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Transcript
Eye induction and retinal cell
specification
Chris Strang Ph.D.
Vision Sciences
WORB 308
[email protected]
975-7222
Outline
• Eye field and optic vesicle induction
• Development of the lens, iris, ciliary body, and anterior
chamber.
• Retina induction
• Retinal cell proliferation
– Birth order of retinal cell types
– Developmental potency
– Intrinsic and extrinsic factors influencing cell fate
• Specification of fovea
Eye field induction:
The eye field begins to be defined as early as the morula (32
cell) stage. In morula stage frog embryos, only a subset of
cells is competent to become retina. Wnt signaling is required
for diencephalic fate. It is also specifically required for eye
induction. Wnt signaling causes expression of the eyespecific transcription factors Pax6 and Rx1. These, along with
Six3, are initially expressed in a single domain across the
anterior neural plate of many species. Overexpression of the
Wnt receptor ‘frizzled’ results in overexpression of Pax6 and
Rx1.
The separation of the single domain into two bilateral eye
fields depends upon Shh. Shh protein from the prechordal
plate suppresses Pax6 expression in the center of the
embryo, dividing the field in two. Mutations in the SHH gene
or inhibition of protein processing results in cyclopia. The
phenotype results in a single eye in the center of the face
(and usually below the nose).
The primary optic vesicles arise from the frontal eye fields
as an evagination of neural tube epithelium at the 5 vesicle
stage. The optic vesicle is connected to the diencephalon
by the optic stalk, which will become the optic nerve. The
eye arises from several types tissues. Neural ectoderm
gives rise to retina and retinal pigment epithelium (RPE),
the lens is derived from surface ectoderm, while the sclera
and anterior chamber are derived from migrating cells. In
humans, eye development begins around E22, but isn’t
complete until several months after birth.
The lens placode is induced by contact between the optic
vesicle and the overlying ectoderm. The lens placode then
invaginates and pinches off to form a hollow lens vesicle. The
lens vesicle is subsequently filled with differentiating primary
fiber cells that elongate from the posterior. Elongation
involves changes in cell structure and shape as well as the
synthesis of crystallins, transparent, lens-specific proteins. In
the mature lens an anterior layer of proliferative epithelial cells
remains and the remainder of the lens is composed of fiber
cells.
The surface ectoderm from which the lens vesicle forms
gives rise to the cornea. The iris and ciliary body develop at
the periphery of the retina. Unlike the other muscles of the
body (which are derived from the mesoderm), part of the iris
is derived from the ectodermal layer. Specifically, this region
of the iris develops from a portion of the optic cup that is
continuous with the neural retina, but does not make
photoreceptors. Migrating mesenchymal tissues form the
sclera, trabecular meshwork, and anterior chamber.
The optic vesicle infolds, forming a bilayered optic cup. The
inner wall of the optic cup becomes the neural retina, while
the outer wall becomes the pigment epithelium. The cells of
the outer layer produce melanin pigment (one of the few
tissues other than the neural crest cells that can form
melanin) and ultimately becomes the RPE. Initially, the
blood supply is from the hyoid artery. After the hyoid artery
is disassembled, the ophthalmic artery and veins provide
the blood supply to the retina.
The role of Pax6 in eye formation is conserved across many
species, including human and fruit fly. If the mouse Pax6
gene is inserted into the fruit fly genome and activated
randomly, eyes form in cells where mouse Pax6 is expressed.
The Pax6 protein remains important in the development of
the lens and retina. It is also expressed in the mouse
forebrain, hindbrain, and nasal placodes. However, the eyes
seem to be most sensitive to its absence. Pax6 knockouts
lack eyes, and heterozygotes have small eyes.
The developing retina consists of the RPE and the neural
layer. The cells of the neural layer proliferate rapidly to form
the retina proper. Retinal progenitor cells give rise to the
ganglion cells, bipolar cells amacrine cells, photoreceptors
and Muller glia. In contrast, the optic stalks may give rise to
only one cell type, the astrocytes of the optic nerve.
www.ucl.ac.uk/zebrafish-group/research/eye.php
One of the first steps in retina
formation is to enlargement of the
progenitor pool.
S-phase: DNA synthesis/chromosome
duplication
G2: interphase 2
M-phase: mitotis
G1: interphase 1
G0: terminal division
The outer surface, the ventricular zone, is apposed to the RPE,
while the inner layer is at the vitreal side. As with developing
cortex, prolifererating cell undergo interkinetic nuclear
migration.
Cell division takes place at the outer ventricular surface.
During G1 cells migrate inward. They undergo S-phase at the
inner, vitreal surface, and migrate outward in G2. Cells that exit
the cell cycle do so at the outer surface, and then migrate to
final location.
Although cells leave the cell cycle
at the outer surface, retinal layers
are generated from the inner
retina to the outer. The GCL
arises first, followed by the INL
and then the ONL. Cells destined
for each of these layers may be
born concurrently, but timing
influences the probability of a
cell taking on one fate or
another. Fate may be defined by
cell ‘birthday’. Cells may be fate
committed at the point they leave
the cell cycle. Competence
becomes more restricted as
development progresses.
Ganglion cells are born first. Small GCs are born earliest, and
large GCs are later. GCs whose axons will not cross the optic
chiasm are born earlier than those that will develop axons that
will cross contralaterally. GCs are followed by the development
of horizontal cells and then cones. Cone genesis usually begins
and ends before rod genesis.
Amacrine cells, bipolar cells, rods and Müller glia cells are born
later. Different AC types may also be born at different times. For
example, amacrine cells born earliest may migrate to the GCL,
and give rise to the population of displaced amacrine cells.
The birth orders of cones and rods varies slightly by species
and whether or not the retina is rod or cone dominated. If more
of a given cell type is needed, that type may be born later. This
allows more progenitors to be formed prior to exiting the cell
cycle.
Retinal progenitor cells are
multipotent. A single progenitor
cell can give rise to each of the cell
types in the retina. However,
competence may become
increasingly restricted over time.
That is, specific fates may only be
available at different time periods.
There appears to be a combination
of internal programming at time of
birth and environmental exposure.
There is a balance between
intrinsic and extrinsic factors that
influence cell fate.
Intrinsic factors:
The primary intrinsic factor
is birthday. Cells dissociated
at the time that GCs are
usually born tend to become
RGCs. That is, progenitor
cells dissociated early in
development have a higher
probability of differentiating
into GCs. In contrast, cells
that are dissociated later in
development have a higher
probability of differentiating
into rods.
While there are interactions between multiple intrinsic
factors, the importance of a cell’s birthday appears to be
due to transcription factors expressed at different time
points. Expression of members of bHLH proneural
genes appear to drive specific cell fate. For example,
Chx10 promotes bipolar cells, while Prox1 is involved in
HC fate
Extrinsic factors:
Feedback from postmitotic neurons
is a primary example. If early
embryonic retinal progenitors are
cultured with other early
progenitors, they tend to
differentiate into GCs. However, if
early cells are either mixed with
late progenitors, or exposed to
media from cultures of later
progenitors they tend to take on
the later fate.
Notch-delta signaling is another extrinsic feedback
mechanism. Inactive notch promotes neuronal differentiation
and upregulates the ligand delta. Delta from a differentiated
neural cell binds and activates notch in a neighboring cell.
The active notch delays differentiation of the second cell
which increases the likelihood of it becoming a Müller cell.
Development of the fovea: The
primate fovea is specialized for
high acuity color vision. It is
avascular, with an extremely
high density of cones and an
absence of inner retinal layers,
due to displacement to the
periphery. Rods are also
absent from the center 300 mm
and S-cones are sparse in the
central 100 mm .
Although the fovea is thinner overall, the ONL over the center
of the foveal pit is very thick, due to the high density of cone
cell bodies. The cones are very long and thin. The axons,
fibers of Henle, extend away from fovea. There is also a bias
towards cells of the midget pathway which is responsible for
detailed color vision.
During development, all of the cell types are produced,
except for rods and possibly S-cones. Over time the cones in
the fovea centralis become more tightly packed, while the
GCL, IPL and INL are moved toward periphery. This causes a
thickening of the inner layers around the pit and forms the
foveal rim. The long fibers of Henle serve to maintain the
connections between photoreceptors, horizontal cells and
bipolar cells.
Retinal cell differentiation begins
in the center and progresses to
periphery. The order of cell
differentiation is same and
synaptic connections are
formed. Thus, prior to pit
formation, the central retina has
all five layers. At the beginning
of fovea differentiation, packing
density in the cone foveal
mosaic is 12,000/mm2, by the
time the fovea has matured
packing density is 30,000/mm2 .
This occurs via displacement
not generation of new cells.
Pre- pit stage:
The OPL is thin, while the INL
IPL, and GCL are thick. At this
point the early fovea is thicker
than the rest of the retina,
because the peripheral retina is
not as far advanced
developmentally. There is a
central bulge and rods can be
identified surrounding the cone
mosaic. Structurally, the cone
pedicle is adjacent to the cell
nucleus, and only a short axon
is needed to make synaptic
connections in the OPL.
The foveal depression begins to
form as the GCL and IPL are
displaced toward the periphery
and axons from the INL to the
GCL are angled to maintain
synaptic connections. The INL
still has several layers of cell
bodies. The ONL remains thin
with only short Henle fibers from
the ONL to the OPL. As the INL
thins and cells are displaced to
the sides, the photoreceptor
cells become packed more tightly
in the fovea. At birth, the human
fovea is still incompletely
developed.
Cone packing results important
morphological changes. Cone cell
bodies and the fibers of Henle
elongate. Initially cones are
cuboidal and 8-10 mm in diameter.
At birth (for people), the synaptic
pedicle and the inner and outer
segments have developed, and
the cell has become ~11-14 mm in
length. Adult foveal cones will be
80-100 mm long and the diameter
will decrease to ~2 mm. Cones tilt
so the that long end is tipped
toward the center, and the Henle
fiber is angled away from the
center to maintain OPL
connections.
Outline
• Eye field and optic vesicle induction
• Development of the lens, iris, ciliary body, and anterior
chamber.
• Retina induction
• Retinal cell proliferation
– Birth order of retinal cell types
– Developmental potency
– Intrinsic and extrinsic factors influencing cell fate
• Specification of fovea