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Visual System
Development in
Vertebrates
Introductory article
Article Contents
. Introduction
. The Retina
. The Retinal Axon Pathway
Eloisa Herrera, Instituto de Neurociencias de Alicante, CSIC-UMH, Alicante, Spain
Lynda Erskine, University of Aberdeen, Aberdeen, Scotland, UK
Online posting date: 20th September 2013
Based in part on the previous version of this eLS article ‘Visual System
Development in Vertebrates’ (2005) by Karl G Johnson, Derryck
Shewan and Christine E Holt.
The development of a functional visual system involves a
complex series of inductive and signalling interactions
essential for the formation of the eye and its central
connections with the brain. Outpouchings from the
forebrain, together with the overlying surface ectoderm
and neural crest cells give rise to the major structures of
the eye (neural retina, pigmented epithelium, lens and
cornea). Central connections between the eye and brain
regions that receive direct connections from the eye
(visual targets) are formed by the axons of retinal ganglion cells. Attractive and repulsive cues in the extracellular
environment guide the retinal axon along specific pathways in the brain. Gradients of signalling molecules,
together with spontaneous neural activity, drive a pointto-point mapping of retinal axons in visual targets,
ensuring accurate reconstruction of the visual image. The
cellular, molecular and inductive mechanisms that sculpt
each of these key developmental processes essential for
normal visual system development are beginning to be
understood.
Introduction
The ability to see accurately and to interpret the external
environment confers an enormous selective advantage on
virtually all motile organisms. As a consequence, after the
evolution of pigments capable of being excited by light,
functional visual systems have evolved using a wide variety
of light-bending and light-detecting structures. Although,
many structural differences exist between the mature eyes
eLS subject area: Developmental Biology
How to cite:
Herrera, Eloisa; and Erskine, Lynda (September 2013) Visual System
Development in Vertebrates. In: eLS. John Wiley & Sons, Ltd:
Chichester.
DOI: 10.1002/9780470015902.a0000789.pub3
of different vertebrate species, the developmental
mechanisms that mould them are well conserved. These
include the development of the neural retina from the
anterior neural plate, the induction of the lens from the
ectoderm overlying the optic vesicle, the navigation of
axons from the retina to postsynaptic neurons and the final
refinement process to form precise maps in target regions in
the brain. The focus of this article is on the mechanisms
controlling the development of the eye and its central
connections.
The Retina
Embryonic origin and morphological
development
The mature vertebrate retina detects and relays light signals from the external environment to specific regions of
the brain. It is derived from the neuroepithelium of the
anterior neural tube that bulges laterally (evaginates) soon
after neural tube closure to give rise to balloon-shaped
optic vesicles (Figure 1). The optic vesicles maintain continuity with the neural tube via the optic stalk and continue
to evaginate until they reach the overlying skin ectoderm.
Contact with the surface ectoderm induces the central part
of the optic vesicle to flatten and subsequently fold inwards
(invaginate) to form a double-layered structure, termed the
optic cup. The inner layer of the optic cup becomes the
neural retina and the outer layer forms the pigmented
epithelium that covers the back of the retina. The invagination process eventually brings the neural retina into
contact with the presumptive retinal pigmented epithelium, obliterating the original lumen of the optic vesicle.
The pigment epithelial cells then begin to synthesise large
amounts of melanin, a dark pigment that prevents peripheral light from entering the eye, and sinuous processes
from these cells envelop the photoreceptors. Concurrent
with optic cup formation, the optic stalk diminishes in size
to become a narrow bridge of cells linking the eye to the
ventral diencephalon. This structure provides the scaffold
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Visual System Development in Vertebrates
Lens placode
Neuroepithelium Epithelium
Lens pit
Optic vesicle
Optic vesicle
Optic cup
(b)
(a)
(c)
Pigment epithelium
Retina
Cornea
Lens
Optic nerve
Optic stalk
Rostral
Lens
vesicle
(d)
Medial
(e)
Lateral
Caudal
Figure 1 Development of the vertebrate eye. (a) The lateral neuroepithelium evaginates towards the overlying presumptive lens ectoderm, creating an
optic vesicle. (b) When this bulge contacts the overlying ectoderm, it begins to invaginate, while the overlying ectoderm thickens to form a lens placode.
(c) Progressive invagination of the neuroepithelium generates the optic cup (d) while the invaginating lens placode forms the lens pit. The presumptive lens
tissue eventually buds off from the overlying epithelium to form a lens vesicle, and the neuroepithelium behind the optic cup constricts to form the optic
stalk. (e) Differentiation of the neural retina then takes place, and the pigmented epithelium forms around the retina. Axons from retinal ganglion cells at the
innermost surface of the retina exit the eye on their way to the brain, forming the optic nerve.
along which blood vessels enter the eye and retinal axons
travel out of the eye to establish the optic nerve. See also:
Eye Anatomy; Eye Development: Gene Control
Genetic control of eye development
During the early stages of development, a small subset of
cells in the anterior neural plate becomes fated to form the
retinae. Accumulating evidence indicates that a hierarchy
of genes that are strictly regulated in space and time controls eye development. The earliest known genes to be
expressed in the eye-forming neuroepithelium include a
number of highly conserved transcription factors, such as
SIX3, PAX6 and RX1. These genes are first expressed in a
discrete region in the most anterior part of the neural plate.
Later, this single expression domain separates into two
bilateral spots that will form the optic vesicles. Disruptions
in the function of these transcription factors cause dramatic retinal defects. For example, mutations in the pax6
gene cause a reduction in eye size, and in severe cases
animals lack eyes altogether. Despite Drosophila and
2
vertebrate eyes being anatomically different, many of the
genes responsible for controlling the development of the
vertebrate eye are also crucial in eye development in the
fruit fly Drosophila. The vertebrate SIX3 gene, for example,
is a homologue of Drosophila sine oculus, and vertebrate
PAX6 is a homologue of fly eyeless.
The separation of one medial eye-field into two lateral
structures appears to depend on Sonic hedgehog signalling
from the mesoderm at the anterior midline, possibly
modulated by a transforming growth factor (TGF)b
pathway. Studies in zebrafish and mice have demonstrated
that the loss of Sonic hedgehog, or the loss of certain
members of the TGFb superfamily, can cause a cyclopic
phenotype, presumably by blocking the separation of the
central eye-field into bilaterally symmetrical spots (Chiang
et al., 1996; Müller et al., 2000). See also: Drosophila Eye
Development and Photoreceptor Specification; Drosophila Retinal Patterning; Eye Development: Gene Control;
Hedgehog Signalling; Inherited Retinal Diseases: Vertebrate Animal Models; Pax Genes: Evolution and Function; Regulation of Neuronal Subtype Identity in the
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Visual System Development in Vertebrates
Vertebrate Neural Tube (Neuronal Subtype Identity
Regulation); Transcription Factors
R
R
R
R
R
R
ONL
Retinal polarity
OPL
At the optic cup stage, signalling molecules secreted from
the patterning centres induce the expression of specific
transcription factors that will establish retinal polarity
along the temporal–nasal (T–N) and dorsal–ventral (D–
V) axes. The winged-helix transcription factors Foxg1 and
Foxd1, expressed along the T–N axis, determine the temporal and the nasal retina, respectively. Soon after T–N
polarity is determined, initial D–V polarity develops
through the actions of morphogens such as Sonic hedgehog
and BMPs. The dorsalising effect of BMP4 appears to be
counteracted by ventroptin, a BMP4 antagonist expressed
in the ventral retina. Although the direct regulatory interactions are largely unknown, ectopic expression of ventroptin in the dorsal retina represses the expression of
BMP4 and the transcription factor Tbx5, both of which are
normally expressed in the dorsal retina, and promotes
expression of Vax2, the major determinant of ventral
retina. See also: Bone Morphogenetic Proteins and Their
Receptors; Transcription Factors
Lamination: formation of retinal layers
The optic vesicle, like the developing neural tube, consists
of a morphologically homogeneous population of columnar epithelial cells. During the morphogenetic changes that
accompany optic cup formation (Figure 1), the cells in the
neural retina divide repeatedly. These proliferating cells
typically extend throughout the entire width of the retina,
with their nuclei migrating from the outer surface (adjacent
to the retinal pigmented epithelium) to the inner (vitreal)
surface and back again during the cell cycle, creating a
pseudostratified epithelium. After several rounds of division, cells begin to exit from the cell cycle and migrate to
their final positions. The order of cell birth dates is well
defined in the mammalian retina, with the different classes
of cells generated in overlapping waves. The first cells to be
born are the retinal ganglion cells (RGCs) that migrate to
the vitreal surface and elaborate a single axon and several
dendrites (Figure 2). The next neurons to be born are the
cones, amacrine cells and horizontal cells, followed by the
rods and bipolar cells. Mueller glia, the only nonneuronal
cells within the neural retina, are the last cells to differentiate from the precursor cell pool. See also: Drosophila
Retinal Patterning; Photoreceptor Cell Development
Regulation; Vertebrate Neurogenesis: Cell Polarity
As these cells continue to divide and differentiate, they
begin to form three layers of cell bodies separated by two
plexiform or synaptic layers. The RGC layer lies at the
vitreal surface of the retina, and the fibre layer comprising
of RGC axons lies superficial to the cell bodies. The inner
plexiform layer separates the RGC layer from cells in the
inner nuclear layer, which contains amacrine, bipolar,
horizontal and Mueller cell bodies, and provides a site for
H
Mu
INL
A
B
I
IPL
GCL
OFL
RGC
To optic
disc
Figure 2 Diagram of a section through the vertebrate neural retina. At the
top are the photoreceptors (R) in the outer nuclear layer (ONL). Below this
is the outer plexiform layer (OPL) where the photoreceptors synapse with
cells in the inner nuclear layer (INL) such as horizontal cells (H) or bipolar
cells (B). In the inner nuclear layer are amacrine cells (A) and Mueller glia
(Mu). The bipolar cells and inner plexiform cells (I) form synaptic
connections with retinal ganglion cells (RGC) in the inner plexiform layer
(IPL). RGCs, which have their cell bodies located in the ganglion cell layer
(GCL), send their axons along the optic fibre layer (OFL) to the optic disc,
where they emerge from the back of the eye in the optic nerve (not
shown). Vitreal surface down. Adapted with permission from Dowling
(1970). & Association for Research in Vision and Ophthalmology.
synaptic connections between the cells in these two layers.
The outer plexiform layer separates the inner nuclear layer
from the outer nuclear layer that contains the photoreceptors. Synaptic connections between the photoreceptors and the bipolar and horizontal cells form in the
outer plexiform layer (Figure 2). Thus, the process of lamination separates the retina into three functionally distinct
but synaptically connected layers of neurons involved in
receiving, integrating and relaying visual information.
However, the mechanisms that segregate the cells into their
appropriate layers remain unknown.
Although often viewed as homogeneous populations,
each retinal cell type has multiple subtypes that perform
distinct functions and make specific patterns of synaptic
connections. Thus, photoreceptors consist of both rods
and cones that differ in their sensitivity to light, morphology and distribution in the retina. Moreover, at least 15
different subtypes of RGCs, as well as multiple types of
amacrine and bipolar cells have been described. Synaptic
connections form between specific subtypes of retinal
interneurons and RGCs, with the connections arranged
within distinct sublaminae of the inner plexiform layer. The
mechanisms controlling the formation of these precise
patterns of synaptic connections are beginning to be
understood.
In the chick retina selective adhesion has been shown to
play an important role. Four highly related adhesion
molecules (Sidekick-1, Sidekick-2, DSCAM and
DSCAML1) are expressed in nonoverlapping subsets of
amacrine and RGCs. These molecules bind to themselves,
but not to other, even highly related, proteins creating a
potential mechanism for the pairing together of appropriate synaptic partners – selective adhesion promotes
pairs of cells expressing the same molecule to interact and
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Visual System Development in Vertebrates
form synapses, whereas cells expressing different proteins
do not. In support of this idea, ectopic expression or loss of
function of any of these molecules impairs synaptic specificity in the retina, in a pattern consistent with the disruption of a homophilic adhesion ‘code’ between presumptive
synaptic pairs (Yamagata and Sanes, 2008). Although an
attractive model to date, this adhesion code has not been
described in any other species. Indeed in mice carrying
mutations in either Dscam or DscamL1 no defects in
synaptic specificity have been found. Instead, the main
function of DSCAM in the murine retina appears to be to
control the regular spacing of neuronal cell bodies and
dendrites, ensuring appropriate distribution of the different cell types over the entire retina (Fuerst et al., 2008;
Fuerst et al., 2009).
The mechanisms that act to constrain the synaptic connections to the plexiform layers and their targeting to
specific sublaminae are also beginning to be elucidated.
During early postnatal stages in mouse, when the synaptic
connections in the retina are beginning to develop, several
inhibitory signalling molecules of the semaphorin family
(Sema5A, Sema5B and Sema6a), are expressed either in the
outer retina bordering the region where the inner plexiform
layer will develop or in specific sublaminae of the inner
plexifrom layer. In the absence of these molecules, many
neurites mistarget resulting in visual function abnormalities (Matsuoka et al., 2011a, 2011b). Thus, inhibitory cues
play an important role in the initial targeting of neurites,
with local adhesive interactions acting subsequently to
control synaptic pairing. See also: Developmental Biology
of Synapse Formation; Semaphorins; Synapse Formation
The vertebrate lens
The vertebrate lens is derived from the ectoderm that
overlies the evaginating optic vesicle (Figure 1). After contact with the optic vesicle, the induced ectoderm thickens to
form the lens placode, invaginates forming the lens cup and
finally pinches off from the surrounding ectoderm forming
the lens vesicle. The lens vesicle initially has a large central
cavity into which cells from the posterior lens vesicle (the
primary lens fibres) elongate. These fibres expand to fill the
cavity and terminally differentiate, losing their mitochondria and nuclei, but maintaining a hard, transparent
cytoplasm consisting almost exclusively of crystallin proteins. The cells on the anterior side of the lens vesicle continue to divide, producing cells that will differentiate into
secondary fibre cells. Eventually, the cells overlying the
primary lens fibres become quiescent, and division is
restricted to a germinative ring surrounding the central
lens. Secondary lens fibres develop continuously in the
germinative ring. These cells differentiate similarly to the
primary fibre cells, forming concentric layers around the
central primary fibres, with the oldest fibres being in the
centre of the lens and the youngest fibres at the periphery.
See also: Regeneration of the Vertebrate Lens and Other
Eye Structures; Synapse Formation
4
The formation of the lens is a classical example of an
inductive interaction. In seminal experiments performed in
the early 1900s, Hans Spemann first demonstrated the
importance of the optic vesicle for lens induction using
tissue ablation approaches in amphibians. Cauterisation of
the developing optic vesicle before contact with the surface
ectoderm resulted in loss of both the eye and the lens.
Moreover, in cases where the ablation was incomplete, and
a portion of the optic vesicle remained, lenses formed only
in those cases where contact occurred between the optic
vesicle remnant and surface ectoderm. These findings
suggested that contact of the optic vesicle with the surface
ectoderm is both necessary and sufficient for lens induction. However, we now know that the situation is actually
more complicated, and direct interaction of the optic
vesicle and lens forming ectoderm is the final step in a series
of sequential inductive interactions that act to restrict head
ectoderm towards a lens fate. See also: Lens Induction;
Spemann, Hans
The vertebrate cornea
Contact with the developing lens triggers the differentiation of the overlying ectoderm into the cornea. This ectoderm forms a multilayered structure with a highly complex
extracellular matrix. As the basal ectodermal cells begin
secreting collagen to form the primary stroma, migrating
neural crest cells arrive at the developing cornea and form
the corneal endothelium. These cells secrete hyaluronic
acid into the extracellular matrix, causing the matrix to
swell and allow a second wave of migrating neural crest
cells to invade the cornea. These cells secrete collagen type 1
and hyaluronidase, initiating the shrinking of the stroma.
The corneal stroma is dehydrated by thyroxine, a hormone
from the thyroid gland, and the collagen-rich extracellular
matrix becomes the transparent cornea. See also: Extracellular Matrix; Neural Crest: Origin, Migration and Differentiation; Vertebrate Embryo: Patterning the Neural
Crest Lineage
The Retinal Axon Pathway
RGC axon guidance in the retina
RGC bodies are situated close to the vitreal surface of the
retina and generate long axons that navigate to the optic
disc and exit the retina through the optic nerve. A variety of
cell adhesion molecules expressed both by RGC axons and
the surrounding neuroepithelial cells and the glial endfeet
combine with permissive extracellular matrix molecules to
support axon growth. But what makes axons set off
towards the optic disc (their exit point form the eye) rather
than growing elsewhere within the retina? In rodents, a
receding ring of a repulsive protein, chondroitin sulphate
proteoglycan (CSPG) coincides with initial axon growth,
starting close to the centre of the retina, and creating an
environment where CSPG is expressed peripheral to the
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Visual System Development in Vertebrates
first-born RGCs and not centrally. Through inhibitory
signalling, this prevents growth towards the retinal periphery, and helps ‘push’ the axons in their correct direction
towards the optic disc (Brittis et al., 1992). CSPGs do not
act alone to repel axons away from the retinal periphery.
Other inhibitory signalling molecules, such as Slit proteins,
are localised at relatively higher concentrations in the retinal periphery and, by repelling axons away from the retinal periphery, help ensure that axons normally grow
centrally from the outset (Thompson et al., 2006). Conversely, relatively higher expression of attractive signalling
molecules, such as Sonic hedgehog, central to the front of
growing axons play an opposite role, and are important for
promoting growth towards the central retina (Kolpak
et al., 2005). Thus, the spatial and temporal localisation
and function of axon guidance cues underlie the RGC axon
guidance towards the optic disc. See also: Axon Growth;
Axon Guidance; Cytoskeleton in Axon Growth; Immunofluorescence; In Situ Hybridization; Knockout and
Knock-in Animals; Specific Neural Connection Formation in the Developing Nervous System; Transgenic Mice
What induces axons to turn away from the retinal surface and into the optic nerve? Netrin-1, a secreted axon
guidance molecule, is expressed in the eye only at the optic
disc. In vitro, retinal axons that express the receptor deleted
in colorectal cancer (Dcc) can be attracted to a gradient of
netrin-1. Mice deficient in Dcc or netrin-1 show optic nerve
hypoplasia, as many axons fail to leave the eye and grow
haphazardly around the optic disc (Deiner et al., 1997). The
chemoattractive effect of netrin-1 on RGC axons depends
on the levels of an important signalling molecule, cyclic
adenosine monophosphate (cAMP), within the axons.
When cAMP levels are high axons turn towards netrin-1,
but if cAMP levels are decreased axons are instead repelled
by netrin-1. Netrin-1 itself can induce an increase in growth
cone cAMP levels, but when laminin-1 is simultaneously
detected by the axons the rise in cAMP levels is prevented,
and under these circumstances RGC axons are repelled by
netrin-1 in vitro. At the optic disc, netrin-1 is expressed all
around the RGC axons, but laminin-1 is only expressed on
the vitreal side. This spatial distribution could lead to
higher cAMP levels within axons on the optic nerve side,
leading the axon to preferentially turn into the optic nerve
(Höpker et al., 1999). See also: Netrins
Development of the optic chiasm
The optic chiasm is the midline site where retinal axons
from each eye meet and undergo a selective crossing process such that visual information from the right eye projects
exclusively to target regions on the left side of the brain,
and information from the left eye goes to the right side of
the brain. It develops at the midline of the ventral diencephalon (developing hypothalamus) where RGC axons
from the two eyes intersect. Developing astrocytes are the
predominant cell type in this region, but their morphology
varies, such that more stellate astrocytes occupy positions
in the lateral region of the optic chiasm, whereas the radial
glia form a dense network immediately surrounding the
midline where future RGC axon divergence will occur.
Differentiating hypothalamic neurons are also present
within the optic chiasm before RGC axon invasion. These
neurons are some of the earliest to differentiate in the
central nervous system, and are arrayed in an inverted ‘V’
shape that demarcates the posterior boundary of the optic
chiasm.
The precise position of the chiasm is conferred, at least in
part, by inhibitory factors, such as the Slit proteins, which
surround the fascicles of axons. By creating barriers to
axon growth, these inhibitory molecules delimit permissive
channels, through which the axons normally grow (Figure
3). In mice or zebrafish lacking Slit proteins or their
Roundabout (Robo) receptors, the retinal axons are no
longer channelled along their correct pathway and can
grow in aberrant directions, including crossing the midline
in ectopic locations (Fricke et al., 2001; Plump et al., 2002).
Indeed, this widespread ‘straying’ of axons from their
normal pathway in the absence of Slit-signalling lead to the
name ‘astray’ being given to zebrafish carrying a mutation
in robo2, the main Slit receptor expressed by retinal axons.
Axon divergence at the optic chiasm
The visual world is segregated into two halves at the optic
chiasm. Stimulation in the left visual field is transmitted to
the right side of the brain, whereas stimulation in the right
visual field is conducted to the left side of the brain. Many
lower vertebrates have largely nonoverlapping visual fields
because their eyes are located on the sides of their heads. In
such organisms, axons from the left eye cross the chiasm
and project exclusively to the right optic tract, whereas
axons from the right eye project into the left optic tract.
Primates, and other mammals and birds with forwardfacing eyes, have predominantly overlapping visual fields:
an object in the left half of the visual world will stimulate
RGCs in the nasal half of the left eye, and in the temporal
half of the right eye. Thus, in order to segregate the visual
world into two halves, axons must separate at the optic
chiasm so that nasal retinal axons project to contralateral
structures, whereas the temporal axons project to ipsilateral structures (Figure 3). The proportion of axons
remaining in the mature ipsilateral optic tract correlates
with the amount of overlap in the visual fields; primate eyes
have large overlapping visual fields and approximately
50% of RGC axons cross at the chiasm, whereas in rodents,
only a small proportion of RGCs in the ventrotemporal
retina send axons to the ipsilateral tract, consistent with the
reduced overlap in their visual field.
The mechanisms that segregate the visual world at the
optic chiasm are beginning to be understood. Ephrin-B, a
ligand for the EphB family of receptor tyrosine kinases, is
expressed by glial cells at the optic chiasm midline and acts
as a repellent signal for ipsilateral RGC axons that express
EphB. In frogs, Ephrin-B is upregulated at the midline of
the chiasm at metamorphosis. At this stage, a population of
ventrotemporal RGCs expressing EphB, begin to project
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Visual System Development in Vertebrates
Retina
Temporal
Nasal
Nasal
-Optic chiasm
---
- ++
+ +
-+ +
- --
Temporal
----
Lateral geniculated nucleus
Superior colliculus
(+)
Contralateral axons: Isl2, Nrp1, NrCam, PlexinA1 and Robo
Ipsilateral axons: Zic2, EphB1 and Robo
Cells sourrounding the optic nerve fascicles: Slits (−)
Midline cells: EphrinB2(−), VEGF-A (+) and NrCam/Sema6D (+)
Attractive signalling
(−)
Repulsive signalling
Figure 3 Molecular mechanisms underlying axonal guidance at the optic chiasm of mice. RGC axons expressing Robo receptors exit the retina via the
optic nerve. Diffusible Slit molecules delimit a repulsion-free corridor that demarcates the point where the optic chiasm must form. Axons from the temporal
retina (blue lines) that express the EphB1 receptor (induced by the transcription factor Zic2) are repelled by ephrin-B2 that is expressed by glial cells at the
midline. As a consequence of EphB1/ephrin-B2 interaction, ipsilateral axons turn to project to targets in the same side. Contralateral axons (red lines) do not
express EphB1 and ignore Ephrin-B2. Instead, because they express Neuropilin1 (Nrp1) they are attracted by VEGF-A expressed at the midline. Additionally,
contralateral but not ipsilateral axons express NrCAM and PlexinA1. PlexinA1, NrCAM and Sema6D are also expressed at the midline and interact to help
promote midline crossing. Contralateral RGCs express the transcription factor Isl2, although a link between this transcription factor and expression of the
crossing axon guidance molecules remains to be established.
ipsilaterally, whereas all axons in premetamorphic
embryos project contralaterally. If metamorphic RGCs
from the ventrotemporal (i.e., ipsilateral-projecting) retina
are transplanted into embryonic eyes, their axons now
cross the chiasm. Furthermore, precocious expression of
Ephrin-B at the embryonic chiasm induces premetamorphic axons to be deflected at the midline (Nakagawa et al., 2000). Similarly, mice in which the gene for
EphB1 (expressed specifically by ipsilateral RGCs) has
been knocked out have a reduced ipsilateral projection,
and when Ephrin-B2 signalling is blocked at the midline of
the optic chiasm the ipsilateral projection does not develop
(Williams et al., 2003). In general, species with binocular
vision express ephrin-B at the chiasm whereas animals
6
without binocularity lack ephrin-B at the chiasm. See also:
Ephrins
The expression of EphB1 in ipsilaterally projecting
RGCs is controlled by the zinc finger transcription factor,
Zic2 (Figure 3). Zic2 is expressed in ipsilateral but not in
contralateral RGCs. In mice carrying a mutation in this
transcription factor the ipsilateral projection does not
develop properly. Conversely, ectopic expression of Zic2 in
the RGC that normally cross the midline switches the
laterality of their axons to project ipsilaterally (Herrera
et al., 2003; Garcia-Frigola et al., 2008). Zic2 expression in
ventrotemporal retina is conserved both in mammals and
amphibians, precisely mirroring the extent of binocularity.
Thus, Zic2/EphB/ephrin-B-based axon sorting at the
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Visual System Development in Vertebrates
chiasm appears to have been conserved through evolution,
suggesting that it plays a key role in the decision not to
cross the midline. See also: Albinism: Genetics; Electroporation; Transcription Factors
More puzzling has been the mechanisms that induce
contralateral axons to grow towards and through the
chiasm midline. Recently, however, the first growth-promoting factor essential for contralateral growth at the
chiasm midline was identified. Surprisingly, this factor was
found to be vascular endothelial growth factor (VEGF)-A,
a molecule best known for its role in blood vessel development, and not shown previously to play a physiological
role in axon outgrowth. VEGF-A is expressed at the
chiasm midline and, independent of its role in blood vessels, provides growth-promoting and chemoattractive
signals to contralateral retinal axons that express one of its
known receptors, neuropilin-1 (Figure 3). In mice carrying a
mutation in neuropilin-1 or a form of VEGF-A that cannot
signal through neuropilin-1, many axons that would normally project contralaterally fail to cross the midline
(Erskine et al., 2011). However, it is unclear currently if the
role of VEGF-A in contralateral retinal axon growth is
conserved among species. Although neuropilin-1 has been
found to play an important role in ensuring contralateral
growth at the optic chiasm in zebrafish, the identified
ligand in this case is a member of the class 3 semaphorin
family of inhibitory signalling molecules (Sakai and Halloran, 2006). Whether VEGF-A also acts in this system, or
indeed any species other than mice, has not yet been
investigated.
A second growth-promoting mechanism, involving a
transmembrane semaphorin, Sema6D, has also been
identified recently. In mice, Sema6D is expressed at the
chiasm midline and in combination with PlexinA1 and
NrCAM, which are expressed both on the retinal axons
and in their environment, generates an attractive signalling
complex that may act in concert with VEGF-A to promote
contralateral growth (Kuwajima et al., 2012; Figure 3).
See also: Axon Guidance at the Midline
RGC targets in the brain: the lateral
geniculate nucleus (LGN) and the superior
colliculus (SC)
After leaving the optic chiasm, and entering either the
ipsilateral or contralateral optic tract, retinal axons project
to multiple targets in the thalamus and midbrain. In lower
vertebrates, the majority of axons terminate in the optic
tectum of the midbrain. In mammals, retinal axons project
to two main targets, the SC, the tectum homologue and the
LGN in the thalamus (Figure 3).
Topographical mapping of retinal axons at
the visual targets
The visual image projected on to the retina is inverted,
owing to the shape and refractive properties of the lens. The
inverted retinal image is reinverted when transmitted to the
brain, without sacrificing detailed or positional information. Each RGC axon terminates in a position within the
targets that correlates precisely to the position of its
ganglion cell body in the retina relative to its neighbouring
cells. In this way, axons originating in the most nasal retina
project to the most posterior areas in the targets, whereas
those originating more temporally terminate progressively
more anteriorly (Figure 4). In addition, axons from the
dorsal retina innervate the lateral targets, and axons from
the ventral retina innervate the medial targets. Thus, the
entire retinal image is mapped point-to-point on to the
targets. This form of organisation, termed topographical
mapping, is the most parsimonious way of transferring
spatially structured information from one group of neurons to the next. See also: Topographic Maps in the Brain
To establish a specific and accurate topographical map,
mechanisms must exist to provide each axon with positional
information relative to its neighbours. Roger Sperry first
proposed the presence of ‘cytochemical tags’ after his classical experiments in the 1940s. Sperry detached and rotated
frog’s eyes by 1808, and analysed the effects on their behaviour. After regeneration, the frogs behaved as though their
visual maps had been inverted; leaping backwards for lures
presented ahead of them, and diving down for lures presented above them. This suggested to Sperry that a given
location in the retina connected to a given location in the
tectum. A stimulus presented above the frog will stimulate
ventral RGCs. However, after eye inversion, these ventral
RGCs bear the ‘cytochemical tag’ of a dorsal RGC, and
connect to the brain as a dorsal RGC would. Thus, the frog
interprets a stimulus above as a stimulus below and dives in
the wrong direction. See also: Sperry, Roger Wolcott
The molecular nature of the ‘cytochemical tags’ predicted by Sperry are being unravelled. Gradients of ligands
that guide axon growth have been identified in the LGN
and the SC, whereas complementary gradients of receptors
for these ligands are expressed by RGC axons (Figure 4). In
particular, Ephrin-A ligands of the highly conserved EphA
family of receptor tyrosine kinases exist in the visual targets. In the SC ephrin-A expression is highest in the posterior areas and progressively decreases more anteriorly. In
the retina, EphA receptors are more highly expressed in the
most temporal retina and decrease towards the nasal
retina. Ephrin-A ligands induce a concentration-dependent response by RGC axons, such that axon growth is
inhibited by higher concentrations of ligand, but axon
growth is supported at lower concentrations. Axon
responses thus depend on RGC position in the retina, given
the gradients of receptor expression. Thus, temporal axons
that innervate the anterior colliculus express more receptor
and require less ligand to cause termination in the target
tissue. Axons originating more nasally in the retina express
fewer receptors, and therefore require a higher level of
ligand to cause termination. Consequently, more nasal
axons grow through to the posterior SC where there are
higher ligand concentrations, whereas temporal axons are
kept out of the posterior SC by an increasing gradient of
repulsive molecules. Similar rules apply to the LGN.
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Visual System Development in Vertebrates
EphA
Dorsal
Temporal
EphB
Nasal
Ventral
Anterior
Medial
Lateral
Posterior
EphrinA
EphrinB
Figure 4 Topographical mapping of retinal axons on to the SC. The retinocollicular connection is highly organised, such that axons from the neighbouring
neurons in the retina terminate in neighbouring positions in the SC. Axons originating in the nasal retina terminate in the caudal SC, whereas temporal RGCs
send axons to the rostral colliculus. In addition, axons originating in the dorsal retina map to the lateral SC, whereas ventral RGC axons terminate in the
medial SC. Retinocollicular mapping is mediated by reciprocal molecular gradients in the retina and SC. EphA receptors are expressed by RGCs in an
increasing nasal-temporal gradient. Nasal neurons express few EphA receptors, whereas neurons located more temporally express progressively more
receptors. Ephrin-A ligands, which inhibit RGC elongation, are expressed in the SC in an increasing anterior–posterior gradient. Nasal RGC axons project
further into the SC because they express fewer EphA receptors and are subsequently less sensitive to the repulsive Ephrin-A ligands. Conversely, the axons of
temporal RGCs invade only a short distance into the SC because of their high sensitivity to Ephrin-A ligands. In the dorsoventral axis, a gradient of EphB
receptors exists in the retina with highest expression ventrally, while a gradient of Ephrin-B, highest medially, is expressed in the colliculus. In this case,
however, EphB/ephrin-B signalling seems to mediate attraction.
The reciprocal expression of ligands and receptors in the
retina and colliculus also contributes to topographical
mapping in the dorsal–ventral axis, but in this case pathfinding is regulated by graded attraction of retinal axons.
In frogs, EphB1-expressing cells in the ventral tectum
appear to attract Ephrin-B expressing axons from the
dorsal retina. In mice EphB-expressing axons from the
ventral retina project to medial collicular areas that express
high levels of Ephrin-B1 ligands. In cooperation with the
molecular organisation underlying the anterior–posterior
axon targeting, this three-dimensional spatiotemporal
regulation of Eph/ephrin, together with other guidance
8
molecules, provides an extremely accurate mechanism for
the topographic mapping of RGC axons to their target
tissues in the brain. See also: Ephrins
The graded expression of EphAs and EphBs along the T–
N and D–V retinal axis required for topographic mapping
results from the early events that determine retinal polarity.
For instance, the expression of the transcription factors
Foxg1 and Foxd1 along the T–N axis early in the development is essential for the later gradual expression
of retinal EphA/ephrinAs. Similarly, early expression
of Vax2 or Tbx5 is critical for determining a correct
EphB/ephrin-Bs retinal gradient (McLaughlin et al., 2003a).
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Visual System Development in Vertebrates
Further refinement of the retinotopic map before eye
opening is thought to require spontaneous electrical
activity within the RGCs themselves. RGCs in the developing retina, even before the formation of functional
photoreceptors, exhibit spontaneous activity. This activity,
in the form of retinal waves, are spontaneous bursts of
action potentials that originate in one specific subtype of
amacrine cells, the starburst amacrine cells, and propagate
in a wave-like fashion across the RGC layer, such that the
neighbouring RGCs fire nearly synchronously. Genetic or
Retina
Optic chiasm
LGN
Primary visual
cortex
(a)
Ocular dominance
columns
Blobs
C
Layers
I
1
2
3
C
I
4A
4B
4C
4C
5
6
Orientation
columns
(c)
5I
3I
6C 2I
4C From
1C
3I 4C
5I
6C
2I
LGN
1C
(b)
Figure 5 Diagram of the retinogeniculocortical projection in primates. (a) An outline of the pathway, going from the retina (top) through the optic chiasm
to the lateral geniculate nucleus (LGN). Second relay neurons in the LGN project to layer 4 of the primary visual cortex, maintaining a rough topographical
map of the visual world. (b) Projections from the retina to the LGN. Retinal ganglion cells project to eye-specific layers within the LGN. Axons from the nasal
half of the contralateral eye (C) project to layers 1C, 4C and 6C, whereas temporal axons from the ipsilateral eye project to layers 2I, 3I and 5I. (c) Structure
of hypercolumns in the primary visual cortex. LGN inputs arrive in layer 4, where they are segregated into ocular dominance columns, blobs and
orientation-selective columns. Adapted from Kandel et al. (1991). & McGraw-Hill.
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9
Visual System Development in Vertebrates
pharmacological blockage of retinal waves produces a
poorly defined retinotopic map at the targets (McLaughlin
et al., 2003b; Huberman et al., 2008). See also: Neural
Activity and the Development of Brain Circuits
Eye-specific layering of retinal axons at the
visual targets
In addition to receiving retinal input in a topographic
manner, the main visual targets are innervated in an eyespecific fashion in mammals. The LGN and SC are formed
by layers that receive input from each eye (see Figure 5a).
The right targets receive input from the nasal half of the left
retina and the temporal half of the right retina, whereas the
left targets receive input from the nasal half of the right
retina and temporal half of the left retina. Each layer of the
mature targets contains positionally mapped input from
one eye, such that the adjacent layers receive input from
opposite eyes. RGCs responsive to topographically identical coordinates in the visual field project to precisely the
same location in the LGN, but in different eye-specific
layers (Figure 5). See also: Brain Imaging: Localization of
Brain Functions
This layer specificity is not established from the beginning. Early in development ganglion cell axons from each
eye intermingle, with separation mediated by activitydependent mechanisms. Inhibition of retinal waves prevents the formation of eye-specific layers in the targets.
Similarly, if one eye is removed early in development,
axons from the remaining eye will invade the layer corresponding to the other eye. Thus, patterned activity occurring independently in each eye drives the layer-specific
segregation of axons via a competitive mechanism
(Huberman, 2007). Ipsilateral but not contralateral axons
express the serotonin transporter (Sert) in a Zic2-dependent manner. In the absence of Sert eye-specific refinement
is perturbed, resembling the phenotype observed when
retinal waves are blocked (Garcia-Frigola and Herrera,
2010). It is unclear, however, whether and how the serotonin pathway interacts with spontaneous activity to
shape eye-specific layering. See also: Neural Activity and
the Development of Brain Circuits
The primary visual cortex: the main visual
processing centre
The second relay neurons in the LGN that receive synaptic
input from the retina send their axons to layer 4 of the
ipsilateral primary visual cortex, maintaining a rough
topographical map (Figure 5). Thalamocortical topography
is also established by guidance molecules such as EphrinAs that are expressed in a rostral-low to caudal-high gradient within the ventral telencephalon and EphA receptors
that are expressed in a complementary gradient within the
dorsal thalamus, rostromedial-high to caudolateral-low.
Interactions between ephrin-A ligands and EphA receptors
are essential to establish the initial topographic
10
arrangement of thalamocortical connections as the axons
enter the ventral telencephalon.
In addition to being topographically arranged in the
visual cortex, LGN inputs are segregated into right and left
eye-specific patches as a consequence of activity-dependent
competition between the axons representing each eye.
Similar to the development of connections in the LGN,
establishment of these ocular dominance columns depends
on differential temporal patterns of retinal activity.
Blocking neural activity, or synchronous stimulation of
retinal ganglion cells in both eyes, disrupts the formation of
ocular dominance columns.
Information in the visual cortex is also segregated
according to stimulus orientation. Orientation columns are
organised cortical regions perpendicular to the cortex
containing neurons that respond to a stimulus oriented at
the same angle. Initially, orientation columns form as a
low-contrast map, where sensitivity to different orientations is not clearly segregated. However, before ocular
dominance column formation, these orientation columns
become exquisitely tuned, so the neighbouring locations in
the cortex are sensitive to similar angles of stimulus
orientation. Unlike ocular dominance columns, however,
orientation-selective columns develop normally in the
absence of visual stimulation. Orientation column formation can be blocked by inhibiting activity in the visual
cortex, suggesting that interactions within the cortex help
to establish these columns (White and Fitzpatrick, 2007).
As development proceeds, the visual cortex further
refines the segregation of visual information into its component parts, resulting in a repeating pattern of complex
functional units called hypercolumns (Figure 5). Hypercolumns contain not only orientation-selective columns and
ocular dominance columns, but also a third system of
columns called blobs, which receive information on object
colour. These functional subunits are refined via activitydependent mechanisms. Within the primary visual cortex,
a visual object is separated into information regarding its
shape, movement and colour. The formation of synaptic
connections between neurons in different layers within the
primary visual cortex, as well as connections between different visual cortical areas, is likely to help reassemble a
visual image, for we do not see simply patterns of shapes
and colours, but rather an intact representation of the
visual world. See also: Cerebral Cortex Development
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Further Reading
Adler R and Canto-Soler MV (2007) Molecular mechanisms
of optic vesicle development: complexities, ambiguities and
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Donner AL, Lachke SA and Maas RL (2006) Lens induction in
vertebrates: variations on a conserved theme of signaling
events. Seminars in Cell and Developmental Biology 17:
676–685.
Erskine L and Herrera E (2007) The retinal ganglion cell axon’s
journey: insights into molecular mechanisms of axon guidance.
Developmental Biology 308: 1–14.
Gilbert SF (2010) Developmental Biology, 9th edn, Sunderland,
MA, USA: Sinauer Associates Inc.
Harada T, Harada C and Parada LF (2007) Molecular regulation
of visual system development: more than meets the eye. Genes
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Kandel ER, Schwartz JH, Jessell TM, Siegelbaum SA and
Hudspeth AJ (2012) Principles of Neural Science, 5th edn, New
York, USA: McGraw-Hill Medical.
Petros TJ, Rebsam A and Mason CA (2008) Retinal axon growth
at the optic chiasm: to cross or not to cross. Annual Review of
Neuroscience 31: 295–315.
Price D, Jarman AP, Mason JO and Kind PC (2011) Building
Brains: An Introduction to Neural Development, 1st edn. Chichester, West Susses, UK: Wiley-Blackwell.
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