Download Ear manipulations help model neuroplasticity limitations

University of Iowa
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Theses and Dissertations
Fall 2013
Ear manipulations help model neuroplasticity
limitations
Karen Louise Elliott Thompson
University of Iowa
Copyright 2013 Karen Louise Elliott Thompson
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/4969
Recommended Citation
Thompson, Karen Louise Elliott. "Ear manipulations help model neuroplasticity limitations." PhD (Doctor of Philosophy) thesis,
University of Iowa, 2013.
http://ir.uiowa.edu/etd/4969.
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EAR MANIPULATIONS HELP MODEL NEUROPLASTICITY LIMITATIONS
by
Karen Louise Elliott Thompson
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Biology
in the Graduate College of
The University of Iowa
December 2013
Thesis Supervisor: Professor Bernd Fritzsch
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Karen Louise Elliott Thompson
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Biology at the December 2013 graduation.
Thesis Committee: ___________________________________
Bernd Fritzsch, Thesis Supervisor
___________________________________
Douglas Houston
___________________________________
Michael Dailey
___________________________________
Joshua Weiner
___________________________________
Daniel Weeks
To my husband, Nathan, and my son, Noah
ii
ACKNOWLEDGMENTS
I would like to first thank my husband, Nathan, for his continued support for me
and helping me pursue my goals. I would also like to thank my advisor, Dr. Bernd
Fritzsch, for his guidance throughout my years in graduate school. In addition, I would
like to thank members of the Fritzsch lab, both past and present: Jeremy Duncan, Israt
Jahan, Jennifer Kersigo, Benjamin Kopecky, Hannah Maher, Ning Pan, and Tian Yang.
Finally, I would like to thank members of my committee: Dr. Bernd Fritzsch, Dr. Michael
Dailey, Dr. Douglas Houston, Dr. Daniel Weeks, and Dr. Joshua Weiner for their
guidance, suggestions, and support.
iii
TABLE OF CONTENTS
LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
LIST OF ABBREVIATIONS .......................................................................................... viii
CHAPTER I INTRODUCTION ..........................................................................................1
Connecting Sensory Organs with the Central Nervous System .......................1
Inner Ear Anatomy and Function .....................................................................2
Neurosensory Development of the Inner Ear ...................................................4
Evolution of the Inner Ear ................................................................................7
Motor Neuron Targeting .................................................................................10
Inner Ear Sensory Neuron Guidance ..............................................................15
Hindbrain Neurons..........................................................................................20
Experimental Approach for Generating and Manipulating a ‘Novel’
Sensory System ...............................................................................................27
CHAPTER II METHODS .................................................................................................28
Animals ...........................................................................................................28
GFP mRNA injections ....................................................................................28
Dextran amine injections ................................................................................29
Transplantations ..............................................................................................29
Swimming Behavior .......................................................................................31
Dextran amine label ........................................................................................32
Lipophilic dye label ........................................................................................32
Quantification of percent overlap of sensory neurons in the vestibular
nucleus of ‘three-eared’ frogs .........................................................................34
In situ hybridization ........................................................................................35
Immunohistochemistry ...................................................................................36
Plastic imbedding for light and transmission electron microscopy ................37
Three-dimensional reconstruction of Mauthner cells .....................................37
Dendritic Branching Analysis.........................................................................38
CHAPTER III TRANSPLANTATION OF XENOPUS LAEVIS EARS REVEALS
THE ABILITY TO FORM AFFERENT AND EFFERENT
CONNECTIONS WITH THE SPINAL CORD .............................................39
Abstract ...........................................................................................................39
Introduction.....................................................................................................40
Results.............................................................................................................42
Assessment of transplantations................................................................42
Transplanted ears develop hair cells and ganglia ....................................43
Afferent innervation of transplanted ears ................................................44
Efferent innervation of transplanted ears ................................................45
Cranial and lateral line nerves develop nearly normal ............................47
Discussion .......................................................................................................47
iv
CHAPTER IV TRANSPLANTATION OF XENOPUS LAEVIS TISSUES TO
DETERMINE THE ABILITY OF MOTOR NEURONS TO
ACQUIRE A NOVEL TARGET ...................................................................63
Abstract ...........................................................................................................63
Introduction.....................................................................................................64
Results.............................................................................................................67
Completion of eye removal and assessment of transplantation
success .....................................................................................................67
Afferent innervation of transplanted ears ................................................69
Efferent innervation of transplanted ears ................................................70
Innervation of transplanted somite, eye muscle, and jaw muscle ...........72
Innervation of transplanted heart and liver ..............................................73
Discussion .......................................................................................................74
CHAPTER V GENERATION OF ‘THREE-EARED’ FROGS REVEALS
MOLECULAR AND ACTIVITY-BASED GUIDANCE OF
CENTRAL PROJECTIONS ...........................................................................91
Abstract ...........................................................................................................91
Introduction.....................................................................................................92
Results.............................................................................................................95
Success of transplantation .......................................................................95
Analysis of Swimming Behavior.............................................................96
Analysis of Afferent Projections to the Hindbrain ..................................97
Discussion .......................................................................................................99
CHAPTER VI EAR MANIPULATIONS REVEAL A CRITICAL PERIOD OF
HINDBRAIN DEPENDENCE ON THE EAR FOR DEVELOPMENT
AND SURVIVAL ........................................................................................109
Abstract .........................................................................................................109
Introduction...................................................................................................110
Results...........................................................................................................115
Effect of stage of ear removal on the degree of ear regeneration ..........115
Effect of stage of ear removal on the presence of the Mauthner cell ....115
Effect of stage of ear removal on the dendritic branching of
surviving Mauthner cells .......................................................................116
Effect of increasing afferent input on the dendritic branching of
Mauthner cells .......................................................................................120
Discussion .....................................................................................................121
Viability of the Mauthner cell depends on ear input .............................122
Dendrite growth and branching depends on vestibular afferents ..........123
CHAPTER VII CONCLUSIONS AND FUTURE DIRECTIONS .................................132
Motor Neuron Acquisition............................................................................132
Inner Ear Sensory Neuron Guidance ............................................................135
Hindbrain Neurons........................................................................................143
Proposed Mechanism ....................................................................................148
REFERENCES ................................................................................................................152
v
LIST OF TABLES
Table 3.1. Success of transplantation of otic placodes. .....................................................53
Table 3.2. Formation of residual ears as related to success of transplantation. .................54
Table 4.1. Success of tissue transplantation.......................................................................80
Table 5.1. Analysis of tadpole swimming behavior. .......................................................102
vi
LIST OF FIGURES
Figure 3.1. Stage 46 X. laevis showing transplanted ears. .................................................55
Figure 3.2. Development of the transplanted ear. ..............................................................57
Figure 3.3. Afferent and efferent innervation of transplanted ears. ...................................59
Figure 3.4. Development of cranial and lateral line nerves. ..............................................61
Figure 4.1. Stage 46 Xenopus laevis. .................................................................................81
Figure 4.2. Afferent projections to transplanted ears. ........................................................83
Figure 4.3. Efferent projections to transplanted ears. ........................................................85
Figure 4.4. Transplanted muscle tissue. .............................................................................87
Figure 4.5. Transplantated tissue lacking nicotinic acetylcholine receptors such as
heart and liver. ..........................................................................................................89
Figure 5.1. Stage 46 Xenopus laevis 'three-eared' frogs. .................................................103
Figure 5.2. Inner ear afferent projections.........................................................................105
Figure 5.3. Overlap and segregation of inner ear afferents from transplanted and
native ears. ..............................................................................................................107
Figure 6.1. Success of ear removal. .................................................................................126
Figure 6.2. Mauthner cell survival. ..................................................................................128
Figure 6.3. Dendritic development of Mauthner cells following ear manipulation.........130
Figure 7.1. Proposed mechanism to guide swimming behavior in three-eared frogs. .....150
vii
LIST OF ABBREVIATIONS
ACh
Acetylcholine
Akt
Protein Kinase B
Atoh1
Atonal homolog 1
Atoh7
Atonal homolog 7
BDNF
Brain Derived Neurotrophic Factor
bHLH
basic Helix-Loop-Helix
BMP
Bone Morphogenetic Protein
Chrna9
Acetylcholine Receptor subunit α9
Chrna10
Acetylcholine Receptor subunit α10
CNS
Central Nervous System
E
Embryonic day
Eya1
Eyes absent homolog 1
FGF
Fibroblast growth factor
Fgf3
Fibroblast growth factor 3
Fgf8
Fibroblast growth factor 8
Fgf10
Fibroblast growth factor 10
Gata3
GATA binding protein 3
GFP
Green Fluorescent Protein
Islet-1
Insulin gene enhancer protein 1
LIM
LIM homeodomain
miR
MicroRNA
MMR
Marc’s Modified Ringers Solution
viii
MuSK
Muscle Specific Receptor Tyrosine Kinase
MyoVI
Myosin VI
nAChR
Nicotinic Acetylcholine Receptor
Neurod1
Neuronal differentiation 1
Neurog1
Neurogenin 1
NGS
Normal Goat Serum
NT-3
Neurotrophic Factor 3
P
Postnatal day
Pax2
Paired box gene 2
Pax5
Paired box gene 5
Pax6
Paired box gene 6
Pax8
Paired box gene 8
PaxB
Paired box gene B
PBS
Phosphate Buffered Saline
PFA
Paraformaldehyde
Rapsyn
Receptor-Associated Protein of the Synapse
RGC
Retinal Ganglion Cell
RIC-3
Resistance to Inhibitors of Cholinesterase 3
Six1
Sine oculus-related homeobox 1 homolog
Sox3
SRY-box containing gene 3
TrkB
Tyrosine Kinase B
VAChT
Vesicular Acetylcholine Transporter
VOR
Vestibulo-Ocular Reflex
ix
Wnt
Wingless-related MMTV Integration Site
x
1
CHAPTER I
INTRODUCTION
Connecting Sensory Organs with the Central Nervous
System
Sensory organs send information about the outside world to specified nuclei in the
central nervous system (CNS). In turn, the CNS sends information back to certain
sensory organs to modulate the incoming signal. For example, the retina of many
vertebrates receives a centrifugal or efferent innervation (Fritzsch, 1991) and this is even
more conserved for the vertebrate ear (Fritzsch, 1999, Köppl, 2011). As sensory organs,
such as the ear, diversified over time to change from a vestibular to a mixed
vestibular/auditory system, new connections evolved to process that information
distinctly. This process of forming contacts between the CNS and sensory organs or
subsystems is incompletely understood.
The inner ear, one of the sensory organs located in the head, evolved in early
vertebrates (Retzius, 1881, Fritzsch and Straka, 2013). The inner ear transforms
mechanical stimulation of sensory-transducing hair cells into electrical impulses to
transmit sound, gravity, balance, and acceleration information through afferent neurons to
the hindbrain which in turn sends a signal back to the inner ear through efferent neurons
to modulate the periphery. How these afferent and efferent processes of neurons navigate
to the hindbrain or hair cells, respectively, is not well understood at the molecular level
beyond a small set of experimental data (Simmons et al., 2011, Yang et al., 2011). It has
been suggested that efferents to the ear are rerouted facial branchial motor neurons that
2
may do so because of the unique expression of one or more transcription factors
(Simmons et al., 2011).
In my thesis I examine the ability of the nervous system to adapt to a ‘novel’
vestibular system through transplantation of frog inner ears. Specifically, I ask the
following questions:
1) Can inner ear hair cells recruit any motor neuron as an efferent innervation
through their conserved nicotinergic acetylcholine receptor?
2) Can inner ear afferents find their targets in the hindbrain if they enter through a
different cranial nerve or enter into a foreign territory?
3) What mechanisms do inner ear afferents use to find their targets in the hindbrain?
4) How will alteration of afferent input, either by ear removal or addition of ‘extra’
ears, affect neurons in the CNS dedicated to inner ear sensory input?
My thesis will first provide a background on the inner ear anatomy, neurosensory
development and evolution, followed by motor neuron acquisition and sensory neuron
guidance. Finally I will discuss how sensory neurons can shape second-order neurons in
the hindbrain that influence motor control.
Inner Ear Anatomy and Function
The inner ear is comprised of the vestibular and auditory systems. The more
dorsal, vestibular portion contains sensory epithelia for the perception of gravity, balance,
and acceleration, whereas the more ventral, auditory portion contains sensory epithelia
for the perception of sound. The sensory epithelia contain mechanosensory hair cells and
supporting cells. The hair cells transduce mechanical stimulation of their stereocilia into
electrical signals that are relayed to the brain through afferent (sensory) neurons (Torres
3
and Giráldez, 1998, Rubel and Fritzsch, 2002, Fritzsch and Beisel, 2003). The brain, in
turn, sends signals through efferent (motor) neurons to modulate the sensitivity of the hair
cells (Simmons et al., 2011) that are highly conserved across vertebrates (Köppl, 2011).
The vestibular sensory epithelia consist of the utricle and saccule for the
perception of linear acceleration and gravity and the three semicircular canals: anterior
crista, posterior crista, and horizontal crista for the perception of angular acceleration.
The utricle and saccule maculae are covered by calcium carbonate crystals, known as
otoconia. The otoconia are partially imbedded in a matrix that is tethered to the
kinocilium of the underlying hair cells. This dense otoconial complex provides inertia to
generate shearing forces on the hair cells in response to linear acceleration or gravity
(Riley and Phillips, 2003, Lundberg et al., 2006). Each semicircular canal cristae is
located within the ampulla associated with the respective semicircular canal. The long
hair cell stereocilia within each crista are covered by a gelatinous material. Movement of
endolymph fluid within the canal moves the gelatinous material and deflects the hair cell
stereocilia (Lewis et al., 1985, Riley and Phillips, 2003)
The auditory sensory epithelia consist of the cochlea in mammals or the basilar
papilla in most other organisms. Frogs and other amphibians have an amphibian papilla
in addition to the basilar papilla. Furthermore, in amphibians, the saccule also has an
auditory role in addition to a vestibular one (Bever et al., 2003, Riley and Phillips, 2003,
Quick and Serrano, 2005). Fish, amphibians, and reptiles also have a lagena, which may
provide auditory information in addition to gravitational information (Lewis and Narins,
1999, Bever et al., 2003).
4
Neurosensory Development of the Inner Ear
The vertebrate inner ear arises from a single otic placode adjacent to
rhombomeres 5 and 6 of the hindbrain (Fritzsch et al., 1998, Rinkwitz et al., 2001, Riley
and Phillips, 2003, Groves, 2005, Ohyama et al., 2007, Schlosser, 2010, Pieper et al.,
2011). The otic placode originates as part of a pan-placodal domain (Groves, 2005,
Ohyama et al., 2007, Schlosser, 2010, Pieper et al., 2011). The pan placodal domain is an
area of non-neuronal ectoderm bordering the neural plate that gives rise to the various
sensory placodes in the vertebrate head (Schlosser, 2010). This pan placodal domain is
defined by Six1, Eya1 and Pax2/8 expression (Bouchard et al., 2010) and these genes are
suggested to promote generic placodal properties (Schlosser and Ahrens, 2004, Schlosser,
2005, 2010).
The pan-placodal domain is later subdivided into the anterior and posterior
placodal area; the otic placode originating from the latter (Schlosser and Ahrens, 2004,
Schlosser, 2010). The otic placode is induced by signals from the underlying paraxial
mesoderm and the adjacent neural plate (Yntema, 1950, Gallagher et al., 1996, Torres
and Giráldez, 1998, Rinkwitz et al., 2001, Groves, 2005). Members of the fibroblast
growth factor (FGF) family, such as Fgf3, Fgf8, and Fgf10, have been shown to play a
role in otic placode induction, although the specific FGF varies across vertebrates
(Groves, 2005, Schlosser, 2010). Mice mutant for these Fgf genes have absent or
reduced otic placodes and overexpression of FGFs in chick, mouse, Xenopus laevis, and
zebrafish results in otic vesicle expansion and extra otic vesicles (Lombardo et al., 1998,
Vendrell et al., 2000, Alvarez et al., 2003, Pauley et al., 2003, Wright and Mansour,
2003, Groves, 2005, Schimmang, 2007). These FGFs induce Pax8, Pax2, and Sox3
5
expression, among others, in the otic placode (Hans et al., 2004, Solomon et al., 2004,
Mackereth et al., 2005, Sun et al., 2007). Since FGFs also play a role in epibranchial and
lateral line placode induction (Baker and Bronner-Fraser, 2001, Sun et al., 2007,
Schlosser, 2010), additional factors are needed to further specify the otic placode. Other
factors, such as Wnts, bone morphogenetic proteins (BMPs), and Notch, have been
shown to play an additional role in otic placode induction (Ladher et al., 2000, Rinkwitz
et al., 2001, Solomon et al., 2004, Ohyama et al., 2006, Ohyama et al., 2007, Park and
Saint-Jeannet, 2008, Urness et al., 2010). In Xenopus, the otic placode is first visibly
recognized at stage 21 (Nieuwkoop and Faber, 1994), though otic placode markers, for
example Pax8 and Pax2, are expressed earlier (Schlosser and Ahrens, 2004).
Following induction, the otic placode undergoes invagination to form a cup and
later closes off to form the otic vesicle that is completely separated from the overlying
ectoderm (Alvarez et al., 1989, Torres and Giráldez, 1998, Rinkwitz et al., 2001, Fekete
and Wu, 2002, Bever et al., 2003). In Xenopus, the vesicle forms by stage 27 and has
separated from the ectoderm by stage 28 (Nieuwkoop and Faber, 1994). All inner ear
cell types: hair cells, supporting cells, and inner ear afferents arise from the otic vesicle
(Fekete and Wu, 2002). Afferent sensory neurons that will innervate vestibular and
auditory sensory epithelia differentiate and delaminate relatively early from the otic
vesicle (Torres and Giráldez, 1998, Rubel and Fritzsch, 2002, Riley and Phillips, 2003,
Alsina et al., 2009) following upregulation of neurogenin 1(Neurog1) and Neuronal
differentiation 1 (Neurod1) (Ma et al., 1998, Ma et al., 2000, Jahan et al., 2010a).
Expression of the earliest neuronal marker, Neurog1, is first upregulated in the otic
placodes at stage 20-22 in Xenopus embryos (Nieber et al., 2009). The otic, or eighth,
6
ganglion (VIII) is first detected by stage 31 in Xenopus (Nieuwkoop and Faber, 1994,
Quick and Serrano, 2005) and axons reach the hindbrain by stage 34 in axolotls (Fritzsch
et al., 2005a), and at comparable stages in Xenopus. The ganglion will later separate into
the vestibular and auditory ganglia (Torres and Giráldez, 1998).
After the otic vesicle has formed, the walls undergo growth and folding to create a
series of interconnected chambers that will eventually form the various components of
the inner ear (Riley and Phillips, 2003). The initial process of forming the ear is
relatively conserved across most vertebrates, but differences arise in the formation of
specific epithelia in various organisms. The formation of the pars superior, comprised of
the utricle and semicircular canals, is highly conserved in vertebrates, whereas the
formation of the pars inferior, comprised of the saccule and auditory component(s), has
more variation across different animals (Riley and Phillips, 2003). In general, the
endolymphatic duct, which is used to maintain proper levels of endolymph in the inner
ear (Riley and Phillips, 2003), is the first structure to develop (Morsli et al., 1998, Bever
et al., 2003, Riley and Phillips, 2003) and in Xenopus, this occurs by stage 37 (Quick and
Serrano, 2005). A single sensory epithelia patch develops in the otic vesicle and
eventually separates into the individual components (Fritzsch et al., 2001a). From this
sensory patch, the semicircular canal cristae, utricle, and saccule are the first to develop
followed by the auditory sense organ(s) (Morsli et al., 1998, Bever et al., 2003, Riley and
Phillips, 2003, Quick and Serrano, 2005). In a study involving paint filling of Xenopus
ears, the formation of the semicircular canals is initiated at stage 43 and is completed by
stage 47; the utricle and saccule are prominent by stage 46, the amphibian papilla and
basilar papilla are prominent by stage 47, and lagena is prominent by stage 49-50 (Bever
7
et al., 2003). However, histological evidence puts sensory epithelia development at
slightly earlier stages for each (Paterson, 1949, Quick and Serrano, 2005). The earliest
hair cells, observed by F-actin staining for stereocilia bundles, are observed as early as
stage 31 in Xenopus and the appearance of a separate pars superior and pars inferior are
formed by stage 42 (Quick and Serrano, 2005). It is also at this time when the animals
begin to swim (Quick and Serrano, 2005). Between stages 45 and 47, the vestibular
sensory epithelia components differentiate and between stages 47 and 50, the auditory
sensory epithelia components differentiate (Quick and Serrano, 2005).
Evolution of the Inner Ear
Evolution of new cells, tissues, and organs likely occurs as a duplication event at
the sensory cell level, followed by segregation and diversification (Duncan and Fritzsch,
2012) comparable to the well-known molecular events. The perception of gravity,
movement, and sound by the inner ear depends upon the mechanosensory hair cell. It is
thought that the mechanosensory cell is at the base of the evolution of the ear and other
mechanosensory systems (Duncan and Fritzsch, 2012). In other words, the ciliated
mechanosensory cell existed prior to the evolution of the inner ear. The ancestor for the
mechanosensory cell is the single-celled choanoflagellates. These choanoflagellates have
a single kinocilium surrounded by microvilli (Fritzsch et al., 2007, Fritzsch and Straka,
2013). Similarly, during development, the vertebrate hair cell initially forms a central
kinocilium surrounded by stereocilia, but later adopts the asymmetric organization of a
polarized kinocilium and stereocilia (Duncan and Fritzsch, 2012). Mechanosensory cells
exist in other non-vertebrates such as ascidians, molluscs, cnidarians, and insects; each
8
serving a unique purpose, and are most likely homologous to the vertebrate hair cell
(Burighel et al., 2011).
The sister group to vertebrates within Deuterostomes are the tunicates (Delsuc et
al., 2006, Burighel et al., 2011). In this group, the ascidians have two organs, the
capsular organ and the coronal organ. The capsular organ has a sensory macula
containing 5-6 cilia-containing primary sensory cells (contain both cilia and an axon) that
are believed to perceive vibrations. These capsular organs were thought to have evolved
from isolated or clustered primary sensory cells aggregating into the organ. Due to their
location, organization and basic function, these primary mechanosensory cells are
thought to be homologous with the vertebrate inner ear (Burighel et al., 2011). In fact,
these ascidian cells originate from a Pax2/5/8-expressing region, similar to that of the
inner ear (Wada et al., 1998, Bassham et al., 2008, Bouchard et al., 2010). The other
organ, the coronal organ, consists of a row of ciliated secondary sensory cells that make
both afferent and efferent synapses with neurons (Burighel et al., 2003, Burighel et al.,
2011). These ciliated cells are flanked by supporting cells (Burighel et al., 2011). This
organization is quite similar to that of the vertebrate inner ear, which consists of separate
secondary mechanosensory cells and neurons rather than a single primary sensory cell
(Fritzsch and Beisel, 2004, Pan et al., 2012). It is thought that the secondary
mechanosensory cells and neurons are derived from the primary mechanosensory cell
(Duncan and Fritzsch, 2012).
The derivation of a separate mechanosensory hair cell and neuron from an
ancestral primary mechanosensory cell with an axon is thought to have arisen from the
duplication and diversification of the basic helix-loop-helix (bHLH) atonal transcription
9
factor. Atonal bHLH genes evolved with multicellular organisms (Seipel et al., 2004)
and are associated with photo-and mechanosensor development (Fritzsch et al., 2007).
The atonal bHLH family consists of Atonal homolog 1 (Atoh1), Neurog1 and Neurod1.
The ancestral atonal gene multiplied and split giving rise to Atoh1 and Neurog1 (Fritzsch
and Beisel, 2003, 2004). The newly-generated genes could then be assigned novel
functions (Fritzsch and Beisel, 2001, 2004, Pan et al., 2012). In invertebrates, the
primary mechanosensory cells that contain an axon rely on atonal for their development.
In vertebrates, hair cells depend on Atoh1 for their development and the afferent neurons
depend on Neurog1 (Ma et al., 1998, Bermingham et al., 1999, Ma et al., 2000, Fritzsch
et al., 2010). It is thought that the sensory precursor cells underwent an additional round
of cell division to give rise to both the hair cell through Atoh1 expression and the sensory
neuron through Neurog1 expression, thus suggesting a clonal relationship between the
two (Fritzsch and Beisel, 2003, 2004). In addition, neurons must upregulate Neurod1 to
differentiate as neurons (Jahan et al., 2013). Neurod1 inhibits Atoh1 expression and in
the absence of Neurod1, some neurons differentiate as hair cells (Jahan et al., 2010b). It
is thought that hair cells then became restricted to the placodal region to form an
aggregation of hair cells in a sensory epithelia that over time would evolve to form the
first ear (Duncan and Fritzsch, 2012).
The earliest ear likely existed as a single sensory epithelia, likely a gravistatic
sensor, found in most free-swimming animals (Markl, 1974, Fritzsch et al., 2007, Duncan
and Fritzsch, 2012) and it is from this simple gravistatic-sensing ear that all other
vertebrate ears arose. Of the vertebrate ears, the ear of the hagfish is the most simple and
thought to be the closest to the ancestral ear (Fritzsch and Beisel, 2004). To achieve up
10
to the nine distinct sensory epithelia in gymnophionans (Fritzsch and Wake, 1988), or
any of the more derived vertebrate ears, duplication, segregation, and specialization of
the existing sensory epithelia occurred (Fritzsch et al., 2007).
Motor Neuron Targeting
The vertebrate inner ear receives two kinds of fibers: afferents that come from
placodally-derived sensory neurons (Rubel and Fritzsch, 2002) and efferents which come
from brainstem neurons (Simmons, 2002, Simmons et al., 2011). Whereas afferents
carry information from the ear to the brain, efferents provide a feedback loop to modify
mechanical stimuli information processing at the periphery. In mammals, efferents
project bilaterally from their origin in the hindbrain, through the vestibulocochlear nerve,
to innervate both inner ears; however, in other vertebrates, projections are mostly
ipsilateral, but with some contralateral innervation (Holt et al., 2011). The origin of the
auditory efferents in mammals is the superior olivary complex, whereas the vestibular
efferents originate outside the vestibular nucleus, near the facial nerve, in the hindbrain
(Brown, 2011, Holt et al., 2011). All other non-mammalian vertebrates have a single
population of efferent neurons originating partially within or near the facial motor
nucleus in the hindbrain (Holt et al., 2011, Köppl, 2011). In amphibians and other lateral
line-containing vertebrates, the efferent nucleus referred to as the octavolateralis nucleus
and gives rise to efferents innervating both the lateral line neuromasts as well as the
vestibular and auditory hair cells (Hellmann and Fritzsch, 1996, Fritzsch, 1999, Holt et
al., 2011).
The close proximity of the efferents to the facial branchial motor neurons
(Roberts and Meredith, 1992) and their similarities during development, has led to the
11
suggestion that the inner ear efferents are rerouted facial branchial motor neurons
(Fritzsch and Nichols, 1993). The inner ear efferents and the facial branchial motor
neurons both exit the cell cycle near the floorplate in rhombomere 4 of the hindbrain and
later migrate to their final location (Simmons et al., 2011). Although their nuclei migrate
away from each other, the inner ear efferents and facial branchial motor neuron axons run
together through the facial genu before diverging to the eighth and seventh cranial nerves,
respectively (Simmons et al., 2011). In the absence of inner ear afferent neurons, the
inner ear efferents project with the facial nerve (Ma et al., 2000). Inner ear efferents may
be guided to reach the ear by the transcription factor, GATA3, which is uniquely
expressed in the inner ear efferents and is absent from the facial branchial motor neurons
(Karis et al., 2001). Knock out of Gata 3 results in most inner ear efferents projecting
along the facial nerve (Duncan et al., 2011). Thus the efferent population was likely
rerouted from the facial branchial motor neurons after the emergence of Gata3 and of the
inner ear.
In vertebrates, the ear develops at the rostral boundary of the anteriormost, first
forming somite (Cooke, 1978, Chung et al., 1989, Huang et al., 1997) and paraxial
mesoderm anterior to the ear does not form somites (Noden and Francis-West, 2006) but
may form somitomeres. Furthermore, in vertebrates, the most rostral somite is reduced in
size to accommodate the ear. In animals without ears, such as amphioxus, somites are
found more rostrally (Bardet et al., 2005). Taken together, these data could be interpreted
to imply that in vertebrates, the ear develops in place of a somite or somitomere. If true,
the facial branchial motor neurons that were destined to innervate the somite- or
12
somitomere-derived muscle fibers may have been rerouted to innervate the ear when the
ear evolved in ancestral vertebrates (Fritzsch et al., 2007).
That a subpopulation of facial branchial motor neurons was able to acquire a
novel target, the inner ear, proposes a bigger question of how motor neurons acquire a
novel target in general. Synaptic contacts likely originated in evolution as connections
between sensory-motor neurons and muscle tissue in diploblastic animals, such as
jellyfish, (Seipel et al., 2004) forming a monosynaptic reflex network from a sensory cell
directly to a motor cell. As animal cell types diversified, the sensory-motor interface
evolved in complexity, forming muscle fibers, motor neurons, sensory neurons, and
interneurons (Fritzsch and Glover, 2007). As tissues diversified in the course of
evolution, motor neurons acquired novel targets. Vertebrate motor neurons have evolved
to form synapses on a variety of targets originating from developmentally different
sources including mesoderm-derived muscle fibers, epithelial placode-derived
neurosensory cells and neurons (i.e. the inner ear hair cells and neurons), and neural
crest-derived autonomic ganglia (Eisen, 1999, Fritzsch, 1999, Takano-Maruyama et al.,
2010, Simmons et al., 2011).
Of all cranial motor neurons, only the facial nerve innervates targets from three
different developmental origins: branchial arch-derived muscle by branchial motor
neurons, neural crest-derived ganglia by visceral motor neurons, and placodally-derived
inner ear hair cells by inner ear efferents (Fritzsch and Northcutt, 1993). While the inner
ear efferents are now classified as separate from the facial nerve as discussed above, their
proposed origin as rerouted facial branchial motor neurons merges them with this group.
The glossopharyngeal nerve also innervates more than one tissue type: muscle, ganglia,
13
and in aquatic vertebrates such as Xenopus laevis, hair cells of the placodally-derived
posterior lateral line (Hellmann and Fritzsch, 1996). Given the ability of some motor
neurons to innervate targets from a variety of origins, the possibility exists that conserved
core molecular machinery exists that allows multiple motor neuron types to recognize a
particular target. However, some tissues are only innervated by one motor neuron type,
even when other motor neuron types project to proximal territories as is the case with the
innervation of the trapezius muscle by the spinal accessory nerve rather than spinal motor
neurons (Boord and Sperry, 1991, Sienkiewicz and Dudek, 2010, Dudek et al., 2011).
This suggests that there must be some limitation to the potential promiscuity of motor
neurons for targets of different embryological origin.
In order for a vertebrate effector cell to be able to respond to input from a motor
neuron, it must have the ability to detect acetylcholine (ACh) input. One thing in
common that all targets receiving direct motor input have is the presence of nicotinic
ACh receptors (nAChRs) (Zuo et al., 1999, Vernino et al., 2009). nAChRs are comprised
of a pentamer of various subunits: α, β, δ, ε, and γ (Tsunoyama and Gojobori, 1998, Jones
and Sattelle, 2004, Albuquerque et al., 2009). Of these subunits, the α subunit is
required for the receptor to bind ACh (Jones and Sattelle, 2004). The original α sequence
has diversified, giving rise to the 10 different isoforms present today (Jones and Sattelle,
2004). Of the 10 α subunits, α9 and α10 are the most ancestral (Sgard et al., 2002,
Franchini and Elgoyhen, 2006, Katz, 2011, Lipovsek et al., 2012). The α9 and α10
subunits are expressed only in the ear, pituitary gland, lymphocytes, and keratinocytes
(Sgard et al., 2002, Katz, 2011, Kawashima et al., 2012) and activation of nAChRs
containing these subunits results in cell hyperpolarization or desensitization instead of
14
depolarization as with other nAChRs (Elgoyhen and Katz, 2012, Kawashima et al., 2012,
Lipovsek et al., 2012). The more derived α subunit, α1, is present in muscle tissue
(Tsunoyama and Gojobori, 1998, Albuquerque et al., 2009, Katz, 2011, Elgoyhen and
Katz, 2012). The remaining α subunits are expressed in neurons (Tsunoyama and
Gojobori, 1998, Albuquerque et al., 2009, Katz, 2011, Elgoyhen and Katz, 2012). Given
that all motor neuron targets contain nAChRs, and tissues that are not directly innervated
by motor neurons do not contain these receptors, it is likely that the presence of nAChRs
and associated molecules to form synaptic contacts is necessary for general motor neuron
development, much like Islet-1 (Ericson et al., 1992, Inoue et al., 1994).
However, while the presence of nAChRs might be necessary for motor
innervation, it is not sufficient as nAChR-containing lymphocytes are never innervated.
Instead, in lymphocytes, ACh present in the bloodstream binds to nAChRs and
muscarinic AChRs to modulate immune function by downregulation or upregulation of
proinflammatory cytokines respectively (Kawashima et al., 2012). In contrast, tissues
that do receive direct motor innervation, such as muscle, autonomic neurons, and inner
ear hair cells, contain three accessory molecules: muscle specific receptor tyrosine kinase
(MuSK), receptor-associated protein of the synapse (rapsyn), and a transmembrane
protein RIC-3. These molecules have been shown to be important for synapse formation
(Apel et al., 1997, Sanes et al., 1998, Lin et al., 2001, Osman et al., 2008) as they are
necessary for receptor assembly and clustering at the neuromuscular junction (Apel et al.,
1997, Sanes et al., 1998, Lin et al., 2001). In addition, rapsyn and RIC-3 have been
shown to be necessary and sufficient for functional nAChRs containing α9 and α10 at the
cell surface of inner ear hair cells (Osman et al., 2008). Thus, not only are nAChRs
15
required for motor neuron innervation, but accessory molecules are needed as well. From
this it follows that the specific α nicotinergic subtype present in the accessory moleculecontaining target tissue, together with the specific LIM code in different motor neuron
populations (Tsuchida et al., 1994), could determine the degree of normal motor neuron
to target specificity or plasticity, in addition to other unknown molecular contributions.
Inner Ear Sensory Neuron Guidance
Vestibular and auditory neurons delaminate from the developing otic vesicle
(Torres and Giráldez, 1998, Rubel and Fritzsch, 2002, Riley and Phillips, 2003, Alsina et
al., 2009) and migrate away to form the vestibular and auditory ganglia (Maklad and
Fritzsch, 2003). The neurons differentiate as bipolar neurons, sending their dendrites and
axons to the inner ear hair cells and to the CNS, respectively (Fritzsch et al., 2002).
Vestibular sensory neuron axons enter the hindbrain at the level of the lateral vestibular
nucleus, bifurcate, and send an ascending branch to the superior vestibular nucleus and
the cerebellum and a descending branch to the medial and inferior vestibular nuclei
(Büttner-Ennever, 1992). Auditory sensory neurons enter the cochlear nuclei, bifurcate,
and send axons to the anteroventral cochlear nucleus, the posteroventral cochlear nucleus,
and the dorsal cochlear nucleus (Rubel and Fritzsch, 2002). The auditory sensory
neurons project to the cochlear nuclei in a tonotopic manner that maintains the frequencyspecific organization of the cochlea (Rubel and Fritzsch, 2002). Similar to the highlyspecific organization of the auditory nuclei, vestibular sensory neurons innervating the
various sensory epithelia partially segregate (Kevetter and Perachio, 1986, Maklad and
Fritzsch, 1999, 2003), though it is not a complete segregation as fibers from different
sensory epithelia do overlap in the vestibular nuclei (Birinyi et al., 2001, Maklad and
16
Fritzsch, 2002). It is possible that the partial overlap functions to integrate semicircular
canal input with that of the utricle and/or saccule for a given movement (BüttnerEnnever, 1992, Maklad and Fritzsch, 2002, Straka et al., 2002, Maklad and Fritzsch,
2003, Newlands and Perachio, 2003) to allow for precise and coordinated compensatory
eye, head, and body movements (Büttner-Ennever, 1992, Maklad and Fritzsch, 2002).
While much is known about where the inner ear vestibular sensory neurons project to, the
mechanisms used by these neurons to project to their specific location in the vestibular
nuclei remain largely unknown.
The eye has been extensively studied and is developmentally related to the ear
through shared molecular processes. For example, jellyfish express PaxB in both the
statocyst and eye (Kozmik et al., 2003). PaxB has evolved into Pax 6 and Pax 2/5/8
(Fritzsch and Piatigorsky, 2005, Galliot et al., 2009). The highly-conserved Pax 6 is
involved in eye development across nearly all species (Gehring and Ikeo, 1999), whereas
Pax 2/5/8 is involved in ear development (Pfeffer et al., 1998, Heller and Brändli, 1999,
Bouchard et al., 2010). Elimination of Pax 6 or Pax 2/5/8 leads to the absence or severe
reduction of the eye and ear, respectively (Hoge, 1915, Gehring and Ikeo, 1999,
Bouchard et al., 2010). Retinal ganglion cells (RGCs) also depend upon Pax 2 for proper
guidance (Torres et al., 1996, Mansouri et al., 1999). In addition to Pax, the bHLH
Atonal transcription factor is important for both eye and ear development. In Drosophila
melanogaster, atonal is expressed in both photoreceptors and chordotonal organs (Jarman
et al., 1995). Atonal has since duplicated and diversified. In the eye, Atoh7 is expressed
in the RGCs and is important for their development (Kanekar et al., 1997, Brown et al.,
1998, Brown et al., 2002, Skowronska-Krawczyk et al., 2009). Overexpression of Atoh7
17
in Xenopus resulted in an increase in RGCs (Brown et al., 1998) and inactivation of
Atoh7 in zebrafish and mouse results in the near absence of RGCs in the retina (Brown et
al., 2001, Kay et al., 2001). In the ear, Atoh1 is important for hair cell formation,
whereas other atonal family members, Neurog1 and Neurod,1 are important for the
development of sensory neurons (Fritzsch et al., 2010). Conditional deletion of Neurod1
in the mouse inner ear of mice resulted in a reduction in neuron number, aberrant central
projections, and transformation of neurons into hair cells as a result of disinhibition of
Atoh1 (Jahan et al., 2010a, Jahan et al., 2010b). These data suggest that Neurod1 plays a
role in axon guidance to central targets, in addition to its role in neuronal differentiation,
which is supported by the ability of Neurod1 to replace Atoh7 in retinal ganglion cells for
normal central projections (Mao et al., 2008).
The molecular similarities between the ear and the eye would suggest that the ear
may use similar mechanisms as the eye for guiding axons to their proper locations in the
CNS. In the eye, vertebrates use concentration gradients of EphA and ephrin-A along the
nasal-temporal axis of the retina and anterior-posterior axis of the superior
colliculus/tectum to guide retinal ganglion axons to the appropriate region of the CNS
(Clandinin and Feldheim, 2009) to maintain appropriate representation of the visual field.
Eph receptors and their ligands, ephrins, are involved in axon guidance, among other
roles. Binding of ephrin to an Eph receptor generally results in repulsion (Kullander and
Klein, 2002). In the eye, EphA concentrations are the greatest in the temporal retina and
lowest in the nasal retina. In the superior colliculus/tectum, ephrin-A concentrations are
higher in the posterior and lower in the anterior. Thus, RGCs from the temporal retina,
containing high EphA receptor are repulsed by the higher concentration of ephrin-A in
18
the posterior superior colliculus/tectum and terminate in the anterior superior
colliculus/tectum, where ephrin-A concentrations are low. In contrast the RGCs from the
nasal retina are not repulsed by ephrin-A and terminate in the posterior superior
colliculus/tectum (Clandinin and Feldheim, 2009).
The retinal projections to the superior colliculus/tectum are further refined in
some vertebrates to account for input from both eyes. In higher mammals, (e.g. cats and
primates), the eyes are located in the front of the head, resulting in a significant overlap
of part of the visual field between the two eyes (Schmidt and Tieman, 1985, Leamey et
al., 2009). This overlap of the visual field results in the segregation of eye-specific
projections to the visual cortex into stripes, referred to as ocular dominance columns
(Hubel and Wiesel, 1977, Wiesel, 1982), whereas when there is no overlap of the visual
field, there is no formation of ocular dominance columns (Schmidt and Tieman, 1985).
The formation of ocular dominance columns can be induced in lower vertebrates with
lateral eyes, (i.e. frogs and fish), by transplantation of an extra eye such that two eyes
project to the same tectum (Constantine-Paton and Law, 1978, Springer and Cohen, 1981,
Constantine-Paton, 1982, Easter, 1983). Various experiments have demonstrated that the
formation of ocular dominance columns is primarily activity-based (Hubel and Wiesel,
1965, Swindale, 1981, Meyer, 1982, Swindale and Cynader, 1983, Boss and Schmidt,
1984, Mower et al., 1984, Reh and Constantine-Paton, 1985) and presents a compromise
between the molecularly-specified targeting and the individual activity of the neurons.
It seems logical to suggest that the ear may use both molecular and activity-based
mechanisms to guide cochlear afferents (Rubel and Fritzsch, 2002) and projections from
the various end organs of the vestibular system (Maklad and Fritzsch, 2003, Newlands et
19
al., 2003) to the proper location in the CNS, comparable to the eye. Ephs and Ephrins
have been identified in the auditory system and have been demonstrated in mice and
chicken to play some role in axon guidance (Allen-Sharpley and Cramer, 2012, AllenSharpley et al., 2013, Defourny et al., 2013). Ephs and Ephrins have also been shown to
be expressed in vestibular neurons and their peripheral vestibular hair cell targets
(Bianchi and Liu, 1999), though a specific role in vestibular guidance, especially
centrally, has not been determined. Support for molecular targeting in the vestibular
system comes from selective labeling of various vestibular sensory epithelia. Maklad and
Fritzsch (2002) demonstrated that vestibular afferents made no large errors, such as
targeting extra-vestibular areas. This initial targeting was activity-independent as
observations were made prior to the onset of vestibular sensation (Maklad and Fritzsch,
2002, Maklad and Fritzsch, 2003, Maklad et al., 2010). However, activity-based
guidance may play a later role in refining projections since changes in the pattern of
connectivity in the vestibular nucleus from sensory neurons innervating individual
sensory epithelia occur around the onset of a functional vestibular system (Maklad and
Fritzsch, 2003). Exposure of rats to microgravity resulted in decreased branching in the
vestibular nucleus from the gravity-sensing saccule (Ronca et al., 2000, Fritzsch et al.,
2001a), which suggests that activity plays a role in refining vestibular processes. Thus
the hypothesis is that sensory neurons may first be guided by molecular mechanisms to
the vestibular nuclei, which are further refined by the specific activity from each sensory
epithelia with particular head and/or body movements.
20
Hindbrain Neurons
Once inside the hindbrain, vestibular afferents from the various inner ear sensory
epithelia synapse on neurons in the vestibular nuclei. Vestibular nuclei neurons project
both caudally to the spinal cord and rostrally to the cerebellum and extraocular motor
nuclei where they are involved in several reflexes with the proprioceptive and visual
systems to maintain balance, posture, and gaze during movement. To maintain balance
and posture, vestibular neurons project rostrally to spinal motor neurons, which synapse
on extensor and flexor muscles in the neck and on extensor muscles of the limbs to
control head and body movements (Glover, 1996, Uchino and Kushiro, 2011). To
maintain proper gaze, vestibular nuclei neurons project both contralaterally and
ipsilaterally to neurons in the extraocular motor nuclei neurons to coordinate eye
movement in a process known as the vestibulo-ocular reflex (VOR) (Glover, 2003,
Straka, 2010). The VOR is an open loop system: vestibular sensory neuron to vestibular
nuclei neuron to extraocular neuron. The loop is closed by relay of the retinal image
back to the extraocular motor nuclei where they also integrate the intended body
movement from the spinal cord and actual body movement from the vestibular nucleus
(Straka, 2010). In addition, vestibular neurons project to the floccular lobe of the
cerebellum (Uchino and Kushiro, 2011). Purkinje cells in the flocculus receive head and
eye movement information via the parallel fibers and retinal image motion through the
climbing fibers where they play a role in VOR learning (Gittis and du Lac, 2006).
Auditory neurons synapse on neurons in the cochlear nuclei. From there, cochlear
nuclei neurons project to the superior olivary complex to localize sound and to the lateral
lemniscus and superior colliculus, which are part of the auditory pathway leading to the
21
cortex (Cant and Benson, 2003). In addition, cochlear nuclei neurons project to the
ventrolateral medullary nucleus to control autonomic functions (Kamiya et al., 1988), and
in some mammals, to the pontine and medullary reticular formation to control
cardiovascular and respiratory function as well as an auditory startle response (Cant and
Benson, 2003).
Neurons in the cochlear nuclei are dependent upon input from the inner ear for
their survival (Levi-Montalcini, 1949, Parks, 1979, 1981, Goodman and Model, 1988,
Fritzsch, 1990). Unilateral removal of the inner ear early in chick development led to a
reduction in the volume and number of neurons in the cochlear nuclei, as compared to the
unoperated side, after embryonic day (E) 11 (Levi-Montalcini, 1949). The cochlear
nuclei neurons continued to die over time. By E21, there were 32% and 82.5% fewer
neurons than observed on E11 in two of the cochlear nuclei (Levi-Montalcini, 1949).
This reduction in volume and number of neurons was later confirmed and extended
(Parks, 1979, Ryugo and Parks, 2003). The time point at which the number of neurons in
the cochlear nuclei began to diminish corresponded to the onset of afferent activity via
action potentials (Jackson et al., 1982). Blocking action potentials by application of
tetrodotoxin to the sensory neurons resulted in a reduction of volume and number of
neurons in the cochlear nuclei, similar to that seen with ear ablation (Pasic and Rubel,
1989, Sie and Rubel, 1992). Together, these data would suggest that most of the initial
neuronal development occurs in the hindbrain without excitatory input from primary
sensory neurons and that later, without this activity and/or activity-related neurotrophin
release, cell death and atrophy of remaining hindbrain neurons occurs (Rubel and
Fritzsch, 2002).
22
However, other studies have shown that this dependence on a presynaptic neuron
is not permanent, which would imply that there is a critical period for cell survival (Rubel
and Fritzsch, 2002). Removal of the cochlea in chicks 2 and 6 weeks post-hatch resulted
in a 30% loss of cochlear nucleus magnocellularis neurons (Born and Rubel, 1985),
similar to that seen when the ear was ablated at embryonic stages (Parks, 1979); however,
in adult chickens (66 weeks post-hatch), there was no significant loss of cochlear nucleus
neurons following cochlear removal (Born and Rubel, 1985). Similar critical periods
exist in mammals. Removal of the cochlea in postnatal day (P) 5 mice resulted in the loss
of 61% of neurons in the cochlear nucleus compared with only 1% loss when the cochlea
was removed at P14 (Mostafapour et al., 2000). In gerbils, removal of the cochlea prior
to P7 resulted in cell death in 45-88% of cochlear nucleus neurons, whereas removal of
the cochlea after P9 resulted in virtually no cell death (Tierney et al., 1997) The loss of
susceptibility to presynaptic neuron ablation occur around the time of hearing onset, P12
in gerbils (Woolf and Ryan, 1984, 1985, Tierney et al., 1997), which supports at least an
early necessity of activity for cell survival. As mentioned previously, early embryonic
removal of the chick ear resulted in death of most cochlear nuclei and severe atrophy of
the remaining neurons (Levi-Montalcini, 1949, Parks, 1979); however, virtually no cell
death occurred in the vestibular nuclei (Levi-Montalcini, 1949), whose neurons exit the
cell cycle much earlier (Altman and Bayer, 1980), suggesting they may be past the
critical period at the time these manipulations took place. In this context it is important
to note that even though early ear removal results in significant cochlear nucleus cell loss,
some cells do survive. Unfortunately, the reason for why some cells die and others do
not is not well understood.
23
The leading hypothesis to explain why some post-synaptic neurons survive while
others die following early afferent deprivation is that two competing intracellular
responses are initiated following afferent loss: apoptotic-like and survival pathways
(Garden et al., 1994, Hyde and Durham, 1994a). This hypothesis is supported by several
studies. For example, early processes of cellular degradation following afferent loss are
constant across all cells, not just some (Steward and Rubel, 1985, Born and Rubel, 1988,
Garden et al., 1994, Garden et al., 1995, Kelley et al., 1997), and oxidative enzyme
activity first increases and then decreases following afferent loss (Durham and Rubel,
1985, Durham et al., 1993, Hyde and Durham, 1994b). Thus the cells that survive
depend upon the effectiveness of the survival pathway to overcome apoptosis. Afferent
neurons rely on neurotrophic factors provided by the hair cells of the ear and the target
neurons for cell survival (Fritzsch et al., 2001b). In the ear, the neurotrophic factors are
BDNF and NT-3, and loss of BDNF or NT-3 in the inner ear results in embryonic
sensory neuron cell death (Fritzsch et al., 2001b). Changes in the amount of receptor for
BDNF, Tyrosine Kinase B (TrkB), occur in the cochlear nucleus following ear ablation
(Suneja and Potashner, 2002). The nature of the neurotrophic substance by which
afferents support cochlear nucleus neurons has not been determined, although
neurotrophic factors have been suggested to play a role. Levi-Montalcini proposed that
loss of ‘neurotrophic’ support from the afferents following ear removal may cause the
cell death in the cochlear nucleus (Levi-Montalcini, 1949). Surviving cells must either
receive enough neurotrophic support elsewhere for survival or are past a critical phase
when inner ear sensory neurons provide support.
24
These above-mentioned studies have increased our understanding of the
importance of primary sensory input on higher-order neuron survival; however, these
studies looked only at whole populations of cells when determining the critical period of
cell survival and when determining why some cells survive while others do not following
ear removal. Furthermore, due to technical limitations, ears can be removed in the chick
only embryonically, and in the mouse, only postnatally, which prevents a complete
analysis of the loss of afferent input over a longer period of time in these species. In fact,
mice genetically engineered to lose all neurons (Ma et al., 2000) do not survive past birth
and thus cannot be used to study the effect of neuronal loss in postnatal development. In
addition, the effects on higher-order neurons following ear removal at early embryonic
placodal stages have not been well studied, largely due to the unpredictable tendency of
partial or complete otic placode regeneration (Levi-Montalcini, 1949, Waldman et al.,
2007). In the hindbrain of the frog, an animal easily accessible across all developmental
time points due to its external development, exists a single pair of cells that receive inner
ear input, the Mauthner cells. Developmental effects of ear removal can thus be studied
on this single neuron.
Mauthner cells are a pair of large, easily identifiable, reticulospinal neurons at the
level of the ear in the hindbrain of many aquatic animals (Herrick, 1914, Bartelmez,
1915). These cells are an important component of the escape reflex (Korn and Faber,
2005). Inner ear vestibular and auditory neurons of fish and premetamorphic amphibians
form synapses on the lateral dendrite of the ipsilateral Mauthner cell. The Mauthner cell
axon crosses the midline of the hindbrain and projects caudally to synapse onto
contralateral spinal motor neurons to activate the C-start escape response (Korn and
25
Faber, 2005, Sillar, 2009). In addition to the ear, Mauthner cells also receive input from
the lateral line and the eye that can add to the C-start escape response (Zottoli et al., 1987,
Bezgina et al., 2000, Korn and Faber, 2005, Sillar, 2009).
Several studies suggest that the Mauthner cell is dependent upon input for its
development and/or survival. A study in axolotls (Ambystoma mexicanum) has shown
the absence of the Mauthner cell in one-third of embryos following otic vesicle ablation
at stage 27 (Piatt, 1969). In one embryo observed by Goodman and Model following ear
ablation, the entire Mauthner cell was absent (1988). While Piatt (1969) suggested that
the inner ear afferents are important for, and often a decisive factor for, the development
of the Mauthner cell, Goodman and Model (1988) suggested that surgical perturbations
may be the cause of the occasional Mauthner cell absence. In contrast to early ear
removal, no Mauthner cells were absent when the ear was extirpated at later stages (stage
34), although dendritic branching was reduced (Kimmel et al., 1977, Fritzsch, 1990).
Since early ear removal, before the critical period, results in significant loss of the
cochlear nuclei neurons (Levi-Montalcini, 1949, Parks, 1979, Born and Rubel, 1985,
Tierney et al., 1997, Mostafapour et al., 2000). These data would suggest that removal of
the ear at the earlier stages (Piatt, 1969, Goodman and Model, 1988) prevented the
differentiation and/or survival of the Mauthner cell, hinting at a critical period for afferent
innervation on Mauthner cell survival and development.
The inner ear not only affects the development of the Mauthner cell, but may have
a role in the development of the lateral dendrite as well. In a study in axolotls, removal
of the ear of midtailbud animals (stage 28-30) prior to the outgrowth of the VIIIth nerve
resulted in significant reduction of dendritic branching of the Mauthner cell lateral
26
dendrites in areas normally receiving vestibular input (Goodman and Model, 1988). Ear
removal at a slightly later stage in axolotls (stage 34) also resulted in reduction of
dendritic branching (Kimmel et al., 1977), though the degree of dendritic loss across
other stages has not been investigated. Removal of otic vesicles in zebrafish resulted in
reduced branching of the Mauthner cell lateral dendrites (Kimmel, 1982). Likewise,
ablation of the otic vesicle in stage 38 X. laevis embryos resulted in reduced Mauthner
cell lateral dendrites (Fritzsch, 1990). The eye synapses on another Mauthner cell
dendrite, the ventral dendrite. Enucleation of the eye resulted in delayed development of
the Mauthner cell and diminished ventral dendrite volume in X. laevis and goldfish
(Bezgina et al., 1999, Bezgina et al., 2000, Grigor'eva et al., 2010, Mikheeva et al., 2011).
Increase in the lateral dendrite size in combination with reduction in the ventral dendrite
was observed occasionally following eye enucleation (Grigor'eva et al., 2010, Grigorieva
et al., 2012). Blocking nerve impulse activity in axolotls by grafting to them
tetrodotoxin-containing newts did not affect Mauthner cell dendritic branching patterns,
suggesting that that innervation itself, rather than neural activity, is important for
dendritic development (Goodman and Model, 1990). However, spontaneous local
synaptic vesicle release could still play a role in this tetrodotoxin model. Taken together,
these data suggest that the ingrowing axons stimulate growth and development of the
dendrites. This is further supported by a study in axolotls where an additional ear was
transplanted rostral to the native ear. The Mauthner cells in these animals were claimed
to display enhanced branching of the lateral dendrite (Goodman and Model, 1988)
although no quantitative data to support this conclusion were provided.
27
Experimental Approach for Generating and
Manipulating a ‘Novel’ Sensory System
Ears from the frog, Xenopus laevis, were either transplanted from the same
embryo or removed from a different same staged embryo to generate a ‘novel’ sensory
system or remove an existing one, respectively. The manipulated ears provide data to
better understand how the ear becomes innervated during development and, by logical
inference, through evolution. Specifically, ears were transplanted caudally to the trunk to
replace a somite or rostrally to the orbit to replace the eye to test whether any motor
neuron can become an efferent to the inner ear. To further elucidate the properties of
motor innervation, other tissues were transplanted to test for the ability of any tissue to
receive motor input. In addition, ears were transplanted from a donor to a host embryo
rostral to the native ear to create a three-eared frog with two tandem ears on one side.
These ears were transplanted either in the native orientation to maintain similar activity
between the two ears or rotated by 90 degrees to set up differential activity between the
two ears in order to determine the extent to which molecular cues and activity-based
guidance directs sensory axons to their locations within the vestibular nucleus. Finally,
ears were removed or additional ears were transplanted to determine the effects of loss of
input or additional input on the developing hindbrain target neuron, the Mauthner cell.
Ears were removed over a time course to establish a potential critical period of necessity
of input for Mauthner cell survival. Detailed descriptions and results of these
experiments will be outlined in the following chapters.
28
CHAPTER II
METHODS
Animals
Xenopus laevis embryos were obtained through induced breeding using human
gonadotropin injection and fertilized with a sperm suspension in 1X Marc’s Modified
Ringer’s Solution (MMR, see below), pH 7.8. Fertilized embryos were maintained in
stock cultures in Petri dishes. Other than during manipulations, embryos were kept in
90mm Petri dishes containing 0.1X MMR (diluted from 1x MMR) until stage 46. X.
laevis stages were as described by Nieuwkoop and Faber (1994).
GFP mRNA injections
For synthesis of green fluorescent protein (GFP) mRNA, plasmid template
(pβGFP/RN3P) was linearized using SfiI and transcribed using T3 RNA polymerase from
the mMessage mMachine kit (Ambion). Protocol was followed according to
manufacturer’s directions.
The jelly coat was removed using 2% cysteine in 0.1X MMR (diluted from 1X
MMR) shortly after fertilization for embryos used in injections. Embryos were placed in
a Ficoll solution (2% Ficoll 400, GE/Pharmacia, in 0.5X MMR) for 5 min. GFP mRNA
was diluted with distilled water so that the final amount injected was 1 ng. Embryos
were injected at the 2 to 4 cell stage using a calibrated glass needle controlled by a PicoInjector (Harvard Apparatus, Holliston, MA). Injections were made into each cell,
keeping the total amount of mRNA per embryo constant.
29
Dextran amine injections
Dextran amine (Fluorescein, 3000MW (Fritzsch, 1993)) was dissolved in water to
make a 1% solution. The jelly coat was removed as described above. Embryos were
placed in the Ficoll solution (2% Ficoll) for 5 min prior to injection. Embryos were
injected at the one cell stage using a glass needle controlled by a Pico-Injector.
Transplantations
All transplantations were performed in 1x MMR pH 7.6-7.8, diluted from 10x
stock (1M NaCl, 18mM KCl, 20mM CaCl 2 , 10mM MgCl 2 , 150mM Hepes) at room
temperature. To determine the ability of various motor neurons to acquire a novel target,
tissues were transplanted. For ear to trunk transplantations, otic placodes from the right
side of stage 24-26 embryos were removed and transferred ipsilaterally more caudal,
replacing a somite. For ear to orbit transplantations, otic placodes from the right side of
stage 24-26 embryos were removed using fine tungsten needles and transferred
ipsilaterally to the orbit, replacing the eye. In addition, otic placodes were removed from
stage 24-26 embryos previously injected with GFP mRNA to label donor tissue and
transplanted to the orbit of a non-injected host embryo of the same stage to replace the
eye. To check the success rate of the ear transplantations, the ear was removed and
reimplanted, but rotated by approximately 180°. Those rotated ears were monitored and
scored as above (Table 3.1). In contrast to the caudal transplantation our success rate of
normal differentiation of rotated ears was greater (approximately 92% vs. 67%).
For somite transplantations, donor somite tissue from stage 24-25 embryos previously
injected with GFP was removed using fine needles and transplanted to the orbit of a host
stage 24-25 embryo to replace the eye. Alternatively, donor somite tissues from stage
30
24-25 embryos were placed in a Fluorescein dextran amine solution (10,000MW, 10%)
for 5 min, rinsed and transplanted into host stage 25 embryos to replace the ear. For eyeto-muscle transplantations, donor eyes and surrounding tissue from stage 25 embryos
previously injected with GFP were removed using fine needles and transplanted to the
trunk of a host stage 25 embryo to replace approximately three to four somites (the size
of the eye). For jaw muscle transplantations, donor jaw muscle from stage 45-46 donor
embryos previously injected with dextran amine (1%) was removed and processed into
small pieces. A small jaw muscle fragment was transplanted to the trunk of a host stage
25 embryo to replace a somite. For heart transplantations, donor heart tissue from stage
27 embryos, some having been previously injected with GFP mRNA, was removed using
fine needles and transplanted to the orbit of a host stage 24-26 embryo to replace the eye.
For liver transplantations, donor liver tissue from anesthetized (0.02% Benzocaine)
(Crook and Whiteman, 2006) stage 42 embryos was removed and transplanted to stage
24-26 embryos to replace the eye.
To determine whether inner ear sensory neurons use molecular and activity-based
cues, ‘three-eared’ frogs were generated. Single otic placodes from donor X. laevis
embryos were transplanted rostral to the native ear in host embryos between stages 25-27.
Otic placodes were transplanted either in the native orientation or rotated by 90 degrees.
The rationale being that ears in alignment should have little difference in activity,
whereas when one is rotated by 90 degrees, the sensory epithelia will be offset from each
other and there should be differential activity between the two ears in tandem.
To study the effects of ear removal on the Mauthner cell, single otic placodes or
otic vesicles were removed from stage 24, 25, 26, 27, 28, 30, 32, 34, 36, 38, and 40
31
embryos. Single otic placodes or vesicles were removed from stage 24-26 embryos and
then immediately replaced to serve as a surgical control.
Embryos were kept in 1xMMR for about 10-15 min to promote healing before
transfer to 0.1xMMR. Healing was confirmed visually as a fusion of the ectoderm
superficial to the otocyst with the ectoderm of the insertion side. Animals were reared
and the transplantation was checked daily for continued growth. Appearance of otoconia
was monitored and compared with the remaining untouched control ear. Success of ear
to trunk transplantation was scored as three levels of success as follows: normal ear
development with formation of otoconia; formation of an ear without recognizable
otoconia; no ear development (Table 3.1). Completeness of transplanted ear formation in
the orbit was observed to determine the success of transplantations (Fig. 4.1A-4.1C).
Success of GFP-positive ear to orbit transplantations was observed as the presence of a
third ear (Fig. 4.1D) and was confirmed to contain GFP using an epifluorescence
microscope. Success of other tissue transplantations was monitored (Fig. 4.1E-4.1H).
Movies were recorded of beating hearts in the orbit using a camera mounted to a
dissecting microscope (Leica). Prior to fixation, swimming behavior was observed and
dextran amine dyes were injected where applicable (see below). After embryos reached
stage 46, they were anesthetized in 0.02% Benzocaine and fixed in 10%
paraformaldehyde (PFA) by immersion.
Swimming Behavior
Animals were dropped into a 3”x4” swimming arena to observe their initial startle
and swimming behavior. All initial behaviors were recorded and scored as the following:
32
normal (swimming upright), swiming on side, swimming upside down, swimming
vertical, spinning, looping (Ferris wheel formation), spiraling (corkscrew formation).
Dextran amine label
Texas Red, rhodamine, or fluorescein 3000 molecular weight dextran amines
were used to backfill from the spinal cord to the transplanted ear, from the transplanted
ear to the spinal cord, and from the spinal cord into the hindbrain. Dextran amines were
dissolved in distilled water and recrystallized on a tungsten needle (Fritzsch, 1993).
Stage 46 embryos were anesthetized in 0.02% benzocaine (Crook and Whiteman, 2006)
For dye filling of afferents and efferents to the ear, a small cut was made into the spinal
cord adjacent to the transplanted ear and the dextran amine dye was applied. For
transganglionic filling of afferents and retrograde filling of efferents, a small opening was
made into the transplanted ear and the dextran amines were applied to the sensory
epithelial surface inside the ear, which rapidly filled the entire ear vesicle with dye (Fig.
3.1I). For labeling of the Mauthner cell in the hindbrain, a small cut was made into the
spinal cord rostral to the hindbrain and dextran amine dye was applied. The embryos
were transferred to the above outlined 0.1X MMR buffer in a Petri dish to rinse off
excess dye and then were transferred to a fresh Petri dish for 1-3h, depending upon the
distance the dye needed to travel (2mm/h (Fritzsch, 1993)). The wash dish was changed
frequently. Finally, the embryos were re-anesthetized and fixed in 4 or 10% PFA by
immersion.
Lipophilic dye label
For ears transplanted caudally to the trunk, lipophilic dye-soaked filter paper
(Fritzsch et al., 2005b, Tonniges et al., 2010) was used to backfill from the spinal cord to
33
the transplanted ear. Small pieces of dye-soaked filter paper were implanted into the
spinal cord immediately rostral (NeuroVue™ Green) and caudal (NeuroVue™ Maroon)
to the transplanted ear (Fig. 3.1H). For other embryos, small pieces of flattened dyesoaked filter paper (NeuroVue™ Red) were implanted immediately ventral to the spinal
cord, entering from the contralateral side so not to destroy the nerves innervating the ear
(Fig. 3.1J). For this injection, the dye would only contact the spinal motor neurons
exiting the ventral spinal cord. For some embryos in which dextran amines were injected
into the ear, lipophilic dye (NeuroVue™ Maroon) was implanted rostral and caudal to the
ear into the nerves innervating the muscle to backfill to the spinal cord. For tissues
transplanted to the orbit, small pieces of dye-soaked filter paper were flattened and
implanted longitudinally into the midbrain (NeuroVue™ Maroon) and transversely into
the hindbrain (NeuroVue™ Red) at the level of the native ear (Fig. 4.2A), thus primarily
filling the oculomotor nerve as it decussates the midline of the midbrain and the
trigeminal nerve as it exits the hindbrain respectively. Both oculomotor and trigeminal
nerves normally send projections to or near the orbit and thus near the transplanted tissue
in the orbit. For some embryos, dye-soaked filter papers were implanted into the native
and transplanted ears to label fibers projecting to the brain. For tissues transplanted to the
trunk, small pieces of dye-soaked filter paper (NeuroVue™ Red) were flattened and
implanted immediately ventral to the spinal cord as described above for the ear to trunk
transplantation. For somite tissue transplanted to replace the ear, small pieces of dyesoaked filter paper were implanted transversely into the hindbrain at the level of the
trigeminal nerve (NeuroVue™ Red) and at the level of the vagus nerve (NeuroVue™
Maroon). For three-eared frogs, small pieces of dye soaked filter paper were flattened
34
and implanted into the native ear (NeuroVue™ Maroon) and transplanted ear
(NeuroVue™ Red) to label fibers projecting to the hindbrain. Dyes were allowed to
diffuse at room temperature for 15-24 hours, 36°C for 15-18 hours, or 60°C for 7 hours.
Filter paper was removed prior to imaging. Embryos or dissected tissues were mounted
on a slide in glycerol and images were taken with a Leica TCS SPE confocal microscope
or TCS SP5 multi-photon confocal microscope.
Quantification of percent overlap of sensory neurons in
the vestibular nucleus of ‘three-eared’ frogs
The percent overlap of sensory neurons in the vestibular nucleus of ‘three-eared’
frogs when the transplanted ear was either in the native orientation or rotated by 90
degrees was calculated using the Leica software. Three optical sections were selected for
each animal, near the top, middle, and bottom of the stack. A region of interest line was
drawn perpendicularly through the sensory neurons at the approximate midpoint rostralcaudal of the descending tract of the vestibular nucleus. Using the line profile intensity
tool, an intensity histogram was provided for the channels representing the two
populations of sensory neurons, belonging to the native and transplanted ear. The medial
and lateral boundaries of the two histograms were determined manually by using the
boundaries generated from a threshold omitting the bottom 25% of the relative intensity.
Animals in which either of the fluorescent signals was weak, making it difficult to
generate an accurate intensity profile, were not included in the analysis. The percent
overlap was calculated by determining the percent of the narrower histogram contained
within the wider histogram. The percent overlap obtained for each of the three optical
sections was pooled to provide a percent overlap per animal. Animals were pooled to
35
provide the mean percent overlap for the two orientations of ‘three-eared’ frogs. The
means and standard errors were calculated. Students t-test was used as a test for
significance.
In situ hybridization
Whole-mount In situ hybridization for micro RNA (miR)-183 and miR-124 was
performed on control and transplanted ears from stage 46 embryos as previously
described (Pauley et al., 2003, Pierce et al., 2008). Briefly, locked nucleic acid probe for
miR-183 and miR-124 (Integrated DNA Technologies, Coralville, IA) was labeled with
digoxigenin (Roche Diagnostics GmbH, Mannheim, Germany). Fixed embryos were
bleached overnight in 2ml Eppendorf tubes containing 3% hydrogen peroxide. Embryos
were re-fixed with 4% PFA, washed in RNAse free PBS 3 times, digested with proteinase
K treatment for 40 min for miR-183 or 5 min for miR-124 and then fixed again in
4%PFA. The embryos were washed again with PBS. After 1h incubation in
hybridization mix at 60°C, 200 μl salmon sperm and 100ng riboprobe were added and the
embryos were kept overnight at 60°C. Embryos were then washed with 2X SSC and then
PBS, after which 2 μl RNAse A Enzyme (5mg/ml, 83U/mg) was added and embryos
were kept at 37°C for 90 min. Embryos were then washed several times with 1X Wash
solution (Roche), then incubated in 1X blocking buffer (Roche) for 1h and finally
overnight in anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche;
diluted 1:2000 in 1X blocking buffer). The antibody solution was discarded and replaced
by 1X wash solution once per hour and then kept overnight in wash solution. Embryos
were rinsed with 1X detection buffer (Roche) and then detected with BM Purple.
36
Immunohistochemistry
Control and transplanted ears, somites, hearts, and liver were immunostained with
antibody against acetylated tubulin (Farinas et al., 2001), myosin (Myo) VI (Kaiser et al.,
2008), and vesicular acetylcholine transporter (VAChT) (de Castro et al., 2009).
Hindbrains were immunostained with antibody against 3A10 to label reticular neurons,
including the Mauthner cell(s) (Liu et al., 2003, Patten et al., 2007). Since the 3A10
antibody did not label all of the dendrites filled with dextran amine dye (Fig. 5.3A-A”),
this antibody was used only to confirm the presence or absence of the Mauthner cell.
Cranial and lateral line nerves were immunostained with antibody against acetylated
tubulin. Embryos were defatted in 70% ethanol from 30 min to overnight and then
rehydrated in PBS for 1h. Embryos were blocked with 500μl 1% Triton X-100 and 500μl
5% normal goat serum (NGS) for one hour. Embryos were washed 3 times briefly in
PBS and then incubated in either tubulin primary antibody (1:800; Cell Signaling
Technology), Myo VI (1:400; Proteus Biosciences; a specific hair cell marker), VAChT
(1:500; Sigma; identifies motor neuron terminals), or 3A10 (1:500; Developmental
Studies Hybridoma Bank, University of Iowa) in diluted block (0.01% Triton X-100,
0.25% NGS in PBS) for 72h. Embryos were washed in PBS 3 times for an hour each and
blocked for an hour. Embryos were then washed 3 times briefly in PBS and incubated in
species-specific secondary antibody (1:500; Alexa) for 24h in the diluted block solution.
Embryos were washed 3 times for an hour each. The embryos or tissues of interest were
mounted on slides in glycerol. Confocal images were taken with a Leica TCS SPE
confocal microscope or TCS SP5 multi-photon confocal microscope.
37
Plastic embedding for light and transmission electron
microscopy
Transplanted ears along with surrounding muscle tissue were fixed in 2.5%
glutaraldehyde overnight and then washed in 0.1M phosphate buffer three times. Ears
were then fixed in 1% osmium tetroxide for one hour and washed again in 0.1M
phosphate buffer. Ears were then dehydrated in 70% ethanol overnight. Next, ears were
further dehydrated in absolute ethanol with 5 washes, 10 min each, then put in a 1:1
solution of absolute ethanol to propylene oxide for 5 min, and finally propylene oxide, 5
washes, 10 min each. Ears were then infiltrated with a 1:1 solution of propylene oxide to
resin overnight. Next, the propylene oxide was allowed to evaporate for 4 hrs and the
resin was poured into a mold. This was incubated at 60°C for 24 hrs. Sections were cut
with an ultratome using a diamond blade, 2μm thick for light microscopy and 100nm
thick for electron microscopy. Thick sections were counterstained for light microscopic
imaging with Stevenel’s blue (2% potassium permanganate and 1.3% methylene blue) at
60°C (del Cerro et al., 1980). Ultrathin sections were counterstained with uranyl acetate
and lead citrate and imaged in a Jeol 1230 TEM microscope.
Three-dimensional reconstruction of Mauthner cells
Embryos injected with dextran amines into the spinal cord were used for threedimensional (3D) reconstruction of Mauthner cells (Kopecky et al., 2012). Briefly,
brains were mounted ventral-side up in glycerol on a microscope slide. Confocal z-series
images at 1-3 µm were taken of the hindbrain using a Leica TCS SP5 confocal
microscope. Z-series stacks were loaded into Amira Version 5.4 software for manual
segmentation and volume rendering as described previously (Kopecky et al., 2012).
38
Dendritic Branching Analysis
The total number of terminal branches for each Mauthner cell lateral dendrite was
counted. Those with the greatest and the least difference between left and right Mauthner
cells were removed in all populations as outliers for the calculations of mean and
standard error. The actual difference between left and right Mauthner cells was counted
by subtracting the number of branches on the left from the right Mauthner cell. In
addition, the absolute difference was calculated by subtracting the smaller branch number
from the larger regardless of side. Students paired t-test was used to determine whether a
significant difference in branch number occurred between control (left) and treated (right)
Mauthner cells. A Bonferonni multiple comparison adjustment was performed following
an ANOVA on the differences between left and right Mauthner cells across all groups.
Sholl analysis (Sholl, 1953) was performed to determine the branching pattern of
the lateral dendrite of Mauthner cells. Concentric circles spaced 25 µm were drawn
around the cell soma. The number of lateral dendritic branches that crossed each circle
were counted for the right Mauthner cell. Students t-test was used as a test for
significance.
39
CHAPTER III
TRANSPLANTATION OF XENOPUS LAEVIS EARS REVEALS
THE ABILITY TO FORM AFFERENT AND EFFERENT
CONNECTIONS WITH THE SPINAL CORD 1
Abstract
Previous comparative and developmental studies have suggested that the
cholinergic inner ear efferent system derives from developmentally redirected facial
branchial motor neurons that innervate the vertebrate ear hair cells instead of striated
muscle fibers. Transplantation of Xenopus laevis ears into the path of spinal motor
neuron axons could show whether spinal motor neurons could reroute to innervate the
hair cells as efferent fibers. Such transplantations could also reveal whether ear
development could occur in a novel location including afferent and efferent connections
with the spinal cord. Ears from stage 24-26 embryos were transplanted from the head to
the trunk and allowed to mature to stage 46. Of 109 transplanted ears, 73 developed with
otoconia. The presence of hair cells was confirmed by specific markers and by general
histology of the ear, including TEM. Injections of dyes ventral to the spinal cord
revealed motor innervation of hair cells. This was confirmed by immunohistochemistry
and by electron microscopy structural analysis, suggesting that some motor neurons
rerouted to innervate the ear. Also, injection of dyes into the spinal cord labeled
vestibular ganglion cells in transplanted ears indicating that these ganglion cells
connected to the spinal cord. These nerves ran together with spinal nerves innervating
1 This chapter is adapted from Elliott KL, Fritzsch B (2010) Transplantation of Xenopus laevis ears reveals
the ability to form afferent and efferent connections with the spinal cord. Int. J. Dev. Biol. 54: 1443-1451.
40
the muscles, suggesting that fasciculation with existing fibers is necessary. Furthermore,
ear removal had little effect on development of cranial and lateral line nerves. These
results indicate that the ear can develop histologically normal in a new location, complete
with efferent and afferent innervations to and from the spinal cord.
Introduction
The vertebrate inner ear receives two kinds of fibers: afferents that come from
placodally-derived sensory neurons (Rubel and Fritzsch, 2002) and efferents which come
from brainstem neurons (Simmons, 2002). Whereas afferents carry information from the
ear to the brain, efferents provide a feedback loop to modify mechanic stimuli
information processing at the periphery. Efferents have been described in all vertebrates
(Fritzsch, 1999) and the extensive branching of single fibers to reach both lateral line and
inner ear hair cells has been extensively studied in amphibians (Hellmann and Fritzsch,
1996). The nature of these efferent cells has long been enigmatic and they have been
referred to as reticular formation cells or branchial motor neurons given their close
proximity in many vertebrates to the facial branchial motor neurons (Roberts and
Meredith, 1992). More recent developmental studies have shown that efferent
innervation of the ear is derived from facial branchial motor neurons (Fritzsch and
Nichols, 1993) and that efferents may be guided to reach the ear by the transcription
factor Gata3 that is uniquely expressed in these motor neurons and nearby reticular
formation neurons (Karis et al., 2001). In vertebrates, the ear develops at the rostral
boundary of the anteriormost, first forming somite (Cooke, 1978, Chung et al., 1989,
Huang et al., 1997) and paraxial mesoderm anterior to the ear does not form somites
(Noden and Francis-West, 2006) but may form somitomeres. Furthermore, in
41
vertebrates, the most rostral somite is reduced in size to accommodate the ear. Combined
these data could be interpreted to imply that in vertebrates, the ear develops in place of a
somite or somitomere. If true, the facial branchial motor neurons destined to innervate
the somite- or somitomere-derived muscle fibers may have been rerouted to innervate the
ear when the ear evolved in ancestral vertebrates (Fritzsch et al., 2007).
This raises the question as to whether there is a unique property of cranial motor
neurons to innervate hair cells of the ear or whether any motor neuron can innervate the
ear if placed in its trajectory. In other words, is formation of efferents to the ear a chance
event that happened to capture facial branchial motor neurons simply because the ear
evolved in the trajectory of these motor neurons? Supporting evidence comes from the
lateral line system in which branchial motor neurons associated with the
glossopharyngeal nerve become efferent to the hair cells of the posterior lateral line
(Hellmann and Fritzsch, 1996).
In the present study, we transplanted Xenopus laevis ears into the path of spinal
motor neuron axons, revealing that spinal motor neurons have the ability to innervate the
ear as efferent fibers and end on hair cells instead of muscle cells and probably modify
hair cell function via the now well characterized nicotinergic acetylcholine receptors
expressed in hair cells (Sugai et al., 1992, Jagger et al., 2000, Katz et al., 2004, Derbenev
et al., 2005). Such transplantations could also reveal the potential for normal ear
development in a novel location, including afferent connection with the spinal cord, a
part of the CNS that never receives inner ear afferent innervations which is normally
restricted to the alar plate of the brainstem (Maklad and Fritzsch, 2003).
42
Results
Assessment of transplantations
Success of transplantations is shown in Table 3.1 and in Figure 3.1. Residual ear
formation is shown in Table 3.2 for embryos in which the success of transplantation and
ear regeneration were monitored. In some cases in which only a transplanted vesicle
without otoconia formed, a new otic vesicle was observed in the native location (Fig.
3.1E). This was more frequent in transplantations of earlier embryos (stage 24) than in
later stages (stage 26). It remains unclear whether this indicates a residual capacity for
parts of the otocyst to regenerate. However, in many cases, the residual native ‘ear’
consisted of nothing more but an endolymphatic duct filled with calcium carbonate
crystals (Fig. 3.1C and 3.1I). In transplanted ears which formed otoconia, 75% did not
form a residual ear nor endolymphatic duct in the original location (Fig. 3.1B and 3.1H).
However, formation of a residual native ear in transplants that had developed otoconia
occasionally occurred (17%).
To determine whether manipulation of the otic placode affects further
development, ears were removed, rotated 180°, and replaced. Even though for
transplantations we sought to maintain orientation, it was not always the case and often
the ears underwent some degree of rotation. Thus rotation of the ear and replacing it
back into the head replicated many of the manipulations performed during transplantation
and could serve as an additional control for the ability of a manipulated ear to develop
normally. Success of ear rotations is shown in Table 3.1. Eight of the 13 ears were
normal or near normal in appearance (Figs. 3.1D and 3.1G) when compared with control
embryos (Fig. 3.1A). Otoconia in nearly half of the ears were even found to be re-
43
oriented so that they resumed native orientation. In the chick, anterior-posterior
specification occurs after the formation of the otic placode (Bok et al., 2005), thus this
might explain the re-orientation of rotated X. laevis ears. Only one ear rotation failed to
form otoconia. Thus we conclude that our manipulations themselves have little effect on
normal ear development.
Transplanted ears develop hair cells and ganglia
To assess normal development of the sensory epithelia in ears containing
otoconia, in situ hybridization for miR-183, a conserved hair cell marker of ears (Pierce
et al., 2008), was performed on ears containing otoconia. In the untransplanted ears,
miR-183 was localized in the sensory end organs: the utricle, saccule, and anterior,
horizontal and posterior canals as well as in the placodally-derived ganglion cells:
Trigeminal, Vestibular and Vagus (Fig. 3.2A). Transplanted ears were also miR-183
positive in the utricle, saccule, the 3 semicircular canals, and in the placodally-derived
vestibular ganglion cells (Fig. 3.2B). To confirm the presence of hair cells, transplanted
ears were immunostained with antibody against myoVI and acetylated tubulin. MyoVIpositive hair cells in patches of sensory epithelia were observed in transplanted ears (Fig.
3.2C), although some sensory epithelia contained only a few hair cells. These hair cells
had kinocilia positive for tubulin (Fig. 3.2D). Serial microtome sections of plasticimbedded transplanted ears also revealed hair cells with apical specializations as seen
with a light microscope (Fig. 3.2F) and with transmission electron microscopy (Fig.
3.2G). These data suggest that the transplanted ears developed relatively normally with
all sensory epithelia present that occur at that stage.
44
To confirm the presence of sensory ganglia in the transplanted ears, in situ
hybridization for miR-124 was performed on transplanted ears. MiR-124 is expressed in
the central nervous system (Mishima et al., 2007) and in inner ear vestibular ganglion
cells (Weston et al., 2006). Cells positive for miR-124 were present in transplanted ears
(data not shown). To further confirm the presence of ganglion cells, otic placodes from
embryos injected with GFP were transplanted into uninjected individuals. Daily
observation using a dissection microscope equipped with epifluorescence revealed GFPpositive delaminating ganglion cells migrating away from the ear. After a week, these
delaminated cells were imaged with confocal microscopy (Fig. 3.2E), indicating that the
ganglion cells originate from the transplanted otic placode and migrate outward during
embryo development.
Afferent innervation of transplanted ears
Dextran amine and lipophilic dyes were injected into the spinal cord of embryos
(Fig. 3.1H) containing otoconia in their transplanted ears to label innervations of these
ears. Both types of dye labeled ganglion cells in the transplanted ear (Figs. 3.2E, 3.3A
and 3.3B). The lack of dye in some ganglion cells in Figures 3.2E and 3.3A indicates
that not all ganglion cells sent projections to the spinal cord. Ganglion cells making
connection with the spinal cord appear to do so by fasciculation with existing nerves
innervating the surrounding muscle tissue (Figs. 3.3A and 3.3B). In addition to making
projections to the spinal cord, immunohistochemistry for acetylated tubulin revealed
fibers (presumably from ganglion cells) closely apposed to hair cells (Fig. 3.3C).
Transmission electron microscopy confirmed the presence of afferent terminals on hair
cells (Fig 3.3K).
45
Efferent innervation of transplanted ears
To determine whether spinal motor neurons project to hair cells in the
transplanted ear, several methods were used. Transplanted ears were injected with
Rhodamine dextran amine dyes to backfill to the spinal cord (Fig 3.1I). In addition, for
some embryos Flurosceine dextran amine dye or lipophilic dye (NeuroVue™ maroon)
was applied rostral and caudal to the ear to label motor neurons that innervate muscle
tissue to determine if any uncut nerves outside of the ear took up Rhodamine dextran
amine that might then give a false positive; these cells would be double labeled. Cell
bodies in the ventral spinal cord positive for only Rhodamine dextran amines were
present in 10 of 16 ventral spinal cords examined, suggesting that motor neurons can
innervate the ear if placed in their trajectory (Fig. 3.3D).
To further demonstrate that motor neurons do innervate the ear, lipophilic dye
was injected immediately ventral to the spinal cord from the contralateral side as to not
damage the ipsilateral nerves innervating the ear (Fig. 3.1J). The dye would only come
into contact with spinal motor neurons exiting the spinal cord via ventral roots and thus
any nerves labeled in the transplanted ear should be motor neurons that had rerouted to
innervate the ear. Lipophilic dye label was observed in a few nerve fibers innervating
several of the transplanted ears (Figs 3.2E and 3.3G). In one example, lipophilic-dye
filled nerve fibers that traveled around the ear on each side and also filled nerve fibers
which sent projections to the hair cells (Fig. 3.3G). To further visualize the extent to
which this ear was innervated, beyond the contribution from the spinal motor neurons,
this ear was immunostained with antibody against tubulin. This revealed more extensive,
presumably afferent innervations (Fig. 3.3H). The deeper ventral roots observed in
46
Figure 3.3G were faintly stained with tubulin, but not easily detected in the collapsed
image (Fig. 3.3H). The reduction of staining of deeper neurons is likely due to the
inability of the antibody to fully penetrate the tissue. In another example, a few of the
ganglion cells sent projections along the presumed motor nerves as they colocalized with
the dye (Fig. 3.2E, see G*) therefore, it was necessary to confirm motor innervation even
when co-localization with ganglion cells was not apparent as in Figure 3.3G.
To confirm that motor neurons innervate some transplanted ears, antibody against
VAChT was used to show motor terminals on hair cells. Since antibody against VAChT
had not yet been characterized in amphibians, we tested it on lateral line neuromasts and
hair cells in untransplanted ears, two sources with known motor innervation (Will, 1982).
The neuromasts and control ears were found to have puncta positive for VAChT on the
hair cells (Figs. 3.3E and 3.3F). In the transplanted ears, we found terminals positive for
VAChT on hair cells (Figs. 3.3I and 3.3J). Specifically, the transplanted ear that had
shown extensive spinal motor neuron innervation when dye was injected ventral to the
spinal cord (Fig. 3.3G) also had VAChT-positive terminals on hair cells that co-localized
with areas labeled by the lipophilic dye (Fig. 3.3I). To further validate the presence of
efferent terminals on hair cells, transplanted ears that were positive for VAChT terminals
on hair cells were imbedded in resin and sectioned for transmission electron microscopy.
Efferent terminals were confirmed on hair cells by the presence of axon terminals
containing vesicles (Fig. 3.3L), thus demonstrating that spinal motor neurons can
innervate an ear if placed in its trajectory.
47
Cranial and lateral line nerves develop nearly normal
To assess the effect otocyst removal had on the development of the cranial nerves
and lateral line nerves, heads from both control embryos and those having an ear
transplanted were immunostained with antibody against acetylated tubulin. Figure 3.4A
shows the normal distribution of the cranial nerves. In embryos in which an ear was
removed, near normal distribution of the cranial nerves was observed although the cranial
nerves that would normally circle the ear now traverse the empty space (Fig. 3.4B and
3.4C). Similar effects were observed with the lateral line nerves. Compared to control
embryos (Figs. 3.4D and 3.4F), in transplanted embryos the lateral line neuromasts
spread out to cover the space not occupied by the ear (Fig. 3.4E, 3.4G, and 3.4H). In
some instances, and more often when the ear was transplanted during the later stages, the
parietal lateral line neuromasts failed to develop or were reduced (Fig. 3.4E). Otherwise
the development of the lateral line nerves in transplanted embryos was normal and
showed almost no quantitative differences in the number of neuromasts, in particular the
supraorbital line (10.1 ± 0.5 neuromasts posterior to the parietal line for the control side
versus 10.2 ± 0.5 for the transplanted side (mean ± standard error of the mean, n = 11).
Discussion
Our results confirm and extend previous studies on ear transplantation (Fritzsch,
1996, Fritzsch et al., 1998, Groves, 2005) in amphibians but add the hitherto unstudied
aspect of afferent and efferent innervations. Below we will first discuss the degree of
development we achieved in transplanted Xenopus ears, followed by a discussion of our
tracing data.
48
Evidence that these transplanted ears could develop relatively normally in a novel
location is the presence of hair cells in the transplanted ears, identified by morphology as
well as in situ hybridization and immunohistochemistry for hair cell markers. This
suggests that the epithelial end organs all develop and that either their development is
likely not dependent upon the tissue surrounding the placode or that it was specified prior
to transplantation, as recent work in chicks has demonstrated that the otic ectoderm
becomes specified prior to placodal formation (Abelló et al., 2010). On the other hand,
while care was taken to remove only the otic placode, it cannot be ruled out that some
surrounding mesoderm and ectoderm was also transplanted, thus influencing further
development of the ear. Furthermore, in the GFP-labeled ears, while migration of
ganglion cells was observed, it is also possible that some of the green cells observed
surrounding the ear may be transplanted mesoderm in addition to the GFP-labeled
delaminating ganglion cells. Taken together, these sets of data confirm that welldeveloped sensory epithelia form with hair cells. Thus, many transplanted ears develop
fully normal with respect to otoconia formation, hair cell differentiation and segregation
of several sensory epithelia (Fritzsch and Wake, 1988).
That the transplanted ear can develop normally would suggest that the fates of the
cells in the otic placode were determined by stage 24-26. In most of the embryos where
only a vesicle formed on the trunk, a partial or near normal ear redeveloped in the
original location. Waldman et al. (2007), using X. laevis, ablated otic placodes at various
stages to determine the ability of the surrounding tissue to regenerate an ear. Placodes
ablated at early stages (21-23) were able to regenerate completely normal ears whereas
beyond stage 28, the ear rarely regenerates. Ablation at stages 24-27 sometimes resulted
49
in regenerated, although abnormal ears (Waldman et al., 2007). In a study with
salamanders, ablation of the otic placode could result in regeneration of the inner ear, but
only when ablation was done at early stages (Kaan, 1926). Therefore, in the present
study, it could be that in the embryos in which an ear redeveloped, the embryo was at a
stage in which the remaining tissue was competent to regenerate an ear. On the other
hand, it is also possible that since the transplanted placode only formed a vesicle on the
trunk and not a complete ear, that not the entire placode was completely transplanted.
Waldman et al. (2007) also demonstrated that partial ablations could result in
regeneration of the ears in some instances. Therefore, it could also be that the partial
placode remaining in the head regenerated an ear. The fact that the partial placode in the
trunk did not form a complete ear might be due to the novel environment that it
developed in and that perhaps additional cues from the head might be necessary for
restoring the otocyst. Support for the latter possibility, that residual ears formed when the
placode was not completely transplanted, was that most of the transplanted ears that
contained otoconia did not have a residual ear in the native location (Fig. 3.1B).
Whereas, for transplanted ears containing only a vesicle and no otoconia or when no
transplant was observed, some component of the residual ear, whether it be just the
endolymphatic duct or an entire ear, formed in the native location (Figs. 3.1C and 3.1E).
In addition to the ability of the ears to develop in a novel location, many ears were
also innervated. Dye injections into the spinal cord or transplanted ears revealed afferent
and efferent innervations respectively. Using these dye injections, we observed that some
afferent ganglion cells sent projections to the hair cells and to the spinal cord along
trajectories that likely do not bear any informational molecules used by inner ear
50
afferents to navigate during development to reach the hindbrain (Rubel and Fritzsch,
2002). Furthermore, the random dispersal of delaminating ganglion cells away from the
ear is also explained by an absence of these normal guidance molecules as there did not
appear to be any formal organization of their migration (Fig. 3.2E). The observation that
some, but not all, ganglion cells were filled with dye when injections were made into the
spinal cord (Figs. 3.2E and 3.3A), suggests that connections to the spinal cord happen by
chance and are only made if the growing ganglion cell axons happen to fasciculate with
existing nerves. However, since many ganglion cells axons were apposed to hair cells,
synaptic connections are likely made thus indicating that the mechanism for hair cell
innervation develops normal in the novel location.
Immunostaining of transplanted ears with antibody against VAChT revealed
puncta on the basal surface of the hair cells, demonstrating the presence of motor
terminals on these hair cells. For transplanted ears that were also labeled from dye
injections ventral to the spinal cord, VAChT puncta were found only on hair cells that
were in close proximity to the labeled spinal motor neuron projections (Figs. 3.3G and
3.3I). Furthermore, transmission electron microscopy confirmed efferent terminals on
hair cells in transplanted ears as the presence of many synaptic vesicles in the axon
terminal is a property of efferent axons, but is not found in afferent axons (Jones and
Eslami, 1983, Simmons, 2002). The reduced integrity of the membranes was a result of
utilizing ears previously processed for immunohistochemistry to confirm the presence of
VAChT. Thus, the evidence presented here of efferent innervation of the transplanted ear
demonstrates that some spinal motor neurons have the ability to reroute to innervate a
new target if the new target replaces the previous target and is in the direct trajectory of
51
the growing axon. This finding is consistent with the hypothesis that efferent innervation
of the ear arose in evolution from rerouted facial branchial motor neurons.
Finally, immunohistochemistry for acetylated tubulin was performed to determine
the effect, if any, transplantation had on the development of the cranial and lateral line
nerves of the head. The results presented here demonstrated that ear transplantation had
little to no effect on further cranial and lateral line development other than, for both the
cranial nerves and lateral line neuromasts, they spread out to either traverse or fill in,
respectively, the space left void by the ablated ear (Fig. 3.4). It is possible that for the
lateral line neuromasts, either the ear suppresses expansion of the anterior and posterior
lateral lines and that, in the absence of the ear, the neuromasts spread out to cover that
space, or simply as the skin closes following the removal of the ear, the lateral line
placode is stretched as the wound heals. One aberrant effect that was occasionally
observed was the absence of the parietal lateral line on the transplanted side (Fig 3.4E).
In X. laevis, the lateral line placodes is formed by about stage 24 (Winklbauer and
Hausen, 1983). At this stage, the inducibility of surrounding tissue to form the lateral
line begins to decline (Schlosser, 2002). Therefore any disruption of the placode at stage
24 likely results in reinduction of surrounding tissue to form a new placode, whereas at
stage 26, the surrounding tissue is less inducible, resulting in a possible loss of
neuromasts in some places, such as the parietal line.
In conclusion, the results presented here demonstrate the potential for a motor
neuron to reroute to innervate a novel target, supporting the idea that efferents may have
arisen from rerouted facial branchial motor neurons during the evolutionary formation of
the ear. Such rerouting to novel targets can be induced by ablation of innervations of a
52
given muscle fiber inducing the attraction of nearby motoneuron axons to expand to
denervated targets in X. laevis (Fritzsch and Sonntag, 1990, 1991), mice (Fritzsch et al.,
1995, Porter and Baker, 1997) and man (Engle et al., 1997, Miyake et al., 2008). We
cannot exclude such near range attractive signaling from denervated hair cells. However,
since hair cell innervation by spinal motor neurons occurred only when the hair cells
were in the direct trajectory of the growing axons, this suggests that the rerouting of the
facial branchial motor neurons occurred as a chance event when the ear developed in
their trajectory. Future tests will determine whether reinnervation of the ear can occur by
other cranial motor neurons and thus reflects a general motor neuron ability to engage in
synaptic innervations in placode derived hair cells.
53
Table 3.1. Success of transplantation of otic placodes.
Development of
Development of ear No development of
ear with otoconia
without otoconia
ear
Transplanted ears (109)
73*
29*
7
Rotated ears (13)
12
1
0
Note: Numbers of transplants or rotated ears performed are indicated in parentheses.
*Examples of transplants with and without otoconia are shown in Figures 1B/1F and 1C
respectively.
54
Table 3.2. Formation of residual ears as related to success of transplantation.
Residual ear
Development of
Development of
No development of
formation
transplanted ear with
transplanted ear
transplanted ear
otoconia
without otoconia
No residual ear
30
0
0
ED only
3
10
0
Residual ear, no ED
7
0
0
Residual ear + ED*
0
2
3
Note: ED, endolymphatic duct
* Residual formation of ear plus endolymphatic duct is shown in Figure 1E.
55
Figure 3.1. Stage 46 X. laevis showing transplanted ears. (A) Control, untransplanted
embryo, ears are circled in black. (B) Embryo with a transplanted ear containing
otoconia, outlined in a dotted red line. (C) Embryo with a transplanted empty vesicle,
outlined in a dotted red line. (D) Embryo that had its ear rotated, outlined in a dotted
yellow line. (E) Untransplanted ear, left, and residual ear formation, right. (F) Higher
magnification of transplanted ear in B. (G) Higher magnification of rotated ear in D. (H)
Lipophilic dye injections into the spinal cord rostral and caudal to the ear. (I) Dextran
amine dye injection into the transplanted ear. (J) Lipophilic injection ventral to the spinal
cord and contralateral to the ear. Native, unmanipulated ears are labeled. U, utricle; S,
saccule; ED, endolymphatic duct. Asterisks indicate sites of dye injections. Scale bar is
0.5 mm for A-D,H,I and 0.25 mm for E,F,G,J.
56
57
Figure 3.2. Development of the transplanted ear. (A) MiR-183 in sensory epithelia in
the native ear and in surrounding cranial ganglia as well as ganglia next to the ablated ear
(i.e. Trigeminal ganglia, V). (B) Lateral view of the transplanted ear with miR-183
positive sensory epithelia and ganglion cells; anterior is to the right. (C) Acetylated
tubulin (green) and MyoVI (red) immunostaining of a transplanted ear showing hair cells.
(D) Magnification of region in C show kinocilia (white arrows) on hair cells. (E) Green
fluorescent protein (GFP)-labeled transplanted ear reveals GFP-positive delaminating
sensory ganglion cells (G), a few of which (G*) are colocalized with lipophilic dye (red).
Lipophilic dye injections were ventral to the spinal cord. (F) 2μm thick transverse
section through the dorsal and lateral view of transplanted ear showing a sensory
epithelium with hair cells and an endolymphatic duct. (G) Transmission electron
micrograph of two hair cells in the transplanted ear with synaptic ribbons (black arrows).
The boxed areas are shown at higher magnification in Figure 3.3K and 3.3L. U, utricle; S,
saccule; Ac, anterior canal crista; Hc, horizontal canal crista; Pc, posterior canal crista; G,
ganglion cell(s); HC, hair cells K, kinocilium; S, stereocilia; ED, endolymphatic duct.
Scale bar is 0.5 mm in A,B; 25 µm in C,D; 50 µm in E, 0.1 mm in E inset; 10 µm in F;
and 5 µm in G.
58
59
Figure 3.3. Afferent and efferent innervation of transplanted ears. (A) Transplanted
ear that had dextran amine (red) injected into the spinal cord. Autofluorescence of the
tissue is green. Inset: higher magnification of boxed area. (B) Transplanted ear that had
lipophilic dyes injected into the spinal cord rostral (red) and caudal (green) to the ear. (C)
Acetylated tubulin immunostaining (red) of boxed area in B showing ganglion cells
apposed to hair cells. Autofluorescence of the tissue is green. (D) Cells in the ventral
spinal cord positive for dextran amine dyes that had been injected into the transplanted
ear. (E) Neuromasts in the skin showing vesicular acetylcholine transporter (VAChT)positive terminals on the hair cells. Autofluorescence is blue. (F) Hair cells in a control
ear immunostained for acetylated tubulin and VAChT. (G) Transplanted ear that had
lipophilic dye injected ventral to the spinal cord show nerve fibers extending to the ear.
(H) Transplanted ear in G immunostained for acetylated tubulin. (I) Transplanted ear in
G immunostained for acetylated tubulin and VAChT. Inset: higher magnification of
boxed area. Red arrows indicate VAChT immunostaining. (J) Transplanted ear
immunostained for acetylated tubulin and VAChT. Inset: single optical section of
VAChT staining (red arrows) at the base of hair cells. (K) Electron micrograph of an
afferent terminal on a hair cell. A synaptic ribbon (arrow) is observed on the hair cell
above the afferent terminal (L) Electron micrograph of an efferent terminal with vesicles
(circled) making a synapse on a hair cell. These TEM images were obtained from ears
previously processed for immunohistochemistry to confirm the presence of VAChT,
hence poor preservation of membranes. GC, ganglion cell; HC, hair cell; MN, motor
neuron cell body; NM, neuromasts; Aff, afferent terminal; Eff, efferent terminal; V,
vesicles; Mt, mitochondria. Scale bar is 100 µm in A,B,C,D,G,H; 25 µm in E,F,I,J and 1
µm K,L.
60
61
Figure 3.4. Development of cranial and lateral line nerves. Acetylated tubulin
immunostaining of cranial nerves in a control embryo (A) and in embryos in which the
ear was transplanted (B, C). (D) Acetylated tubulin (green) immunostaining of lateral line
nerves in the cranial skin of a control embryo (E) Acetylated tubulin (green) and Hoechst
(blue) immunostaining of a later-stage transplanted embryo. Acetylated tubulin (green)
immunostaining of lateral line nerves in the cranial skin of a control embryo (F) and an
embryo in which the ear was transplanted (G). (H) Higher magnification of boxed area in
E. Areas where native ears are present and where ablated ears had occupied are circled
with solid and dotted lines, respectively. Native ears are labeled ‘Ear’. Eyes (A,B) and
skin over the eyes (D,E,F,G) are labeled ‘Eye’. Cranial nerves are labeled with Roman
Numerals. LL, lateral line ; U, utricle; S, saccule; L, lagena; Ac, anterior canal; Hc,
horizontal canal; Pc, posterior canal. Scale bar is 250 µm.
62
63
CHAPTER IV
TRANSPLANTATION OF XENOPUS LAEVIS TISSUES TO
DETERMINE THE ABILITY OF MOTOR NEURONS TO
ACQUIRE A NOVEL TARGET 2
Abstract
The evolutionary origin of novelties is a central problem in biology. At a cellular
level this requires, for example, molecularly resolving how brainstem motor neurons
change their innervation target from muscle fibers (branchial motor neurons) to neural
crest-derived ganglia (visceral motor neurons) or ear-derived hair cells (inner ear and
lateral line efferent neurons). Transplantation of various tissues into the path of motor
neuron axons could determine the ability of any motor neuron to innervate a novel target.
Several tissues that receive direct, indirect, or no motor innervation were transplanted
into the path of different motor neuron populations in Xenopus laevis embryos. Ears,
somites, hearts, and lungs were transplanted to the orbit, replacing the eye. Jaw and eye
muscle were transplanted to the trunk, replacing a somite. Applications of lipophilic dyes
and immunohistochemistry to reveal motor neuron axon terminals were used. The ear,
but not somite-derived muscle, heart, or liver, received motor neuron axons via the
oculomotor or trochlear nerves. Somite-derived muscle tissue was innervated, likely by
the hypoglossal nerve, when replacing the ear. In contrast to our previous report on ear
innervation by spinal motor neurons, none of the tissues (eye or jaw muscle) was
innervated when transplanted to the trunk. Taken together, these results suggest that
2 This chapter is adapted from Elliott KL, Houston, DW, Fritzsch B (2013) Transplantation of Xenopus
laevis tissues to determine the ability of motor neurons to acquire a novel target. PLoS ONE 8(2): e55541.
doi:10.1371/journal.pone0055541.
64
there is some plasticity inherent to motor innervation, but not every motor neuron can
become an efferent to any target that normally receives motor input. The only tissue
among our samples that can be innervated by all motor neurons tested is the ear. We
suggest some possible, testable molecular suggestions for this apparent uniqueness.
Introduction
Synaptic contacts likely originated in evolution as connections between sensorymotor neurons and muscle tissue in diploblastic animals, such as jellyfish, (Seipel et al.,
2004) forming a monosynaptic reflex network from sensory cell directly to effector cell.
As animal cell types diversified, the sensory-motor interface evolved in complexity,
forming muscle fibers, motor neurons, sensory neurons, and interneurons (Fritzsch and
Glover, 2007). As tissues diversified in the course of evolution into the over 200 cell
types recognized in metazoans, motor neurons acquired novel targets during this
diversification process. As new targets evolved to provide novel synaptic contacts, motor
neurons evolved into novel sub-categories (Fritzsch and Northcutt, 1993, Murakami et
al., 2004, Dufour et al., 2006). Although all motor neurons share a common
developmental transcription factor, Islet 1 (Ericson et al., 1992, Inoue et al., 1994), they
have developed unique LIM code molecular signatures for each population (Tsuchida et
al., 1994). Vertebrate motor (efferent) neurons have evolved to form synapses on a
variety of targets originating from developmentally different sources including
mesoderm-derived muscle fibers, epithelial placode-derived neurosensory cells and
neurons (i.e. inner ear hair cells and neurons), and neural crest-derived autonomic ganglia
(Eisen, 1999, Fritzsch, 1999, Takano-Maruyama et al., 2010, Simmons et al., 2011).
Among all cranial motor neurons, motor neurons of the facial nerve are unique in that
65
they innervate targets from three different developmental origins: branchial arch-derived
muscle by branchial motor neurons, neural crest-derived ganglia by visceral motor
neurons, and placodally-derived inner ear hair cells by inner ear efferents (Fritzsch and
Northcutt, 1993). Although the efferents to the inner ear hair cells project in mammals
along the vestibulocochlear nerve, it has been shown that efferent innervation of the inner
ear is ontogenetically derived from the facial branchial motor neurons, as inner ear
efferent neurons exit the cell cycle in the same area as the facial branchial motor neurons
in early embryonic mice prior to segregation of the two neuron populations (Fritzsch and
Nichols, 1993, Köppl, 2011, Simmons et al., 2011) and can project with the facial nerve
in the absence of afferent neurons (Ma et al., 2000). Only one other branchial motor
nerve, the glossopharyngeal nerve, innervates more than one tissue type: muscle, ganglia,
and in aquatic vertebrates such as Xenopus laevis, hair cells of the placodally-derived
posterior lateral line (Hellmann and Fritzsch, 1996). Given the ability of some motor
neurons to innervate targets from a variety of origins, the possibility exists that there is
conservation of the core molecular machinery that allows for recognition of a particular
target by multiple motor neuron types.
In contrast to the ability for few populations of motor neurons to innervate a
variety of targets, most tissue types have only one source of motor input, even when other
motor neuron types project to adjacent territories. Such is the case with the innervation
of the trapezius muscle by the spinal accessory nerve rather than spinal motor neurons
(Boord and Sperry, 1991, Sienkiewicz and Dudek, 2010, Dudek et al., 2011). Further
support for specificity between motor neurons and targets includes studies in some
vertebrates showing that spinal motor neurons can reinnervate the correct muscles
66
following nerve transection (Landmesser, 1980). In addition, spinal motor neurons can
find the correct muscle following anterior-posterior reversal of a few lumbar spinal cord
segments, demonstrating innervation selectivity (Lance-Jones and Landmesser, 1980,
Landmesser, 1980). However, there must be some degree of plasticity in the system, as
the oculomotor nerve has been shown to innervate the lateral rectus muscle, a normal
target of the abducens nerve, in Duane’s retraction syndrome where the abducens nerve is
absent (Demer et al., 2007) or the abducens nerve expands its territory when the
oculomotor nerve is absent (Fritzsch et al., 1995). Furthermore, motor neurons were able
to reroute to innervate a novel target, as demonstrated by Xenopus laevis spinal motor
neurons rerouting to innervate hair cells of an ear transplanted to the trunk to replace a
somite (Elliott and Fritzsch, 2010). This result indicates the presence of a common
molecular denominator between some targets of motor neurons, in this case between the
inner ear hair cells and somites, that allows for recognition by the same population of
spinal motor neurons. It is likely that the ability of different motor neurons to form
synaptic contacts with hair cells of the inner ear uses an evolutionary conserved
molecular mechanism for target recognition and for maintenance of innervation.
Whether this applies to other motor neuron populations or other tissues is not yet known.
Thus, our goal is to determine the extent to which various nerves can gain affinity for a
novel target.
In the present study, we expand upon the previous X. laevis transplantation study
(Elliott and Fritzsch, 2010) by transplanting X. laevis ears, which are directly innervated
by motor (efferent) neurons into the path of various cranial motor neurons: oculomotor,
trochlear, trigeminal, and, abducens to test for further acceptance of motor neuron
67
innervation by hair cells. In addition, other tissues that receive direct motor innervation
(somite-derived muscle and branchial arch-derived eye muscle and jaw muscle) were
transplanted into the path of various cranial motor neurons or into the path of spinal
motor neurons, respectively. Finally, tissues that are not directly innervated by motor
neurons (heart, liver) were transplanted into the path of oculomotor, trochlear, and
abducens motor neurons. Transplantation of the ear to the orbit revealed that motor
neurons destined to innervate the muscles for eye movement could innervate hair cells of
the transplanted ear if placed in their trajectory; however, comparable transplantations of
other tissues revealed no innervation in somite-derived muscle, hearts, and livers
transplanted to the orbit. In addition, neither eye muscle nor jaw muscle transplanted to
the trunk to replace a somite was supplied by axons from spinal motor neurons.
However, when somite-derived muscle was transplanted to replace an ear, there was
supply of axons from what appears to be the hypoglossal nerve. Together these data
suggest that the ear differs from other motor neuron targets in that it can receive motor
neuron axons from a variety of sources and thus has a built in ability for novel motor
neuron innervation.
Results
Completion of eye removal and assessment of transplantation success
Completeness of transplanted tissue formation was scored to determine the
effectiveness of transplantations (Figure 4.1A-1H; Table 4.1). Success for all tissues was
defined as the detection of transplanted tissue, whereas completeness of transplantation
was defined as the amount and/or degree of normality of transplanted tissue. Of the 159
successful ear to orbit transplantations (out of 173), almost two-thirds of the ears
68
transplanted to the orbit contained otoconia (n = 97), whereas others were just a vesicle
and lacked otoconia (n = 62) (Figure 4.1B-4.1C). Hair cells were present in ears that
lacked otoconia (data not shown). Only ears containing otoconia were considered more
complete ears and were used for further analysis. Removal of the lens placode and
developing eye still lead to the regrowth of parts of the eye in all but 36 of the 159
successful ear to orbit transplantations (Compare inset in Fig. 4.1A with insets in Figs.
4.1B and 4.1C). Eye muscles, which develop from surrounding mesoderm, accompanied
residual eyes. All embryos containing any remaining portion of the eye were used for
further analysis.
Success of muscle tissue transplantation (somite, eye muscle, and jaw muscle)
was also quantified by the presence of transplanted tissue (Figure 4.1D-4.1F). All but
one transplant (out of 10) involving somites, whether to the orbit or otic region, were
successful. Eyes with attached muscle transplanted to the trunk were present in 6 of 7
embryos. Attempts to transplant only eye muscle was not successful as no extra muscle
was detected in the trunk when eye muscle was transplanted without the eye. Jaw muscle
transplanted to the trunk was present in 12 of 13 embryos. Embryos with larger muscle
transplants were used for further analysis. Success of heart and liver transplantations was
also investigated (Figure 4.1G-4.1H). There was variation in the amount of heart tissue
that developed in the orbit ranging from none detectable to a large, beating heart (Fig.
4.1G). Heart tissue was present in 23 of 31 transplants. Slightly less than half (10 of 23)
of the hearts transplanted were beating regularly and apparently autonomously in the
orbit. One transplanted heart beat at 130 beats per min (Fig. 4.1G), which was near the
rate of native hearts (146 beats per min, n = 7). All 10 embryos containing beating
69
transplanted hearts were used for further analysis. Liver tissue was present in the orbit in
all 13 transplants and all were used for further analysis.
Afferent innervation of transplanted ears
Lipophilic dyes implanted into the brain revealed sensory vestibular ganglion
cells, detected by their cell bodies, labeled with dyes from both midbrain (blue) and
hindbrain (red) implantations (Fig. 4.2A) when the ear was transplanted to the orbit.
Most delaminating sensory ganglion cells sent projections along the nearby trigeminal
nerve and into the hindbrain (Figs. 4.2B and 4.2C). Fewer ears sent projections along the
oculomotor nerve and into the midbrain (Fig. 4.2D). Of the 25 transplanted ears analyzed
with lipophilic dye tracing, 23 had lipophilic dye labeling of axons projecting to the
transplanted ears. Twenty-one of these 23 ears had lipophilic dye labeling of sensory
vestibular ganglion cells. Of these, 16 sent sensory axons along the trigeminal nerve
only, 2 had ganglion cell axons projecting along the oculomotor nerve only, and 3 had
projections along both trigeminal and oculomotor. However, not all ganglion cells may
have sent projections to the hindbrain or midbrain as was evident in the transplanted ears
labeled with GFP (Fig. 4.2C). In these ears (n=4), some GFP-positive ganglion cells did
not project to the brain areas where dye was implanted (Fig. 4.2C) as they showed no
lipophilic dye label. It is not known where these unlabeled ganglion cells project to and
if they reach the brain at all. Afferent axons that do reach the brain, as observed
following implantations of lipophilic dye into the ear, project along either the trigeminal
oculomotor or optic nerve to enter the brain; however, there was no consistency between
different transplants in the trajectory of afferent projections (Fig. 4.2F-I). Of the eighteen
ears in the orbit labeled with lipophilic dyes that sent afferents to the brain, one projected
70
to the forebrain, midbrain, and hindbrain, five projected to the forebrain and midbrain,
three to the midbrain alone, five to the midbrain and hindbrain, and four to the hindbrain
alone. Afferent axons from three of the transplanted ears appeared to project to the
ipsilateral vestibular nucleus (Fig. 4.2I, 4.2I’), a native target of the ear. These data
suggest that projections of inner ear afferents primarily grow randomly along adjacent
nerves with little evidence for preferences.
Efferent innervation of transplanted ears
Efferent innervation of transplanted ears from the oculomotor and trochlear
nerves was demonstrated with lipophilic dye implantation into the midbrain. Of the 25
embryos implanted with lipophilic dyes into their brains, 9 transplanted ears had
projections to them from the oculomotor nerve (Fig. 4.3A) and 1 from the trochlear nerve
(Fig. 4.3F). These projections were without obvious co-labeling of vestibular sensory
ganglion cells, though a pure motor innervation required additional confirmation (see
below). In 8 of 9 embryos in which the oculomotor nerve sent axons to the transplanted
ear, the surrounding eye muscle was also innervated; the remaining embryo had no eye or
eye muscles remaining to be innervated. In the embryo in which the trochlear nerve sent
axons to the transplanted ear, eye muscles were present, but were not innervated. For the
13 embryos lacking oculomotor or trochlear nerve projections to the transplanted ear but
had regrowth of part of the eye, the motor nerves innervated only the eye muscles. The
most noticeable difference between embryos whose transplanted ears had projections to
them and those whose did not was the position of the transplanted ear relative to the
native eye muscle. All transplanted ears imaged that were more medial than the eye
muscle received projections from motor nerves (5 of 5 ears). Half of the transplanted
71
ears that were equidistant from the brain as the eye muscle received projections from
motor nerves (5 of 10 ears). Finally none of the ears located lateral to the eye muscles
were innervated (0 of 8 ears). These data imply that the ear is as good a substrate for
motor neurons as are eye muscles with the decisive difference being driven by the
relative position following a simple first encountered, first innervated rule.
It was necessary to confirm that there were indeed motor neuron axons projecting
to the transplanted ear and not just afferent projections of sensory ganglion cells from the
ear back to the midbrain along the oculomotor nerve, since ganglion cells were shown to
occasionally project along the oculomotor nerve into the midbrain (Figs. 4.2D, 4.3I).
Sensory ganglion cells were detected in these ears by the existence of neuronal cell
bodies in a putative vestibular ganglion that were also labeled with lipophilic dye. Their
axons could be traced back to the oculomotor nerve (Fig. 4.3I), or in other cases were
indistinguishable from the nerve itself (Fig. 4.2D). We classified ears as receiving
projections from oculomotor or trochlear nerve based on the absence of lipophilic
labeling of sensory ganglion cells. These ear transplants without lipophilic dye-labeled
neuronal cell bodies along the oculomotor or trochlear nerves were selected for further
analysis. To further confirm that these axons projecting to the transplanted ear were of
motor origin, we used an antibody against VAChT. Our previous data demonstrated that
ears containing VAChT-positive axon terminals on hair cells observed with electron
microscopy, had motor neurons with synaptic vesicles terminating at the base of hair cells
(Elliott and Fritzsch, 2010), thus VAChT is a good indicator of motor innervation. We
tested 12 ears that had lipophilic label when dye was inserted into the midbrain: 7 with
projections from the oculomotor or trochlear nerve without labeled ganglion cells and 5
72
that had ganglion cells that were labeled (Fig. 4.2D) to serve as a control since these were
not expected to have VAChT labeling. VAChT-positive motor axon terminals were
confirmed on hair cells in the 7 transplanted ears determined to have motor neuron
innervation based on lipophilic labeling (Figs. 4.3C-4.3E, 4.3H). The 5 ears that had
ganglion cells labeled by midbrain lipophilic dye implantation (Figs. 4.2D, 4.3I) were not
positive for VAChT (Fig. 4.3K). Immunohistochemistry for tubulin revealed innervation
of the transplanted ears, demonstrating that axons from the oculomotor nerve was at least
a subset of the total innervation of the ear, the remainder likely being afferents (Figs.
4.3B, 4.3G).
Innervation of transplanted somite, eye muscle, and jaw muscle
Somite-derived muscle transplanted to the orbit failed to be innervated by
oculomotor or trochlear nerves (0 of 14 transplants) as demonstrated by absence of
innervation when lipophilic dyes were implanted into the midbrain. Even when the
somite-derived muscle was located medial to the native eye muscles as was the case for 3
transplants, the oculomotor nerve bypassed the somite-derived muscle to innervate the
remaining eye muscles (Figs. 4.4A-4.4C). In contrast, when the somite-derived muscle
was transplanted to the otic region to replace the ear, 3 of 5 transplants showed some
projections of possibly motor neuron axons to the somite-derived muscle, likely by the
hypoglossal nerve. This suggestion derives from implantations of lipophilic dyes
implanted into the hindbrain rostral and caudal to the transplanted somite (Fig. 4.4D).
The remaining 2 transplants were not innervated.
Eye muscle transplanted with the eye to the trunk failed to be innervated by spinal
motor nerves (0 of 4 transplants) as demonstrated when lipophilic dyes were implanted
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ventral to the spinal cord to label motor neurons as they exit the spinal cord (Fig. 4.4E).
In addition, jaw muscle transplanted to the trunk failed to be innervated by spinal motor
nerves (0 of 9 transplants) as demonstrated when lipophilic dyes were implanted ventral
to the spinal cord (Figure 4.4F). For both eye muscle and jaw muscle transplants, the
spinal motor neuron axons exiting the spinal cord navigated around the transplanted
tissue and innervated neighboring native somite-derived muscle.
Innervation of transplanted heart and liver
Lipophilic dyes implanted into the midbrain and hindbrain revealed some axons
projecting to transplanted heart tissue. Seven of 9 hearts had projections from the
trigeminal nerve, as observed by dye implantations into the hindbrain (Fig. 4.5A). Five
of the 9 hearts had projections from oculomotor neurons, as observed by implantations
into the midbrain (Fig. 4.5B); however closer examination of these latter hearts showed
that oculomotor neurons may be terminating on autonomic ganglia associated with the
heart rather than on heart muscle itself (Fig. 4.5C). Thus, there were no clear examples
of direct heart muscle innervation by motor neurons. Likewise, the heart beat was not
changing when tadpoles moved around, suggesting limited effectiveness of oculomotor
neurons to change the autonomous heartbeat frequency, when compensatory eye
movements are initiated (Straka et al., 2009, Rössert et al., 2011).
Lipophilic dyes implanted into the hindbrain revealed very little innervation of the
transplanted liver and no innervation of the liver was observed when lipophilic dyes were
implanted into the midbrain. If there were apparent projections to the liver from the
hindbrain, it was from a subset of trigeminal nerve axons. Trigeminal projections to the
liver occurred in 11 of the 13 transplants. In 7 livers a subset of axons from the
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trigeminal just passed over the surface of the liver (Fig. 4.5E). The oculomotor nerve
failed to innervate the transplanted liver in the 13 transplanted livers imaged and if it
approached the liver, it would pass around or over to innervate the nearby eye muscles, if
the latter were present (Fig. 4.5D and 4.5E).
Discussion
The results here extend our previous work (Elliott and Fritzsch, 2010) by
demonstrating that, not only spinal somatic motor neurons, but other subsets of motor
neurons such as oculomotor motor neurons can also reroute and innervate the hair cells of
the transplanted ear. In addition, we have tested for the ability of other targets and nontargets of motor neurons to receive novel innervation when similarly transplanted. Here
we will discuss the range of innervation of novel tissues and its likely implications.
Using lipophilic dye implantations, we were able to demonstrate the ability of the
oculomotor nerve, and in one case the trochlear nerve, to extend motor neuron axons to
hair cells of an ear transplanted to the orbit to replace the eye. This finding, together with
that of Elliott and Fritzsch (2010), that spinal somatic motor neurons can innervate the
ear when their normal target is no longer present, supports the idea that facial branchial
motor neurons may have been rerouted to innervate the ear when the ear evolved in place
of somites or somitomeres in ancestral vertebrates (Fritzsch and Nichols, 1993, Fritzsch
et al., 2007, Simmons et al., 2011). Together, these data demonstrate that spinal,
branchial, oculomotor and thus likely any motor neuron have the ability to become an
efferent to the ear; however, whether this is also true for visceral motor neurons
projecting axons to neural crest derived ganglia (Fritzsch and Northcutt, 1993, TakanoMaruyama et al., 2010) remains to be seen. Unlike the ear, transplantation of somite,
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heart, and liver tissue into the orbit did not result in innervation by either the oculomotor
or the trochlear nerve, with the exception of the autonomic parasympathetic ganglia
associated with the heart which received axons from the oculomotor nerve in a few cases,
possibly reflecting the parasympathetic component of the oculomotor nerve. Overall, this
suggests that not all motor neurons can become efferents to any target, even if that tissue
normally receives motor innervation, for example the somites. This is in line with
previous reports of selective reinnervation of eye muscles after trochlear nerve
transection which showed that in Xenopus, the trajectory of the nerve combined with the
timing of innervation formation, determines the pattern of innervation by only one ocular
nerve or several (Fritzsch and Sonntag, 1990).
Unlike the facial branchial motor neurons which can innervate a variety of tissue
types, including hair cells as efferents (Simmons et al., 2011), the oculomotor and
trochlear motor neurons normally innervate only specific eye muscles, with the exception
of a parasympathetic branch of the oculomotor nerve that innervates the parasympathetic
ciliary ganglion (Adams et al., 2008). It is possible that this branch of the oculomotor
was responsible for the innervation of parasympathetic ganglia associated with the heart
tissue when transplanted to the orbit.
The inability of the oculomotor or trochlear axons to innervate somite-derived
muscle tissue or of the spinal motor neurons to innervate branchial-arch-derived muscle
tissue are likely due to differences in the origin of the tissues and may reflect an inability
of cranial nerves to supply somite-derived tissue and of spinal motor neurons to supply
branchial-arch-derived tissue. Such is the case with the trapezius muscle in normal
development. The branchial-arch-derived trapezius muscle sits in the trajectory of spinal
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motor neurons, yet is innervated by motor neuron axons of the spinal accessory nerve or
by a branch of the vagus (Boord and Sperry, 1991, Sienkiewicz and Dudek, 2010, Dudek
et al., 2011). Thus, it seems plausible that cranial motor neurons and spinal motor
neurons rely on unique molecular signatures adapted by their target tissues to prevent
cross-innervation at the head-neck boundary. What this signature is remains unclear.
Data on experimental reorganization of ocular innervation in cases of loss of abducens or
oculomotor innervation (Fritzsch et al., 1995) suggests that some hierarchy of crossinnervation possibilities exist that need to be further investigated.
The ability of the hypoglossal motor axons to innervate somite-derived muscle
tissue transplanted to the region previously occupied by the ear may reflect the natural
ability of the hypoglossal motor neurons to innervate the somite-derived tongue muscle
(Leperchey, 1979). In aquatic organisms, such as X. laevis, the tongue is absent
(Cannatella and de Sá, 1993). The hypoglossal nerve, without a target, may degenerate
as nerve or motor neurons have not been found in adult X. laevis (Nikundiwe and
Nieuwenhuys, 1983); however, given the chance to innervate a target of similar origin
(somite-derived muscle), the hypoglossal motor neurons apparently does so at least
transiently. In a sense, motor neuron axons supplies of somite-derived muscle via the
hypoglossal nerve, also recapitulates the original efferent innervation paradigm of the ear
previously suggested which forced some facial motor neurons to innervate the ear in the
absence of their likely somite derived original target (Fritzsch, 1999).
As was observed in our previous work (Elliott and Fritzsch, 2010), afferents from
transplanted ears can project to a novel area in the CNS. Although most afferents
projected along the trigeminal nerve into the hindbrain as seen from both hindbrain and
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ear implantations with lipophilic dyes, some afferents from ears transplanted to the orbit
projected back to the midbrain along the oculomotor or optic nerve. From there, the
occasional axons projected into the forebrain. It appears that these afferents fasciculated
with the nearest cranial nerve. That more axons followed the trigeminal nerve than any
of the other nerves may be due to the larger territory of trigeminal projections than that of
the others (Borges and Casselman, 2010). However, even with the same entry point into
the brain along a given cranial nerve, there was no consistency once inside the brain for
afferent axon projections.
The ability of the oculomotor and trochlear nerves to send projections to hair cells
of the ear but not the other tissues directly suggests that there is something unique about
the ear to allow for cross-innervation. One thing in common that all targets receiving
direct motor input have is the presence of nicotinic acetylcholine receptors (nAChRs)
(Zuo et al., 1999, Vernino et al., 2009). nAChRs are comprised of a pentamer of various
subunits: α, β, δ, ε, and γ (Tsunoyama and Gojobori, 1998, Jones and Sattelle, 2004,
Albuquerque et al., 2009). Of these subunits, the α subunit is required for the receptor to
bind ACh (Jones and Sattelle, 2004). The original α sequence has diversified, giving rise
to the 10 different isoforms present today (Jones and Sattelle, 2004). Of the 10 α
subunits, α9 and α10 are the most diverged (Sgard et al., 2002, Franchini and Elgoyhen,
2006, Katz, 2011, Lipovsek et al., 2012). Hair cells contain these most divergent of
nAChR alpha subunits, α9 and α10 (Sgard et al., 2002, Franchini and Elgoyhen, 2006,
Katz, 2011, Lipovsek et al., 2012). It is possible that all motor neurons retain the ability
to form synapses on α9 and α10-containg nAChRs. Mice in which the gene encoding the
α9 nAChR subunit (Chrna9) or α10 nAChR subunit (Chrna10) was knocked out showed
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that these receptors were in part necessary for the development of synaptic connections
between the olivocochlear efferents and the inner ear hair cells (Vetter et al., 1999, Vetter
et al., 2007, Katz, 2011). In mice lacking either α9 or α10 nAChR subunits, efferent
synaptic contacts were larger in size but reduced in number compared to wild type
littermates (Vetter et al., 1999, Vetter et al., 2007). No α9 or α10 double null mouse
efferent innervation has been reported, leaving it open whether absence of both receptors
eliminates all efferent synaptogenesis on hair cells. We are currently planning to
knockdown both α9 and α10 nAChR subunits in X. laevis and transplant ears from these
embryos into control embryos, either replacing the native ear or transplanting to the trunk
to replace a somite or the orbit to replace the eye. The prediction would be that there is
no efferent innervation of hair cells by any motor neurons, including an absence of hair
cell innervation in the untransplanted ear. Future work would require also misexpressing
α9 and α10 nAChR subunits in tissues that did not receive motor innervation when
transplanted to either the orbit or trunk such to determine whether it is these nAChRs that
allow for hair cell innervation by other motor neuron types.
In conclusion, the results presented here demonstrate the potential for a motor
neuron to reroute to target a novel tissue that is transplanted into its trajectory; however,
the nervous system is not completely plastic and not every motor neuron can interact with
any target. Our data suggest that it is only the ear (and possibly parasympathetic ganglia)
that can receive motor input from any motor neuron when placed in the trajectory of all
motor neuron types tested here. The next step is to determine what properties present in
the ear that are lacking in the other tissues transplanted that allow for synaptic formation
by motor neurons. As previously mentioned, one strong candidate is the presence of the
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ancestral α9 and α10 nAChR subunits in the ear. Additionally there could be other
unique components involved in synaptic formation as well as short range guidance cues
to guide axons to the hair cells. Determining these factors may provide an additional
understanding of the evolution of motor neuron innervation specificity of distinct
peripheral targets. Furthermore, the insights generated here may be applicable in helping
individuals with motor neuron damage regain function by rerouting other motor neurons
to the denervated target tissue.
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Table 4.1. Success of tissue transplantation.
Transplantation
Transplanted Tissue Transplanted Tissue
Total
Present
Absent
Ear to Orbit
159
14
173
Somite to Orbit
9
1
10
Eye/Eye muscle to Trunk
6
1
7
Jaw muscle to Trunk
12
1
13
Heart to Orbit
23
8
31
Liver to Orbit
13
0
13
Note: Success of transplantation is defined as the visual detection of transplanted tissue.
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Figure 4.1. Stage 46 Xenopus laevis. (A) Embryo with a transplanted ear containing
otoconia completely replacing the eye. Inset shows higher magnification of the
transplanted ear. (B) Embryo with a transplanted ear containing otoconia medial to the
reformed eye. A residual ear (RE) regrew in the native location. Inset shows higher
magnification of the transplanted ear and remaining portion of the eye (circled). (C)
Embryo with a transplanted, empty vesicle medial to the eye, which has formed a
secondary eye more caudal. A residual ear regrew in the native location. Inset shows
higher magnification of the transplanted ear and remaining portion of the eye (circled).
(D) Embryo with transplanted GFP-expressing somite-derived muscle tissue medial to
the eye. (E) Embryo with transplanted somite-derived muscle tissue to replace the ear.
(F) Embryo with a transplanted donor GFP-expressing eye to the trunk, replacing a
somite. Inset shows the GFP expression in the transplanted eye and surrounding
transplanted eye muscle. (G) Embryo with a transplanted heart completely replacing the
eye. (H) Embryo with a transplanted liver completely replacing the eye. Native,
unmanipulated ears are labeled ‘Ear’ and are circled with a black dotted line. Eyes,
native and reformed, are indicated by ‘Eye’. Arrows indicate transplanted tissues;
transplanted ears are circled in addition with a white dotted line. Scale bar is 1 mm in A,
B, C, F, H; 0.5 mm in D, E.
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Figure 4.2. Afferent projections to transplanted ears. (A) Embryo showing
implantation of lipophilic dyes into the midbrain (blue) and hindbrain (red). The
transplanted ear is noted by the arrow. (B) Transplanted ear with ganglion cells (GC)
projecting to hair cells (HC) in the inner ear and along the trigeminal nerve (V, red) back
to the hindbrain. The optic nerve (II) is green. (C) Transplanted ear labeled with GFP
reveals delaminated ganglion cells (GC), some of which project back to the brain (*)
along the trigeminal nerve (V) as noted by colocalization with lipohilic dye. Other
ganglion cells (**) did not colocalize with lipophilic dyes. (D) Transplanted ear labeled
with GFP reveals delaminated ganglion cells (GC) which project back to the brain along
the oculomotor nerve (III). Inset is higher magnification of boxed area showing the GFP
labeled otic ganglion cells. (E) Embryo demonstrating implantations of lipophilic dyes
into the native ear (blue) and transplanted ear (red, arrow). (F-I) Brains from embryos
following lipophilic implantation into the native ear (green) and transplanted ear (red)
reveal variation in afferent projections from the transplanted ear. Note: some of the
lipophilic dye-labeled projections are from cranial nerves that were labeled transcellularly
from the afferents. (I’) Stack of eight z-series confocal images from I showing hindbrain
projections from the transplanted ear to the alar plate, probably the vestibular nucleus.
Scale bar is 1 mm in A and E, 50 µm in B and C, 100 µm in D, F, G, H, I, and I’.
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Figure 4.3. Efferent projections to transplanted ears. (A) Implantations of lipophilic
dyes into the midbrain (green) and hindbrain (red) revealed axon projections from the
oculomotor nerve (III) to hair cells of the transplanted ear (circled). (B)
Immunohistochemistry for tubulin of the ear in A shows all innervation. (C-E)
Immunohistochemistry for VAChT (red) confirms motor terminals on hair cells (HC) of
boxed areas in B. Insets show higher magnification of VAChT staining at the base of hair
cells. (C’) Single z-series images at the base of the hair cells (lower left) showing
VAChT-positive terminals and at the apex (upper right) devoid of VAChT staining. (F)
Implantations of lipophilic dyes into the midbrain (green) and hindbrain (red) revealed
axon projections from the trochlear nerve (IV) to hair cells in the transplanted ear
(circled). Afferent axons projected along the trigeminal nerve to the hindbrain,
demonstrated by the colocalization of ganglion cells (GC) with the red lipophilic dye. (G)
Immunohistochemistry for tubulin of the ear in G shows all innervation. (H)
Immunohistochemistry for VAChT (red) confirms motor terminals on hair cells of boxed
area in G. Inset shows higher magnification of VACHT staining at the base of hair cells.
(I) Implantations of lipophilic dyes into the midbrain (green) and hindbrain (red) revealed
ganglion cells (GC) projecting along the oculomotor nerve (III). For this ear, the
oculomotor nerve innervated the eye muscles ventral to the transplanted ear. (J)
Immunohistochemistry for tubulin of the ear in I shows all innervation. (K)
Immunohistochemistry for VAChT (red) shows the absence of motor terminals on hair
cells (HC) of boxed area in I. Scale bar is 100 µm in A, B, F, G, I, J; 25 µm in C, C’, D,
E,H, K.
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Figure 4.4. Transplanted muscle tissue. (A) Implantations of dye into the midbrain
(red) and hindbrain (blue) revealed axon projections from oculomotor nerve to the eye
muscles but not to the transplanted somite-derived muscle (GFP, green). (B-C) Single z
series showing innervation to the eye muscle but not the transplanted somite-derived
muscle (GFP, green). (D) Implantations of dye into the hindbrain at the level of the
trigeminal (red) and the glossopharyngeal, vagus, and hypoglossal (green) revealed axons
projecting to transplanted somite-derived muscle tissue, likely from the hypoglossal
nerve. (E) Implantations of dye ventral to the spinal cord revealed spinal motor neuron
innervation of surrounding somite-derived muscle but not to the GFP-positive eye muscle
(green) transplanted with the eye. (F) Implantations of dye ventral to the spinal cord
revealed spinal motor neuron innervation of surrounding somite-derived muscle, but not
to the jaw muscle transplanted from a dextran-injected embryo. Scale bar is 100 µm.
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Figure 4.5. Transplantated tissue lacking nicotinic acetylcholine receptors such as
heart and liver. (A) Implantations into the midbrain (red) and hindbrain (blue) revealed
trigeminal innervation of the transplanted GFP-positive heart. The oculomotor (III) only
innervated nearby eye muscle tissue. (B) Example of axons from the oculomotor nerve
(III) projecting to a transplanted heart in addition to eye muscle. (C)
Immunohistochemistry for tubulin (green) and VAChT (red) demonstrate that axons from
the oculomotor nerve project to ganglion cells associating with the heart but not on the
heart muscle itself. (D) Implantations into the midbrain (green) and hindbrain (red)
revealed no axons projecting to the liver. (E) Implantations into the midbrain (green) and
hindbrain (red) showed nerve fibers passing over the liver, but not innervating it.
Transplanted tissue is circled. Scale bar is 50 µm in A, C; 100 µm in B, D, E.
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CHAPTER V
GENERATION OF ‘THREE-EARED’ FROGS REVEALS
MOLECULAR AND ACTIVITY-BASED GUIDANCE OF
CENTRAL PROJECTIONS
Abstract
As new sensory organs, such as the ear, arose in evolution, connections between
the sensory system and CNS were formed. How these inner ear neurons find their central
target in the hindbrain to transmit sound and movement information is not yet known.
Much more is known about the developmentally-related visual system. In the eye, retinal
ganglion cells use both molecular cues (Eph/Ephrin) and activity-based mechanisms
(formation of ocular dominance columns) to guide axons to the proper location in the
CNS.
In order to determine whether the ear also uses molecular mechanisms and
activity-based guidance, we generated ‘three-eared’ frogs.
An additional ear was
transplanted rostral to the native ear, either maintaining the native orientation, or rotating
it by 90 degrees. The rationale was that in the native orientation, the gravistatic and
angular acceleration-detecting sensory epithelia would be in line and thus there should be
little or no difference in activity between the two ears. On the other hand, when one ear
is rotated 90 degrees, its sensory epithelia will be offset from the native ear and will not
respond the same to a given stimulus. Swimming behavior of tadpoles indicated that
embryos in which the transplanted ear was in line with the native ear swam more
normally, whereas embryos in which the transplanted ear was rotated by 90 degrees
swam aberrantly. Injections of lipophilic die tracers indicated that afferent axons from
the two ears projected to overlapping areas when the transplanted ear was in line with the
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native ear. In contrast, when the transplanted ear was rotated by 90 degrees, afferent
axons from the two ears were segregated from each other, forming ‘vestibular dominance
columns’ reminiscent of the ocular dominance columns formed from varying activity
between the two eyes. These results suggest that afferents of transplanted ears rotated 90
degrees interfere with vestibular processing as indicated by the aberrant swimming. In
addition, the partial overlap and segregation of afferent innervation in the two ‘threeeared’ frog models implies that both molecular and activity-based mechanisms affect
central projections.
Introduction
During normal development, the central nervous system (CNS) makes very
stereotyped connections with peripheral sensory targets allowing for proper transmission
of information. Such evolutionarily stable connections project to molecularly specified
locations and nuclei within the brain (Brodmann, 1909). For instance, retinal ganglion
cells project to the superior colliculus/tectum (Clandinin and Feldheim, 2009), olfactory
receptor neurons to the olfactory bulb (Adam and Mizrahi, 2010), vestibular afferents to
the vestibular nucleus and cerebellum (Barmack, 2003), and auditory afferents to the
auditory nucleus (Meredith, 1988).
While these connections are rather refined in
organisms that possess these sensory systems now, the brain has evolved such
connections and sensory organs over time.
How those sensory organs and their
projections evolved in vertebrates is unknown.
Proper connections need to be made from a novel sensory system to the CNS to
allow meaningful interpretation of the data provided by the sensory organ. In the wellstudied visual system, projections of the retinal ganglion cells to the superior
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colliculus/tectum are guided by molecular cues in order to produce a topographical map
of the environment. Vertebrates use concentration gradients of the EphA receptor and its
ligand, ephrin-A, along the nasal-temporal axis of the retina and anterior-posterior axis of
the superior colliculus/tectum to guide retinal ganglion axons to the appropriate region of
the CNS (Clandinin and Feldheim, 2009) to maintain appropriate representation of the
visual field. These projections to the superior colliculus/tectum are further refined in
some vertebrates to account for input from both eyes. In higher mammals, (i.e. cats and
primates), the eyes are located in the front of the head, resulting in a significant overlap
of part of the visual field between the two eyes (Schmidt and Tieman, 1985, Leamey et
al., 2009). The same part of the visual field from both eyes project to the same area in the
CNS, resulting in the segregation of eye-specific projections to the visual cortex into
stripes, referred to as ocular dominance columns (Hubel and Wiesel, 1977, Wiesel,
1982). In animals with laterally-located eyes, there is no or very limited overlap of the
visual field and there is no formation of ocular dominance columns (Schmidt and
Tieman, 1985). The formation of ocular dominance columns can be induced in lower
vertebrates with lateral eyes, (i.e. frogs and fish), by transplantation of an extra eye such
that two eyes project to the same tectum (Constantine-Paton and Law, 1978, Springer and
Cohen, 1981, Constantine-Paton, 1982, Easter, 1983).
Various experiments have
demonstrated that the formation of ocular dominance columns is primarily activity-based
(Hubel and Wiesel, 1965, Swindale, 1981, Meyer, 1982, Swindale and Cynader, 1983,
Boss and Schmidt, 1984, Mower et al., 1984, Reh and Constantine-Paton, 1985) and
presents a compromise between the difference in activity between the two eyes and the
same molecular map for both eyes.
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Much less is known about axon guidance in the ear. The ear has been shown to
be developmentally related to the eye. In Jellyfish, Pax B is expressed in both the eye
and the statocyst (Kozmik et al., 2003). PaxB has evolved into Pax 6 and Pax 2/5/8
(Fritzsch and Piatigorsky, 2005, Galliot et al., 2009).
Pax 6 is involved in eye
development across nearly all species (Gehring and Ikeo, 1999), whereas Pax 2/5/8 is
involved in ear development (Pfeffer et al., 1998, Heller and Brändli, 1999, Bouchard et
al., 2010).
In addition, in Drosophila melanogaster, atonal is expressed in both
photoreceptors and chordotonal organs (Jarman et al., 1995). Atonal has since duplicated
and diversified. In the eye, Atoh7 is expressed in the RGCs and is important for their
development (Kanekar et al., 1997, Brown et al., 1998, Brown et al., 2002, SkowronskaKrawczyk et al., 2009), whereas in the ear, Atoh1 is important for hair cell formation, and
other atonal family members, Neurog1 and Neurod1 are important for the development of
sensory neurons (Fritzsch et al., 2010). Given these developmental similarities between
the eye and ear and other similarities including the ability to form a space map
(topographical map in the visual system, tonotopic in the auditory system) and similar
cortical plasticity in cross-modal innervation studies (Mao et al., 2011), we hypothesized
that the ear may use both molecular and activity-based mechanisms to order cochlear
afferents (Rubel and Fritzsch, 2002) and terminate specified projections from the various
end organs of the vestibular system (Maklad and Fritzsch, 2003, Newlands et al., 2003)
comparable to the eye.
To test this hypothesis, we generated for the present study ‘three-eared’ frogs to
determine whether the ear uses molecular and activity-based cues for axon guidance by
transplanting an additional ear rostral to the native ear, either maintaining its orientation
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or rotating it by 90 degrees in the horizontal plane. The rationale is that in the native
orientation, the gravistatic and angular acceleration-detecting sensory end organs would
be in line and thus there should be little or no difference in activity between the two ears.
On the other hand, when one ear is rotated 90 degrees, its sensory end organs will be
offset from the native ear and will not respond the same way to a given stimulus. If the
axons are guided using molecular cues only, afferents from both ears should project to
the same areas in the brain with little segregation between axons from the two ears,
regardless of the orientation of the transplanted ear. If there is both molecular and
activity-based guidance, then in animals in which the transplanted ear was in line with the
native ear, afferents should project as above; however, when the transplanted ear was
rotated 90 degrees, afferents should project to the same areas in the brain, but with
segregation between axons, forming “vestibular dominance columns” reminiscent of the
ocular dominance columns formed from varying activity between the two eyes.
Results
Success of transplantation
Success for ear transplantation was defined as the detection of a third ear, whereas
completeness of transplantation was defined as the amount and/or degree of normality of
transplanted tissue. Of the 195 successful ear transplantations in which a third ear could
be identified, 86% of the ears transplanted contained otoconia (n = 167), whereas 14%
were just a vesicle and lacked otoconia (n = 28). Of the 167 ears that contained otoconia,
105 were nearly indistinguishable from a normal ear, showing two otocnia-bearing
maculae and three semicircular canal cristae. In 49 transplantations, the transplanted ear
fused with the native ear, forming a larger ear with multiple sets of otoconia. Fusion
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occurred more frequently with earlier transplantations (stage 25 vs stage 27).
Transplanted ears were not detected in 17 animals. Only animals in which a complete
third ear formed (Fig. 5.1), containing otoconia over the utricle and saccule, and either in
the normal orientation (n = 38) or rotated by 90 degrees (n = 55), were used for further
analysis.
Analysis of Swimming Behavior
Initial swimming behaviors, normal (swimming upright), swimming on side,
swimming upside down, swimming vertical, spinning, looping (Ferris wheel formation),
and spiraling (corkscrew formation), were determined in animals for animals in which the
third ear was in line with the native ear or rotated by 90 degrees (Table 5.1). In animals
in which the transplanted ear was in the native orientation, animals primarily swam
upright. Slightly less than half (45%) had one additional swimming behavior, though
most of the time was spent swimming upright (data not shown). Of these 45%, only two
animals showed more than one additional swimming behavior. The most common
behavior, besides normal swimming, in these animals was spinning. This was usually
one of the first behaviors observed when the embryos were dropped into the arena, and
often they quickly re-oriented themselves. This initial spinning behavior is also seen in
approximately half of control, unmanipulated animals. Also for control animals, most
time was spent swimming upright (data not shown). The remaining behaviors were seen
in 3% to 11% of animals with the transplanted ear in the native orientation.
In animals in which the transplanted ear was rotated by 90 degrees, most animals
were able to eventually swim upright (93%), but not all. Normal upright swimming was
the first behavior observed in 31% of these animals. For those that were able to swim
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upright at some point, all but two displayed additional swimming behaviors. Of the
animals displaying additional swimming behaviors besides normal swimming, 43%
showed at least two additional swimming behaviors. The most common behavior,
besides normal swimming, in these animals was swimming on the side (49% of animals),
swimming vertically (42% of animals), and spinning (35% of animals). The remaining
behaviors were seen in 5% - 23% of animals. The swimming behavior of most animals
in which the transplanted ear was rotated by 90 degrees is similar to animals with
unilateral ear ablation, resulting in an ear only on one side. As with animals with a
rotated transplanted ear, most animals with an ablated ear were able to swim upright
(85% of animals); however, of the ablated ear animals eventually able to swim upright,
71% displayed at least two additional swimming behaviors, which was a little higher than
the 43% of animals with a rotated transplanted ear. The most common behaviors in
animals with only one ear, besides the ability to eventually swim upright, were spinning
and swimming on the side (70% and 60% of animals, respectively).
Analysis of Afferent Projections to the Hindbrain
Lipophilic dyes implanted into the native and transplanted ears of fourteen
animals revealed afferent projections in the hindbrain. The transplanted ear projected
either with the native VIIIth ganglion, completely with its own ‘VIIIth’ ganglion, or in a
mixture of both with the native and with their own ‘VIIIth’ ganglion (Fig. 5.2). In one
animal, the transplanted ear, in the native orientation, projected entirely with the native
VIIIth ganglion, although fibers from the two ears were mostly segregated within the
ganglion (Fig. 5.2A). In seven animals, the transplanted ear projected with its own
‘VIIIth’ ganglion (Fig. 5.2B). Five of these animals had rotated ears; two had ears in the
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native orientation. In the remaining six animals, the transplanted ear projected partially
with the native ganglion and partially with its own ganglion (Figs. 5.2C and 5.2D). Two
of these animals had rotated ears; four had ears in the native orientation. Axons from the
additional ‘VIIIth’ ganglion from the transplanted ears entered the hindbrain at the
approximate level of the trigeminal ganglion.
Afferents from all seven embryos with ears transplanted in line with the native ear
project to the same area in the hindbrain with nearly complete overlap of fibers from the
two ears (Figs. 5.3A and 5.3B). In contrast afferents from all seven embryos with ears
transplanted 90 degrees offset from the native ear project in general to the same area in
the hindbrain, but with nearly complete to complete segregation of fibers from the two
ears (Figs. 5.3C and 5.3D). Projections into the hindbrain from the rotated ears were
always located medial to the native ear projections (Figs. 5.3C and 5.3D), but lateral to
the trigeminal tract.
The percent overlap of sensory neurons from the native and transplanted ears was
calculated from intensity histograms obtained for the two sensory neuron populations
(Figs. 5.3E-5.3F). When the transplanted ears were in line with the native ear, the
percent overlap of the narrower histogram with the wider histogram was 97.7 ± 2.3% (n =
5) (Fig. 5.3G). The range of overlap for a single optical section in these animals was
between 66-100%. When the transplanted ears were rotated by 90 degrees with respect
to the native ear, the percent overlap of the narrower histogram with the wider histogram
was 21.3 ± 6.4 (n = 5) (Fig. 5.3G). The difference in overlap profiles between animals
with normally oriented transplanted ears compared with rotated transplanted ears was
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significant (p<0.0001). The range of overlap for a single optical section in these animals
was between 0-57%.
Discussion
The results presented here on ‘three-eared’ frogs establish the basic mechanisms
used by inner ear afferents to project to the proper location in the hindbrain. In addition,
we have examined the functionality of the third ear to influence the swimming behavior
of the tadpoles.
The transplanted ear developed completely normally in 105 of 195 cases,
supporting our previous data in frogs and data from chicks showing future sensory
epithelia have already been specified by the otic placode stage (Abelló et al., 2010, Elliott
and Fritzsch, 2010). The fusion of the transplanted ear and native ear may be a sideeffect of the early transplantation, as this was observed less frequently when the ear was
transplanted at stage 27 rather than at stage 25.
Using lipophilic dyes injected into the native ear and transplanted ear, we showed
that both ears, regardless of orientation project to the same approximate region of the
hindbrain. This suggests that initial guidance of inner ear sensory afferents is molecularbased. While the exact molecular nature of the guidance remains unknown, the most
logical candidate is the Eph/Ephrin system used by RGCs to guide axons to the
tectum/superior colliculus to maintain the visual field. Ephs and Ephrins have been
shown to play some role in axon guidance in the auditory system (Allen-Sharpley and
Cramer, 2012, Allen-Sharpley et al., 2013, Defourny et al., 2013). Given that Ephs and
Ephrins are also expressed in vestibular neurons (Bianchi and Liu, 1999, Cowan et al.,
2000), suggests that Ephs and Ephrins may play a role in guiding sensory afferents to the
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proper location centrally in the vestibular nucleus, though this would need to be
confirmed.
While sensory neurons from the two ears projected to the same approximate
region, the precise targeting depended upon the orientation of the transplanted ear with
respect to the native ear. The nearly complete overlap in projections from the two ears
when the transplanted ear was in the native orientation and the nearly complete
segregation of projections from the two ears when the transplanted ear was rotated by 90
degrees suggest that activity-based mechanisms also play a role in axon guidance in the
vestibular system. The nearly complete segregation of sensory axons in the vestibular
nucleus when the transplanted ear was rotated by 90 degrees formed ‘vestibular
dominance columns’ reminiscent of the ocular dominance columns found in the visual
system naturally (Hubel and Wiesel, 1977, Wiesel, 1982) and experimentally ‘three eyed
frogs’ (Constantine-Paton and Law, 1978, Springer and Cohen, 1981, Constantine-Paton,
1982, Easter, 1983) as a result of differential activity between two eyes. This activitybased guidance likely refines the initially set up by molecular-based guidance. Initial
targeting by vestibular afferents is thought to be molecular since initial observations of
vestibular afferent targeting were made prior to the onset of vestibular sensation (Maklad
and Fritzsch, 2002, Maklad and Fritzsch, 2003, Maklad et al., 2010). Later changes in
the pattern of vestibular nucleus connectivity from the various sensory epithelia occur
around the onset of a functional vestibular system (Maklad and Fritzsch, 2003).
The ability of ‘three-eared’ frogs to swim normally depended upon the
manipulation, which suggests that projections into the vestibular nucleus from the
transplanted ear are functional. Animals in which the transplanted ear was in alignment
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swam similar to control, unmanipulated, animals most of the time. In contrast, animals in
which the transplanted ear was rotated by 90 degrees displayed more aberrant swimming,
similar to that of animals with only one ear. These interpretations are consistent with our
control experiments in which we remove and reinsert one ear, forcing afferents to reestablish their connections. Such manipulations result in completely normal swimming
behavior indistinguishable from unmanipulated animals. Given that the latter
manipulations results in asymmetrical and mismatched gravitational sensation suggests
that bilateral symmetry in sensory epithelia orientation is required for proper sensing of
the animals’ orientation to proper guide its swimming.
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Table 5.1. Analysis of tadpole swimming behavior.
Animal Treatment
Control (n = 28)
One Ear (n = 20)
Normal Third Ear (n = 38)
Rotated Third Ear (n = 55)
Upright
Side
28
17
38
51
1
12
4
27
Swimming Behavior
Vertical Upside
Spin
Down
1
0
10
5
9
14
1
3
12
12
23
19
Spiral
Loop
0
8
1
7
0
7
1
5
Note 1: Numbers in parenthesis are the total number of animals for each condition.
Note 2: Numbers represent total animals observed for each swimming behaviors. Some
animals displayed multiple behaviors and are included in each total.
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Figure 5.1. Stage 46 Xenopus laevis 'three-eared' frogs. (A) Embryo with a
transplanted third ear in the native orientation. (B) Embryo with a transplanted ear rotated
90 degrees from the native ear. (C) Higher magnification of the natively-oriented
transplanted ear and the right native ear in A. (D) Higher magnification of the 90 degree
rotated transplanted ear and the right native ear in B. (E) Three-dimensional
reconstruction of a 90 degree rotated transplanted ear next to the native ear.
Endolymphatic space is magenta, endolymphatic duct is cyan, and the hair cells are
green. Native, unmanipulated ears are labeled ‘Ear’ and are circled with a black dotted
line. Transplanted ears are indicated with a white arrow and are circled with a white
dotted line. U, utricle; S, sacule. Blue and yellow arrows indicate ear orientation. Scale
bar is 0.5 mm.
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105
Figure 5.2. Inner ear afferent projections. (A) Animal in which inner ear afferents
projected entirely together in the VIIIth ganglion. (B) Animal in which inner ear afferents
from the natively-oriented transplanted ear projected in their own ‘VIIIth’ ganglion and
entered the hindbrain separate from the inner ear afferents from the native VIIIth
ganglion. (C) Animal in which the inner ear afferents from the 90 degrees rotated
transplanted ear entered the hindbrain from both in its own ‘VIIIth’ ganglion and with the
native VIIIth ganglion. (D) Animal in which the inner ear afferents leave the nativelyoriented transplanted ear both in its own ‘VIIIth’ ganglion and along with the native
VIIIth ganglion. Green arrowheads indicate projections from the transplanted ear. Red
arrowheads indicate projections from the native ear. Scale bar is 100 µm.
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107
Figure 5.3. Overlap and segregation of inner ear afferents from transplanted and
native ears. (A-B) Hindbrain from two animals in which the transplanted ear was in the
native orientation showing overlap of sensory neurons from the native (red) and
transplanted (green) ears. (C-D) Hindbrain from two animals in which the transplanted
ear was rotated by 90 degrees with respect to the native ear showing segregation of
sensory neurons from the native (red) and transplanted (green) ears. (E) Intensity
histogram from an animal with the transplanted ear in line with the native ear shows an
overlap of intensity profiles in a single optical section. (F) Intensity histogram from an
animal with the transplanted ear rotated by 90 degrees with respect to the native ear
shows a segregation of intensity profiles in a single optical section. (G) Mean percent
overlap and standard error of sensory neurons from the native and transplanted ears for
animals in which the transplanted ear was in line with or rotated by 90 degrees with
respect to the native ear. Numbers represent the number of animals. Each animal is the
mean of measurements taken from 3 different optical sections. ***, p<0.001. Scale bar
is 25 µm.
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109
CHAPTER VI
EAR MANIPULATIONS REVEAL A CRITICAL PERIOD OF
HINDBRAIN DEPENDENCE ON THE EAR FOR DEVELOPMENT
AND SURVIVAL
Abstract
Many second-order neurons rely on afferents from sensory organs during a
critical period for survival and differentiation. Research describing these effects has
mostly focused on whole populations of neurons, rather than a single cell. We studied
the effects of partial deafferentation on a single identifiable cell in the hindbrain of a frog,
the Mauthner cell. Some previous work hinted on a critical period in Mauthner cell
viability without ear input while others found that surviving Mauthner cells had reduced
branching of the lateral dendrite. The suggestion that axons from the ear stimulate the
growth and development of the dendrites is further supported when transplantation of an
additional ear allegedly increased dendritic branching. Extirpation of the ear at various
stages of Xenopus laevis development defines a critical period of progressively reduced
dependency of Mauthner cells on afferents lasting to about stage 36. In addition, earlier
ear removal results in a greater reduction in the number of dendritic branches as
compared with later ear removal. However, some rudimentary lateral dendrites form,
indicating a limited autonomous development of the lateral dendrite. The greater
reduction in the number of dendrites when the ear was removed at earlier stages indicates
that inner ear afferents play a role in further dendritic development. The latter is
confirmed quantitatively in frogs receiving a third ear: the lateral dendrite is larger and
shows significantly more branches than the control dendrite of the unmanipulated side.
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Introduction
The ablation of a primary sensory organ results in hypoplasia of higher-order
neuronal centers (Harrison, 1935). Early studies in amphibians demonstrated that
removal of an eye results in a reduction in the size of the opposite midbrain and removal
of the nasal pit results in a reduction in the olfactory center in the forebrain (Harrison,
1935). In addition, several studies have shown that removal of the ear and associated
ganglion neurons prior to axonal outgrowth alters the development of various target
neurons in the hindbrain (Levi-Montalcini, 1949, Parks, 1979, 1981, Goodman and
Model, 1988, Fritzsch, 1990). Removal of an ear during embryonic development lead to
the eventual reduction in the volume and number of neurons in the cochlear nuclei (LeviMontalcini, 1949, Parks, 1979, Ryugo and Parks, 2003) at the time point at which
afferent activity could be detected (Jackson et al., 1982). Blocking afferent activity with
tetrodotoxin resulted in a reduction of volume and number of neurons in the cochlear
nuclei, similar to that seen with ear ablation (Pasic and Rubel, 1989, Sie and Rubel,
1992). Together, these data would suggest that most of the neuronal development occurs
without excitatory activity and that later, without this activity and/or activity related
neurotrophin release, cell death and atrophy of remaining neurons occurs (Rubel and
Fritzsch, 2002). This dependence on a presynaptic neuron for survival is not permanent,
implying there is a critical period in which input is necessary for cell survival (Rubel and
Fritzsch, 2002). In gerbils, cochlea removal before postnatal day (P) 7 resulted in cell
death in 45-88% of cochlear nucleus neurons, whereas removal after P9 resulted in
virtually no cell death (Tierney et al., 1997). Other species also have critical periods, but
with varying critical time points and degrees of cell loss (Born and Rubel, 1985,
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Mostafapour et al., 2000). In addition, while removal of the ear early resulted in death of
most cochlear nuclei and severe atrophy of the remaining neurons (Levi-Montalcini,
1949, Parks, 1979), virtually no cell death occurred in the vestibular nuclei (LeviMontalcini, 1949). Vestibular nuclei neurons exit the cell cycle much earlier than
cochlear nuclei neurons (Altman and Bayer, 1980), suggesting that the vestibular nuclei
neurons may be past the critical period at the time these manipulations took place. Even
though early ear removal results in significant cochlear nucleus cell loss, some cells do
survive. Unfortunately, the reason for why some cells die and others do not is unclear.
The leading hypothesis to explain why some post-synaptic neurons survive while
others die following early afferent deprivation is that two competing intracellular
responses are initiated, apoptotic-like and survival pathways, following afferent loss, and
one pathway overtakes the other (Garden et al., 1994, Hyde and Durham, 1994a). This
hypothesis is supported by several pieces of data. For example, early degradative
processes following afferent loss are constant across all cells, not just some (Steward and
Rubel, 1985, Born and Rubel, 1988, Garden et al., 1994, Garden et al., 1995, Kelley et
al., 1997), and oxidative enzyme activity first increases and then decreases following
afferent loss (Durham and Rubel, 1985, Durham et al., 1993, Hyde and Durham, 1994b).
Thus the cells that survive depend upon the effectiveness of the survival pathway to
overcome apoptosis. Levi-Montalcini proposed that loss of ‘neurotrophic’ support from
the afferents following ear removal may cause the cell death in the cochlear nucleus
(Levi-Montalcini, 1949); however, the exact molecular nature of the factors by which
afferents support cochlear nucleus neurons is still unknown.
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These above-mentioned studies have increased our understanding of the
importance of primary sensory input on second-order neuron survival; however, these
studies looked only at whole populations of cells when determining the critical period of
cell survival and determining why some cells survive while others do not following ear
removal. Furthermore, due to technical limitations, ears can be removed in the chick
only embryonically, and in the mouse, only postnatally, which prevents a comprehensive
analysis of the loss of afferent input over a longer period of time in these species. While
afferents or ears can be genetically removed in mice, the resulting mutants are typically
not viable blocking investigations on long term auditory nucleus viability (Ma et al.,
2000). To overcome limitations, we have studied the effects of early otic placode/ear
extirpation at various developmental stages, focusing on a single cell in the hindbrain of
the externally developing frog, the Mauthner cell.
Mauthner cells are a pair of large, easily identifiable, reticulospinal neurons at the
level of the ear in the hindbrain of many aquatic animals (Herrick, 1914, Bartelmez,
1915) that are an important component of the escape reflex (Korn and Faber, 2005).
Inner ear vestibular and auditory neurons of premetamorphic amphibians and fish form
synapses on the lateral dendrite of the ipsilateral Mauthner cell, which in turn synapses
on contralateral spinal motor neurons to activate the C-start escape response (Korn and
Faber, 2005, Sillar, 2009). In addition to the ear, Mauthner cells also receive input from
the lateral line and the eye that can add to the C-start escape response (Zottoli et al., 1987,
Bezgina et al., 2000, Korn and Faber, 2005, Sillar, 2009). A study in axolotls has shown
the absence of the Mauthner cell in one-third of embryos following otic vesicle ablation
at stage 27 (Piatt, 1969); however, all Mauthner cells were present when the ear was
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extirpated at a later stage (stage 34), although dendritic branching was reduced (Kimmel
et al., 1977). In one embryo observed by Goodman and Model following ear ablation,
the entire Mauthner cell was absent (1988). While Piatt (1969) suggested that the inner
ear afferents are important for, and often a decisive factor for, the development of the
Mauthner cell, Goodman and Model (1988) suggested that surgical perturbations may be
the cause of the occasional Mauthner cell absence. Since early ear removal, before the
critical period, results in significant loss of the cochlear nuclei neurons (Levi-Montalcini,
1949, Parks, 1979, Born and Rubel, 1985, Tierney et al., 1997, Mostafapour et al., 2000),
we reason that removal of the ear at the earlier stages (Piatt, 1969, Goodman and Model,
1988) may have prevented the differentiation and/or survival of the Mauthner cell,
indicating a yet to be defined critical period for a single cell.
The inner ear not only affects the development of the Mauthner cell, but may also
have a role in the development of the lateral dendrite that receives ear afferents. In a
study in axolotls (Ambystoma mexicanum), removal of the ear of midtailbud (stage 2830) prior to the outgrowth of the VIIIth nerve resulted in significant reduction of dendritic
branching of the Mauthner cell lateral dendrites in areas normally receiving vestibular
input (Goodman and Model, 1988). Ear removal at a slightly later stage in axolotls (stage
34) also resulted in reduction of dendritic branching (Kimmel et al., 1977). Likewise,
removal of otic vesicles in zebrafish resulted in reduced branching of the Mauthner cell
lateral dendrites (Kimmel, 1982) and ablation of the otic vesicle in stage 38 X. laevis
embryos resulted in reduced Mauthner cell lateral dendrites (Fritzsch, 1990). The ear is
not the only sensory system that, when removed, affects dendritic branching of the
Mauthner cell. Enucleation of the eye resulted in delayed development of the Mauthner
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cell and diminished ventral dendrite volume in X. laevis and goldfish (Bezgina et al.,
1999, Bezgina et al., 2000, Grigor'eva et al., 2010, Mikheeva et al., 2011). Increase in
the lateral dendrite size in combination with reduction in the ventral dendrite was
observed occasionally following eye enucleation (Grigor'eva et al., 2010, Grigorieva et
al., 2012). Blocking nerve impulse activity in axolotls by grafting to them tetrodotoxincontaining newts did not affect Mauthner cell dendritic branching patterns, suggesting
that that innervation itself, rather than neural activity, is important (Goodman and Model,
1990), the latter being in stark contrast to later experiments using other means to block
activity (Pasic and Rubel, 1989, Sie and Rubel, 1992). Taken together, these data suggest
that the ingrowing axons from the ear stimulate growth and development of the dendrites.
This is further supported by a study in axolotls where an additional ear was transplanted
rostral to the native ear. The Mauthner cells in these animals displayed unspecified
‘enhanced branching’ of the lateral dendrite (Goodman and Model, 1988).
In the present study, we alter the sensory input to the developing Mauthner cell in
various ways in order to determine how sensory input shapes the development of the cell
and of its lateral dendrites. First, we explore the effects of otic placode/vesicle
extirpation on the developing Mauthner cells and their lateral dendrites by removing the
ear at both early stages (stage 24-26), when the ear is a placode, and later stages (stage
27-40), when the ear has become a closed vesicle, in X. laevis. We also explore the
effects of additional otic placode transplantations on the developing Mauthner cell and
lateral dendrite. This study defines the critical period during which the Mauthner cell is
dependent upon input from the ear for cell survival. In addition, this study determines the
dependence on input from the ear for lateral dendrite development and also the degree of
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autonomous development of the lateral dendrite in the absence of the ear or with
diminished afferent input.
Results
Effect of stage of ear removal on the degree of ear regeneration
Removal of ears at otic placode stages (stages 24-26) led to regrowth of a part or
of nearly all of the ear by stage 46 in over one-third of the embryos (Fig. 6.1A), the
remaining embryos did not have ear regrowth (Fig. 6.1D), as confirmed with tubulin and
Myo VI staining (Fig. 6.1E). The amount of ear regrowth in these early stages varied,
ranging from an endolymphatic duct, to a small otic vesicle, or to a nearly complete ear.
Removal of ears at otic vesicle stages (stages 27-40) resulted in fewer instances in which
the ear regrew in embryos by stage 46 as compared to ears removed at placode stages
(stages 24-26); most had no ear regrowth when the ear was removed at stage 27 and
older. In these later stages, if there was any regrowth, it was nearly always just the
endolymphatic duct. No regrowth of any part of the ear was detected in embryos in
which the ear was removed at stages 38-40. These data confirm previous work indicating
placodal induction extends over a lengthy period (Yntema, 1950).
Effect of stage of ear removal on the presence of the Mauthner cell
Prior to determining the effect of ear removal on the Mauthner cell, we wanted to
establish that the physical process of ear removal has no effect on the survival of the
Mauthner cell, as was previously suggested (Goodman and Model, 1988). In order to test
this, ears were removed between stages 24-26, and immediately replaced. The replaced
ear developed normally in 23 of 25 embryos (Compare Fig. 6.1C with Fig. 6.1B). For
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one embryo, the replaced ear had a single otoconia, the other was only a vesicle. The
Mauthner cell was present at stage 46 for all embryos examined in which their ears were
removed and replaced immediately (n = 14) (Figure 6.2B) and was morphologically
similar to that of control animals (Figure 6.2A).
The stage of ear removal had an effect on the development and/or maintenance of
the Mauthner cell. The earlier the stage of ear removal, the less likely it was that the
ipsilateral Mauthner cell would be present at stage 46 (Figure 6.2C). To confirm that the
absent Mauthner cell was not an artifact of incomplete Dextran amine dye application, an
antibody against reticular neurons, including the Mauthner cell, 3A10, was also used
(Figs. 6.2D-D”). To determine the percentage of Mauthner cell survival, the number of
animals with a Mauthner cell present on both the ablated side as well as control side was
divided by the total number of animals analyzed in each group (Fig. 6.2C). When the ear
was removed at otic placode stages, between stages 24 to 26, the Mauthner cell was
present on the ablated side only 38% of the time (n = 91). When the ear was removed at
early otic vesicle stages, between stages 27 to 30, the Mauthner cell was present on the
ablated side 64% of the time (n = 39). When the ear was removed at later otic vesicle
stages, the Mauthner was present on the ablated side 95% of the time (n = 40). The
Mauthner cell was always present when the ear was removed between stages 36 to 40 (n
= 24).
Effect of stage of ear removal on the dendritic branching of surviving
Mauthner cells
Prior to determining the effect ear removal has on dendritic branching of the
lateral dendrite of surviving Mauthner cells, we wanted to establish the innate variability
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in dendritic branching between left and right Mauthner cells within the same animal as
well as variability in dendritic branching between animals for control animals and for
those whose ear was removed and then immediately replaced. The total numbers of
dendritic branch terminals were counted for three-dimensionally (3D) reconstructed left
and right Mauthner cells (Figs. 6.3B-6.3E) obtained from confocal images of dextran
amine dye-labeled cells. 3D reconstructions were made from dextran amine-labeled
Mauthner cells as opposed to 3A10-labeled cells due to a more complete filling of the
dendrites (Figs. 6.3A-6.3A”). The mean number of dendritic branches in the lateral
dendrite of control animals was 22.2 ± 1.8 for the left Mauthner cell and 21.1 ± 1.4 for
the right (n = 14) (Figure 3F). There was no significant difference between left and right
Mauthner cell dendritic branch number in control animals (p > 0.05). There was more
variation in the number of dendritic branches between different control animals than
within the same animal. The range of dendritic branches between control animals was
from 11 branches to 33 branches for a single Mauthner cell. The greatest difference
between the dendritic branch number of left and right Mauthner cells within the same
animal was 7 branches. Both the difference, subtracting the number of branches on the
left from the right Mauthner cell, and the absolute difference, subtracting the smaller
branch number from the larger regardless of side, were calculated. The mean difference
and mean absolute difference between left and right Mauthner cell dendritic branch
number were 1.1 ± 1.2 and 3.9 ± 0.6 (n = 14), respectively.
For animals in which the right ear was removed and immediately replaced, the
mean number of dendritic branches in the lateral dendrite was 23.9 ± 1.9 for the left
Mauthner cell and 19.7 ± 1.3 for the right (n = 14) (Figure 6.3F). Though the Mauthner
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cell on the side in which the ear was removed and immediately replaced had fewer
dendritic branches compared with the control side, there was no significant difference
between left and right Mauthner cell dendritic branch number when one ear was removed
and immediately replaced (p > 0.05). The mean difference and mean absolute difference
between left and right Mauthner cell dendritic branch number for embryos in which the
right ear was removed and immediately replaced were 4.1 ± 2.1 and 6.3 ± 1.6 (n = 14),
respectively. These data would suggest that the surgical procedure to remove an ear has
little effect on Mauthner cell lateral dendrite development. Importantly, no animal with
ear replacement had lost the ipsilateral Mauthner cell despite the fact that needed
manipulations exceed simple otocyst removal.
When ears were removed at any of the stages, the dendritic branching of the
ipsilateral Mauthner cell lateral dendrite was always reduced compared to the control
side. This reduction in branching for the ipsilateral Mauthner cells was significant for
each of the stages of ear removal: early placode, early otic vesicle, and later otic vesicle
(p < 0.001 for each). For animals in which the right ear was removed at placode stages
(stages 24-26), the mean number of dendritic branches in the right lateral dendrite was
4.9 ± 0.6 compared with 23.1 ± 3.1 branches on the control side (n = 14) (Figure 6.3F).
For animals in which the right ear was removed at early otic vesicle stages (stages 27-30),
the mean number of dendritic branches in the right lateral dendrite was 7.3 ± 1.0
compared with 21.3 ± 2.5 branches on the control side (n = 14) (Figure 6.3F). For
animals in which the right ear was removed at later otic vesicle stages (stages 31-40), the
mean number of dendritic branches in the right lateral dendrite was 8.8 ± 0.9 compared
with 23.8 ± 2.2 branches on the control side (n = 14) (Figure 6.3F). The difference in
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branch number between left and right Mauthner cells was calculated by subtracting the
number of dendritic branches of the right Mauthner cell from the left Mauthner cell.
These differences were compared across groups using an ANOVA. Following a
Bonferonni multiple comparison adjustment, there was no significant difference between
the control and remove and replace groups(p > 0.05). While there was a trend showing a
more severe reduction in dendritic branching at the earliest stage of ear removal when
compared to ear removal at later stages, no stage of ear removal was significantly
different from each other (p < 0.05); however, all stages of ear removal were significantly
different from the control and remove and replace groups (p < 0.005). These data suggest
that the lateral dendrite of surviving Mauthner cells may depend over a lengthy period on
vestibular input for normal development.
Sholl Analysis was performed to compare branching patterns. The numbers of
dendritic crossings for Mauthner cells from animals in which the ipsilateral ear had been
removed and replaced were significantly less than control Mauthner cells for 25 µm and
50 µm distances away from the soma (p<0.05), but were similar 75 µm away from the
soma and beyond (Fig. 6.3G). The numbers of crossings for Mauthner cells from animals
in which the ipsilateral ear had been removed were fewer than control (Fig. 6.3G). The
number of crossings for Mauthner cells from animals in which the ipsilateral ear had been
removed at otic placode stages (stages 24-26) was significantly fewer than the number of
crossings for control Mauthner cells at 25 µm and 50 µm distances away from the soma
(p<0.05). The number of crossings for Mauthner cells from animals in which the
ipsilateral ear had been removed at early otic vesicle stages (stages 27-30) was
significantly fewer than the number of crossings for control Mauthner cells at 25 µm and
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50 µm distances away from the soma (p<0.05). The number of crossings for Mauthner
cells from animals in which the ipsilateral ear had been removed at later otic vesicle
stages (stages 31-40) was significantly fewer than the number of crossings for control
Mauthner cells at only 50 µm away from the soma (p<0.05). While there were no
crossings in control right side Mauthner cells beyond 100 µm away from the soma, four
animals with removed ears had crossings at 125 µm away from the soma, though only by
one dendrite.
Effect of increasing afferent input on the dendritic branching of
Mauthner cells
The addition of an ear rostral to the native ear (Fig. 6.1F) lead to growth of the
vestibular nerve fibers of these ears into the brain that mostly end in the vestibular fiber
tract and nuclei with the unmanipulated control ear (Fig. 6.1G). In those cases were
afferents of the ‘third’ transplanted ear reached into the brain there was a significant
increase in dendritic branching in the ipsilateral Mauthner cell of approximately 30%
more branches (p < 0.001). The mean number of dendritic branches in the right lateral
dendrite was 31.1 ± 1.7 compared with 22.5 ± 2.1 branches on the control side (n = 10)
(Fig. 6.3F). These data suggest that the upper limit of lateral dendrite branching is
determined by yet to be understood interactions of all vestibular afferent fibers with the
growing lateral dendrite.
Sholl Analysis was also performed. The numbers of dendritic crossings for
Mauthner cells from animals in which an additional ear was transplanted rostral to the
native ear were significantly more than Mauthner cells from control animals between 75
µm and 100 µm away from the soma, at which point there were no additional crossings in
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control Mauthner cells (Fig. 6.3G) (p<0.05). In one animal with an extra ear, crossings
were detected 175 µm away from the soma. Five additional animals with an extra ear had
crossings 150 µm away from the soma.
Discussion
The results presented here extend previous work on studying the effects of ear
removal on second order neurons in the hindbrain by focusing on a single hindbrain
neuron, the Mauthner cell. Below, the effect of the stage of ear removal on the regrowth
of the ear will be addressed. In addition, the effect of ear removal on Mauthner cell
survival as well as the effect of ear removal or addition of an extra ear on the dendritic
development of the Mauthner cell will be put in perspective.
The later the stage of ear removal, the increased likeliness was that all or part of
the ear would not regenerate. Ablation of ears in embryos between stages 24-27 had
previously been shown to have the capacity to regenerate a new ear or partial ear
(Waldman et al., 2007). Similar to this study, our results showed complete or partial ear
regeneration during these stages. Beyond stage 27 the ear rarely regenerated; if there was
any part of the ear present, it was nearly always only the endolymphatic duct. We did not
find any instance of regeneration beyond stage 38. That the ear regenerated more often
when removed at the early placode stages indicates that the remaining tissue was
competent to regenerate a new ear (Waldman et al., 2007), consistent with earlier
suggestions (Yntema, 1950). Alternatively, while care was taken to remove the entire
ear, it cannot be ruled out that a small portion was left behind. This may be the likely
scenario for endolymphatic ducts being present at later stages of ear removal, when the
tissue is not believed to be as likely to regenerate (Waldman et al., 2007).
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Viability of the Mauthner cell depends on ear input
By using two independent methods to label neurons, Dextran amine dye tracers
and immunohistochemistry, we have shown that the development and/or survival of the
Mauthner cell is dependent upon the presence of the ear. Moreover, the ability of the
Mauthner cell to survive depends on the stage at which the ear is removed. The earlier in
development that the Mauthner cell is deprived of input from the ear, the more likely the
cell does not survive. The Mauthner cell was always observed beyond stage 36,
indicating that stages earlier than stage 36 are in a critical period of dependence on the
ear for survival. Stage 36 corresponds with the stage at which afferents from the ear were
observed projecting into the hindbrain of axolotls (Fritzsch et al., 2005a). As with
neurons in the cochlear nucleus (Levi-Montalcini, 1949, Parks, 1979, Ryugo and Parks,
2003), not all Mauthner cells are absent following ear ablation. Perhaps, as has been
proposed for neurons in the cochlear nucleus (Garden et al., 1994, Hyde and Durham,
1994a), both the apoptotic and survival pathways are initiated in the Mauthner cell and
whether the cell survives or not depends upon which pathway overtakes the other.
Clearly, our data are consistent with earlier suggestions of a dependency of the Mauthner
cell on the ear. Moreover, since we always found a Mauthner cell when we simply
removed and replaced the ear, we reject the suggestion that loss of Mauthner cells is
simply a function of ear removal manipulation (Goodman and Model, 1988). The
Mauthner cell provides a unique opportunity to study initiation of degeneration as a
consequence of partial and delayed denervation in a single cell.
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Dendrite growth and branching depends on vestibular afferents
Our data suggests that the significant reduction in branching following ear
removal was a result of the loss of input from the ear and not from the physical process of
ear removal, since there was no significant difference in the total number of dendritic
branches of the lateral dendrite between left and right Mauthner cells when the ear had
been removed and immediately replaced on the right side. The numbers of dendritic
crossings from the Sholl analysis were less for Mauthner cells in which the ear was
removed and immediately replaced when compared with Mauthner cells from control
animals for shorter distances from the soma. Since there was no significant difference in
total numbers of dendrites, we conclude the effect of ear removal itself has a potential
effect on dendritic morphology, but not as much on the total number of dendritic
branches. Perhaps the slightly different morphology was due to a short delay in the
arrival of sensory afferents from ears that were removed and immediately replaced as
compared to sensory neuron afferents from control ears, though this has not been
confirmed.
In all cases in which the ear was removed and not replaced, the dendritic
branching on the ablated side was always less than the dendritic branching on the
unoperated side. Furthermore, in all cases in which an extra ear was transplanted rostral
to the native ear, there was increased dendritic branching on the side with an extra ear
compared with the unoperated side. Taken together, these quantitative data suggest that
the degree of dendritic branching is related to the number of presynaptic sensory
afferents, consistent with earlier exclusively qualitative findings (Kimmel et al., 1977,
Kimmel, 1982, Goodman and Model, 1988, Fritzsch, 1990). Our study investigates not
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only at the effect of ear removal on dendritic branching, but also the effect of the timing
of ear removal. The earlier the ear was removed, the fewer dendritic branches were
present, suggesting that the degree of dendritic development depends upon the time at
which the ear was removed. While there was no significant difference between the three
stages of ear removal when comparing the difference between left and right Mauthner
cells following ear removal, there was a trend in which the number of dendritic branches
was slightly higher when the ear was removed at later otic vesicle stages (stages 31-40)
compared with early otic vesicle stages (stages 27-30), and the number of dendritic
branches was slightly higher when the ear was removed at early otic vesicle stages
(stages 27-30) than when the ear was removed at placodal stages (stages 24-26). Since
the otic ganglion is only recognized after stage 31 (Nieuwkoop and Faber, 1994), the
slightly more dendritic branches in the earlier otic vesicle stages (stages 27-30) than in
the otic placode stages (stages 24-26) may indicate that there are other mechanisms for
dendritic development in addition to direct innervation from the presynaptic sensory
afferents. That any dendrites developed in these two groups in the complete absence of
any innervation could support additional mechanisms such as short range diffusible
factors released from the ear or alternatively suggest that there is some autonomous
development of dendritic branches in the direction of the future sensory neurons, but that
contacts between the dendrites and sensory neurons are necessary for further
development and maintenance of the dendritic branches.
The increase in dendritic branching following the addition of an extra ear rostral
to the native ear was also observed in axolotls (Goodman and Model, 1988), though not
quantified. An increase in dendritic branching indicates that additional sensory neurons
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entering the hindbrain from the transplanted ear stimulate the growth of additional
dendritic branches. That the number of dendritic branches on the side with the extra ear
was not double the number on the control side suggests that an upper limit of dendritic
branching may occur. This could be tested by grafting additional ears to a single side.
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Figure 6.1. Success of ear removal. (A) Percentage of animals with any form of ear
regrowth at each stage of ear removal. (B) Control Xenopus laevis at stage 46. (C)
Embryo in which the right ear was removed and replaced. (D) Embryo in which the right
ear was removed. (E) Immunohistochemistry for acetylated tubulin showing cranial
nerves (roman numerals) and myoVI showing the absence of the ear on the right side as
indicated by the absence of hair cells. (F) Embryo in which an additional ear was added
rostral to the native right ear. (G) Lipophilic dye labeling showing sensory neuron
projections from both the native (red) and transplanted (green) ears into the vestibular
nucleus in the hindbrain. Ears are circled and labeled, Ear. Scale bar is 0.5 mm in B-D
and F; 200 µm in E; 25 µm in G.
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Figure 6.2. Mauthner cell survival. (A) Dextran amine dye-labeled Mauthner cells in a
control animal. (B) Dextran amine dye-labeled Mauthner cells in an animal in which the
right ear was removed and immediately replaced. (C) Percentage of Mauthner cells
present on the right side at the different stages of ear removal. (D) Dextran amine dyelabeled and (D’) 3A10 antibody immunohistochemistry showing the absence of the
ipsilateral Mauthner cell following the removal of the right ear at stage 28. (D’’) Merge
of D and D’. M, Mauthner cell. Scale bar is 50 µm.
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Figure 6.3. Dendritic development of Mauthner cells following ear manipulation.
(A) Dextran amine dye labeling and (A’) 3A10 immunohistochemistry of a Mauthner cell
(M) showing filling of dendrites by dextran amine dye. (A”) Merge of A and A’. (B) 3D
reconstruction of a pair of Mauthner cells from a control embryo. (C) 3D reconstruction
of a pair of Mauthner cells in which the right ear was removed and immediately replaced
show little difference in the number of dendritic branches between Mauthner cells. (D)
3D reconstruction of a pair of Mauthner cells from an animal in which the right ear was
removed at stage 26 show a reduction in dendritic branching in the ipsilateral Mauthner
cell. (E) 3D reconstruction of a pair of Mauthner cells from an animal in which an
additional ear was transplanted rostral to the native ear at stage 26 show an increase in
dendritic branching in the ipsilateral Mauthner cell. (F) Number of dendritic branches
following ear removal or ear addition. Dark shaded bars are left (control) Mauthner cells,
light shaded bars are right (treated) Mauthner cells. ***, p<0.001. (G) Sholl Analysis of
the Mauthner cells on the right (treated) side in F. The number of dendritic branch
crossings were counted at 25 µm intervals. Scale bar is 50 µm.
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CHAPTER VII
CONCLUSIONS AND FUTURE DIRECTIONS
As outlined in Chapter 1, in order for a novel sensory system to become
functional, it needs to send information it gathers about the outside world to the CNS for
processing. In turn, the CNS will send information back to several sensory organs to
modulate the incoming signal. How new sensory organs evolved over time and how the
process of forming contacts between the CNS and the newly evolving sensory organs
develops has been poorly understood. The goal of this work was to understand how the
nervous system adapts to a novel sensory system through transplantation of frog inner
ears to specifically understand how inner ear hair cells acquire motor innervation, how
inner ear sensory neurons find their CNS targets, and how manipulation of the inner ear
affects CNS targets of sensory neurons.
Motor Neuron Acquisition
It has been suggested that the motor neurons innervating the inner ear hair cells as
efferents are rerouted facial branchial motor neurons (Fritzsch and Nichols, 1993) given
their close proximity of the efferents to the facial branchial motor neurons (Roberts and
Meredith, 1992) and similarities with the efferents during development (Simmons et al.,
2011). The inner ear develops at the rostral boundary of the anteriormost, first forming
somite (Cooke, 1978, Chung et al., 1989, Huang et al., 1997) and in animals without ears,
such as amphioxus, somites are found more rostrally, indicating that the vertebrate ear
develops in place of a somite or somitomere. This implies that the facial branchial motor
neurons that were destined to innervate the somite- or somitomere-derived muscle fibers
may have been rerouted to innervate the ear (Fritzsch et al., 2007). We first tested this
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theory by transplanting otic placodes caudally to the trunk to replace a somite,
recapitulating the likely original scenario that arose in vertebrates during ear evolution.
These transplanted ears grew normally in their novel location in the trunk and in some
instances their hair cells received motor neuron terminals, as was shown by
immunostaining with VAChT antibody and confirmed with electron microscopy (Chapter
3). That spinal motor neurons could reroute to innervate hair cells of a transplanted ear
as efferents indicates that the original rerouting of facial branchial motor neurons to the
inner ear of vertebrates is a possible scenario. Given that spinal motor neurons only
contacted hair cells when the ear was transplanted in the direct trajectory of growing
axons suggests that the initial innervation of the vertebrate ear by rerouted facial
branchial motor neurons was likely a chance occurrence and became a permanent fixture
through selection of those genes responsible for rerouting a subset of facial motor
neurons to the ear. Neurons that lose their target, and subsequent neurotrophic factors,
often degenerate and die (Gould and Enomoto, 2009). An alternate target, the ear, would
have provided neurotrophic support (Fritzsch et al., 2004, Green et al., 2012) to maintain
the displaced facial branchial motor neurons.
The ability to become an efferent to the inner ear is not a unique property of
neurons once destined to innervate somite-derived tissues, since the ear transplanted to
the orbit to replace the eye receives motor neuron terminals on hair cells from both the
oculomotor and trochlear nerves (Chapter 4), suggesting that any motor neuron has the
ability to become an efferent to hair cells of the inner ear. However, this is a property
unique to the inner ear as other targets of motor innervation are not always crossinnervated by other motor neurons when transplanted (Chapter 4). That any target of
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motor innervation is not innervated by any motor neuron is likely due to differences in
the origin of the tissues and may reflect an inability of cranial nerves to innervate somitederived tissue and of spinal motor neurons to innervate branchial-arch-derived tissue.
This is in line with the selective innervation of the trapezius muscle. The branchial-archderived trapezius muscle is located in the trajectory of spinal motor neurons; however, it
is not innervated by them but by the spinal accessory nerve or by a branch of the vagus
(Boord and Sperry, 1991, Sienkiewicz and Dudek, 2010, Dudek et al., 2011), suggesting
that cranial motor neurons and spinal motor neurons rely on unique molecular signatures
to prevent cross-innervation at the head-neck boundary.
The ability of all nerves tested to directly innervate hair cells of the ear but rarely
innervate other tissues directly suggests that there is something unique about the ear to
allow for all tested cross-innervations. Hair cells contain the most primitive nAChR
alpha subunits, α9 and α10 (Sgard et al., 2002, Franchini and Elgoyhen, 2006, Katz,
2011, Lipovsek et al., 2012). Perhaps all motor neurons retain the ability to form
synapses on the primitive α9 and α10-containg nAChRs in addition to the more-derived
versions specific to that motor neuron type. Mice in which the gene encoding the α9
nAChR subunit (Chrna9) or α10 nAChR subunit (Chrna10) was knocked out showed
that these receptors were in part necessary for the development of synaptic connections
between the olivocochlear efferents and the inner ear hair cells (Vetter et al., 1999, Vetter
et al., 2007, Katz, 2011). In mice lacking either α9 or α10 nAChR subunits, efferent
synaptic contacts were larger in size but reduced in number compared to wild type
littermates (Vetter et al., 1999, Vetter et al., 2007). No α9 or α10 double null mouse
efferent innervation has been reported, leaving it open whether absence of both receptors
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eliminates all efferent synaptogenesis on hair cells. Future directions would be to
knockdown both α9 and α10 nAChR subunits in X. laevis and transplant ears from these
embryos into control embryos, either replacing the native ear or transplanting to the trunk
to replace a somite or the orbit to replace the eye. The prediction for both would be that
there is no efferent innervation of hair cells by any motor neurons, including an absence
of hair cell innervation in the untransplanted ear. Future work would require also
misexpressing α9 and α10 nAChR subunits in tissues that did not receive motor
innervation when transplanted to either the orbit or trunk such to determine whether it is
these nAChRs that allow for hair cell innervation by other motor neuron types.
Additionally there are likely other unique components involved in synaptic formation as
well as short range guidance cues that will guide any motor axon to hair cells. A
comparison of deep sequencing profiles of ears and of the other tissues transplanted to
foreign areas may give insights into the molecular properties that are either present in the
ear and lacking in the other tissues transplanted or properties that are uniquely expressed
in only the somatic-derived tissue but not in the branchial arch derived tissue, or vice
versa, but are also expressed in the ear. Candidate genes would be those with known
roles in short-range axon guidance or synaptic formation. A test for functionality would
be to knock these genes down in X. laevis and again transplant ears from these embryos
into control embryos to either replace the native ear, to the trunk to replace the somite, or
to the orbit to replace the eye.
Inner Ear Sensory Neuron Guidance
The mechanism by which inner ear vestibular afferents project to their specific
targets in the vestibular nuclei is not well known. What is known is that Neurod1 is
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necessary for normal vestibular afferent projection. In mice lacking Neurod1, vestibular
afferents project to cochlear nuclei in addition to vestibular nuclei (Jahan et al., 2010a).
Transplantation of the ear to the orbit resulted in sensory neurons projecting along
existing nerves into the brain (Chapter 4). Sensory neurons from the transplanted ear
projected either along the trigeminal nerve into the hindbrain or along the optic or
oculomotor nerve into the midbrain and forebrain. Of those fibers that projected along
the trigeminal nerve, some were able to reach the vestibular nucleus. However, the
inability of other sensory neurons entering the midbrain to find the vestibular nucleus
indicates the absence of long-range diffusible signals. Based on the differential ability of
afferents to reach vestibular nuclei only upon entering into the hindbrain, it is plausible
that any molecular cues are at most short-ranged. Since all vestibular afferents of
transplanted ‘third’ ears will end up in the vestibular nuclei, no matter their entry point
into the hindbrain alar plate, we can reject the alternative assumption that they reach the
vestibular nucleus by pure chance. If this were the case we would have encountered
many animals with afferents from transplanted ‘third’ ears being associated with fiber
tracts other than the vestibular nucleus.
In animals in which vestibular afferents enter the midbrain, there was no
consistency in sensory neuron projections. This indicates a random interaction of
vestibular afferents with local neurons, indicating axons were unable to re-organize these
neurons to form a ‘vestibular nucleus’. This is in contrast to the olfactory system where
transplantation of olfactory placode results in the ability of olfactory sensory neurons to
form glomeruli in any location in the brain that has been tested (Graziadei et al., 1978,
Graziadei et al., 1979, Graziadei and Samanen, 1980, Magrassi and Graziadei, 1985,
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Morrison and Graziadei, 1996) with the notable exception of the hindbrain. A critical
experiment would be to swap olfactory eptihelia with ears to see how each of these
placodal derived neurons can handle the challenge of interacting with a very different set
of target neurons.
Given the developmental similarities between the ear and the eye, we
hypothesized that the ear may use the same mechanisms as the eye: molecular cues, as
suggested above, and activity-based guidance, to route inner ear afferents to the proper
location in the hindbrain. The inconsistencies in sensory neuron guidance when the ear
was transplanted to the orbit made this difficult to test. Instead, we tested whether
molecular mechanisms and activity-based guidance played a role in guiding inner ear
sensory neurons by transplanting an additional ear immediately rostral to the native ear,
either in the native orientation or rotated by 90 degrees to generate ‘three-eared’ frogs
(Chapter 5). Transplanting the ear rostral to the native ear puts the transplanted ear in the
territory of any potential molecular cues that may be guiding native inner ear sensory
neurons to the vestibular nucleus as the vestibular nucleus extends through all
rhombomeres and continuous into the cerebellum as much as the vestibular afferents do.
Furthermore, the addition of an ear rostral to the native ear would force two ears to the
same side hindbrain, much like two eyes either normally, or experimentally (ConstantinePaton and Law, 1978), project to the same superior colliculus/tectum. This allows us to
determine whether the sensory neurons are guided to the same territory as the native
sensory neurons, indicating a molecular mechanism. By transplanting the ears either in
the native orientation or rotated by 90 degrees allows us to determine whether activitybased mechanisms play a role in fiber competition as well. The rationale is that when
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rotated 90 degrees, the sensory epithelia would not be aligned between the two ears in
tandem and they would respond differently to the same stimulus, essentially setting up a
differential activity scenario. The transplanted ears in either orientation projected to the
same approximate area of the hindbrain as the native ear (Chapter 5), suggesting
molecular cues play a role in axon guidance in the vestibular system. In addition, when
the transplanted ear was in alignment with the native ear, there was nearly complete
overlap of sensory axons from the two ears in the vestibular nucleus. In contrast to this,
when the transplanted ear was rotated by 90 degrees compared with the native ear, there
was nearly complete segregation of sensory axons from the two ears in the vestibular
nucleus (Chapter 5), forming ‘vestibular dominance columns’ reminiscent of the ocular
dominance columns found in the visual system naturally (Hubel and Wiesel, 1977,
Wiesel, 1982) and in experimentally generated three eared frogs (Constantine-Paton and
Law, 1978, Springer and Cohen, 1981, Constantine-Paton, 1982, Easter, 1983). These
data suggest that in addition to molecular mechanisms, activity also plays a role in axon
guidance in the vestibular system.
The addition of an additional ear, rostral to the native ear, in a sense, recapitulates
ear evolution. It appears that ear evolution starts with the duplication of a sensory
epithelia, followed by further spatial segregation and diversification (Duncan and
Fritzsch, 2012). In this experiment, we have effectively duplicated the sensory epithelia
of the ear by adding a second ear. Early studies proposed that a common placode gave
rise to the inner ear and lateral line and that neurons from these tissues project to the
same nuclei in the hindbrain (Duncan and Fritzsch, 2012). However, it is now known
that not only do the inner ear and lateral line originate from their own placode (Schlosser,
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2010), but they project to molecularly-distinct regions within the hindbrain (Fritzsch et
al., 2006, Duncan and Fritzsch, 2012). Furthermore, neurons of the trigeminal and
solitary tract also project to molecularly-specified regions within the hindbrain (Qian et
al., 2001). It has been shown that hindbrain nuclei boundaries are established by
differential bHLH gene expression (Fritzsch et al., 2006) and that axons from the various
sensory systems project to these molecularly-defined regions from the start (Fritzsch et
al., 2005a), indicating that sensory afferents are guided to these regions, possibly by
reading molecular cues from the brain. The same bHLH genes that regulate neuronal and
hair cell development, Neurog1 and Atoh1, establish boundaries for the vestibular and
auditory nuclei, respectively (Fritzsch et al., 2006). Thus the genes that are important for
inner ear neurosensory development are again utilized for axon targeting in the brain.
Conditional deletion of Neurod1, a bHLH gene downstream of Neurog1, resulted in
vestibular and cochlear sensory afferents projecting to both vestibular and cochlear nuclei
(Jahan et al., 2010a).
While all vestibular neurons project entirely to the vestibular nuclei, within the
vestibular nuclei, there is a partial overlap in projections from the different sensory
epithelia (Birinyi et al., 2001, Maklad and Fritzsch, 2002, Straka et al., 2002). However,
it is thought that the partial overlap functions to integrate semicircular canal input with
that of the utricle and/or saccule for any given movement (Büttner-Ennever, 1992,
Maklad and Fritzsch, 2002, Straka et al., 2002, Maklad and Fritzsch, 2003, Newlands and
Perachio, 2003). While overlap exists in the vestibular nuclei, it is not a complete
overlap, as some vestibular neurons innervating the various sensory epithelia partially
segregate (Kevetter and Perachio, 1986, Maklad and Fritzsch, 1999, 2003), likely due to
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differential activities set up by the perceptions of different movements. That both the
transplanted and native ears project to the same approximate area suggests that during the
evolution of a new inner ear sensory epithelia, molecular cues initially guide the sensory
afferents to the same area of the brain. However, once a duplicated sensory epithelia has
acquired a novel function, for example the perception of linear acceleration in an
different direction upon formation of a perpendicular gravistatic sensory epithelia or
sound perception by the basilar papilla upon loss of otoconia from a gravistatic function,
the sensory afferents from the new inner ear epithelia project to a unique location in the
brain within the molecularly-defined boundary for those neurons (Duncan and Fritzsch,
2012, Fritzsch and Straka, 2013). Inner ear sensory afferents apparently are guided to the
vestibular area which forms a likely molecularly defined space that captures all vestibular
afferents. The transplanted and rotated ears mimic the diversification of a newly evolved
sensory epithelia, in this case perception in a different orientation. The segregation of
afferents from these rotated ears from the native ears into ‘vestibular dominance
columns’ should logically mimic similar processes when additional epithelia form. In
other words, I suggest that as sensory epithelia diversified in evolution to have unique
perceptions, it is the differential activity in these epithelia that drove the afferents to
project to unique locations within the molecularly-specified vestibular region.
The formation of ‘vestibular dominance columns’ is comparable to ocular
dominance columns. In the retina projection of the eye, the formation of ocular
dominance columns is a compromise between the molecularly-specified targeting of each
eye to a specific location in the tectum/superior colliculus (Clandinin and Feldheim,
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2009) and the differences in retinal activity between the two eyes (Hubel and Wiesel,
1977, Wiesel, 1982).
In contrast to the ear, for the retina projection, the molecular basis is well
understood. In the eye, concentration gradients of EphA and ephrin-A along the nasaltemporal axis of the retina and anterior-posterior axis of the superior colliculus/tectum
guide retinal ganglion axons to the appropriate region of the CNS (Clandinin and
Feldheim, 2009). Given that Ephs and ephrins have been shown to play some role in
axon guidance in the auditory system (Allen-Sharpley and Cramer, 2012, Allen-Sharpley
et al., 2013, Defourny et al., 2013) and that Ephs and ephrins are expressed in vestibular
neurons and their peripheral vestibular hair cell targets (Bianchi and Liu, 1999, AllenSharpley et al., 2013), suggests that Ephs and ephrins may play a role in guiding sensory
afferents to the proper location centrally in the vestibular nucleus. It has recently been
shown that expression of different Eph classes in auditory and vestibular neurons
conbributes to boundary formation in the hindbrain for the two populations of neurons
(Allen-Sharpley et al., 2013), but whether or how Ephs and ephrins play a role in
vesbibular axon pathfinding centrally has not been established. Future directions would
be to determine the specific Ephs and ephrin(s) expressed in the frog vestibular nucleus.
Likely candidates would be those of the three ephrin-B ligands or ephrin-A5 since
vestibular axons have been shown to express the EphB2 receptor (Himanen et al., 2004,
Siddiqui and Cramer, 2005, Allen-Sharpley et al., 2013). Of these ligands, ephrin-B2 has
been shown to be expressed in auditory and vestibular nuclei in mice (Liebl et al., 2003).
Since misexpression of EphA4 also affected the boundary of the vestibular nucleus
(Allen-Sharpley et al., 2013), ephrin-A ligands may also guide vestibular axons and their
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expression centrally would also be determined. The ephrin(s) expressed in the vestibular
nucleus would be knocked down or misexpressed in one half of a X. laevis embryo and
the axon targeting of the vestibular nuclei by vestibular sensory axons on the manipulated
side would be compared with the vestibular axon targeting of the vestibular nuclei on the
unaltered side to determine whether Ephs and ephrins play a role in neuronal guidance in
the vestibular system as in the auditory system (Allen-Sharpley and Cramer, 2012).
Initial targeting by vestibular afferents is thought to be molecular since initial
observations of vestibular afferent targeting were made prior to the onset of vestibular
sensation (Maklad and Fritzsch, 2002, Maklad and Fritzsch, 2003, Maklad et al., 2010).
This would logically imply that the activity-based guidance refines the initially set-up
molecular-based guidance. Support comes from observed changes in the pattern of
vestibular nucleus connectivity around the onset of a functional vestibular system
(Maklad and Fritzsch, 2003). This order of axon guidance could be experimentally
demonstrated using ears transplanted from X. laevis embryos previously injected with tau
GFP mRNA (Brand, 1995) or tdTomato mRNA (Blackiston and Levin, 2013). Otic
placodes from embryos injected with tau GFP and otic placodes from embryos injected
with tdTomato would be transplanted in tandem into an uninjected host, either both in the
native orientation, or one rotated by 90 degrees from the other. The daily growth and
projection of the sensory afferents could be monitored with confocal microscopy. I
would expect to see afferents from the two ears projecting to the same area initially in
both conditions, but only in those with an ear rotated by 90 degrees, over time see
segregation of the axons.
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The ability of ‘three-eared’ frogs to swim normally depended upon the
manipulation. Animals in which the transplanted ear was in alignment swam similar to
control, unmanipulated, animals most of the time. In contrast, animals in which the
transplanted ear was rotated by 90 degrees displayed more aberrant swimming, similar to
that of animals with only one ear (Chapter 5). Given that the latter manipulations results
in asymmetrical and mismatched gravitational sensation suggests that bilateral symmetry
in sensory epithelia orientation is required for proper sensing of the animals’ orientation
to proper guide its swimming. A proposed mechanism for how the specific ear
manipulations leads to the swimming behavior observed will be discussed in the last
section.
Hindbrain Neurons
It has long been known that ablation of the inner ear results in a reduction in the
number and volume of neurons in the cochlear nucleus (Levi-Montalcini, 1949, Parks,
1979, Ryugo and Parks, 2003) at the time point at which afferent activity could be
detected (Jackson et al., 1982). It has been shown that activity is necessary for neuronal
survival (Pasic and Rubel, 1989, Sie and Rubel, 1992) but only for a certain critical
period, after which there is little to no reduction in the number and volume of cochlear
nucleus neurons following ear ablation (Born and Rubel, 1985, Tierney et al., 1997,
Mostafapour et al., 2000). Rather than focus on an entire population of neurons as was
done in these studies, we instead looked at the effect of ear removal on a single target
neuron, the Mauthner cell. The Mauthner cell is one of the first cells to become postmitotic at stage 10-12 in Xenopus laevis (Vargas-Lizardi and Lyser, 1974), several stages
before the onset of ear development (Schlosser, 2010). Previous data had shown that ear
144
removal results in the occasional loss of the Mauthner cell, indicating a dependence on
the ear for cell survival after its initial development. In surviving Mauthner cells, a
decrease in dendritic branching was observed following ear removal (Piatt, 1969,
Kimmel et al., 1977, Kimmel, 1982, Goodman and Model, 1988, Fritzsch, 1990).
However, these studies only looked at a few time points. From the few time points
available, we hypothesized that we would see a critical period of dependence upon the
ear for Mauthner cell survival. By using two independent methods to label neurons,
Dextran amine dye tracers and immunohistochemistry, we have shown that the Mauthner
cell is dependent upon the presence of the ear for its development and/or survival, but
only for a critical time in development (Chapter 6). The earlier in development that ear is
removed, the more likely the Mauthner cell does not survive. The Mauthner cell was
always present after stage 36, indicating that by stage 36, the critical period of
dependence on the ear for survival has ended. Stage 36 corresponds with the stage at
which afferents from the ear were observed projecting into the hindbrain of axolotls
(Fritzsch et al., 2005a). The exact nature of the early support provided by the ear to the
Mauthner cell for survival and/or maintenance is unknown. Loss of an ear prior to any
vestibular sensory neuron outgrowth often results in the loss of a Mauthner cell. This
suggests that a short-range diffusible factor released by the ear itself is a possible
candidate. Future directions would be to determine the nature of this factor promoting
cell survival and/or maintenance. I would look at expression profiles of known shortrange diffusible factors expressed by the inner ear, for example the FGFs. Fgf10 is
nearly exclusively expressed in the otic placode and otic vesicle ranging from stages 23
through 40 in Xenopus laevis (Lea et al., 2009). To test whether FGF10 plays a role in
145
Mauthner cell survival/ and or maintenance, otic placodes would be removed at stages
24-26, when the Mauthner cell was most absent, and beads soaked in FGF10 would be
inserted in its place to determine whether FGF10 can functionally rescue the Mauthner
cell in the absence of the ear.
Not all Mauthner cells are absent following ear ablation, as was observed for
neurons in the cochlear nucleus following ear ablation (Levi-Montalcini, 1949, Parks,
1979, Ryugo and Parks, 2003). Perhaps, as has been proposed for neurons in the
cochlear nucleus (Garden et al., 1994, Hyde and Durham, 1994a), both the apoptotic and
survival pathways are initiated in the Mauthner cell and whether the cell survives or not
depends upon which pathway overtakes the other. The Mauthner cell provides a unique
opportunity to study initiation of these pathways in a single cell that could potentially be
applied to any neuron. Future directions would be to look at markers for
survival/apoptosis, such as activated Akt or cleaved caspace-3 (Ferrer and Planas, 2003,
Zhang et al., 2011), in Mauthner cells following ear removal at the early placodal stages
(stages 24-26), when Mauthner cell absence was high, and at early otic vesicle stages
(stages 27-30) when there was more Mauthner cell survival. Both of these stages are
within the critical period of dependence of the ear for cell survival. Following ear
removal, groups of animals will be observed daily for survival or apoptotic pathway
presence in the Mauthner cell. I would expect to see initial upregulation of both
apoptotic and survival pathway markers and over time, more apoptotic in stages 24-26
removal, more survival in stages 27-30 removal.
For animals in which the Mauthner cell survived following ear removal, the
dendritic branching on the ablated side was always less than the dendritic branching on
146
the unoperated side. Furthermore, in all cases in which an extra ear was transplanted
rostral to the native ear, there was increased dendritic branching on the side with an extra
ear compared with the unoperated side. Together, these data suggest that the degree of
dendritic branching is related to the number of presynaptic sensory afferents, supporting
the few earlier findings (Kimmel et al., 1977, Kimmel, 1982, Goodman and Model, 1988,
Fritzsch, 1990). The timing of ear removal may be relevant. The earlier the ear was
removed, the fewer dendritic branches were present, suggesting that the degree of
dendritic development depends upon the time at which the ear was removed. While there
was no significant difference between the three stages of ear removal when comparing
the difference between left and right Mauthner cells following ear removal, there was a
trend in which the number of dendritic branches was slightly higher when the ear was
removed at later otic vesicle stages (stages 31-40) compared with early otic vesicle stages
(stages 27-30), and early otic vesicle stages (stages 27-30) being slightly higher than
when the ear was removed at placodal stages (stages 24-26). The dendritic branching in
the latter two groups occurs prior to the formation of the otic ganglion in stage 31
(Nieuwkoop and Faber, 1994). That there were slightly more dendritic branches in the
earlier otic vesicle stages (stages 27-30) than in the otic placode stages (stages 24-26)
may indicate the presence of other mechanisms for dendritic development in addition to
direct innervation from the presynaptic sensory afferents. The development of dendrites
in general in these two groups in the complete absence of any innervation could support
the presence of additional mechanisms or alternatively suggest that there is some
autonomous development of dendritic branches toward the future sensory neurons, but
147
that contacts with the neurons are necessary for further development and maintenance of
the dendritic branches.
The increase in dendritic branching following the addition of an extra ear rostral
to the native ear was also proposed in axolotls (Goodman and Model, 1988), though the
authors did not quantify the exact increase in dendritic branching. Using
neuroanatomical techniques we quantify this here for the first time and show that indeed
by all measures an extra ear causes an increased lateral dendrite in Mauthner cells. The
increase in Mauthner cell dendritic branching on the side with an extra ear indicates that
additional sensory neurons entering the hindbrain from the transplanted ear stimulate the
growth of additional dendritic branches. That the number of dendritic branches on the
side with the extra ear was not double the number on the control side suggests that an
upper limit of dendritic branching may occur. Future directions would be to transplant
ears both rostral and caudal to the native ear, thus further increasing the number of
sensory afferents from the current study, and determining the percent increase in the
number of dendritic branches of the ipsilateral Mauthner cell compared to the
contralateral control Mauthner cell. If the percent increase is similar, then it would
indicate there is a maximum increase in the number of dendritic branches possible for a
given Mauthner cell. Alternatively, molecular knockdown of neuronal development
could be achieved through inhibition of Neurod1 function, thereby generating ears with a
continuous set of reduced afferents to establish quantitative relationships with the
Mauthner cell dendrites beyond our data.
148
Proposed Mechanism
As mentioned previously, afferents from three-eared frogs are guided to their
proper locations using both molecular and activity-based mechanisms. Projections into
the hindbrain from the two ears guide the swimming of the tadpoles, such that when the
transplanted ear is in alignment with the native ear, afferents from both ears overlap in
the vestibular nucleus and the animal swims relatively normally. In contrast, when the
transplanted ear is rotated by 90 degrees from the native ear, afferents from both ears
segregate in the vestibular nucleus and the animal shows more aberrant swimming
behaviors.
Furthermore, we have shown that the dendritic branching of a target neuron of
inner ear afferents, the Mauthner cell, is increased with the addition of an ear. Blocking
afferent activity has been shown not to affect dendritic branching in amphibians
(Goodman and Model, 1990), therefore the dendritic branching pattern is independent of
the orientation of the ear and should be similarly increased for animals with both
normally-orientated or rotated transplanted ears.
Thus, we propose that the molecular cues guide the inner ear sensory neurons to
target neurons (Fig. 7.1A). The increased dendritic branching from the additional ear
allows for contacts from both ears (Figs. 7.1B and 7.1C). When the ears are in alignment
with each other, the incoming signal from the ears would be the same and the cell would
respond (Fig. 7.1B); however, when the transplanted ear is rotated, the incoming signals
would likely be in opposition, essentially cancelling each other out (Fig. 7.1C). Thus, an
additional but rotated ear would have the same effect as having no ear on one side and
149
would explain the aberrant behavior seen similarly for both three-eared frogs with a
rotated transplanted ear and one-eared frogs.
Future directions would be to test this model by determining whether ear
manipulations determine the initial direction of movement following low-level startle
stimulation using a high speed camera. The Mauthner cell is an important component of
the C-start escape response (Korn and Faber, 2005). Inner ear vestibular and auditory
neurons of premetamorphic amphibians and fish form synapses on the lateral dendrite of
the ipsilateral Mauthner cell, which in turn synapses on contralateral spinal motor
neurons to activate the C-start escape response (Korn and Faber, 2005, Sillar, 2009). We
would expect to see that in control animals, there is no bias in the initial direction of the
startle response since the incoming signal to the Mauthner cell and its dendritic branching
should be symmetrical. In animals in which the ear was removed, generating one-eared
animals, there should be a bias in turning toward the ablated side. This would be a result
of the contralateral Mauthner cell firing due to the absence of signal reaching the
ipsilateral Mauthner cell. In animals in which the transplanted ear is in alignment with
the native ear, we would expect that if there is a bias in direction, it would be in the
direction opposite the transplanted ear since the ipsilateral Mauthner cell should have
increased dendritic branching and may have a slightly higher tendency to fire than the
contralateral Mauthner cell. In contrast, in animals in which the transplanted ear is
rotated by 90 degrees, if the incoming signals cancel each other out, than like the oneeared animals, the initial direction should be a bias toward the ablated side.
150
Figure 7.1. Proposed mechanism to guide swimming behavior in three-eared frogs.
(A) In normal, 2-eared embryos, molecular cues guide the inner ear sensory neurons to
target neurons. (B-C) The increased dendritic branching from the additional ear allows
for contacts from both ears. (B) The transplanted ear (green) is in alignment with the
native ear (red) and sensory neurons overlap. When the ears are in alignment with each
other, the incoming signal from the ears would be the same and the cell would respond.
(C) The transplanted ear (green) is rotated 90 degrees with respect to the native ear (red)
and sensory neurons segregate, but still contact dendrites of the same cell. When the
transplanted ear is rotated, the incoming signals would likely be in opposition, essentially
cancelling each other out. Thus, an additional but rotated ear would have the same effect
as having no ear on one side.
151
152
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