyes returned to
normal levels by 7 days after the last injection (Figure 2.8). It must be noted that
in treated eyes where the anterior chamber depth has been diminished and the
lens is closely opposed to the cornea, the measurements of IOP may not
accurately represent the IOP within the vitreous chamber. Nevertheless, we feel
that the measured increases in IOP are representative of increased IOP, but we
cannot be certain that the increased IOP was indeed, on average, 11 mm Hg.
74
Figure 2.8: Intraocular injections of the combination of IGF1 and FGF2 increase
intraocular pressure (IOP) in a dose-dependent manner. Histograms indicate the
mean and standard deviation for the IOP of eyes treated with 4 consecutive daily
doses of 300ng IGF1 alone, 200ng FGF2 alone, 1µg insulin + 200ng FGF2, 50ng
IGF1 + 200ng FGF2, 200ng IGF1 + 200ng FGF2, or 300ng IGF1 + 200ng FGF2.
IOP measurements were made at 1 or 7 days after the last injection. Significance
of difference (p-value; n=6 at day1, n=5 at day 7) was determined using a twotailed student t-test.
Discussion
We report here that the combination of IGF1 and FGF2 has a profound
influence on ocular growth, the refractive state of the eye, and IOP. The mean
increases in axial length were more than 1.2 mm for 200 ng IGF1 and 200 ng
FGF2, compared to increases of nearly 2.5 mm for 800 ng IGF1 and 200 ng
FGF2. These IGF1/FGF2-induced increases in eye size over 4 days of treatment
are similar to those induced by 7 days of form-deprivation (Gottlieb, FugateWentzek et al. 1987; Wallman, Gottlieb et al. 1987; Vessey, Cottriall et al. 2002).
Interestingly, IGF1 alone, FGF2 alone, nor the combination of insulin and FGF2
75
influenced axial elongation, refractive error or IOP. Furthermore, we find that the
combination of IGF1 and FGF2 stimulates the reactivity of retinal glia, the
proliferation of CMZ progenitors, and hypertrophy of NPE cells in the ciliary body.
By comparison, the combination of insulin and FGF2 stimulates the reactivity of
retinal glia (Fischer, McGuire et al. 2002; Fischer, Scott et al. 2009), the
proliferation of CMZ progenitors (Fischer and Reh 2000; Fischer, Dierks et al.
2002), the hypertrophy of NPE cells in the ciliary body (Fischer and Reh 2003),
but not elevated IOP. The precise mechanisms underlying the synergistic
activities of IGF1 and FGF2 remain uncertain, and this combination of factors has
outcomes distinct from the combination of insulin and FGF2.
Insulin and IGF1 have similar effects upon retinal cells; activation of
microglia, NIRG cells and Müller glia (Fischer, Scott et al. 2009; Fischer, Scott et
al. 2010). The IGF1 receptor is expressed by cells scattered across the inner
retinal layers (IPL and GCL), NIRG cells and/or microglia, but not by cells in the
inner and outer nuclear layers (Fischer, Scott et al. 2010). With Müller glia,
microglia and NIRG cells stimulated by IGF1, there are elevated levels of cell
death and wide-spread focal retinal detachments in response to an excitotoxic
insult (Fischer, Scott et al. 2010). The increased cell death was prominent within
areas of retinal detachment which were coincident with a stark loss of Müller glia
and an accumulation of NIRG cells. In the current study, we find evidence for
activation of Müller glia by the combination of IGF1 and FGF2; these factors
caused the delamination of Müller glia nuclei away from the center of the INL.
76
The presence of Sox9-positive glial nuclei in peripheral regions of the ONL is
consistent with the interkinetic nuclear migration and proliferation of Müller gliaderived progenitor-like cells that occurs in response to growth factors (Fischer,
McGuire et al. 2002; Fischer, Scott et al. 2010; Ghai, Zelinka et al. 2010). The
actions of IGF1 and FGF2 at extra-retinal cells within the eye remains poorly
understood and require further investigation. Our data indicate that receptors for
IGF1 and FGF2 are expressed by many different ocular tissues, including the
sclera. Furthermore, our data suggest that IGF1/FGF2-induced increases in axial
length and vitreous chamber depth are long lasting; implying long-lasting
changes in the sclera.
IGF1 and FGF2 may override or bypass the growth-slowing actions of the
glucagonergic amacrine and bullwhip cells to increase growth of the vitreous
chamber. Glucagonergic amacrine cells are known to differentially respond to
plus and minus defocus, produce increased levels of glucagon peptide in
response to growth-slowing visual stimuli, and glucagon peptide is known to
potently inhibit FDM and lens-induced myopia (Fischer, McGuire et al. 1999;
Bitzer and Schaeffel 2002; Feldkaemper and Schaeffel 2002; Fischer, Omar et
al. 2005; Vessey, Lencses et al. 2005; Fischer, Ritchey et al. 2008). In addition,
unconventional types of glucagon-expressing neurons, the bullwhip and minibullwhip cells, are known to respond to growth-guiding visual cues to coordinate
the addition of new neurons to the edge of the retina and equatorial eye growth
(Fischer, Omar et al. 2005; Fischer, Ritchey et al. 2008. We report here that IGF1
77
and FGF2 increase vitreous chamber depth and equatorial circumference in eyes
with unrestricted vision. In principle, plus defocus in IGF1/FGF2-enlarged eyes
should have activated the glucagonergic retinal neurons. The glucagonergic
amacrine cells and bullwhip cells appear to remain intact in retinas treated with
IGF1 and FGF2 (data not shown). It is possible that plus defocus stimulated
glucagonergic neurons to produce and release elevated levels of glucagon
peptide to diminish the growth-enhancing effects of IGF1 and FGF2.
Alternatively, the extreme myopia and extreme plus-defocus (>20 diopters) that
results from IGF1/FGF2-treatment may be beyond the capacity of retinal circuitry
to discriminate the sign of defocus from generic image blur (Schaeffel and
Diether 1999), and thereby fail to activate the growth-slowing activity of the
glucagonergic neurons. Future studies will need to investigate the activities of
glucagonergic retinal neurons in eyes treated with IGF1 and FGF2.
The sites of action and the retinal cells that are influenced by insulin, IGF1
and FGF2 have recently been investigated. We have reported how insulin and
FGF2 influence retinal cells (Fischer, McGuire et al. 2002; Fischer, Scott et al.
2009). Insulin and FGF2 induce the phosphorylation of ERK1/2, p38 MAPK and
CREB, and the expression of immediate early genes, cFos and Egr1.
Accumulations of pERK1/2, p38 MAPK, pCREB, cFos and Egr1 in response to
insulin alone or FGF2 alone are confined to Müller glia, whereas retinal neurons
do not appear to respond to growth factors. Unlike FGF2, insulin stimulates the
reactivity of NIRG cells and microglia (Fischer, Scott et al. 2009). Similar to
78
insulin, IGF1 selectively stimulates Müller glia to accumulate p38 MAPK and
cFos, but not pCREB or Egr1, and stimulates the reactivity of NIRG cells and
microglia (Fischer, Scott et al. 2009; Fischer, Scott et al. 2009; Fischer and
Bongini 2010; Fischer, Scott et al. 2010). Collectively, our findings indicate that
the primary site of action of insulin, IGF1 and FGF2 in the retina are the Müller
glia, NIRG cells and microglia. Given that glial proliferation and reactivity results
from many different growth factors (Fischer, Scott et al. 2009; Fischer, Scott et al.
2009; Fischer and Bongini 2010; Fischer, Scott et al. 2010 with no influence on
ocular growth, the effects of IGF1 and FGF2 on the retinal glia are unlikely to
have any effect upon axial elongation. The identity of extra-retinal cells that are
directly influenced by insulin, IGF1 and FGF2 requires further investigation.
It seems likely that rapid, transient increases in IOP resulted from acute
angle closure in eyes treated with IGF1 and FGF2. Angle closure is known to be
associated with acute increases in IOP (reviewed in Nongpiur, Ku et al. 2011).
However, we find that significant angle closure persisted for at least 7 days after
treatment, when IOP returned to normal levels. This finding suggests that IOP
may return to normal levels with a partial opening of the angle and/or that
aqueous production was decreased. The mechanisms underlying IGF1/FGF2induced angle closure remain uncertain. We propose that the hypertrophy of
NPE cells adjacent to the lens may cause a lateral expansion and physical,
anterior translocation of the ciliary body and iris to close the angle, and thereby
79
transient increase IOP. To the best of our knowledge, there is no precedent for
the over-growth of tissues flanking the lens to mechanically close the angle.
It is possible that some of the ocular expansion that we observed in
IGF1/FGF2-treated eyes occurs secondary to mechanical pressure imposed by
elevated IOP. However, we have not observed increased eye size with increases
in IOP induced by injections of polystyrene microbeads into the anterior chamber
to inhibit uveoscleral outflow (unpublished observations), suggesting that
elevated IOP may not be sufficient to drive ocular enlargement in the chick.
Conversely, Schmid and colleagues demonstrated that timolol-induced
decreases in IOP do not inhibit form deprivation- or lens-induced myopia
(Schmid, Abbott et al. 2000), suggesting that IOP does not influence visionguided ocular growth. Furthermore, we find that the vitreous chamber of
IGF1/FGF2-treated eyes remained enlarged at 7 days after treatment when IOP
had returned to normal levels. We propose that the persistent increases in the
vitreous chamber of IGF1/FGF2-treated eyes result from remodeling of the
sclera. Scleral remodeling likely underlies the long-lasting changes in vitreous
chamber elongation (Rada, Matthews et al. 1994; Rada, Achen et al. 1998;
Rada, Johnson et al. 2002). We find that receptors for IGF and FGF are
expressed by sclera cells, consistent with the hypothesis that IGF1 and FGF2
may be acting directly on the sclera. Thus, we propose that long-lasting
increases in the size of the vitreous chamber results from changes to the sclera,
and that IGF1 and FGF2 may act directly on sclera cells.
80
In eyes treated with IGF1 and FGF2 we observed ocular enlargement and
elevated IOP, and at some time between 1 and 7 days after treatment IOP
returned to normal, ganglion cells were lost from peripheral regions of the retina
and the Müller glia became reactive. In glaucomatous eyes, the loss of ganglion
cells is prevalent and begins in peripheral regions of the retina. Reports indicate
that ganglion cells in peripheral regions of the retina are more susceptible than
those found in central regions of the retina to increases in IOP (reviewed in Qu,
Wang et al. 2010). It remains uncertain whether the loss of ganglion cells in
IGF1/FGF2-treated retinas result directly from elevated IOP or other effects, such
as glial reactivity. However, glial reactivity likely follows the loss of ganglion cells
in peripheral retinal regions seen at 7 days, but not at 1 day, after treatment and
likely resulted from elevated IOP that persisted beyond 1 day after treatment with
IGF1 and FGF2. Alternatively, factor-induced induced activation microglia could
influence the ganglion cells. For example, IGF1 is known to stimulate retinal
microglia (Fischer, Scott et al. 2010) and activated microglia may have
detrimental effects upon the survival of ganglion cells (Huang, Li et al. 2007;
Nakazawa, Hisatomi et al. 2007). It is unlikely that the loss of ganglion cells in
peripheral retinal regions influences ocular growth given that significant growth
occurred before the loss ganglion cells and reports have indicated that ganglion
cells are not required for vision-guided ocular growth (Troilo, Gottlieb et al. 1987;
Norton, Essinger et al. 1994; McBrien, Moghaddam et al. 1995; Wildsoet 2003).
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Conclusions
Our findings indicate that the combination of IGF1 and FGF2, but neither
factor alone, had a significant impact upon myopic development, ocular
enlargement, angle closure, elevated IOP and retinal damage. Our findings
indicate that IGF1 and FGF2 act at multiple sites within the eye to influence eye
growth, IOP and neuronal survival in the retina. We conclude that IGF1 and
FGF2 act synergistically to have profound transient effects upon IOP, angle
closure and lens integrity, and long-lasting effects upon vitreous chamber
elongation and myopic refractive error.
Acknowledgements
I would like to acknowledge the significant contributions of Christopher
Zelinka, Junhua Tang, Dr. Jun Liu and Dr. Andy Fischer to this project. We thank
Drs. Paul Henion and Paul Linser for providing antibodies to transitin and 2M6,
respectively. We also thank Dr. Andy Hartwick for the use of the TonoLabtm
tonometer. The antibodies developed by Drs. S.J. Kaufman (BrdU), D.M.
Fambrough (Lysosomal membrane glycoprotein), and G. Cole (transitin) were
obtained from the Developmental Studies Hybridoma Bank, which was
developed under the auspices of the NICHD and is maintained by the University
of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Confocal
microscopy was performed at the Hunt-Curtis Imaging Facility at the Department
of Neuroscience of The Ohio State University. This work was supported by
grants (AJF: EY016043-05; ERR: K12EY015447) from the National Institutes of
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Health, National Eye Institute. This paper has been submitted to the journal
Investigative Ophthalmology & Visual Science for publication.
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Chapter 3: The pattern of expression of guanine nucleotide-binding protein
β3 (GNB3) in the retina is conserved across vertebrate species
Abstract
Guanine nucleotide-binding protein β3 (GNB3) is an isoform of the β
subunit of the heterotrimeric G protein second messenger complex that is
commonly associated with transmembrane receptors. The presence of GNB3 in
photoreceptors, and possibly bipolar cells, has been confirmed in murine, bovine
and primate retinas (Lee, Lieberman et al. 1992; Peng, Robishaw et al. 1992;
Huang, Max et al. 2003). Studies have indicated that a mutation in the GNB3
gene causes progressive retinopathy and globe enlargement (RGE) in chickens.
The goals of this study were to 1) examine the expression pattern of GNB3 in
wild-type and RGE mutant chickens, 2) characterize the types of bipolar cells that
express GNB3 and 3) examine whether the expression of GNB3 in the retina is
conserved across vertebrate species. We find that chickens homozygous for the
RGE allele completely lack GNB3 protein. We find that the pattern of expression
of GNB3 in the retina is highly conserved across vertebrate species, including
teleost fish (Carassius auratus), frogs (Xenopus laevis), chickens (Gallus
domesticus), mice (Mus musculata), guinea pigs (Cavia porcellus), dogs (Canis
familiaris) and non-human primates (Macaca fasicularis). Regardless of the
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species, we find that GNB3 is expressed by Islet1-positive cone ON-bipolar cells
and by cone photoreceptors. In some vertebrates, GNB3-immunoreactivity was
observed in both rod and cone photoreceptors. A protein-protein alignment of
GNB3 across different vertebrates, from fish to humans, indicates a high degree
(>92%) of sequence conservation. Given that analogous types of retinal neurons
express GNB3 in different species, we propose that the functions and the
mechanisms that regulate the expression of GNB3 are highly conserved.
Introduction
Guanine nucleotide-binding proteins (G-proteins) are heterotrimeric
proteins, composed of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits. Gproteins are second messengers that interact with 7-transmembrane domain
metabotropic receptors, also known as G-protein-coupled receptors (GPCRs)
(Cabrera-Vera, Vanhauwe et al. 2003; McCudden, Hains et al. 2005; Oldham
and Hamm 2008). Of the 5 different isoforms of the beta subunit that have been
recognized, the β1 and β3 subunits are known to be expressed by
photoreceptors in the retina; with β1 (GNB1) associated with rod photoreceptors
and β3 (GNB3), also known as β-transducin, associated with cone
photoreceptors (Lee, Lieberman et al. 1992; Peng, Robishaw et al. 1992).
Furthermore, there is some evidence that suggests that a subset of bipolar cells
express GNB3 (Peng, Robishaw et al. 1992; Huang, Max et al. 2003)
Mutations in GNB3 have been implicated in the development of multiple
systemic diseases in humans. A polymorphism in the GNB3 gene, C825T, has
85
been associated with hypertension, obesity and depression (Rosskopf, Busch et
al. 2000; Zill, Baghai et al. 2000). In the retina, a mutation in the GNB3 gene has
been shown to impact vision. This was first observed in commercial chicken
breeding stocks in the United Kingdom in the 1980’s with the discovery of the
retinopathy, globe enlarged (RGE) chicken (Curtis, Baker et al. 1987). The RGE
phenotype is known to result from a single codon deletion leading to the loss of a
single aspartic acid residue (Tummala, Ali et al. 2006). The loss of the aspartic
acid is believed to destabilize the GNB3 protein and, consequently, reduce
expression levels (Tummala, Ali et al. 2006). Putative decreases in GNB3
expression levels results in severely reduced visual acuity at hatching that
progressively worsens to complete vision loss in adult animals (Montiani-Ferreira,
Li et al. 2003).
Corresponding to the loss of visual acuity, electroretinograms (ERGs) of
RGE chickens revealed abnormalities compared to wild-type counterparts. These
ERG recordings had an elevated response threshold under dark- and lightadapted conditions, delayed onset of the a-wave and elevated b-wave
amplitudes with bright light (Montiani-Ferreira, Li et al. 2003; Montiani-Ferreira,
Shaw et al. 2007). Despite the reduction in visual acuity at hatching, funduscopic
and histological examination of the RGE retinas revealed no overt retinal
abnormalities during the first 6 weeks of life (Montiani-Ferreira, Fischer et al.
2005). Beyond the first 6 weeks, the eyes of RGE chickens undergo a
progressive globe enlargement involving an increase in vitreous chamber depth,
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increased axial length, decreased anterior chamber depth and flattening of the
cornea. Concurrent with the globe enlargement, the retina slowly degenerates;
this degeneration involves the loss of photoreceptors (Montiani-Ferreira, Li et al.
2003). The precise mechanisms underlying the loss of visual acuity in young
(<P45) RGE chickens, with apparently normal retinas, remains uncertain.
Given the retinopathy that occurs in adult RGE chickens, proper function
of GNB3 is required not only for photoreceptor signal transduction, but also in
maintaining the integrity of the photoreceptors and the maintenance of proper
eye size in adult animals. Although there is compelling evidence for the
expression of GNB3 in cone photoreceptors in murine, bovine and primate
retinas, little is known about the expression of GNB3 in the retinas of other
species. Moreover, expression of GNB3 in bipolar cells has not been well
studied. Thus, the purpose of this study was to examine the expression pattern of
GNB3 in wild-type and RGE mutant chickens, characterize the types of bipolar
cells that express GNB3 and to test whether the expression of GNB3 in the retina
is conserved across vertebrate species.
Methods and Materials
Animals:
The use of animals in these experiments was in accordance with the
guidelines established by the National Institutes of Health and the Ohio State
University. Fertilized eggs and newly hatched wild type leghorn chickens (Gallus
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gallus domesticus) were obtained from the Department of Animal Sciences at the
Ohio State University. The stage of the chick embryos was determined according
to the guidelines established by Hamburger and Hamilton in 1951 (Hamburger
and Hamilton 1992). Postnatal chicks were kept on a cycle of 12 hours light, 12
hours dark (lights on at 8:00 AM). RGE chickens were hatched from fertilized
eggs obtained from a cross of RGE homozygous (RGE-/-) chickens from the
Department of Small Animal Clinical Sciences, Michigan State University. Chicks
were housed in a stainless steel brooder at about 25oC and received water and
Purinatm chick starter ad libitum.
Eyes were obtained post-mortem from goldfish (Carassius auratus; Dr.
Christophe Ribelyaga, Department of Neuroscience, The Ohio State University),
frogs (Xenopus laevis; Dr. Candice Askwith, Department of Neuroscience, The
Ohio State University), mice (Mus musculata; Dr. Karl Obrietan, Department of
Neuroscience, The Ohio State University), guinea pigs (Cavia porcellus; Dr.
Jackie Wood, Department of Physiology and Cell Biology, Ohio State University),
dogs (Canis familiaris; Simon Petersen-Jones, Veterinary Sciences, Michigan
State University) and monkeys (Macaca fascicularis; Dr. John Buford,
Department of Physiology and Cell Biology, The Ohio State University).
Reverse transcriptase PCR:
Retinas from 2 P7 chicks were pooled and placed in 1.5 ml of Trizol
Reagent (Invitrogen) and total RNA was isolated according to the Trizol protocol
88
and resuspended in 50 μl RNAse free water. Genomic DNA was removed by
using the DNA FREE kit provided by Ambion. cDNA was synthesized from
mRNA by using Superscripttm III First Strand Synthesis System (Invitrogen) and
oligo dT primers according to the manufacturer’s protocol. Control reactions were
performed using all components with the exception of the reverse transcriptase
to exclude the possibility that primers were amplifying genomic DNA.
PCR primers were designed by using the Primer-BLAST primer design
tool at NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer sequences
are as follows: GNB3 – forward 5’ GCC CAC GTG GAG AAG CCA CC 3’ –
reverse 5’ CCT GGT CTG CCC GGA GGT CA 3’; GAPDH – forward 5’ CAT CCA
AGG AGT GAG CCA AG 3’ – reverse 5’ TGG AGG AAA TTG GAG GA 3’. The
predicted product size was 812 base pairs for GNB3 and 134 base pairs for
GAPDH. PCR reactions were performed by using standard protocols, Platinumtm
Taq (Invitrogen) and an Eppendorf thermal cycler. PCR products were run on an
agarose gel to verify the predicted product sizes.
Western Blotting
Retinas from 2 P7 wild-type and 2 RGE chicks were harvested on ice in
HBSS+ and immediately sonicated in extraction buffer (Bio-Rad) added with a
protease inhibitor cocktail tablet (Roche). After 5 minute ice incubation, the
sample was centrifuged and the supernatant collected. Protein concentration was
determined using a BCA Protein Assay (Thero Scientific). Samples were loaded
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into 10-well, 4-15% Tris-HCL acrylamide gels (Bio Rad) with Precision Plus
Protein Standard (Bio Rad) for electrophoresis at 95V. Protein transfer was
performed via electrophoresis overnight at 20V onto a nitrocellulose membrane
(162-0117; BioRad). After protein transfer, the membrane was blocked in Trisbuffered saline with 5% (w/v) milk powder and incubated in primary antibodies for
anti-mouse GAPDH at 1:2500 (IMG-5019A-1; Imgenex) or anti-rabbit GNB3 at
1:500 (HPA005645; Sigma-Aldrich) at room temperature overnight. The
membrane was washed in Tris-buffered saline and incubated under horseradishperoxidase conjugated secondary antibodies at 1:4000 (Amersham GE
Healthcare; anti-mouse IgG NA931V; anti-rabbit IgG NA934V) applied for 60
minutes at room temperature. The membranes were washed in Tris-buffered
saline and developed using an ECL™ Western Blotting Detection Reagents
(Amersham GE Healthcare; RPN2106) and UVP BioSpectrum 500 imaging
system.
Fixation, sectioning and immunocytochemistry
Tissues were fixed, sectioned and immunolabeled as described previously
(Fischer, Foster et al. 2008; Fischer, Scott et al. 2009). A summary of the
antibodies used in this study is provided in table 3.1. Working dilutions and
sources of antibodies used in this study included the following. (1) The Islet1
mouse monoclonal antibody was raised to the C-terminus (amino acids 247-349)
of rat Islet1 and used at 1:50 (40.2D6; Developmental Studies Hybridoma Bank –
90
DSHB; University of Iowa). (2) mouse anti-Lim3 was raised to recombinant fulllength murine Lim3 fused to GST and used at 1:50 (67.4E12; DSHB). (3) mouse
anti-visinin was raised to purified bovine visinin and used at 1:100 (7G4; DSHB).
(4) mouse anti-calbindin was raised to calbindin D28k purified from chicken gut
and used at 1:400 (300; Swant Immunochemicals; Bellinzona, Switzerland). (5)
rabbit anti-red/green opsin was raised to recombinant human red/green opsin
and used at 1:400 (AB5405; Chemicon; Temecula, CA). (6) mouse antirhodopsin was raised to purified bovine rhodopsin and used at 1:200 (rho4D2;
Dr. R. Molday; University of British Columbia). (7) mouse anti-PSD-95 was raised
to amino acids 77-299 of human PSD-95/SAP-90 and used at 1:50 (K28/43;
NeuroMab). (8) RetP1, a mouse anti-rhodopsin monoclonal antibody (MAB5316;
Chemicon; Temecula, CA). (9) Rabbit anti-GNB3 antibody was used at 1:400.
The antibody to GNB3 was raised to amino acids 172-317 of human guanine
nucleotide-binding protein β3 subunit and was used at 1:400 (HPA005645;
Sigma-Aldrich). In the current study, we demonstrate the specificity of the GNB3
antibody by using Western blot analysis and absence labeling in a GNB3-mutant
retina.
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Antigen
Species
Concentration
Catalog or
Source
Clone No.
Islet1
mouse
1:50
40.2D6
DSHB
Lim3
mouse
1:50
67.4E12
DSHB
Visinin
mouse
1:100
7G4
DSHB
Calbindin
mouse
1:400
300
Swant
Immunochemicals
Red/Green
rabbit
1:400
AB5405
Chemicon
Rhodopsin
mouse
1:200
rho4D2
Dr. R. Molday
PSD-95
mouse
1:50
K28/43
NeuroMab
RetP1
mouse
1:1000
MAB5316
Chemicon
GNB3
rabbit
1:400
HPA005645
GAPDH
mouse
1:2500
Opsin
Sigma-Aldrich
IMG-5019A-1 Imgenex
Table 3.1: Table of Antibodies used in immunohistochemistry and Western
Blotting
We evaluated the specificity of primary antibodies by comparison with
published examples of results and assays for specificity. None of the observed
labeling was due to non-specific labeling of secondary antibodies or
autofluorescence because sections labeled with secondary antibodies alone
were devoid of fluorescence. Secondary antibodies included donkey-anti-goatAlexa488/568, goat-anti-rabbit-Alexa488/568/647, goat-anti-mouseAlexa488/568/647, goat anti-rat-Alexa488 and goat-anti-mouse-IgM-Alexa568
(Invitrogen) diluted to 1:1000 in PBS plus 0.2% Triton X-100.
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Photography, measurements and cell counts:
Photomicrographs were obtained using a Leica DM5000B microscope
equipped with epifluorescence and Leica DC500 digital camera. Confocal images
were obtained using a Zeiss LSM 510 imaging system at the Hunt-Curtis Imaging
Facility at the Ohio State University. Images were optimized for color, brightness
and contrast, multiple channels overlaid and figures constructed by using Adobe
Photoshop™6.0. Cell counts were performed on representative images. To avoid
the possibility of region-specific differences within the retina, cell counts were
consistently made from the same region of retina for each data set.
Results
Expression of GNB3 in wild-type and RGE chicken retinas:
Reverse-transcriptase PCR (RT-PCR) was used to detect GNB3 mRNA in
retinas from P7 wild-type and RGE chicks. Glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) was used as a positive control. GNB3 mRNA was
detected in both in wild-type and RGE chicks (Figure 3.1a). We probed for GNB3
protein in retinal homogenates using Western blotting. GNB3 protein was
detected, as a single band at a molecular mass of 40kDa, in wild-type retinas but
not in RGE retinas (Figure 3.1b). In wild-type retinas, GNB3 immunofluorescence
was detected in the cytoplasm of photoreceptors and bipolar cells, but was
completely absent in RGE retinas (Figure 3.1c, d). Interestingly, markers for
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bipolar cells (Islet1 and PKC) and photoreceptors (calbindin) have patterns of
expression that were similar in both wild-type and RGE chicks (Figure 3.1e).
This indicates that the loss of GNB3 from bipolar cells and photoreceptors does
not overtly affect the morphology and phenotype of these cells, consistent with
previous reports (Montiani-Ferreira, Fischer et al. 2005).
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Figure 3.1: Expression of GNB3 mRNA and protein in wild-type and RGE -/chickens. mRNA for GNB3 was detected in P7 wild-type and RGE -/- retinas by
using RT-PCR. Each lane represents pools of retinal cDNA from different
animals (a). Protein for GNB3 was detected in P7 wild-type and RGE -/- retinas
by using Western blot analysis (b) and immunofluorescence(c). Vertical sections
of wild-type and RGE-/- retinas were labeled with antibodies to GNB3 (c and d),
Islet1, PKC or calbindin (e). Expression of bipolar cell markers (Islet1 and PKC)
and the cone marker calbindin are identical at P7 in wild-type and RGE-/- retinas
(e). The calibration bar in (50 µm) in panel d applies to panels c, d and e.
Abbreviations: RGE - retinopathy, globe enlargement, GAPDH - Glyceraldehyde
3-phosphate dehydrogenase, ONL - outer nuclear layer, INL - inner nuclear
layer, IPL - inner plexiform layer, GCL - ganglion cell layer.
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GNB3 in the developing chick retina:
RGE mutant chicks have reduced visual acuity at the time of hatching
(Montiani-Ferreira, Li et al. 2003) even though the retina appears normal at this
stage of development (Montiani-Ferreira, Fischer et al. 2005). Thus, it seems
unlikely that GNB3 is expressed during early embryonic stages to significantly
impact retinal development. To test this hypothesis we examined when GNB3
was first expressed during embryonic retinal development. Accordingly, we
labeled sections of embryonic chicken retina at various stages beginning at
embryonic day 5 (E5), shortly after the onset of neuronal differentiation (Prada,
Puga et al. 1991). At E5 and E8 we failed to detect GNB3 expression in wildtype retinas (data not shown). GNB3 expression was not detectable in the retina
until E13 where immunoreactivity was observed in both the inner plexiform layer
(IPL) and the outer plexiform layer (OPL) (Figure 3.2a-c). In order to identify the
cell populations that express GNB3 we probed for the expression of the LIMdomain transcription factor Islet1. This factor is expressed by ganglion cells,
cholinergic amacrine cells, many bipolar cells and about half of all horizontal cells
shortly after fate specification (Edqvist, Myers et al. 2006; Fischer, Foster et al.
2008; Stanke, Lehman et al. 2008). We observed transient Islet1immunofluoresence in the nuclei of cells in the ONL, consistent with a previous
report that the Islet1 antibody cross-reacts with Islet2 in immature photoreceptors
(Fischer, Foster et al. 2008). In addition, Islet1 was detected in the nuclei of
presumptive horizontal cells, bipolar cells and cholinergic amacrine cells in INL
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(Figure 3.2b, e, h, k) consistent with previous observations (Fischer, Stanke et al.
2007). As development progresses, GNB3 appears in the peri-nuclear cytoplasm
of bipolar cells at E15 (Figure 3.2d-f). By E17, immunoreactivity for GNB3 was
observed in the cytoplasm of photoreceptors as expression of Islet2 declines
(Figure 3.2g-i). By E18.5, GNB3 immunoreactivity was concentrated in the outer
segments (Figure 3.2k). We compared GNB3 expression to that of visinin, the
avian homologue of mammalian recoverin, which is expressed by all types of rod
and cone photoreceptors shortly after terminal mitosis (Yamagata, Goto et al.
1990). In E13 chick embryos, visinin was present in the photoreceptors,
concentrated in the immature outer segments, whereas GNB3 immunoreactivity
remained below detectible levels in the photoreceptors (Figure 3.2c). As
photoreceptors mature and become morphologically distinct, GNB3
immunoreactivity becomes concentrated in the cone pedicles and the outer
segments, whereas levels of GNB3 remained low in the peri-nuclear cytoplasm
(Figure 3.2f,i and l).
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Figure 3.2: GNB3 expression in the developing chicken retina. Vertical sections
of retina were labeled with antibodies to GNB3 (green; a-l) and Islet1 (red; b, e, h
and k) and Visinin (red; c, f, i, and l). Retinas were obtained from embryos at
E13 (a-c), E15 (d-f), E17 (g-i) and E18.5 (j-l). The arrowheads indicate colabeling for GNB3 and Islet1 in bipolar cells (k). Areas outlined in yellow
magnified approximately 2.5 times in the lower right corner (k and l). The
calibration bar (50 µm) in panel l applies to all panels. Abbreviations: OPL outer plexiform layer, IPL - inner plexiform layer.
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Figure 3.2
99
Distribution of GNB3 in the postnatal chick retina (Gallus gallus domesticus):
We sought to better characterize the types of cells in the mature chick
retina that express GNB3. Accordingly, we labeled retinal sections with
antibodies to different cell-distinguishing markers. We applied antibodies to PKC,
which is known to be expressed at high levels in rod bipolar cells and at lower
level in cone ON-bipolar cells in the avian retina (Negishi, Kato et al. 1988;
Young and Vaney 1990; Fischer, Seltner et al. 1998; Caminos, Velasco et al.
1999) and in other species (Negishi, Kato et al. 1988; Greferath, Grunert et al.
1990; McCord, Klein et al. 1996; Vaquero, Velasco et al. 1996; Caminos,
Velasco et al. 1999). We found that PKC was co-localized to some of the GNB3positive bipolar cells (Figure 3.3a-c). The rod bipolar cells were intensely
immunoreactive for PKC and weakly immunoreactive for GNB3. Consistent with
the finding of Negishi and colleagues (1988) the axon terminals of the rod bipolar
cells were tristratified, with endings concentrated in layers 1, 3 and 5 of the IPL
(Negishi, Kato et al. 1988). In addition, GNB3-immunoreactivity was detected in
presumptive cone ON-bipolar cells that were weakly immunoreactive for PKC
(Figure 3.3a-c). The GNB3-positive bipolar cells appeared to produce axons that
are bundled together and fasciculate within the inner nuclear layer (INL), and
branch apart upon entry into the IPL (Figure 3.3a & d). Consistent with the notion
that GNB3 is expressed by cone ON-bipolar cells, the axon terminals of these
cells were densely ramified in the ON sub-lamina of the IPL with endings
concentrated in sub-laminae 3 and 4 (Figure 3.3g). In addition, we found GNB3100
positive bipolar cells with somata in the scleral INL that were PKC-negative
(Figure 3.3a-c). We found a high degree of overlap between Islet1 and GNB3 in
bipolar cells (Figure 3.3d-h). Although there was no overlap of GNB3 and Islet1
in the horizontal cells, there was a complete overlap of GNB3 and Islet1 in the
bipolar cells (Fig. 3h). In other words, all of the Islet1-positive bipolar cells were
co-labeled for GNB3 and all of the GNB3-positive bipolar cells were labeled for
Islet1. We next assayed for the co-expression of GNB3 and Lim homeobox gene
3 (Lim3), a transcription factor that is known to be expressed by many bipolar
cells and immature photoreceptors in the chick retina (Edqvist, Myers et al. 2006;
Fischer, Foster et al. 2008). We found weak immunofluorescence for Lim3 in the
nuclei of many bipolar cells that were GNB3-positive (Figure 3.3i-k). In addition,
we found strong immunofluorescence for Lim3 in the nuclei of bipolar cells that
were GNB3-negative (Figure 3.3i-k).
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Figure 3.3: GNB3 is expressed by photoreceptors and bipolar cells in the retinas
of wildtype chicks. Vertical sections of P7 chicken retinas were labeled with
antibodies to GNB3 (green), PKC (red, b and c), Islet1 (red, e-h) and Lim3 (red, j
and k). DRAQ5 (magenta) was applied as a nuclear counter-stain. Images were
obtained using confocal microscopy. Panel h is an orthogonal reconstruction of
a Z-stack of optical sections through central retina labeled for Islet1 and GNB3.
Arrows indicate GNB3-positive bipolar cells that are co-labeled for PKC or Islet1
(a-h). Arrowheads indicate horizontal cells that are labeled for Islet1 alone (d-h).
Carets indicate bipolar cells that are GNB3-negative and intensely
immunoreactive for Lim3 (i-k). Small double-arrows indicate cholinergic amacrine
cells that are labeled for Islet1 (d-f). The calibration bar in (50 µm) in panel g
applies to a-g and i-k, the bar in h applies to h alone. Abbreviations: PRL photoreceptor layer, ONL - outer nuclear layer, INL - inner nuclear layer, IPL inner plexiform layer, GCL - ganglion cell layer.
102
Figure 3.3
103
GNB3 was expressed by most, if not all, types of photoreceptors in the
chick retina. We found that all of the calbindin-positive cone photoreceptors were
immunoreactive for GNB3 which was concentrated in the outer segments (Figure
3.4a-d). However, we identified many GNB3-positive photoreceptor
outersegments that were negative for calbindin (Figure 3.4b-d). The identity of
the GNB3-positive/calbindin-negative photoreceptors remains uncertain.
Examination of the OPL revealed 2 strata of axon terminals, with the most distal
strata containing the terminals of calbindin-positive cone photoreceptors,
consistent with prior findings (Fischer, Foster et al. 2008). Immunolabeling for
visinin and GNB3 revealed significant overlap in outersegments (Figure 3.4e-g),
suggesting that GNB3 is expressed by both rod and cone photoreceptors.
Accordingly, rod photoreceptors were examined with antibodies to rhodopsin and
GNB3. We found some overlap of rhodopsin- and GNB3-immunoreactivity in the
outer segments of photoreceptors (Figure 3.4h-j), consistent with the notion that
GNB3 is expressed by rods in addition to cone photoreceptors.
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Figure 3.4 GNB3 is expressed by all photoreceptors in retinas of wildtype
chicken. Vertical sections of P7 chicken retinas were labeled with antibodies for
GNB3 (green), calbindin (red, a, c & d), visinin (red, f and g) and rhodopsin (red,
i and j). Images were obtained using confocal microscopy. Arrows indicate
photoreceptors labeled for GNB3 and calbindin, visinin or rhodopsin. Arrowheads
indicate photoreceptors labeled for GNB3 alone. The calibration bar in (50 µm) in
panel a applies to a, the bar in b applies to b-d and the bar in g applies to e-j.
Abbreviations: PRL - photoreceptor layer, ONL - outer nuclear layer, INL - inner
nuclear layer, IPL - inner plexiform layer.
GNB3 in teleost fish retina (Carassius auratus):
We next assayed for the expression of GNB3 in the fish retina. Unlike the
pattern of GNB3 expression in the chick retina, there was little immunoreactivity
105
for GNB3 in bipolar cells in the fish retina (Figure 3.5d). We found
immunoreactivity for GNB3 in the outer retina, in putative cone photoreceptors
(Figure 3.5a and d). Immunolabeling for GNB3 was present throughout the
cytoplasm of cone photoreceptors, with a concentration in the outer segments
(Figure 3.5a). The GNB3-positive photoreceptors appeared to be cones based
on their distinctive morphology. To identify the types of photoreceptors that
express GNB3 we used the photoreceptor-specific markers peanut agglutinin
(PNA lectin), RetP1 and immunoreactivity to rhodopsin, which have been shown
to label photoreceptors in a number of species, including several varieties of fish
(Xu and Tian 2008). There was no overlap of labeling for GNB3 and rhodopsin
(Figure 3.5a-c). However, labeling with the RetP1 monoclonal indicated colocalization with GNB3 in photoreceptors (Figure 3.5d-f). Labeling for PNA lectin
and GNB3 overlapped in the cone outer segments, with GNB3 concentrated in
the distal aspects of the outer segments (Figure 3.5g-i).
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Figure 3.5 GNB3 is expressed by cone photoreceptors and bipolar cells in the
goldfish retina. Vertical sections of goldfish retinas were labeled with antibodies
to GNB3 (green), rhodopsin (magenta, b and c), RetP1 (red, e and f), and PNAlectin (red, h and i). Images were obtained using confocal microscopy. Arrows
indicate photoreceptors labeled for GNB3 and RetP1 (d-f) or PNA-lectin (g-i).
Arrowheads indicate bipolar cells labeled for GNB3 alone (d-f). The calibration
bar in (50 µm) in panel b applies to a-c, the bar in f applies to d-f and the bar in i
applies to g-i. Abbreviations: PRL - photoreceptor layer, ONL - outer nuclear
layer, INL - inner nuclear layer, IPL - inner plexiform layer.
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GNB3 in frog retina (Xenopus laevis):
Patterns of GNB3-immunoreactivity in the frog retina were similar to those
seen in fish retina. In Xenopus retinas, intense immunoreactivity for GNB3 was
observed in cone photoreceptors (Figure 3.6a-c), based on the morphology of
these cells. In addition, weak GNB3-immunoreactivity was observed in the outer
segments of rod photoreceptors that were labeled by the RetP1 monoclonal
antibody (Figure 3.6a-c). Further, GNB3-immunoreactivity was localized to Islet1positive nuclei of bipolar cells (Figure 3.6d-f). However, many of the GNB3immunoreactive bipolar cells were negative for Islet1 (Figure 3.6d-f).
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Figure 3.6 GNB3 is expressed by photoreceptors and bipolar cells in the
Xenopus retina. Vertical sections of Xenopus retinas were labeled with
antibodies to GNB3 (green), RetP1 (red, b and c) and Islet1 (red, e and f). Small
triple arrows indicate the outer segments of rod photoreceptors (a-c). Arrows in
panels a-c indicate cone photoreceptors, and arrows in panels d-f indicate
bipolar cells labeled for Islet 1 and GNB3. Arrowheads indicate bipolar cells
labeled for GNB3 alone (d-f). The inset in panel f is enlarged approximately 3fold. The calibration bar in (50 µm) in panel f applies to a-f. Abbreviations: PRL photoreceptor layer, ONL - outer nuclear layer, INL - inner nuclear layer, IPL inner plexiform layer.
GNB3 in mouse retina (Mus musculata):
We next sought to determine whether the patterns of expression of GNB3
observed in birds, fish and frogs were preserved in mammals. GNB3immunoreactivity was concentrated in the outer segments of photoreceptors in
mouse retinas (Figure 3.7a), consistent with a previous report (Huang, Max et al.
2003). In central regions of the retina, the majority of GNB3-positive outer
segments were immunoreactive for red/green opsin, consistent with the notion
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that GNB3 is expressed by cone photoreceptors (Figure 3.7b-d). A minority of
GNB3-positive cone outer segments were negative for red/green opsin in the
photoreceptor layer (PRL), suggesting that these were blue-sensitive cones
(Figure 3.7a-d).
GNB3-immunolabeling was observed in the INL in bipolar cells (Figure
3.7e). Co-labeling for PKC and GNB3 was observed in many bipolar cells (Figure
3.7e-g). A minority of the GNB3-positive bipolar cells were negative for PKC
(Figure 3.7g). The axons terminals of the PKC-positive bipolar cells overlapped
with GNB3-immunoflouresence in the vitread IPL (Figure 3.7e-g). However,
GNB3-positive/PKC-negative bipolar cell terminals were observed in the middle
stratum of the IPL (Figure 3.7e-g). The majority of GNB3-immunoreactive bipolar
cells were positive for Islet1, whereas a minority of the GNB-positive bipolar cells
did not contain detectable levels of Islet1 (Figure 3.7h-j). Our observations are
consistent with a recent report that GNB3 is expressed by a subset of bipolar
cells in mouse retina (Huang, Max et al. 2003). To determine whether GNB3immunoreactivity in the OPL was present in the dendrites of bipolar cells or axon
terminals of photoreceptors, we labeled sections with antibodies to PSD-95, a
MAGUK-family synaptic protein. In the rodent retina, PSD-95 is known to be
present in the axon terminals of photoreceptors (Aartsen, Kantardzhieva et al.
2006). There was little overlap of PSD-95 and GNB3 immunofluorescence in the
OPL (Figure 3.7k-m), suggesting that the PSD-95 was confined to the axon
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terminals of the photoreceptors and the GNB3 was confined to the dendrites of
bipolar cells.
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Figure 3.7: GNB3 is expressed by cone photoreceptors and bipolar cells in the
mouse retina. Vertical sections of mouse retinas were labeled with antibodies to
GNB3 (green), red/ green opsin (red, a, c and d), PKC (red, f and g), Islet1 (red,
i and j) and PSD-95 (red, l and m). Arrows indicate cone photoreceptors labeled
for GNB3 and red/ green opsin (b-d) or bipolar cells labeled for GNB3 and Islet1
(h-j). Arrowheads indicate cholinergic amacrine cells labeled for Islet1 (h-j).
Small double arrows indicate capillaries within the retina that are labeled with
secondary alone (h-m). The calibration bar in (50 µm) in panel b applies to a-b,
the bar in f applies to d-f and the bar in i applies to g-i. Abbreviations: PRL photoreceptor layer, ONL - outer nuclear layer, INL - inner nuclear layer, IPL inner plexiform layer.
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Figure 3.7
113
GNB3 in guinea pig retina (Cavia porcellus):
Examination of the guinea pig retina revealed widespread GNB3
expression in photoreceptors and bipolar cells. GNB3-immunolabeling was
concentrated in the outer segments of presumptive cone photoreceptors (Figure
3.8a-c). Immunolabeling for GNB3 and red/green opsin revealed overlap in the
outer segment in most cones (Figure 3.8c). A minority of GNB3-positive outer
segments were negative for red/green opsin (Figure 3.8a-c). Examination of rod
photoreceptors with antibodies to rhodopsin revealed weak GNB3immunofluoresence in the outer segments of rod photoreceptors (Figure 3.8f). By
increasing the detector gain, GNB3-immunofluorescence was detected in rod
outer segments and in cone inner segments (Fig. 8d). In addition, we found some
immunolabeling for rhodopsin in GNB3-positive cone photoreceptors (Figs. 8d-f),
suggesting that the rhodopsin antibody may cross-react with opsin.
We found significant overlap of GNB3 and Islet1 in the bipolar cells of the
guinea pig retina (Figure 3.8g-l). We found that nearly all (~92%) Islet1-positive
nuclei in the distal INL were rimmed with GNB3-immunofluorescence (Figure
3.8g-l). Many (~35%) of the GNB3-positive bipolar cells were intensely
immunoreactive for PKC (Figure 3.8m-p). However, numerous GNB3-positive
bipolar cells were negative for PKC. Examination of the OPL revealed
stratification of SV2-positive photoreceptor terminals and GNB3-labeling,
suggesting that GNB3 is present in bipolar cell dendrites, not the axon terminals
of photoreceptors (Figure 3.8q-s).
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Figure 3.8: GNB3 is expressed by cone photoreceptors and bipolar cells in the
guinea pig retina. GNB3 immunoreactivity is found in the OPL, IPL, bipolar cells
and photoreceptors. Vertical sections of guinea pig retinas were labeled with
antibodies for GNB3 (green), red/ green opsin (red, b and c), rhodopsin (red, e
and f), Islet1 (red, h-l), PKC (red, m-p) and SV2 (red, r and s). Arrows in panels
a-c indicate cones double positive for red/ green opsin and GNB3, whereas
arrowheads point to GNB3 positive, red/ green opsin negative cones. Insets in
panels c-f are enlarged approximately 3-fold. The areas boxed-out (magenta) in
panels i and m are enlarged approximately 2-fold in panels j-l and n-p,
respectively. Small double arrows indicate Islet1-positive, GNB3-negative
ganglion cells (g-i). Arrows in panels g-l indicate bipolar cells that are labeled for
Islet1 and GNB3, and the arrowheads indicate GNB3 positive bipolar cells.
Hollow arrowheads (q-s) point to the presynaptic terminals of photoreceptors in
the OPL. The calibration bar (50 µm) in panel f applies to panels a-i, bar in s
applies to panels q-s. Abbreviations: ONL - outer nuclear layer; INL - inner
nuclear layer; IPL - inner plexiform layer, GCL - ganglion cell layer.
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Figure 3.8
116
GNB3 in dog retina (Canis familiaris):
We next examined the distribution of GNB3 in the canine retina. In the
distal retina, GNB3-immunoreactivity was observed in presumptive cone
photoreceptors (Figure 3.9a and d). Labeling for cone arrestin and GNB3,
combined with cell morphology, confirmed that GNB3 was expressed by cone
photoreceptors (Figure 3.9b and c). Weak Islet1/2-immunolabeling was
observed in the nuclei of cone photoreceptors, consistent with reports that Islet2
is expressed by developing photoreceptors in the chick retina (Fischer, Foster et
al. 2008) and is maintained at low levels in mature photoreceptors (unpublished
observations). Similar to the distribution seen in other species, GNB3 was
concentrated in the outer segments of the canine cone photoreceptors (Figure
3.9a-c).
Examination of the INL revealed Islet1-positive bipolar cell nuclei
surrounded by a rim of GNB3- immunofluorescence cytoplasm (Figure 3.9e and
3.9f). All of the Islet1-positive nuclei in the distal INL were associated with
GNB3-labeling. These GNB3-positive bipolar cells had axons that terminated in
the proximal, ON sub-lamina, of the IPL. Furthermore, most of the GNB3positive bipolar cells were labeled for PKC (Figure 3.9g-i). However, a minority
(<25%) of the GNB3-positive bipolar cells were negative for PKC (Figure 3.9g-i).
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Figure 3.9: GNB3 is expressed by cone photoreceptors and bipolar cells in the
dog retina. Vertical sections of canine retinas were labeled with antibodies to
GNB3 (green), human cone arrestin (hCAR; red, b and c), Islet1 (red, e and f)
and PKC (red, h and i). Arrows indicate cone photoreceptors labeled for GNB3
and hCAR (a-c) or Islet1 (d-f), arrowheads indicate bipolar cells labeled for
GNB3 and Islet1 (d-f) or PKC (g-i). The calibration bar (50 µm) in panel i applies
to panels a-i. Small double arrows (d-f) indicate Islet1 positive ganglion cells.
Abbreviations: ONL - outer nuclear layer, OPL -outer plexiform layer, INL - inner
nuclear layer, IPL - inner plexiform layer, GCL - ganglion cell layer.
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Figure 3.9
119
GNB3 in the primate retina (Macaca fascicularis):
We found that the pattern of expression of GNB3 in the macaque retina
was similar to that observed in the retinas of other vertebrates. Immunolabeling
for Islet1 was co-localized to GNB3-positive bipolar cells within the INL (Figure
3.10d). Consistent with previous reports (Fischer, Hendrickson et al. 2001), Islet1
was detected in the nuclei of presumptive ganglion cells and cholinergic
amacrine cells , in addition to the nuclei of bipolar cells (Figure 3.10d and 3.10e).
A majority of the Islet1-positive bipolar cell nuclei overlapped with GNB3
immunofluorescence. A minority of the Islet1-positive nuclei in the distal INL was
not associated with detectable levels of GNB3 (Figure 3.10e-g). GNB3-labeling
was also prominent in the distal portion of the outer segments of cones in the
PRL, but was detected at lower levels in the inner segments, including the axon
terminals (Figure 3.10 c), consistent with a previous report (Peng, Robishaw et
al. 1992). Labeling with PNA lectin overlapped with GNB3 in the outer segments
of photoreceptors (Figure 3.10a-d). PNA lectin is known to label cone
photoreceptors in the primate retina (Blanks and Johnson 1984).
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Figure 3.10: GNB3 is expressed by cone photoreceptors and bipolar cells in the
primate retina (Macaca fascicularis). Vertical sections of retina were labeled with
antibodies to PNA-Lectin (red; a and d), GNB3 (green; c, d, f and g) and Islet1
(blue; b, d and e). The large arrows indicate co-labeling for GNB3 and PNA-568
in cone outer segments. The arrow heads indicate co-labeling for GNB3 and
Islet1 in bipolar cells. The calibration bar (50 µm) in d applies to panels a-d.
Panels e-g are enlarged approximately 3-fold. Abbreviations: ONL - outer nuclear
layer; INL - inner nuclear layer, IPL - inner plexiform layer , GCL - ganglion cell
layer.
GNB3 protein sequence homology among species
The near-identical immunolabeling patterns for GNB3 in the retinas of
different species suggest that the primary amino acid sequence is highly
conserved across species. To test this notion, we examined the homology of the
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primary amino acid sequences of GNB3 in different species by using a proteinprotein alignment search tool (BLASTp; http://blast.ncbi.nlm.nih.gov/Blast.cgi).
We found a very high level (83%-100%) of sequence identity among vertebrate
species when compared to human GNB3 protein (Table 3.2). Accounting for
conservative amino acid substitutions, we found very high sequence
conservation, ranging from 92% to 100%, among GNB3 sequences from different
vertebrates. The highly conserved sequence homology observed among species,
as well as the similar expression patterns among retinal cell types, suggests the
importance of GNB3 function in the retina.
Species
Identity
%
Positives
%
Zebrafish
284/340
83
315/340
92
Goldfish
N/A
N/A
N/A
N/A
Gallus Gallus
314/337
93
332/337
98
Xenopus
299/340
87
326/340
95
Mouse
330/340
97
335/340
98
Guinea Pig
283/340
83
315/340
92
Dog
329/340
96
335/340
98
Macaca
340/340
100
340/340
100
Table 3.2: GNB3 protein sequence homology across species referenced to
Homo Sapiens: protein-protein BLAST. Identity refers to identically matched
amino acids. Positives accounts for substitutions with similar amino acids.
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Discussion
In the retinopathy, globe enlarged (RGE) chicken, the mutation of GNB3
results in a profound phenotype featuring a significant loss of visual acuity
beginning at the time of hatching, and photoreceptor degeneration and globe
enlargement in adults (Montiani-Ferreira, Li et al. 2003; Montiani-Ferreira,
Fischer et al. 2005; Montiani-Ferreira, Shaw et al. 2007). Given the significance
of this presentation, we sought to characterize the expression pattern of GNB3 in
the retinas of chicks, as well as in the retinas of different non-avian vertebrates.
The expression of GNB3 in cone photoreceptors was consistent across species
from teleost fish to primates. Furthermore, GNB3 expression was observed in a
subset of bipolar cells, which express Islet1 and PKC in most species. Taken
together, our findings suggest that patterns of GNB3 expression are highly
conserved in cones and bipolar cells across vertebrate species.
GNB3 has been consistently observed in the outer segments of cone
photoreceptors in different mammalian species, including mice, cows and
monkeys (Lee, Lieberman et al. 1992; Peng, Robishaw et al. 1992; Huang, Max
et al. 2003). However there have been no reports of GNB3 expression in rod
photoreceptors. Similar to previous reports, we found GNB3 was exclusively
expressed by cones among primate, murine and canine photoreceptors.
However, in chicken, guinea pig, goldfish and frog retinas, GNB3 was detected in
rod photoreceptors; albeit at levels lower than those observed in cone
photoreceptors. The expression of GNB3 in rod photoreceptors was unexpected
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given previous literature indicating GNB3 expression is restricted to cone
photoreceptors (Lee, Lieberman et al. 1992; Peng, Robishaw et al. 1992;
Tummala, Ali et al. 2006; Dutt and Cao 2009; Haider, Mollema et al. 2009). It is
possible that GNB3 expression occurs in both rods and cones; however, the
GNB3 antibody utilized in this study may lack specificity to cone β-transducin and
could recognize rod β-transducin. Alternatively, GNB3 may be expressed by both
types of photoreceptors. In the chick retina, GNB3 was detected in rod
photoreceptors labeled with the 4D2 rhodospin monoclonal antibody. This
antibody has been shown to label mid-wavelength green-sensitive cones where
the photopigment shares the closest sequence homology with rhodopsin (Xie
and Adler 2000). However, all GNB3-immunolabeling disappeared in RGE
retinas, indicating that the antibody was not cross-reacting with rod β-transducin
in the chick. In guinea pigs, cross-reactivity of the 4D2 antibody with green-opsin
in cones may explain the observation of GNB3 in the outer segments of some
rhodopsin-positive photoreceptors. Despite the presence of GNB3 labeling in the
rod photoreceptors of some species, findings that GNB3 is expressed in cone
photoreceptors in all species examined indicates the highly conserved nature of
the protein and suggests the importance of GNB3 in cone-driven vision.
Examination of the photoreceptor axon terminals revealed the presence of
GNB3 in cone pedicles in most species with the exception of rodents. In mice
and guinea pigs, there was little or no labeling for GNB3 in the cone pedicles,
compared to labeling observed in the outer segments. It is possible that the
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differential distribution of of GNB3 in the axon terminals of the photoreceptors
may result from light-dependent trafficking of GNB3. Examples of light-dependent
trafficking of transducin have been documented in rod photoreceptors, where rod
α-transducin translocates from the outer segments under dark-adapted
conditions to the axon terminal in the OPL following light exposure (Brann and
Cohen 1987). There is also evidence of light-dependent translocation of rod βtransducin in the retinas of mice and rats (Sokolov, Lyubarsky et al. 2002;
Lobanova, Finkelstein et al. 2007). By comparison, there is little evidence of lightdependent translocation of phototransduction-related proteins in cone
photoreceptors. Elias and colleagues reported that, unlike rod α-transducin, cone
α-transducin does not translocate with varying light levels (Elias, Sezate et al.
2004). Rosenzweig and colleagues showed that the subcellular distribution of
transducin was correlated to light-dependent changes in membrane affinity, with
cone α-transducin remaining bound to the outer segment during light activation
(Rosenzweig, Nair et al. 2007). Furthermore, when cone α-transducin is
ecotopically expressed in rod photoreceptors this protein is translocated to inner
segments with changes in light levels, indicating differences in the cell-intrinsic
trafficking mechanisms can occur (Rosenzweig, Nair et al. 2007). Chen and
colleagues reported that cone α-transducin partially translocates in rat retina after
a brief exposure to high-intensity light(Chen, Wu et al. 2007). Reports of cone βtransducin translocation are rare; however, McGinnis and colleagues reported
migration of cone β-transducin with light exposure of dark-adapted retinas in
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mice (McGinnis, Matsumoto et al. 2002). Given this evidence, it is possible that
we failed to detect GNB3 in the axon terminals of cone photoreceptors in mice
and guinea pigs because of light-dependent trafficking and harvesting conditions.
In the inner retina, GNB3 expression was observed in presumptive cone
ON-bipolar cells across species with one exception; goldfish retinas had low
levels of GNB3 expression in bipolar cells. There is some prior evidence for
GNB3 expression in bipolar cells. Peng and colleagues reported, in primate
retina, that GNB3 is expressed in presumptive rod bipolar cells that were colabeled for PKC. This study also noted that the synaptic terminals of the GNB3positive bipolar cells terminated in sublamina b of the INL, suggesting that some
these bipolar cells were cone ON-bipolar cells (Peng, Robishaw et al. 1992).
Furthermore, Huang and colleagues demonstrated GNB3 in the dendrites of
cone bipolar cells and colocalization of GNB3 with PKC in rod bipolar cells in
mouse retina (Huang, Max et al. 2003). In our study, cone ON-bipolar and rod
bipolar cells were identified using antibodies to Islet1 in chicken, macaque,
mouse, canine and guinea pig retinas. It has been previously reported that Islet1,
a LIM-domain transcription factor, is required for the normal differentiation of ONand OFF-bipolar cells in the rodent retina (Elshatory, Everhart et al. 2007). In
chicken, macaque, mouse, canine and guinea pig, the GNB3-positive bipolar
cells expressed Islet1. Supporting the notion that most of the GNB3-positive
bipolar cells are cone ON-bipolars, the axonal processes of these cells terminate
within the ON layer of the IPL, sub-lamina B. In Xenopus retina, GNB3 and Islet1
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did not overlap completely within bipolar cells, suggesting that GNB3 may be
expressed by types of bipolar cells in addition to the cone ON-bipolar cells or that
Islet1 is not exclusively expressed by cone ON-bipolar cells.
Consistent with the hypothesis that GNB3 is expressed by cone ONbipolar cells, we observed significant overlap of GNB3 and PKC. PKC has been
shown to be expressed by both rod bipolar cells and cone ON-bipolar cells in
multiple species (Negishi, Kato et al. 1988; Greferath, Grunert et al. 1990;
McCord, Klein et al. 1996; Vaquero, Velasco et al. 1996; Caminos, Velasco et al.
1999). In the chicken retina, labeling for PKC and GNB3 revealed 3 different sets
of bipolar cells; (1) PKC-negative/GNB3-positive cells, (2) intense PKCpositive/GNB3-positive cells, and (3) weak PKC-positive/GNB3-positive bipolar
cells. These findings suggest that these bipolar cells were presumptive cone ONbipolar (weak PKC-positive/GNB3-positive cells) and rod ON-bipolar cells
(intense PKC-positive/GNB3-positive), whereas the identity of the PKCnegative/GNB3-positive bipolar cells remains uncertain. In the mammalian
retinas, GNB3 was observed in PKC-immunoreactive bipolar cells. The overlap
of expression of PKC and GNB3 in bipolar cells suggests that cone ON-bipolar
and rod bipolar cells utilize GNB3 for signal transduction. In the rodent retina,
some of the axon terminals of the GNB3-positive bipolar cells were clearly
segregated from some of PKC-positive terminals in the IPL, supporting the notion
that GNB3 is expressed by more than one type of bipolar cell. Similar results
were observed in guinea pig and dog retinas, where GNB3-positive/PKC127
negative and GNB3/PKC-positive bipolar cells were observed. Our observations
indicate that the GNB3-positive bipolar cells in the chick retina express Islet1 and
many of these cells express low levels of Lim3. The GNB3-negative bipolar cells
that express high levels of Lim3 are likely to be OFF-cone bipolar cells. Taken
together, these findings suggest that GNB3 is expressed by cone ON-bipolar
cells and in some rod ON-bipolar cells, indicating the importance of GNB3 signal
transduction in the ON pathway.
In the current study, immunofluoresence combined with Western blot
analysis, clearly indicate that GNB3 protein is entirely lost from the retinas of
RGE mutant chicks. The presence of mRNA in both WT and RGE retinas
indicates that the transcriptional mechanisms for the GNB3 expression are intact
in the RGE chicken. A previous report has suggested that the deletion of a single
aspartic acid (D153del) from GNB3 in the RGE chicken reduces levels of
expression by about 70%, as determined by a immuno-slot-blot preparation
(Tummala, Ali et al. 2006). The discrepancy between these studies likely resulted
from the use of different GNB3 antibodies and the application of different
immunological techniques. Our findings indicate that the loss of GNB3 in the
RGE chicken does not overtly affect the formation and maturation of the retina
during embryonic and early postnatal development. This is consistent with a
previous report demonstrating the normal immunohistological profile of retinas in
young (<P30) RGE chicks (Montiani-Ferreira, Fischer et al. 2005).
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Clearly, the loss of GNB3 underlies the phenotype of the RGE-/- chicken
which involves the loss of visual acuity at the time of hatching, and eventually
(after P45) globe enlargement and retinal degeneration (Montiani-Ferreira, Li et
al. 2003; Montiani-Ferreira, Fischer et al. 2005; Tummala, Ali et al. 2006). The
mechanisms underlying the progressive retinopathy and subsequent excessive
ocular growth in this animal model remains unknown. We failed to find GNB3
expression in ocular tissues, with the exception of the retina. Thus, the ocular
phenotypes of the RGE chicken, including uncontrolled eye growth, flattening of
the anterior chamber and progressive retinopathy, result from a complete loss of
GNB3 from retinal bipolar cells and photoreceptors.
The onset of expression of GNB3 during embryonic development
coincides with the late-onset of genes in both photoreceptors and bipolar cells.
The maturation of photoreceptors is delayed for at least 1 week after terminal
mitosis and fate specification in the chick retina (Fischer, Foster et al. 2008). The
onset of GNB3 expression in bipolar cells occurs before the onset of PKC
expression (Caminos, Velasco et al. 1999), and GNB3 expression in
photoreceptors appears before the onset of cone opsin expression, at about E15
(Bruhn and Cepko 1996). Despite an absence of GNB3 expression, the retinas of
RGE -/- chicks show no gross retinal abnormalities, with the exception of subtle
synaptic changes in the OPL (Montiani-Ferreira, Fischer et al. 2005). Moreover,
the late onset of GNB3 in the embryonic chick retina may explain why no severe
ocular abnormalities are observed at hatching in the RGE phenotype; an early
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onset of expression might be expected to have more severe developmental
impact. However, the survival of photoreceptors is compromised in RGE retinas
beginning at about P45, indicating that proper GNB3 function is required to
maintain the photoreceptors in adult animals (Montiani-Ferreira, Fischer et al.
2005).
Conclusions
The pattern of GNB3 expression is very similar in the retinas of multiple
species, suggesting the highly conserved nature of the protein and its importance
for normal visual function. Given the remarkably similar expression patterns of
GNB3 in the retina across species, we propose that the functions and
mechanisms of expression for GNB3 within the retina are highly conserved.
Furthermore, normal GNB3 protein expression is critical for normal signal
transduction in cone photoreceptors and ON-bipolar cells, allowing for proper
retinal function. We propose that the loss of GNB3 from bipolar cells and
photoreceptors in the RGE retina underlies the loss of vision at the time hatching,
and the progressive degeneration and abnormal ocular growth in adult chickens.
Acknowledgements
I would like to acknowledge the significant contributions of Rachel Bongini,
Kimberly Code, Christopher Zelinka, Dr. Simon Petersen-Jones and Dr. Andy
130
Fischer to this project. The antibodies developed by Drs T. Jessell (Islet1) and C.
Cepko (visinin), respectively, were obtained from the Developmental Studies
Hybridoma Bank, which was developed under the auspices of the NICHD and is
maintained by the University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242. Confocal microscopy was performed at the Hunt-Curtis Imaging
Facility at the Department of Neuroscience of The Ohio State University. This
work was supported by grants (AJF: EY016043-04; ERR: K12EY015447) from
the National Institutes of Health, National Eye Institute and was published in the
journal Neuroscience in 2010.
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Chapter 4: Vision-guided ocular growth in a mutant chicken model with
diminished visual acuity
Abstract
The proper regulation of ocular growth is a vision-dependent phenomenon
across multiple species. In this paper, we investigate vision-guided ocular growth
in an animal with congenitally decreased vision, the Retinopathy, Globe Enlarged
(RGE) chicken. The RGE chicken develops an ocular phenotype because of a
spontaneous 3 base pair mutation, D153del, within guanine nucleotide-binding
protein beta-3 (GNB3) (Tummala, Ali et al. 2006). The D153del deletion results in
poor vision in newly hatched chicks that progressively worsens over time.
Beginning at about postnatal day 45, RGE chickens develop a progressive globe
enlargement and retinal degenerations, whereas the retinal and ocular growth
appear normal during the first 3 weeks of postnatal development (MontianiFerreira, Li et al. 2003). The purpose of this study was to investigate whether
growth-regulating visual stimuli influence ocular growth during the early postnatal
development of the RGE chick in a manner similar to that observed in wild-type
(WT) chickens, prior to the development of the globe enlargement phenotype and
retinopathy. In addition, we sought to test whether the glucagon-positive
amacrine cells, which are known to respond to growth-modulating visual cues,
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behave normally in RGE retinas (Fischer, McGuire et al. 1999). We found that
RGE chickens respond to form-deprivation in a manner similar to wild-type
chickens, with increased axial length, globe circumference and myopia
development. When presented with form-deprivation interrupted by 130 minutes
of daily unrestricted vision, growth in axial length, globe circumference and
myopia development was retarded. When treated with minus lenses, the eyes of
RGE chicks showed an increase in axial length, globe circumference and
developed myopia compared to control eyes. When treated with plus lenses, the
eyes of RGE chicks did not differ from control eyes in axial length and developed
hyperopia. Recovery from from-deprivation and minus lens-wear results in an upregulation in Egr1 expression in glucagon-positive amacrine cells, as seen in WT
chicks. We conclude that the eyes of RGE chickens maintain the ability to
respond to vision-guided ocular growth clues prior to the development of the
retinopathy, globe enlarged phenotype. Moreover, RGE retinas alter the
expression of Egr1 in glucagon-positive amacrine cells in response to visual
manipulation in a manner similar to WT chicks. We propose that the RGE
chicken is an effective animal model to examine vision-guided ocular growth in
the presence of congentially reduced vision.
Introduction
The regulation of ocular growth during postnatal development arises from
a sophisticated interplay between the visual environment and the eye. This has
been best demonstrated by the phenomena of form-deprivation myopia and lens133
induced refractive errors. In animals, deprivation of form-vision causes excessive
ocular growth and myopia development (Sherman, Norton et al. 1977; Wiesel
and Raviola 1977; Wallman, Turkel et al. 1978; O'Leary and Millodot 1979;
Norton 1990; Troilo and Judge 1993; Tejedor and de la Villa 2003). Application of
divergent (a.k.a. minus) or convergent (a.k.a. plus) lenses have also been shown
to alter ocular growth, with minus lens-wear inducing ocular growth and plus
lens-wear suppressing ocular growth (Wallman, Wildsoet et al. 1995; Wildsoet
and Wallman 1995; Nickla, Wildsoet et al. 1997). The signaling mechanisms
responsible for regulating changes in ocular growth appear to be confined to the
eye, with no connections to higher visual centers required for vision to influence
ocular growth (Norton 1990; Norton, Essinger et al. 1994; McBrien, Moghaddam
et al. 1995).
Although it is generally accepted that normal visual function is required for
proper emmetropization, the process of ocular growth resulting in the lack of
significant refractive error at maturity, animal studies utilizing toxins to ablate
select populations of retinal neurons have shown that the eye can continue to
respond to form-deprivation. Moreover, the visual acuity of the species in these
experiments can vary greatly, from macaques which have a proper fovea and
visual acuity levels similar to humans and mice which have poor visual acuity
(Hendrickson and Kupfer 1976; Lee and Boothe 1981; Teller 1981; Prusky, West
et al. 2000). These studies, however, are limited by the fact that the application of
exogenous toxins may not be truly selective. The unintended collateral damage
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to retinal neurons and glia that may occur with such treatment makes
interpretation of these studies difficult. An animal model of diminished visual
function, resulting from a well-defined defect without incurring retinal damage,
would be advantageous for the study of the retinal neurons involved in guiding
ocular growth.
The Retinopathy, Globe Enlarged (RGE) chicken may be such a model.
The RGE phenotype in United Kingdom commercial chicken broods was first
reported in 1987. At hatch, the physical appearance of RGE chicks do not differ
from wild-type (WT) chickens; however, obvious behavioral losses in vision
facilitated the identification of the phenotype in commercial chicken broods in the
United Kingdom (Curtis, Baker et al. 1988). These chickens demonstrate
sluggish pupillary responses, abnormal electroretinograms (ERG) and linear
retinal lesions with thinning of the neural retina between 9 and 15 weeks of age
(Montiani-Ferreira, Li et al. 2003). Further examination revealed that RGE chicks
displayed decreased visual acuity at hatching (postnatal day 0 or P0) which
gradually progresses though P30, (Montiani-Ferreira, Li et al. 2003). Biometric
measurements revealed that the eyes of RGE chicks displayed no significant
difference in vitreous chamber depth, axial length, radial diameter, corneal
thickness, corneal curvature or refraction compared to control animals until P33
or later (Montiani-Ferreira, Li et al. 2003). This observation suggest that young
(<P30) RGE chicks are capable of emmetropization despite significant losses in
visual acuity. Electroretinograms in young (approximately P7) RGE chicks
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display decreased retinal sensitivity from hatching, with altered a-waves and
supernormal b-waves in light and dark adapted chicks. Over time, a progressive
reduction was observed in ERG amplitudes (Montiani-Ferreira, Shaw et al.
2007). Histological examination of the eyes of RGE chicks at hatching reveal
normal retinas, with only mild changes in the outer nuclear layer and outer
plexiform layer observed (Montiani-Ferreira, Fischer et al. 2005). The alteration
of the ERG response in RGE chickens without gross morphological changes to
the retinal has been attributed to a lack of guanine nucleotide-binding protein
beta-3 (GNB3), also known as cone β-transducin. The RGE phenotype develops
secondary to a spontaneous 3 base pair mutation, D153del, within the gene
encoding for GNB3 (Tummala, Ali et al. 2006). This leads to improper posttranslational folding of the protein and a complete loss of GNB3 from
photoreceptors and ON-bipolar cells (Ritchey, Bongini et al. 2010). Over time,
progressive retinal changes have been observed in RGE chicks; however,
severe retinopathy was not observed until after day P90 (Montiani-Ferreira,
Fischer et al. 2005). Given the severity of vision loss observed in young (<P30)
RGE chicks with the relatively modest changes to the retina without degredation,
the young RGE chick presents an opportunity to study how an animal with a
reduction in visual acuity, achieved without the use of exogenously applied
toxins, responds to vision-guided ocular growth clues.
Methods and Materials
Animals:
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The use of animals in these experiments was in accordance with the
guidelines established by the National Institutes of Health and the Ohio State
University. RGE chickens were hatched from fertilized eggs obtained from a
cross of RGE homozygous chickens from an established colony at the
Department of Small Animal Clinical Sciences, Michigan State University.
Postnatal wild-type white leghorn chickens (Gallus gallus domesticus) were
obtained at P0 from the Department of Poultry Science at The Ohio State
University. Postnatal chicks were housed in a stainless steel brooder and kept on
a cycle of 12 hours light, 12 hours dark (lights on at 8:00 AM). Chicks received
water and Purina chick starter ad libitum. Daytime luminosity levels between 400
and 700Lux were maintained during the 12 hour light cycle with fluorescent
luminaires (GE Ecolux F32T8SP35Eco). All chicks that were studied were
between age P1 and P30, long before the age of onset for globe enlargement
and retinal degeneration.
Visual manipulations:
Visual manipulations were achieved through the application of formdepriving translucent goggles or lenses. Form-deprivation goggles, –7, +7 or
Plano diopter lenses (Anchor Optics, Barrington, NJ) were applied to the right
eye only of RGE or wild-type chicks using cyanoacrylate adhesive. The left eye
of each animal was untreated. For quantitative real time polymerase chain
reaction (qRT-PCR) experiments, chickens were sacrificed immediately after
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goggle or lens treatment without visual recovery. In experiments assessing Egr1
expression in glucagoneric amacrine cells, chickens were given 2 hours of
unrestricted vision prior to sacrifice. Chickens wearing lenses had lenses cleaned
approximately every 2.5 hours during the light on cycle for the duration of the
experiment.
Ocular Measurements:
Pictographs of enucleated eyes were taken with a Nikon D100 digital SLR
camera. Biometric measurements were acquired from high resolution (>50
pixels/mm) digital pictographs of enucleated eyes using ImagePro Plus, v6.2,
software (Media Cybernetics, Bethesda, MD). Digital biometry of ocular globe
circumference, corneal circumference (Figure 4.1a), axial length, equatorial
diameter and corneal curvature (Figure 4.1b) provides accurate and reproducible
measurements as previously described (Fischer, Ritchey et al. 2008).
Measurements in pixels were converted to millimeters and statistical analyses
were performed using Microsoft Excel.
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Figure 4.1: Biometric measurements obtained from pictographs of enucleated
eyes. Using ImagePro Plus software and pictographs taken along the visual axis,
measurements of ocular globe circumference (panel a, circle a) and corneal
circumference (panel a, circle b) were obtained. Measurement of axial length
(panel b, line e), equatorial diameter (panel b, line f) and corneal curvature
(panel b, arc g) were obtained from profile pictographs of the eye. The calibration
bar equals 5mm.
High-resolution A-scan ultrasonography was used to measure ocular
components along the optical axis in vivo, including axial length, anterior
chamber depth, and crystalline lens thickness. Vitreous chamber depth from Ascan ultrasonography was calculated using the formula:
Corneal anesthesia was achieved using one drop of topical 0.5%
proparacaine hydrochloride ophthalmic solution. After insertion of a 4mmbarraquer pediatric lid speculum, a 20 MHz Panametrics-NDT (Waltham, MA)
transducer with a polystyrene delay line offset (V208-RM) driven by a
Panametrics-NDT 5072 pulser-receiver was coupled to the corneal apex using
ultrasound coupling gel (Medline Industries, Inc.; Mundelein, IL). The acoustic
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reflections were collected and digitized using a PicoScope® 5203 USB-PC
oscilloscope and the PicoScope® 6 PC Oscilloscope software, version 6.3.43.0.
Ultrasonic radio frequency (RF) signals were first filtered with a low-pass filter at
the cutoff frequency of 80MHz to exclude high frequency noise. The envelope of
the signals was then extracted using the analytic signal magnitude (Gammell
1981). Peaks corresponding to anterior cornea surface and retina were selected
for axial length measurement. Times-of-flight were determined and converted to
distance assuming the constant speed of sound of 1540m/s. It is noted that the
speed of sound in cornea and lens may be slightly higher that the assumed
value. Since the goal of the present study was to compare the thickness and
depths between control and treated tissues, the assumption of a uniform speed
of sound should have minimal influence on the outcome measures.
Retinoscopy and Refraction:
Non-cycloplegic streak retinoscopy to the nearest 0.25 diopter was
performed using trial lenses to measure refractive error in control and treated
eyes, similar to previous reports (Fischer, Miethke et al. 1998; Fischer, Seltner et
al. 1998). Retinoscopy was performed by one individual to prevent interindividual variability.
Tissue Preparation for Immunohistochemistry:
Ocular tissues were processed for immunohistochemistry as previously
described (Fischer, Omar et al. 2005). Immunofluorescence was optimized for
Egr1 and glucagon as follows: sections were washed in PBS, blocked for 30
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minutes in antibody diluent (PBS plus 0.2% Triton X-100) plus 5% Normal
Donkey Serum, washed in PBS and goat-anti-human Egr1 (R&D Systems
#AF2818) primary antibody diluted to 1:200 in PBS and incubated overnight.
Sections were then washed in PBS and secondary antibody donkey-anti-goatAlexa488 (Invitrogen), diluted to 1:1000 in antibody diluent, applied for 1 hour.
After washing in PBS, mouse monoclonal anti-glucagon (M. Gregor), diluted to
1:1000 in antibody diluent, was applied overnight. After washing, goat-antimouse-IgM-Alexa568, diluted to 1:1000 in antibody diluent, was applied. The
immunolabeling for Egr1 and glucagon was processed in sequence to avoid
cross-reactivity of the donkey-anti-goat-Alexa488 with the mouse monoclonal
anti-glucagon antibody. The sections were then washed and mounted in 4:1 (v:v)
glycerol in water.
Tissue Preparation for qRT-PCR:
After enucleation, eyes were immediately placed in chilled HBSS+
solution. The retinae were removed and immediately placed in RNAlater
(Ambion, Austin Tx). After RNA stabilization, retinae were placed in 1.5 ml of
Trizol Reagent (Invitrogen; Carlsbad, CA) and total RNA was isolated according
to the Trizol protocol and resuspended in 50 μl RNAse free water. Genomic DNA
was removed by using the DNA FREE kit provided by Ambion (Austin, TX).
cDNA was synthesized from mRNA by using Superscripttm III First Strand
Synthesis System (Invitrogen) and oligo dT primers according to the
manufacturer’s protocol. Primers for preproglucagon and Glyceraldehyde 3141
phosphate dehydrogenase (GAPDH) were designed using the Primer-BLAST
primer design tool at NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).
Primer sequences for preproglucagon and GAPDH are listed in Table 4.1.
qRT-PCR reactions were performed using SYBRGreen PCR mastermix
and the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad,
CA). Control reactions were performed using all components with the exception
of the reverse transcriptase to exclude the possibility that primers were
amplifying genomic DNA. PCR products were run on a 2.0% agarose gel to verify
the predicted product sizes.
Target mRNA
Sequence
Preproglucagon
Forward: AAT GAC AAA TTC CCG GAT CA
Reverse: GGT GTA GGT GCC TTC AGC AT
Forward: GGA ACA CTA TAA AGG CGA GAT
Reverse: TCA CAA GTT TCC CGT TCT CA
GAPDH
Predicted
product size
87
218
Table 4.1: PCR primers (5’ to 3’), target and predicted product sizes
Results
RGE chicks emmetropize in the 1st 2 weeks post-hatching.
Previous reports indicate that RGE chickens become hyperopic; however,
the development of hyperopia occurs after P33 (Montiani-Ferreira, Li et al. 2003).
We sought to determine the refractive error of RGE chickens during the age
range commonly used in visual manipulation experiments (approximately P7P14). The mean spherical equivalent refraction in RGE chicks at P12 was
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determined by using streak retinoscopy. The average refractive error of RGE
chicks at P12 was neutral (–0.03 ± 0.37DS, n=17; Figure 4.2), indicating the lens
powers used in subsequent experiments (–7 & +7) should be sufficient to
investigate whether RGE chicks respond to induced hyperopic and myopic
defocus.
Figure 4.2: Retinoscopy indicates that RGE chickens are emmetropic at age P12.
Non-cycloplegic retinoscopy of a single eye in 17 RGE animals demonstrates
that RGE chickens lack significant refractive error at the time of form-deprivation
or lens experiments. Error bars represent standard deviation.
Form-deprivation causes changes in ocular dimensions in RGE chickens
We sought to determine if the eyes of RGE chicks respond to formdeprivation as an ocular growth stimulus. After 6 days of form-deprivation
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beginning at P7, axial length (AL) was increased in the treated eye when
compared to that of the contralateral control eye (0.20 ± 0.26 mm, n=9; p=0.02).
A control cohort of WT chicks was also examined. As expected, increased axial
length was observed with form-deprivation in WT chickens, with an average
increase of 0.29 ± 0.27 mm after six days of form-deprivation (n=10; p=0.004).
Given that the systemic loss of GNB3 in RGE chickens may lead to animals that
are physically smaller than WT chicks, we compared the axial length of the
control eyes between the two cohorts. Comparison of the axial length in the
untreated control RGE eye and age-matched WT chicks indicates that RGE
animals have smaller eyes compared to WT animals (9.94 ± 0.57mm vs 9.31 ±
0.28mm; p=0.008). Given the variation in animal size between RGE and WT
chickens, we compared the percentage change between the treated eye and the
contralateral control eye. When examining the percentage change in axial length
with form-deprivation, FD-induced changes in the axial length of RGE eyes were
not different compared to the WT chickens (p=0.59; 2 tailed t-test) (Figure 4.3).
WT chickens showed an average 2.97 ± 2.88% increase in axial length with
form-deprivation compared to an average 2.24 ± 2.81% increase in RGE
chickens.
Examination of the growth around the circumference of the globe,
subsequently refered to in the text as circumferential growth, revealed that RGE
eyes grew in response to form-deprivation compared to the contralateral
untreated eye (0.56 ± 0.39mm; p=0.003 paired t-test). We sought to determine if
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this change was consistent with that observed in WT chicks. WT chickens
showed an average increase in circumferential growth of 1.47 ± 0.49mm
(p<0.0001). After conversion to percentage change between the treated and the
control eye, examination of FD-induced increases in circumferential growth in WT
and RGE eyes revealed that the response of RGE chicks was significantly less
(1.47 ± 1.05% RGE vs 3.74 ± 1.26% WT, p<0.001, 2-sided t-test) than that
observed in WT chicks (Figure 4.3). When examining equatorial diameter (Figure
4.1b, line f) the treated eyes of RGE chickens were significantly larger than the
contralateral controls (0.38 ± 0.33mm, p=0.009 2 sided t-test). A significant
increase in equatorial diameter was observed in WT chicks as well (0.45 ±
0.23mm, p<0.001 2 sided t-test). Examination of the percentage change between
WT and RGE chickens revealed that this growth was not statistically significant
(3.57 ± 1.87% WT vs 3.20 ± 2.85% RGE, p=0.74 2 sided t-test) (Figure 4.3).
Form-deprivation does not cause changes in corneal curvature or diameter
Given the observed increases in axial length and in circumferential eye
growth, we examined whether form-deprivation caused changes in the corneal
arc, also known as the radius of curvature, and corneal circumference. Formdeprivation did not significantly change the corneal arc in either WT or RGE
animals based on a 5-point best fit curve matched to the central corneal
curvature. In WT chickens, the average increase in corneal arc with formdeprivation was 0.02 ± 0.29mm (p=0.83; paired t-test). In RGE chickens, the
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average increase was 0.08 ± 0.31mm (p=0.48, paired t-test). For comparison
between experimental groups, the percentage change in corneal arc was
calculated, with the average percentage change being 0.80 ± 8.81% in WT
animals and 2.34 ± 8.84% in RGE animals. This difference was not statistically
significant (p=0.71, 2 sided t-test) (Figure 4.3).
Figure 4.3: Percentage change in ocular growth observed in WT and RGE chicks
with form-deprivation. The right eye of WT (n=10) and RGE (n=9) chickens were
form-deprived for 6 days beginning at P7. No statistically significant difference in
axial length, equatorial diameter, corneal radius of curvature or corneal
circumference between RGE and WT chicks. A statistically significant difference
was observed in circumferential eye growth (p=0.0005, 2 sided t-test). Error bars
represent standard deviation.
Corneal circumference, as defined as a best fit circle around the cornea
(Fig 4.1a, circle b,) was also examined. In WT chickens, there was no difference
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in corneal circumference observed between form-deprived eye and untreated
lateral controls (–0.06 ± 0.53mm, p=0.71, paired t-test). This observation was
also noted in RGE chickens, with an average difference of 0.18 ± 0.42mm
(p=0.24, paired t-test). There was no difference observed between the response
of WT chicks verses RGE chicks in regards to percentage change in corneal
circumference over time with form-deprivation (–0.27 ± 3.10% WT vs 1.21 ±
2.76% RGE; p=0.29 t-test) (Figure 4.3).
RGE Chicks respond to lens-induced ocular growth stimuli
Given the observed increases in axial length in response to formdeprivation, we next tested whether the eyes of RGE chicks would respond to
lens imposed defocus. Spherical lenses with powers of –7 diopters were applied
starting at P7 and continuing through P11.5. When treated with hyperopic
defocus, RGE chickens showed a statistically significant increase in axial length.
The axial length of the treated eye was on average 0.48 ± 0.22mm longer than
the contralateral, untreated control eye (n=10, p<0.001 paired t-test). Identical
treatment in WT chicks also caused a statistically significant increase in axial
length, with an average increase of 0.28 ± 0.21mm (n=10, p=0.002 paired t-test).
To determine if there was a difference in the response to minus lenses between
RGE and WT animals, the percentage change between the treated and control
eyes were calculated. Comparison of the percentage change between the two
cohorts revealed that the response of the RGE chicks to minus lenses was
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statistically more robust than that observed in WT chicks (6.59 ± 2.98% RGE vs
3.75 ± 2.76% WT; p=0.04) (Figure 4.4a).
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Figure 4.4: Percentage change in ocular growth observed in WT and RGE chicks
with lens treatment. The right eye of WT and RGE chickens were treated with –
7DS (n=10/group), +7DS (n=10/group) and Plano (n=6/group) lenses for 4.5
days beginning at P7. –7DS treatment caused significant increase in axial length
in WT and RGE chicks and a significant decrease in axial length in WT chicks
with +7DS lenses (panel a). No significant changes were observed in anterior
chamber depth (panel b) or intraocular lens thickness (panel c). The observed
increase or decrease in axial length with lens treatment was due primarily to
changes in vitreous chamber depth (panel d). * indicate a significant treatment
edffect of p0.05. Error bars represent standard deviation.
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150
Figure 4.4
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The observed increase in ocular growth appears to be due to increased
vitreous chamber depth (VCD), whereas the anterior chamber depth (ACD) and
intraocular crystalline lens (IOL) thickness were unaffected by lens-wear. When
examining anterior chamber depth after minus-lens treatment, no difference was
observed in RGE chicks (–0.05 ± 0.28mm, n=10, p=0.58, paired t-test). When
examining intraocular lens thickness, no difference was observed in RGE chicks
(–0.03 ± 0.30mm, p=0.80, paired t-test). Examination of the WT animals showed
a similar response, with no significant changes observed in the depth of the
anterior chamber (–0.05 ± 0.26mm; p=0.59 paired t-test) and intraocular lens
thickness (0.05 ± 0.37mm, p=0.66 paired t-test). Comparison of RGE and WT
chickens by percentage change treatment verses control revealed no statistically
significant difference in the anterior chamber response (2.13 ± 54.7% RGE vs.
2.66 ± 48.3% WT; p=0.98 2 sided t-test) (Figure 4.4b) or in IOL thickness (–1.36
± 22.8% RGE vs 8.15 ± 31.1% increase WT; p=0.45 2 sided t-test) (Figure 4.4c).
We observed that minus lens-treatment caused a significant increase in
VCD in RGE chicks (0.56 ± 0.50mm, p=0.007, paired t-test). A similar finding was
observed in WT chicks, with an average increase in VCD of 0.28 ± 0.14mm
(p<0.001, paired t-test) with minus lens-treatment. A comparison of the
percentage change treated vs. control between the two cohorts revealed no
statistically significant difference in the percentage increase in VCD (p=0.12, 2
sided t-test) (Figure 4.4d).
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Minus lens-treatment also influenced circumferential eye growth.
Examination of the ocular equator revealed that RGE eyes grew in response to
the lens treatment (0.80 ± 0.53mm, p=0.002 paired t-test). A similar response
was observed in the WT cohort (0.82 ± 0.49mm, p=0.001). Comparison of
circumferential growth between WT and RGE eyes as a percentage change
revealed no statistically significant difference in growth (2.12 ± 1.41% RGE vs
2.17 ± 1.30% WT; p=0.93) (Figure 4.5a). When examining equatorial diameter,
both RGE and WT eyes showed growth in response to –7 lenses (0.45 ± 0.24mm
RGE, p=0.0005; 0.26 ± 0.20mm WT, p=0.005). Comparison of the percentage
change indicated that the response in equatorial diameter was not significantly
different between WT and RGE (3.76 ± 2.04% RGE vs 2.13 ± 1.62% WT;
p=0.07) (Figure 4.5b).
Despite the increase in circumferential eye growth, the corneal
circumference did not change for either the RGE (-0.08 ± 0.53mm; p=0.68 paired
t-test), or WT chicks (0.07 ± 0.41mm; p=0.65 paired t-test). Furthermore, corneal
curvature did not change with minus 7 lens-treatment for either cohort (-0.21mm
± 0.44mm RGE; p=0.18 paired t-test vs. -0.17mm ± 0.58mm WT; p=0.41 paired
t-test).
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Figure 4.5: Percentage change in circumferential ocular growth and corneal
curvature observed in WT and RGE chicks with lens treatment using ImagePro.
The right eye of WT and RGE chickens were treated with –7DS (n=10/group),
+7DS (n=10/group) and Plano (n=6/group) lenses for 4.5 days beginning at P7.
Overall ocular circumference (panel a), equatorial diameter (panel b), corneal
circumference (panel c) and corneal curvature (panel d) are presented. Plano
lens wear caused a significant increase in the percentage change in ocular
circumference in WT chicks compared to RGE animals (panel a). Corneal
circumference was significantly less in RGE chicks compared to WT (panel c).
Error bars represent standard deviation.
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154
Figure 4.5
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Consistent with the observed increases in axial length, eyes treated with –
7 lenses over 4.5 days developed significant (p=0.003) myopia compared to the
untreated control. WT chicks displayed an average of –2.65 ± 2.17D myopia with
treatment. In RGE chicks, significant myopia also developed with lens-treatment
(–1.40 ± 1.24D RGE) (p=0.008, Fig 4.6a).
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Figure 4.6: Non-cycloplegic retinoscopy demonstrates that RGE chickens
develop myopia with 4.5 days of –7 lens wear and hyperopia with 4.5 days of +7
lens wear. After lens removal, retinoscopy was performed using trial lens on the
treatment and control eyes of each animal. Similar to WT animals, RGE animals
developed refractive error subsequent to induced defocus with –7 and +7 lens.
Similar to WT animals, plano lens wear did not induce significant refractive error.
Error bars represent standard deviation.
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157
Figure 4.6
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RGE chicks respond to growth-slowing visual stimuli
Given that RGE eyes respond to growth-promoting stimuli such as formdeprivation and divergent lens wear, we sought to determine if RGE eyes
respond to growth slowing stimuli such as form-deprivation interrupted by brief
periods of unrestricted vision or by plus lens-wear. It is well established that brief
periods of clear vision presented during a form-deprivation treatment allows for
recovery of the growth induced by form-deprivation. One-hundred and thirty
minutes of daily unobstructed vision interrupting constant form-deprivation
resulted in a 95% prevention of excessive ocular growth in WT chickens (Napper,
Brennan et al. 1995). In RGE chickens, we found that the excessive ocular
growth observed with form-deprivation can also be interrupted by brief periods of
clear vision. Two cohorts of RGE chicks were examined, one undergoing 24 hour
form-deprivation for 1 week and a second cohort given 130 minutes of
unobstructed vision per day during the middle of the 12 hour light cycle. After
seven days of constant form-deprivation, the average increase in axial length in
the treated eyes was 0.40 ± 0.41mm (n=13, p=0.004, paired t-test). In the cohort
of RGE chicks given 130 minutes of daily uninterrupted vision, there was no
increase in axial length (0.12 ± 0.27 mm, n=15, p=0.10, paired t-test). To
compensate for inter-animal variability in size, axial length was converted to a
percentage change verses control. Brief periods of normal vision interrupting
form-deprivation caused a statistically significant reduction in the ocular growth
(5.12 ± 5.16% Constant FD vs. 1.59 ± 3.38% interrupted FD; p=0.04, 2 sided t158
test) (Figure 4.7a).This observed difference in axial length resulted from
increases in VCD, where constant FD caused a significant increase in VCD (1.35
± 0.34 mm, n=15; p<0.0001 paired t-test); whereas brief periods of visual
recovery prevented a significant difference in VCD (0.10 ± 0.27 mm, n=13;
p<0.17) (Figure 4.7a). The difference in the percentage change in VCD between
the two treatments was highly significant (22.78 ± 5.89% constant FD vs 1.85 ±
4.65% interrupted FD; p<0.0001).
Constant form-deprivation caused a significant increase in circumferential
ocular growth compared to the contralateral control in the RGE chick, with an
average increase of 1.35 ± 0.41mm (n=12, 39.73 ± 1.10mm treated vs 38.38 ±
1.00mm control; p<0.0001 paired t-test). With 130 minutes of daily visual
recovery, circumferential ocular growth was observed; however, the amount of
growth was reduced, with an average increase in the treated eye of 0.62 ±
0.69mm (38.94 ± 1.67mm treated vs 38.33 ± 1.32mm control; n=14, p=0.005
paired t-test). Comparison of the percentage change showed that the eyes that
underwent constant FD grew more circumferentially than the eyes given a period
of visual recovery (3.53 ± 1.06% constant FD vs. 1.60 ± 1.76% interrupted FD,
p=0.003) (Figure 4.7b). Equatorial diameter was also examined. In both cohorts,
the treated eye showed a statistically significant increase in equatorial diameter,
with the constant form-deprivation cohort showing an average increase of 0.44 ±
0.36mm (n=12, p=0.001 paired t-test) and the visual recovery group showing an
average increase 0.35 ± 0.35mm (n=14, p=0.002 paired t-test). Comparison of
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the percentage change revealed that the observed increase between the 2
cohorts was not significantly different (3.62% ± 3.02% constant FD vs. 2.89% ±
2.87% with visual recovery, p=0.54) (Figure 4.7b). Examination of corneal
circumference revealed that the RGE chicks undergoing constant formdeprivation had a significant increase in corneal circumference compared to the
control eye where animals given visual recovery did not (0.39 ± 0.31mm constant
FD vs. 0.06 ± 0.32mm with visual recovery, p=0.001). This difference as a
percentage change treated to control was significant (2.42 ± 1.93% constant FD
vs 0.44 ± 1.95% with visual recovery, p=0.02) (Figure 4.7b). No statistically
significant changes in corneal curvature were observed in either treatment group
(Figure 4.7b).
Periods of visual recovery prevented the development of myopic refractive
error with form-deprivation. RGE eyes undergoing constant form-deprivation
became significantly more myopic than RGE eyes treated with daily brief periods
of clear vision (–3.04 ± 0.88D constant FD vs. –0.15 ± 1.01D control eye, n=13,
p<0.0001 paired t-test). Animals in the visual recovery group did not develop
myopia, nor did their refractive error differ significantly from the control eye
(+0.17 ± 2.06D FD + visual recovery vs. 0.20D ± 1.72D SD control eye, n=15,
p=0.88 paired t-test) (Figure 4.7c).
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Figure 4.7: Recovery from form-deprivation (FD) slows ocular enlargement and
prevents the development of myopia in RGE chicks. RGE chicks given constant
FD develop increases in axial length (panel a), vitreous chamber depth (panel a),
ocular circumference (panel b), corneal circumference (panel b) and myopia
(panel c) compared to RGE chicks given 130 minutes daily visual recovery from
FD. Error bars represent standard deviation.
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162
Figure 4.7
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Plus Lens-wear slows axial growth in RGE chickens
Convergent lens wear has been shown to be a potent stimulus to reduce
axial ocular growth. To determine if RGE chicks responded in a manner similar to
WT chicks, +7 diopter lenses were applied at P7 for 4.5 days. The axial length of
RGE eyes given 4.5 days of plus lens treatment was not different than the
contralateral control eyes (–0.24 ± 0.43mm, n=10, p=0.11, paired t-test). WT
eyes presented with an identical treatment paradigm revealed a statistically
significant reduction in eye size (–0.27 ± 0.33mm, n=10, p=0.03, paired t-test).
When comparing the percentage change in axial length between RGE and WT
chicks with plus lens treatment, there was no statistically significant difference in
the response between the two cohorts (–3.59 ± 4.47% WT vs. –3.21 ± 5.77%
RGE, p=0.87) (Figure 4.4a). Examination of VCD showed no reduction in VCD in
RGE chicks with plus lens treatment (–0.35 ± 0.86mm, p=0.23). By comparison,
WT chicks had a statistically significant reduction in VCD with plus lens treatment
(–0.28 ± 0.40 mm, p=0.05, paired t-test). There was no statistically significant
difference in the percentage change in VCD of RGE chicks (–5.72 ± 13.8%
reduction VCD) vs WT chicks (–5.05 ± 7.22% reduction VCD) with +7 lens
treatment (p=0.89) (Figure 4.4 d). Plus 7 lens treatment did not have a
statistically significant effect on percentage change in ACD or IOL thickness in
RGE or WT birds (Figures 4.4b & c).
Examination of circumferential eye growth with plus lens treatment
showed that +7 lens treatment failed to inhibit ocular growth around the
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circumference of the eye in RGE or WT chickens. RGE eyes treated with +7
lenses showed a statistically significant increase in circumferential growth (0.47 ±
0.46mm, n=10, p=0.01 paired t-test). Circumferential growth was also observed
in WT animals, with an average increase of 0.75 ± 0.34mm (n=10, p<0.0001
paired t-test). Comparison of the percentage change in circumferential eye
growth with +7 lens wear showed no difference in the response of RGE and WT
animals (1.36 ± 1.23% RGE vs. 2.02 ± 0.90% WT; p=0.13 2 sided t-test). This
finding was also observed in equatorial diameter, with both RGE and WT chicks
showing an increase in growth with plus lens treatment (0.43 ± 0.21mm RGE;
p=0.0001 paired t-test vs. 0.45 ± 0.43mm WT; p=0.008 paired t-test). There was
no difference in the growth in equatorial diameter observed between RGE and
WT chicks (p=0.86). Corneal circumference was unchanged in RGE chicks with
+7 lens wear (–0.21 ± 0.32mm; p=0.07 paired t-test); however, a small
statistically significant decrease in corneal circumference was observed in WT
animals (–0.20 ± 0.28mm; p=0.05 paired t-test). When the percentage change
between the treatment and control eyes was examined, there was no difference
between RGE and WT chicks (p=1.00). Plus lens wear did not change corneal
curvature in RGE (–0.03 ± 0.47mm RGE; p=0.85) or WT chickens (0.04 ±
0.25mm WT; p=0.64); nor did the percentage change in ocular growth differ
between RGE and WT animals for corneal curvature (p=0.73).
Application of +7 lenses induced statistically significant hyperopia in the
treated eye when compared to controls in both WT and RGE chicks. The treated
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eyes of WT chicks were significantly more hyperopic (+3.85 ± 2.26D treated vs.
+0.05 ± 1.23D control, n=10, p<0.0001 paired t-test). RGE chicks displayed a
significant hyperopic shift (+0.78 ± 1.28D treated vs. –0.23 ± 0.43D controls,
n=10, p=0.03 paired t-test); however, the shift was not as robust as that seen in
WT animals (p<0.0001) (Figure 4.6b)
Application of plano lenses does not affect ocular growth
To verify that the growth-altering effects of lens treatment were not due to
the application of a lens to the periorbital tissue, plano lenses were applied to
RGE and WT chicks as a control. After 4.5 days of plano lens wear, no
statistically significant differences were observed between treated and untreated
contralateral control eyes in axial length, ACD, IOL thickness or VCD in RGE
animals or in the WT cohort (Figures 4.4a-d).
Examination of ocular growth around the circumference of the eye
revealed that RGE eyes did not grow significantly with plano lens wear (0.26 ±
0.52mm; p=0.33 paired t-test). Surprisingly, WT eyes showed a significant
increase in circumferential growth (1.05 ± 0.38mm; p=0.001 paired t-test).
However, this difference was not observed in equatorial diameter, where neither
the RGE (0.31 ± 0.34mm RGE, p=0.11 paired t-test) nor the WT animals (0.17 ±
0.24mm WT, p=0.15 paired t-test) showed significant growth. Corneal
circumference was significantly smaller with plano lens wear in RGE chicks (–
0.61 ± 0.33mm RGE, p=0.02 paired t-test); however, no difference was observed
in WT animals (0.01mm ± 0.34mm WT, p=0.95 paired t-test). This resulted in a
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statistically significant difference in the percentage growth between RGE and WT
chicks for corneal circumference (p=0.01, 2 sided t-test). Examination of corneal
curvature revealed no change in RGE (p=0.83) or WT (p=0.33) chicks with plano
lens wear. As expected, application of plano lenses did not cause a significant
shift in refractive error in the WT or RGE animals (Figure 4.6c).
Egr1 expression changes in glucagon-positive amacrine cells with visual
manipulation
In previous reports, form-deprivation caused a decrease in the percentage
of Egr1-positive glucagon-expressing amacrine cells in WT chickens. After
discontinuation of form-deprivation, there was a transient increase in the
percentage of glucagon-positive amacrine cells that co-express Egr1 (Fischer,
McGuire et al. 1999). We sought to determine whether Egr1 expression in
glucagon-expressing amacrine cells is regulated by growth-guiding visual stimuli
in RGE retinas. After undergoing 6 days of form-deprivation, goggles were
removed and RGE chickens were given 2 hours of unrestricted vision. In RGE
chicks, there was a significant (p<0.001, n=8) up-regulation of Egr1 in
glucagonergic amacrine cells in response to recovery from form-deprivation,
consistent with WT animals (p=0.0001, n=10) (Figure 4.8).
Given that the level of Egr1 expression were significantly increased with
recovery from form-deprivation in RGE chickens, we sought to determine if Egr1
expression in glucagonergic amacrine cells was altered in response to lens166
treatment. After 4.5 days of lens-wear, lenses were removed and chickens were
given 2 hours of unrestricted vision. After visual recovery from minus lens wear,
there was a statistically significant increase in the percentage of Egr1-positive
glucagoneric amacrine cells observed (p=0.0004, n=10) compared to the control
eye. This result was consistent with the result observed in WT animals
undergoing minus lens treatment (p<0.0001, n=10). After discontinuation of plus
lens treatment, treated RGE retinas showed no difference in the number of
glucagon-positive amacrine cells that co-express Egr1 compared to contralateral
control retinas (p=0.07; n=10), consistent with the findings observed in WT
retinas (p=0.07; n=10) (Figure 4.8).
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Figure 4.8: The expression of Egr1 in glucagoneric amacrine cells changes with
visual manipulation in WT and RGE chickens. Visual recovery from 6 days formdeprivation or 4.5 days minus lens treatment caused a significant increase (*
p<0.001) in the number of Egr1-positive glucagoneric amacrine cells compared
to control retinas. Retinas recovering from plus lens treatment did not show a
statistically significant difference compared to control retinas in either RGE or WT
chicks. Error bars represent standard deviation.
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Levels of pro-glucagon mRNA are altered with visual manipulation in RGE
chickens
Given the observed vision-induced changes in ocular growth and Egr1expression in glucagonergic amacrine cells in RGE chicks, we sought to
characterize the transcriptional regulation of preproglucagon with visual
manipulation. Form-deprivation goggles or lenses were applied to the right eye of
P7 chicks and lenses were cleaned approximately every 2.5 hours during the
light cycle. After 4.5 days, lenses were removed and the eyes harvested without
a period of visual recovery. After 4.5 days of form-deprivation, WT animals
displayed a decrease in preproglucagon mRNA; whereas RGE animals did not
show a decrease (–21.1± 7.13% WT, n=5, p=0.006 vs.–4.2 ± 7.67% RGE, n=4,
p=0.59). With minus lens wear, both WT and RGE chicks displayed downregulation in preproglucagon (–42.53 ± 4.57% WT, n=5, p<0.0001 vs –27.78 ±
5.92% RGE, n=4, p<0.0001). When presented with a growth slowing stimuli (plus
lenses), RGE eyes showed a down-regulation of preproglucagon mRNA (–12.48
± 5.99% RGE, n=4, p=0.05); where preproglucagon levels in WT chicks did not
change (5.11 ± 18.78% WT, n=4, p=0.79) (Figure 4.9).
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Figure 4.9: Levels of pro-glucagon mRNA are altered with visual manipulation in
RGE chickens. After 4.5 days of minus 7 lens treatment, preproglucagon mRNA
expression is decreased in WT and RGE retinas (* p<0.0001). After treatment
with plus lens treatment, RGE retinas show a decrease in preproglucagon mRNA
(** p=0.05). With form-deprivation, WT animals show a drecrease in
preproglucagon mRNA (***p=0.006), while preproglucagon mRNA levels did not
change in RGE retinas. Error bars represent standard deviation.
Discussion
We report here that RGE chickens respond to vision-guided ocular growth
signals in a manner similar to WT animals. Although early post-natal RGE
chickens are nearly blind at hatching, the eyes responded to form-deprivation
and lens-induced refractive errors. When subjected to form-deprivation through
the use of translucent goggles, the eyes of RGE chicks show an increase in axial
length, a response previously observed in WT chickens (Wallman, Turkel et al.
1978; Gottlieb, Fugate-Wentzek et al. 1987; Wallman, Gottlieb et al. 1987). An
increase in ocular circumference was also observed in RGE chicks under form170
deprivation conditions. When presented with induced hyperopic defocus by –7
lenses, RGE chicks again displayed increases in axial length and circumference,
similar to the effects seen with form-deprivation. When presented with myopic
defocus through convergent lens wear, axial length in RGE chicks was reduced
as previously observed in WT chicks (Wallman, Wildsoet et al. 1995; Wildsoet
and Wallman 1995; Kee, Marzani et al. 2001; Feldkaemper and Schaeffel 2002);
however, circumferential eye growth showed an increase with plus lens wear.
These findings suggest that the retina in the RGE can detect form-deprivation
induced retinal blur. Moreover, the response of RGE chickens to lens-induced
defocus suggests that these animals can detect the sign of imposed defocus,
despite their reduction in vision.
An interesting observation in examining the response of RGE chicks to
lens-induced ocular growth was that with hyperopic defocus, RGE chickens
showed a more robust increase in axial length when compared to age-matched
WT controls. Conversely, the response of RGE eye to myopic defocus was
subdued compared to the WT controls. One potential explanation for the
observed difference may be that an uncontrolled variable in the measurement of
axial length, the globe enlarged phenotype in RGE chicks, may be present. As
the animal ages, progressive globe enlargement would lend to the appearance
that hyperopic defocus is a more potent growth signal in these animals and
myopic defocus a less potent stimulus. Although the RGE phenotype has been
reported to become significant after age P30 (Montiani-Ferreira, Li et al. 2003),
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we can not rule out that progressive globe enlargement did not contribute to the
observed differences in axial length beween RGE and WT chickens. A second
potential explanation would be that the retinas of RGE chickens respond
differentially to different visual manipulations. This would suggest that the
hyperopic defocus induced by minus lens wear is a more potent growthregulation stimulus given the significant increase observed in RGE chicks in axial
growth compared to WT animals. Conversely, myopic defocus could be a less
potent stimulus for regulating ocular growth in RGE animals. A third potential
explanation for the observed difference in axial length between lens groups could
come from reports of the development of hyperopia in RGE chicks secondary to
corneal flattening. The convergent lenses used in our study may have been
rendered less effective over the time course of the experiment with the
development of RGE phenotype-driven hyperopia.
The argument for the development of hyperopia in RGE chicks partially
neutralizing the convergent lens power used in our study is unlikely, given that
we showed that RGE chicks fail to exhibit significant hyperopia at approximately
2 weeks of age when the animals were exposed to visual manipulation.
Furthermore, the reported time point of significant hyperopia development in the
RGE phenotype is at day P33 (Montiani-Ferreira, Li et al. 2003), well after the
age at which our experiments were performed. Assuming that increasing
hyperopic refractive error in RGE chickens did not cause of the different
responses in axial length observed with lens treatment, a potential line of future
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inquiry would be to examine the effectiveness of different lens powers at
promoting or inhibiting axial growth. If a true difference in the sensitivity of the
RGE retina to hyperopic and myopic defocus exists, a growth-response curve to
induced hyperopic and myopic defocus could elucidate whether the difference in
reponse between plus and minus lens wear observed in our study was simply
due to the globe enlargement phenotype. Increasing amounts of hyperopic
defocus would cause increased amounts of ocular growth to compensate for the
induced refractive error. If RGE retinas were truly relatively less sensitive to
induced myopic defocus, the compensation for induced myopia with plus lenses
would lag relative to the compensation response to induced hyperopic defocus
with minus lens wear. If no difference exists in the sensitivity of RGE retinas to
one visual stimulus verses another, the eyes should alter their growth
repsectively to compensate for the induced refractive error. Assuming a relatively
constant rate of ocular growth with the RGE phenotype, a relative consistent lag
in the compensation to convergent lens would be observed over all lens powers.
This could provide some insight to why induced myopic defocus was less
effective at altering axial elongation than induced hyperopia.
Given the rationale above, it is unlikely that the RGE retina is relatively
less sensitive to one ocular growth stimulus, particularly induced myopic defocus.
This was demonstrated by our assay for Egr1 expression in glucagon-positive
amacrine cells. Our data shows that RGE retinas respond in a manner consistent
with WT animals. When allowed 2 hours of visual recovery after form-deprivation
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or induced hyperopia, the percentage of Egr1-positive glucagoneric amacrine
cells in the RGE chicken increased. Conversely, recovery from induced myopia
caused a decrease in the number of Egr1-positive glucagoneric amacrine cells;
however, this number did not reach statistical significance. This does not
preclude the possibility that the transcriptional mechanisms in the RGE chick are
significantly different than those reported in WT chickens. Plus lens wear for 4.5
days in RGE chicks caused a statistically significant decrease in preproglucagon
mRNA; whereas the WT animals showed a slight increase that did not reach
statistical significance, consistent with a previous report (Buck, Schaeffel et al.
2004). Despite the observed difference in preproglucagon levels between WT
and RGE chicks, caution should be used in the interpretation of the data,
particularly given some of the unusual observations reported in PCR assays for
glucagon. For example, Buck reported a short-term down-regulation mRNA
levels in the contralateral control eye with lens treatment (Buck, Schaeffel et al.
2004). Given the data, the most likely reason for the differences observed in axial
length comes from the slowly developing RGE phenotype.
A similarly interesting finding was that not only did minus lenses wear
cause a significant increase in circumferential eye growth, plus lenses also
increased circumferential eye growth. Given that convergent lenses are a potent
stop-grow signal and axial length confirmed the growth slowing effect of the
lenses, why did ocular circumference increase? A possible hypothesis to explain
this finding would be that the imposed myopic defocus in our stuy did not
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effectively stimulate the bullwhip and mini-bullwhip cells in the retina of these
animals, leading to ocular growth. Bullwhip and mini-billwhip cells are an
extremely small subset (<1000) of glucagon-positive neurons residing in the
dorsal and ventral retina. These cells featuring long, unipolar axons that run to
the circumferential marginal zone and express some transcription factors that are
expresses by glucagon-positive amacrine cells, such as Pax6 and AP2α;
however, they express transcription factors not observed in glucagon-positive
amacrine cells such as TrkB and TrkC (Fischer, Skorupa et al. 2006). These cells
have been shown to participate in the regulation of circumferential eye growth in
WT chickens. Bullwhip cells have been shown to up-regulate Egr1 with visual
recovery from form-deprivation, similar to glucagon-positive amacrine cells.
Furthermore, ablation of these cells with colchicine delivered at P7, a timepoint
which spares glucagon-positive amacrine cells from colchicine damage, causes
circumferential eye growth without affecting axial length (Fischer, Ritchey et al.
2008). While bullwhip cells are present in the RGE retina (personal observation,
data not shown), they have not been characterized. It is unknown if they respond
to visual growth stimuli in a manner similar to WT chicks. After form-deprivation,
RGE chicks in our study showed less circumferential eye growth than WT
animals, implying that bullwhip cells were protective for eye growth.
Unfortunately, after minus lens wear, RGE eyes showed equivalent
circumferential growth to WT chicks, indicating no protective effect. The observed
growth with plus lens wear could possibly be due to peripheral deprivation while
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wearing the experimental lenses. Given that the plus lenses used in the
experiment have a smaller diameter than the form-deprivation goggles or minus
lens, this remains a plausible explanation. Regardless, more research is required
to characterize the proteins expressed by these cells and to quantify the
response of these cells to visual stimuli in the RGE chick.
The RGE chicken is an animal model of poor visual acuity; however, they
respond to vision-guided ocular growth signals despite the loss of GNB3 in cone
photoreceptors and ON-bipolar cells. It has been clearly shown that the loss of
GNB3 in photoreceptors and ON-bipolar cells in these animals is complete
(Ritchey, Bongini et al. 2010). This loss of GNB3 negatively affects visual signal
transduction, as GNB3 is part of the heterotrimeric G-protein that regulates
phosphodiesterase activation and subsequent photoreceptor hyperpolarization,
leading to constant cone activation (Shichida and Matsuyama 2009). In ONbipolar cells, GNB3 is involved in metabotropic glutamate receptor 6 (mGluR6)
signaling and should have subsequent abnormal center-on responses (Huang,
Max et al. 2003; Dhingra, Fina et al. 2011). This loss of GNB3 is manifested in
the abnormal a- and b-waves observed with ERG testing (Montiani-Ferreira,
Shaw et al. 2007). GNB3 has not been detected in amacrine cells or ganglion
cells, thus should not be involved in signaling in these cells (Ritchey, Bongini et
al. 2010). The lack of GNB3, combined with our data, further supports the notion
that bipolar cells and photoreceptors are not the primary neurons that detect
retinal blur and begin the mechanisms that drive vision guided ocular growth.
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While phototransduction must be present to detect photons of light so the animal
can see, the processing of light into a vision-guiding ocular growth cue appears
to be a function of cells that are GNB3-negative. Moreover, it appears that ONbipolar cell function can be compromised yet the eye can still respond to visual
growth clues (Pardue, Faulkner et al. 2008). Our data, combined with the
knowledge that ganglion cell function is not required for the eye to respond to
vision-guided ocular growth stimuli (Troilo, Gottlieb et al. 1987; Norton, Essinger
et al. 1994; McBrien, Moghaddam et al. 1995), confirms the importance of
amacrine cells, particularly glucagon-positive amacrine cells in chickens, for
vision guided ocular growth.
Conclusions
Our findings indicate that RGE chickens, despite a congenital reduction in
visual acuity, respond to growth-promoting stimuli by increasing in axial length,
vitreous chamber depth and ocular circumference. RGE chicks fail to develop
form-deprivation myopia and excessive axial length when given brief periods of
daily visual recovery. Glucagon-positive amacrine cells respond to changes in
visual experience by up-regulating Egr1 during the initial recovery period from
form-deprivation or hyperopic defocus and do not alter Egr1-expression during
recovery from myopic defocus. Egr1 chicks down-regulate mRNA levels for
preproglucagon with hyperopic defocus; however, they fail to up-regulate when
presented with myopic defocus. We conclude that RGE chickens behave in a
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similar manner to WT chickens when undergoing visual manipulation; however,
variations in the response occur dependent upon the treatment paradigm.
Acknowledgements
I would like to acknowledge the significant contributions of Christopher
Zelinka, Junhua Tang, Dr. Jun Liu, Kimberly Code, Simon Petersen-Jones and
Dr. Andy Fischer to this project. This work was supported by grants (AJF:
EY016043-05; ERR: K12EY015447) from the National Institutes of Health,
National Eye Institute.
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Chapter 5: Summary and Future Directions
Summary of Findings
While some aspects of the mechanisms that regulate ocular growth are
well established, there are still numerous questions to be addressed in the field.
Despite the advances of our knowledge on ocular growth and myopia,
prevalence rates of myopia continue to increase and potential therapeutic
interventions to prevent myopia development or progression continue to be
sparse. The chicken model has and will continue to play a critical role in myopia
research because of the robust response of the animals to growth-altering
stimuli, the ease of experimental manipulation and the cost-effectiveness of the
model. Given the vast amount of literature on myopia development and the
chicken, this animal model is well positioned to provide answers to the critical
questions of ocular growth in the future. This dissertation addresses aspects of
ocular growth in regards to the effects of molecular modulators of ocular growth,
the conservation of a protein crucial to visual signal transduction and the ability of
an eye that lacks this crucial protein to respond to vision-guided ocular growth
stimuli.
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In chapter 2, we examined the potent effect of the combination of IGF1
with FGF2 on ocular growth. The combination of these growth factors produced
extreme increases in axial length and promoted myopia development while
having significant effects on the anterior chamber, lens and intraocular pressure.
This result is particularly intriguing given that insulin has recently been proposed
as a potential suspect in the increased prevalence of myopia observed globally
and should be a subject of further investigation.
In chapter 3, we examined the conservation of guanine nucleotide-binding
protein β3 across multiple animal species. GNB3 plays a critical role in visual
signal transduction by acting in co-ordination with the GNB gamma (γ) subunit to
bind to GNB alpha (α). Failure of GNB3 to combine with GNBγ and form the
heteromeric β/γ dimer that binds to GNBα leads to constant activation of
phosphodiesterase (PDE) and subsequent constant photoreceptor
hyperpolarization (Shichida and Matsuyama 2009). Moreover, GNB3 is
associated with metabotropic glutamate receptor 6 (mGluR6) signaling in ONbipolar cells (Huang, Max et al. 2003; Dhingra, Fina et al. 2011) Our data shows
that GNB3 is present in cone photoreceptors and in presumptive ON-bipolar cells
in multiple species. This clearly demonstrates the highly-conserved nature of
GNB3 in the retina of vertebrate species and implies that GNB3 is important in
visual signal transduction. In an animal with reduced visual acuity, the RGE
chicken, we demonstrated that GNB3 was not present in any retinal neurons or
glia. Polymerase chain reaction clearly showed that the mRNA transcript for
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production of GNB3 was present; however, Western blotting failed to detect the
presence of GNB3 protein in the retina, confirming reports that the lack of GNB3
in the RGE retinal results from the loss of a single aspartic acid residue
(Tummala, Ali et al. 2006). This loss of GNB3 from the retina leads to abnormal
photoreceptor and bipolar cell function, loss of visual acuity and development of
the retinopathy, globe enlarged phenotype.
In chapter 4, we examined the ability of the RGE chick to respond to
vision-guided ocular growth stimuli. The eyes of young RGE chicks (<P21)
displayed altered ocular growth in response to form-deprivation, hyperopic
defocus and myopic defocus. These animals, similar to WT animals, were also
able to significantly reduce ocular growth from form-deprivation with brief period
of visual recovery per day. In RGE retinas Egr1 was up- or down-regulated in
glucagon-positive amacrine cells with recovery from visual manipulation. The
ability to respond to these treatments in the presence of abnormal photoreceptor
and bipolar cell function demonstrates the importance of amacrine cells in visionguided ocular growth, and that normal function of photoreceptors and ON-bipolar
cells is not required for vision-guided ocular growth.
Future Directions
There are several unanswered questions regarding how the eye regulates
ocular growth and why humans become myopic. Further investigation into the
mechanisms that regulate ocular growth is required.
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Future directions for the role of insulin and IGF in ocular growth.
There has been significant interest in recent years on the role insulin may
play in the development of myopia in humans. Insulin has been implicated as a
candidate in myopia development based on data from animal models and
correlative observations in humans. From animal models, glucagon has been
demonstrated to be a significant factor in the regulation of ocular growth
(Feldkaemper, Wang et al. 2000; Feldkaemper and Schaeffel 2002; Buck,
Schaeffel et al. 2004; Feldkaemper, Burkhardt et al. 2004; Beloukhina, Vessey et
al. 2005; Vessey, Lencses et al. 2005; Vessey, Rushforth et al. 2005; Zhu and
Wallman 2009). This makes insulin an obvious target of investigation given the
complementary roles of insulin and glucagon in maintaining blood glucose levels
(reviewed in Brockman 1978). A review of this evidence is inconclusive as to
whether insulin truly plays a role in regulation of ocular growth. Two primary
papers have examined the role of insulin in regulating eye growth. These papers
are contradictory in some regards, such as the promotion of ocular growth by
insulin without visual manipulation (Zhu and Wallman 2009) or only in
combination with visual manipulation (Feldkaemper, Neacsu et al. 2009). More
damaging to the argument for the role of insulin in myopia is that insulin
treatment in both papers primarily caused changes to the anterior structures of
the eye while minimally affecting the vitreous chamber, where elongation is the
primary course of myopia. A further complication is the relative lack of insulin
receptors in the retina. While insulin can act at IGF1 receptors and IGF1
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receptors are present in the retina, RPE, choroid and sclera; insulin has an
approximate 100-fold less affinity for the IGF1 receptor (Steele-Perkins, Turner et
al. 1988; Waldbillig, Arnold et al. 1991). It remains unknown if insulin binds to
retinal receptors to promote ocular growth, if it acts through IGF1 receptors or if it
acts through an extra-retinal site, such as direct stimulation of the choroid.
Finally, the evidence of insulin as a potent stimulator of ocular growth comes
from the chick model which indicates that insulin can override the growth-slowing
effects of glucagon on form-deprived eyes. The lack of glucagon-positive
amacrine cells in primates requires us to ask the question of whether insulin,
while potentially a potent growth stimulator in chicks, plays a role in ocular growth
in mammals. While IGF2 has been shown to enhance form-deprivation myopia in
guinea pigs, it had no effect on refraction or axial length in an non-occluded eye
(Deng, Tan et al. 2010). The effects of exogenous insulin and insulin/IGF1
receptor antagonists on the development form-deprivation needs to be
addresses in a primate model to test whether insulin has a role in the regulation
of eye growth outside of avian species.
Data from RGE chickens suggest that normal photoreceptor and bipolar
cell function are not critical for vision-guided ocular growth
One of the persistent questions in the study of vision-guided ocular growth
has been identifying which retinal neurons play critical roles in growth regulation.
The identification of the neurons responsible for the local regulation of ocular
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growth would become targets for therapeutic interventions to prevent myopia
development. Photoreceptors were a target for early investigators given their role
in initiating the visual phototransduction cascade. Previous attempts to isolate
photoreceptor function during vision-guided ocular growth through the use of
exogenous toxins have provided contradictory results. This is due, in part, to
studies designed to selectively ablate photoreceptors failing to accomplish the
desired result without altering the surrounding tissue. For example,
formoguanamine, while ablating photoreceptors, alters mitochondrial function in
the RPE (Obara, Matsuzawa et al. 1985). This lack of specificity from
formoguanamine treatment makes interpretation of these experiments using this
treatment paradigm impossible. An example is the work of Westbrook and
colleagues, who demonstrated that formoguanamine-treated chickens show a
reduced growth response to form-deprivation. The formoguanamine treatment;
however, failed to alter, much less obliterate, photoreceptor function. This was
clearly demonstrated by ERG data, which indicated that the function of the outer
retinal circuitry remained intact (Westbrook, Crewther et al. 1995). Assuming that
the ERG a-wave recording accurately reflects outer retinal function, it is difficult
to interpret why the formoguanamine-treated animals failed to show an
equivalent response to form-deprivation compared to the untreated animals.
Many questions regarding formoguanamine-treated retinas and vision-guided
ocular growth remain unanswered. Did the formoguanamine treatment subtly
altered the photoreceptor function and subsequent phototransduction is a way
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undetectable to the electroretinogram? Did the formoguanamine treatment alter
the function of the RPE without altering photoreceptor function? Was the function
of the RPE altered while photoreceptors were left undamaged? These questions
make the interpretation of the early work in photoreceptors and ocular growth
very difficult.
The RGE chicken has provided an opportunity to examine how abnormal
photoreceptor and ON-bipolar cell function affects vision-guided ocular growth.
The lack of GNB3 in RGE animals will prevent the de-activation of
phosphodiesterase and should cause photoreceptors to undergo constant
hyperpolarization. In ON-bipolar cells, the lack of GNB3 affects mGluR6
signaling, altering the ON response. Electrophysiological recordings of RGE
mutant photoreceptors are required to unambiguously determine how the loss of
GNB3 affects photoreceptor and bipolar cell function. Despite the reduction in
vision in these animals, they do have an ERG response that persists for a
prolonged period post-hatch (Montiani-Ferreira, Shaw et al. 2007).
It is unclear why these chicks appear to have some residual vision despite
a complete loss of GNB3. There is the potential that another isoform of GNB,
such as GNB1 (also known as rod transducin), is up-regulated to compensate for
the loss of GNB3 (Peng, Robishaw et al. 1992). In wild-type chickens, some
GNB3 immunoreactivity was observed in rod photoreceptors, indicating that the
possibility of cross reactivity of the isoforms may be possible. However, GNB#immunofluorescence did not persist in rod photoreceptors in the RGE mutant
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retina. Further investigation should determine if levels of GNB1 are elevated in
RGE chicks compared to wild-type retinas. Using enzyme-linked immunosorbent
assay (ELISA), total retinal concentrations of GNB1 can be assessed. If this
assay indicates that GNB1 is up-regulated in RGE chicks, quantitative
immunofluorescent microscopy can be used to determine which cell type(s) in
the retina up-regulates GNB1 in place of GNB3.
Can visually-guided ocular growth stimuli (plus lenses) slow the
progressive globe enlargement of the RGE chick?
As demonstrated in Chapter 4, RGE chicks respond to growth-guiding
visual stimuli in a manner similar to that observed in wild-type chickens. In these
experiments, animals were used during the early postnatal period (age P30 or
younger). In the time course of the development of the retinopathy-globe
enlarged phenotype, RGE chicks present a conundrum in that excessive ocular
elongation occurs with concurrent development of significant hyperopic refractive
error. The development of hyperopia under conditions of globe enlargement
results from flattening of the cornea. While corneal flattening in older RGE (>P33)
animals could be due to passive stretch from overall globe enlargement, it is
unknown if the corneal flattening is a secondary consequence of globe
enlargement or if it actively participates in the development of globe enlargement.
The observed hyperopia in RGE chicks after age P33, averaging 12 diopters,
corresponds with a significant flattening of the cornea, also observed after age
186
P33 (Montiani-Ferreira, Li et al. 2003). The development of hyperopia in RGE
chicks would push the far-point of the eye beyond the retina, potentially
contributing to the globe enlargement phenotype. This would correspond to
observations from wild-type animals that demonstrate that placing the refractive
far-point behind the retina with divergent lenses acts to increase ocular growth.
Moreover, hyperopia of this magnitude could not be easily compensated for by
accommodation. Despite a maximal accommodative range of approximately 1517 diopters due to corneal and lenticular accommodative components, RGE
chickens cannot consistently accommodate to focus incoming light on the retina
(Schaeffel, Howland et al. 1986; Schaeffel and Howland 1987; Troilo and
Wallman 1987). Given that lens-manipulation paradigms demonstrate that ocular
growth can be altered by visual experience, could the flattening of the cornea in
the RGE chicken impact the development of the globe enlargement phenotype?
Future experiments should examine whether growth-slowing stimuli, specifically
plus lenses, can influence the development of axial elongation in the RGE chick
in the P30 to P90 age range. Application of plus lenses to one eye in powers
capable of neutralizing the high myopia of the RGE chick, for example lenses
ranging from +12 to +24 in 4 diopter steps, would allow for a intra-animal
comparison of ocular growth (treatment–control); moreover it would provide a
dose-response curve to the varying levels of myopic defocus. It is anticipated that
the effects of plus lenses would be modest at best. First, with progressive vision
loss as displayed in the ERG data for RGE chicks (Montiani-Ferreira, Shaw et al.
187
2007), the ability to detect retinal blur will diminish over time, potentially reducing
the effect of the stimulus. Secondly, although wild-type chickens can respond to
visual manipulation for up to year, it is anticipated that the RGE animals will show
less capacity for vision-guided ocular growth with advancing age, reducing the
potential effectiveness of plus lenses for preventing globe enlargement
(Papastergiou, Schmid et al. 1998).
Future directions for the Egr1/glucagon pathway in ocular growth
A major limitation in our understanding of myopia is how a growth altering
stimulus, such as form-deprivation, is translated to a molecular pathway that
changes ocular growth. The best known example of a potential molecular
pathway is the association of Egr1 with glucagon peptide in chickens. Previous
studies have suggested that expression levels of Egr1 in glucagon-positive
amacrine cells mirror the expression of mRNA levels for glucagon peptide
production (Buck, Schaeffel et al. 2004; Ashby, Kozulin et al. 2010). This
corresponds with data which demonstrates that competitive inhibition of glucagon
receptors by small molecule inhibitors removes the protective effects of growthslowing stimuli which up-regulate glucagon production. An opposite effect is seen
with small molecule agonists and by application of exogenous glucagon, which
negate the effects of growth stimulation by form-deprivation. Given that alteration
of vision through the application of corrective lenses and form-deprivation
goggles leads to short-term changes in choroidal thickness and longer-term
188
alteration of the sclera, altered glucagon levels inherently must affect these
tissues to cause the changes observed in the chick model of vision-guided ocular
growth. Currently, we do not know how retinal glucagon signals influence the
choroid and sclera to effect changes in ocular growth; nor do we know if
glucagon peptide production is the endpoint of this process or a simple
intermediary up-stream of another retina-derived growth modifying factor. This
lack of knowledge regarding the downstream targets of glucagon limits our
knowledge of the signaling pathways in ocular growth. Future studies should
involve investigation into the identification of the growth-regulating cells that are
influenced by glucagon.
Adeno-associated viral (AAV) gene transfection technology may allow us
further define the glucagon signaling pathway. A potential target for AAV gene
transfection is the RPE, which has been shown to have higher expression levels
of glucagon receptor than either the retina or the choroid, thus implicating the
RPE as a major target of growth process (Buck, Schaeffel et al. 2004). Certain
AAVs, such as AAV1 and AAV2/4, have been shown to be relatively selective for
the RPE when delivered by sub-retinal injection (Le Meur, Stieger et al. 2007;
Lebherz, Maguire et al. 2008; Pang, Lauramore et al. 2008). Delivery of short
hairpin RNA to silence gene expression for glucagon receptor at the RPE using
an AAV vector would provide valuable information on the effect of glucagon
peptide on the choroid and sclera. The knock-down of glucagon receptor in the
RPE should lead to increased rates of ocular growth. If exogenous glucagon,
189
glucagon agonists and antagonists are rendered ineffective in AAV transfected
chicks in visual manipulation paradigms, the implication would be that signaling
through glucagon binding at the RPE causes down-stream effects in the choroid
or retina. Conversely, if AAV transfection to silence gene expression of the
glucagon receptor in RPE does not alter the effectiveness of these agents, then
the assumption would be that glucagon acts as an intermediate, up- or downregulating other factors that alter ocular growth rates. In either case, this would
help provide clarity to the molecular cascade involved in ocular growth regulation.
Concluding Remarks
This dissertation has attempted to add to the body of knowledge regarding
the cellular and molecular pathways involved in vision-guided ocular growth. We
have shown that animals with diminished visual acuity from altered photoreceptor
and bipolar cell function can respond to vision-guided ocular growth stimuli.
Furthermore, we demonstrate that IGF1 acts synergistically with FGF2 to greatly
enhance rates of ocular growth in eyes with unrestricted vision. Better knowledge
of the mechanisms involved in ocular growth will aid in the development of
successful interventions to prevent or dramatically reduce myopia development
in humans.
190
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