1 Molecular Medicine Program, Ottawa Health Research Institute, 501 Smyth Road,
Ottawa, ON K1H 8L6, Canada
2 Department of Molecular and Cellular Biology, The Biolabs, Harvard University,
16 Divinity Avenue, Cambridge, MA 02138, USA
3 Eye Institute, Center for Neuromuscular Disease, and Department of
Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth
Road, Ottawa, ON K1H 8M5, Canada
* Author for correspondence (e-mail: vwallace{at}ohri.ca)
Accepted 31 March 2003
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SUMMARY |
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Key words: Retinal ganglion cells, Shh, Ihh, Pax2, Optic disc/stalk, Neuroepithelial cells, Astrocyte precursor cells, Development, Mouse
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INTRODUCTION |
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Optic stalk neuroepithelial cell development as astroglia requires their
interaction with RGC axons (Juurlink and
Fedoroff, 1980; Huxlin et al.,
1992
). Classical embryological studies demonstrate that RGC axon
invasion of the optic stalk is associated with increased neuroepithelial cell
proliferation, and survival and transformation into glial lineage precursor
cells (Ulshafer and Clavert,
1979
; Navascues et al.,
1985
). Moreover, the failure of axons to invade the optic stalk,
as observed in ZRDCT-An mice with inherited optic nerve aplasia
(Silver and Hughes, 1974
;
Silver et al., 1984
) and
ocular retardation mutant mice (Silver and
Robb, 1979
), as well as in Pax6-/-
(Grindley et al., 1995
) and
Math5-/- (Brown et al.,
2001
) mutant mice, results in abortive neuroepithelial cell
development in the optic stalk. These studies emphasize the critical
requirement of RGC axons in the induction and maintenance of gliogenesis in
the optic stalk. The growth cones of RGCs have been shown to contain clusters
of small axoplasmic vesicles (Kuwabara,
1975
), which might contain factors that signal to cells in the
optic disc and stalk, with which they make tight contacts en route to the
brain (Horsburgh and Sefton,
1986
). Although it is well established that RGC axons are
necessary for the normal development of optic disc and stalk cells, the
signals that mediate this RGC axon-to-neuroepithelial cell interaction are
unknown.
We have investigated the role of Shh from RGCs in optic disc and stalk
neuroepithelial cell development. The Hh gene family encodes secretory
glycoproteins that are required for embryonic tissue patterning and
organogenesis (Ingham and McMahon,
2001; McMahon et al.,
2003
). The three mammalian Hh genes, sonic hedgehog
(Shh), Indian hedgehog (Ihh) and desert hedgehog
(Dhh), share the same signaling pathway components. The Hh receptor
patched (Ptch) and one of the transcriptional adaptors of the
pathway, Gli, are direct targets of Hh signaling, such that
transcript levels of Ptch
(Goodrich et al., 1996
;
Marigo and Tabin, 1996
) and
Gli (Marigo et al.,
1996
; Litingtung and Chiang,
2000
; Bai et al.,
2002
) are upregulated in Hh responsive cells and vice versa.
Hence, Ptch and Gli are established molecular readouts of Hh
signal reception in several tissues.
Hh genes are important regulators of ocular morphogenesis and cellular
diversification in several species examined. In the early somite stage
mammalian embryo, Shh from the prechordal plate, and subsequently
from the ventral forebrain neuroepithelium
(Marti et al., 1995), patterns
ventral forebrain structures including the hypothalamus and optic vesicles
(Chiang et al., 1996
;
Rubenstein and Beachy, 1998
).
At later developmental stages, Shh and Ihh are expressed in
an overlapping temporal fashion but in distinct spatial domains of the rodent
eye (Levine et al., 1997
;
Wallace and Raff, 1999
)
(present study). Although a group of peri-ocular mesenchymal cells express
Ihh at about E12, Shh is expressed in the emerging RGC layer
(present study). Shh signaling from RGCs regulates the proliferation,
differentiation and organization of retinal neuroblasts
(Jensen and Wallace, 1997
;
Levine et al., 1997
;
Stenkamp et al., 2000
;
Zhang and Yang, 2001
;
Wang et al., 2002
), and, in
zebrafish, also drives neurogenesis across the retina
(Neumann and Nuesslein-Volhard,
2000
). Dhh is undetectable, by in situ hybridization, in
or around tissues of the developing rodent eye.
Hh proteins are also axon-associated molecules in the visual systems of
both invertebrate and vertebrate species
(Kunes, 2000). In the fly, Hh
transmitted along retinal axons induces neurogenesis and synaptic cartridge
organization in the brain (Huang and
Kunes, 1996
; Huang and Kunes,
1998
), whereas Shh from RGCs regulates astrocyte proliferation in
the rodent optic nerve (Wallace and Raff,
1999
). Recent biochemical analysis of adult hamster ocular and
brain tissues provides further support for a possible anterograde transport of
Shh in the mammalian visual system
(Traiffort et al., 2001
). At
about E12 of mouse development, neuroepithelial cells in the optic stalk
express Ptch and Gli in the absence of Hh mRNA expression
(Wallace and Raff, 1999
)
(present study). The source of Hh in the optic nerve at this developmental
stage is unclear. However, given that Hh proteins may be axonally transported,
it is not inconceivable that Shh may be associated with the growth cones or
axolema of RGCs and made accessible to neuroepithelial cells in the optic
stalk. In addition, optic disc neuroepithelial cells express Hh target genes
whereas differentiated RGCs express Shh, which suggests that
RGC-derived Shh could signal to neuroepithelial cells at the optic disc. To
investigate these two possibilities, we used a conditional gene ablation
approach because Shh-knockout mice exhibit severe midline patterning
defects and cyclopia (Chiang et al.,
1996
). By successfully disrupting the Shh allele in
regions of the CNS, including retinal precursor cells, prior to RGC
differentiation, we provide genetic evidence for a requirement of RGC-derived
Shh signaling in the differentiation of optic disc and stalk neuroepithelial
cells.
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MATERIALS AND METHODS |
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Histology, RNA in situ hybridization and immunohistochemistry
Tissues for histology, in situ hybridization and immunohistochemistry were
dissected in PBS and fixed overnight in 4% paraformaldehyde in 0.1 M PBS (pH
7.4). After overnight protection in 30% sucrose/PBS, tissues were embedded in
sucrose:OCT (Tissue-Tek) and stored at -80°C until the day of an
experiment, when 10-14 µm cryosections were cut. In situ hybridization was
performed according to Wallace and Raff
(Wallace and Raff, 1999).
Briefly, the sections were air-dried for at least 4 hours before overnight
hybridization at 65°C in a moist chamber with a specific riboprobe
(diluted 1:1000). Following the usual stringency washes and an alkaline
phosphatase-conjugated anti-digoxigenin antibody treatment, staining in nitro
blue tetrazolium/5-bromo-4-chloro-3-indoylphosphate revealed the blue color
indicative of regions of specific in situ gene expression. Templates of
full-length Shh (Shhfl), Shh exon 2
(Shhexon2), Ptch, Gli, Pax2, Pax6, Netrin 1 (Ntn1),
Mitf, Vax2, Pdgfra and Nkx2-1 were in vitro transcribed to
generate the respective digoxigenin-labeled antisense riboprobes.
Anti-neurofilament-associated protein immunohistochemistry was performed
essentially according to Jensen and Wallace
(Jensen and Wallace, 1997),
with a monoclonal antibody-3A10 (Developmental Studies Hybridoma Bank). For
collagen type IV immunoreactivity, sections were fixed in -20°C acetone,
treated with 0.3% H2O2, and blocked in 20% sheep serum
in 0.5% Triton X-100 before incubation for at least 1 hour at room temperature
with polyclonal anti-collagen type IV antibody (1:3000 Biogenesis). Using
diaminobenzidine (DAB) as a substrate, conjugated antibodies were detected
with the Vectorstain ABC Elite avidin/biotin/peroxidase kit (Vector
Laboratories, Burlingame, California).
Retinal Explant Culture
Optic cups of E12 and E14 C57BL/6 embryos were dissected in MEM-HEPES (ICN)
and cultured on 13 mm polycarbonate filters (pore size: 0.8 µm; Nucleopore)
in serum-free conditions as described previously
(Wang et al., 2002). The
culture medium was composed of 1:1 DMEM/F12, insulin (10 µg/ml),
transferrin (100 mg/ml), BSA Fraction V (100 mg/ml), progesterone (60 ng/ml),
putrescine (16 µg/ml), sodium selenite (40 ng/ml) and gentamycin (25
µg/ml). Except for untreated controls, the eyecups were cultured in the
presence of a recombinant myristoylated N-terminal active fragment of Shh
(Shh-N) at 2 µg/ml, an anti-Hh antibody (5E1) at 30 µg/ml
(Ericson et al., 1996
) or an
isotype-matched antibody (1E6) at 30 µg/ml. After 48 hours in culture,
tissues were processed for in situ hybridization, and serial sections cut
through the entire eyecup and analyzed for Pax2 expression. All
stained sections were examined on a Zeiss Axioplan microscope and digital
images were captured with the Axio Vision 2.05 (Zeiss) camera and processed
with Adobe Photoshop, version 7.
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RESULTS |
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Ihh signaling is not required for Gli and Ptch
expression in the retina and optic nerve
Another mammalian hedgehog homolog, Ihh, is expressed by a group
of mesenchymal cells located outside the retinal pigment epithelium
(Wallace and Raff, 1999) (data
not shown), raising the possibility that Ihh could signal to cells in
the retina, optic disc and stalk. To delineate the relative roles of the two
Hh genes in ocular tissue patterning, we examined eye development and Hh
target gene expression in ocular tissues of Ihh-/--mutant
mice at various developmental stages. The eye sizes of Ihh mutants
were comparable to their wild-type littermates
(Fig. 2B,E). However, in
contrast to wild type, Gli and Ptch expression were markedly
downregulated in a layer of peri-ocular mesenchymal cells surrounding the
retinal pigment epithelium (Fig.
2; arrowheads in A,D; data not shown). However, Gli
expression in the neuroretina and optic nerve of Ihh-/-
mice was not different from wild-type littermates
(Fig. 2A,B,D,E). Astrocyte
precursor cells at the optic disc and in the optic nerve also developed
normally in Ihh-/- mice, as indicated by normal
Pax2 expression (Fig.
2F). The loss of Gli expression in the peri-ocular
mesenchyme of Ihh-/- mice
(Fig. 2D) indicates that
RGC-derived Shh does not signal in the peri-ocular tissue. Likewise, Ihh
signaling from the peri-ocular mesenchyme does not induce Gli
expression in the retinal neuroblasts of ThyCreShhn/c mice
with conditional ablation of Shh in RGCs
(Fig. 2C). Taken together,
these findings suggest that Ihh signaling is received by nearby mesenchymal
cells, and is not required for Hh target gene expression in the retina, optic
disc and nerve.
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ThyCreShhn/c embryos have RGC axon guidance defects in the
retina
We showed previously that RGC development is induced across the retina of
ThyCreShhn/c mice, and that it may even be increased
(Wang et al., 2002) (Y.P.W.,
unpublished). Hence, the optic nerve `hypoplasia' of
ThyCreShhn/c mice suggested that not all the axons exited
the retina. We therefore stained retinal sections of perinatal
ThyCreShhn/c mice with an anti-neurofilament-3A10
antibody, which revealed a remarkable axon guidance defect in the retina that
was very similar to that observed in Ntn1-/- mutant mice
(Fig. 6C-F) (Deiner et al., 1997
).
Compared with wild-type mice (Fig.
6A,B), RGC axons of ThyCreShhn/c embryos were
misrouted to sub-retinal spaces in several regions of the retina and at the
optic disc (Fig. 6C-F). Of the
axons that reached the disc, some failed to exit into the optic nerve, and
instead coiled in the sub-retinal space
(Fig. 6; white asterisks in C
and D). The similarity of the retinal phenotype of
ThyCreShhn/c embryos to that of
Ntn1-/- mutants, and the observation that optic disc
astrocyte precursor cells are early targets of Hh signaling, suggested that
netrin signaling was disrupted in optic disc astrocyte precursor cells. We
therefore examined the development of the optic disc astrocyte precursor cells
in ThyCreShhn/c embryos. We were unable to detect any
Ntn1/Pax2-expressing cells at the optic discs of E12
ThyCreShhn/c embryos by in situ hybridization analysis
(Fig. 7A,D; data not shown).
From E15 onwards, the optic disc astrocyte precursor cells express
Pdgfra as they migrate into the retina over RGC axons, such that they
are easily detected in sections at, or within the vicinity of, the optic nerve
head (Fig. 7B,C). In contrast
to wild-type littermates (Fig.
7B,C), three ThyCreShhn/c embryos examined at
this developmental stage consistently demonstrated absence of Pax2,
Pdgfra and Ntn1 expression in the optic disc region
(Fig. 7E,F; data not
shown).
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The ocular phenotype of ThyCreShhn/c mice is not a
consequence of loss of midline Shh early during eye development
Cre-recombinase activity in line 703 Thy1-Cre mice used for this
study was heterogeneous and not restricted to the retina, as some of the
ThyCreShhn/c embryos we recovered had holoprosencephaly,
which indicates that midline Shh expression was affected in these
mice (these mice were not included in our analysis). Given the importance of
midline-derived Shh signaling in proximal-distal patterning of the optic
vesicle, we were concerned that the eye phenotype we observed could have
resulted from loss of Shh at the midline. To address this issue, we first
examined the expression of Shhexon2, Ptch and Gli in the
developing hypothalamus by in situ hybridization. These transcripts were
detectable in ThyCreShhn/c embryos at comparable levels to
their wild-type littermates (Fig.
9A,D; data not shown). We next examined some markers of the
diencephalon such as Nkx2-1 (Titf1 Mouse Genome
Informatics) and Ntn1, whose expression may be regulated by Shh
signaling (Sussel et al.,
1999; Pabst et al.,
2000
; Hynes et al.,
2000
). Analysis of Nkx2-1 and Ntn1 expression in
E13, E16 and E17 ThyCreShhn/c embryos showed comparable
expression patterns to wild-type littermates
(Fig. 9B,C,E,F; data not
shown), which indicates the hypothalamus was present in
ThyCreShhn/c mice, and its ventral molecular identity was
preserved as well. Based on the above findings, and the presence of bilateral
eyes in ThyCreShhn/c embryos, it is unlikely the eye and
optic nerve defects are due to loss of Shh expression at the ventral
midline.
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DISCUSSION |
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RGC-derived Shh and peri-ocular mesenchymal Ihh signal in distinct
domains of the developing eye
There is an overlap in the timing of expression of Shh in the
neuroretina and Ihh in the peri-ocular mesenchyme, which raises the
possibility that either Shh or Ihh, or both, could induce Hh target gene
expression in the retina, optic stalk or peri-ocular mesenchyme. Indeed, there
are several examples where more than one Hh member is responsible for tissue
patterning and/or where one Hh family member compensates for the loss of
another (Pathi et al., 2001;
Zhang et al., 2001
; Wijerde et
al., 2002). As there is currently no blocking antibody available that
discriminates between the two Hh proteins, we employed a genetic approach to
delineate the relative roles of Shh and Ihh signaling in eye development.
Evidence from our analysis conclusively demonstrates that Ihh signaling is not
required for Ptch or Gli expression in the retina and optic
nerve, and probably does not contribute to the development of these
structures. However, Ihh appears to play a role in the development of
peri-ocular structures, as Ihh-/- mice have ocular
phenotypes suggestive of abnormal peri-ocular mesenchymal tissue development
(G.D.D., unpublished). Further evidence for the lack of Ihh signaling in the
retina and optic nerve was accrued from analysis of
ThyCreShhn/c mice. In these conditional mutant mice, Ihh
signaling could induce Gli expression in the peri-ocular mesenchyme,
but not in the retina or optic nerve. Whatever limits the spread of Ihh into
the retina or optic nerve, and RGC-derived Shh into the peri-ocular
environment, is unclear at this time, but may be related to the tight
junctions formed between retinal pigment epithelial cells.
Shh signaling from RGCs is required for Hh signal reception in the
optic stalk
There is ample evidence that axon-derived factors promote glial cell
development (Fields and Stevens-Graham,
2002). Indeed, several experiments conclusively demonstrate that
glial cell numbers in the optic nerve are regulated by RGC axons. Optic nerve
astrocyte proliferation was increased in Bcl2 transgenic mice, which
have more RGC axons than their wild-type littermates
(Burne et al., 1996
).
Furthermore, when RGC axons of wild-type and Wallerian-degeneration-deficient
mutant mice were severed behind the eye, astrocyte proliferation decreased,
which suggests that factors from the RGC body are involved in the induction of
astrocyte proliferation in the optic nerve. Subsequent intra-ocular colchicine
and tetrodotoxin injection experiments revealed that astrocyte proliferation
in the rodent optic nerve depends on fast axonal transport
(Burne and Raff, 1997
).
However, the axonal factors that mediate astrocyte proliferation in the optic
nerve have remained elusive. We showed previously that Shh protein, but not
the mRNA was present in the perinatal optic nerve, and RGC axotomy and
functional anti-Hh antibody blockage abrogated Ptch expression, as
well as astrocyte proliferation, in the perinatal optic nerve, which is
consistent with a model whereby Shh is transported in an anterograde fashion
into the optic nerve (Wallace and Raff,
1999
). We have also observed a marked downregulation of Gli,
Ptch and cyclin D1 expression in postnatal optic nerves of mice with
conditional disruption of Shh in parts of the retina (G.D.D.,
unpublished), which provides further evidence that Hh signaling in the
perinatal optic nerve is dependent on Shh expression in the retina.
Recently, a role for anterograde transport of Shh has been demonstrated in the
visual system of the adult hamster (Triaffort et al., 2001), and in the fornix
of the adult rat forebrain (Lai et al.,
2003
), suggesting that axon-associated Shh signaling occurs in
other parts of the adult CNS as well. The failure of optic stalk
neuroepithelial cells of ThyCreShhn/c mice to
differentiate as glial progenitors at E12 is consistent with an even earlier
requirement of Shh signaling from RGC axons in optic stalk neuroepithelial
cell development. In the fly, in-growing retinal axons contain Hh that induces
patched expression in glial cells along their trajectory and in the
brain, and also triggers neurogenesis in the visual ganglia
(Huang and Kunes, 1996
). Thus,
anterograde axonal transport of Hh proteins may be a conserved phenomenon from
flies to mammals.
Given that ThyCreShhn/c mice develop smaller brains
than their wild-type littermates, it could be argued that the optic nerve
phenotype is caused by insufficient signaling from Shh at the midline, which
results in proximal-distal defects in optic vesicle patterning. However,
evidence from our analysis and other studies strongly disputes this
possibility. ThyCreShhn/c mice express Shh and
Shh-dependent markers at the ventral diencephalon. In addition, it is unlikely
that Shh diffusion from the midline can account for Hh target gene expression
in the optic nerve by E12, and thereafter, as the furthest distance Shh seems
to travel extracellularly is in the range of 20-30 cell diameters
(Lewis et al., 2001, Wijerde
et al., 2002), and from E12 onwards there is no graded Ptch or
Gli expression in the optic stalk to suggest graded Shh signaling
from the ventral midline. The ocular phenotypes associated with loss of
midline Shh, such as cyclopia in Shh-/- mice
(Chiang et al., 1996
), and the
failure of the optic vesicle to transit from vesicle to cup stage, as in
BF1-/- mice (Huh et
al., 1999
), were never observed in
ThyCreShhn/c mice. As cholesterol modified Shh molecules
are associated with the lipid raft machinery in the optic nerve
(Traiffort et al., 2001
), it
is very likely that RGC-derived Shh is associated with their growth cones or
axons in lipid rafts, and signals to cells in the optic disc and stalk. To
directly address the possibility that cholesterol modification is essential
for axonal transport of Shh proteins in the visual system may require, for
example, the generation and analysis of conditional mice that express
functional Shh without the cholesterol moiety in RGCs. Our prediction is that
such mice will develop similar optic nerve phenotype as those observed in
ThyCreShhn/c mice.
Regulation of Pax2 expression and gliogenesis in the optic
stalk requires RGC-derived Shh signaling
The homeobox transcription factor Pax2 plays an important role in
normal development of the proximal optic vesicle, where it is expressed early
in embryogenesis (Favor et al.,
1996; Torres et al.,
1996
; Otteson et al.,
1998
). It also appears that Pax2 is required to suppress
pigment cell formation in the optic disc and stalk, as Pax2
expression is never observed in pigmented cells and
Pax2-/- mice develop a pigmented optic nerve
(Torres et al., 1996
). Indeed
the pigmented optic nerve phenotype of ThyCreShhn/c mice
is histologically indistinguishable from that of
Pax2-/--mutant mice
(Torres et al., 1996
). In an
experiment where RGC axons were prevented from entering the optic stalk, the
neuroepithelial cells became heavily pigmented with increased apoptosis
(Ulshafer et al., 1979). This finding, together with the pigmented optic nerve
phenotype of Pax2-/- mice, suggests that factors from
retinal axons may signal to optic stalk neuroepithelial cells to maintain
Pax2 expression, thereby preventing them from differentiating as
pigmented cells. Hh signaling regulates Pax2 expression
(Macdonald et al., 1995
;
Ekker et al., 1996
), but it is
not known whether this effect is direct or indirect. Misexpression of
shh or twhh in the zebrafish embryo results in the expansion
of the pax2 expression domain at the expense of pax6 in the
optic primodium, with a reduction in eye pigmentation
(Macdonald et al., 1995
;
Ekker et al., 1996
). Thus, the
similarities in the optic nerve phenotypes of ThyCreShhn/c
and Pax2-/- mice provides strong support for a direct link
between RGC-derived Shh signaling in the maintenance of Pax2
expression in the optic nerve.
Optic disc astrocyte precursor cell development depends on
RGC-derived Shh signaling
Optic disc astrocyte precursor cells are first targets of Hh signaling in
the retina, and our findings suggest that RGC-derived Shh signaling promotes
their development. First we show that there is a temporal and spatial
correlation in the pattern of Shh and Gli expression in the
central retina, consistent with local Shh signaling from RGCs. Next we provide
genetic evidence that optic disc astrocyte precursor cells fail to develop in
mice with targeted disruption of Shh in RGCs. Finally, we demonstrate
that Shh signaling modulates the size of the optic disc astrocyte precursor
cell population in vitro. It is probable that the initial requirement for
RGC-derived Shh signaling is to maintain Pax2 expression in optic
disc astrocyte precursors, which is consistent with our in vitro data where we
show that functional blockage of RGC-derived Shh signaling results in a
remarkable decrease in the size of the optic disc Pax2+ cell
population. Later, Shh signaling may also be associated with the induction of
Pdgfra expression in optic disc astrocyte precursor cells, as the
peak of Gli expression by the Pax2+ optic disc cells
coincides with their initial expression of Pdgfra, and Gli has been
shown to directly activate the expression and phosphorylation of
Pdgfra in cell lines (Xie et al.,
2001). The maintenance of Pdgfra expression in more
differentiated retinal astrocyte precursor cells is unlikely to be dependent
upon sustained Shh signaling, as the expression of Pdgfra in
astrocyte precursor cells at the disc is followed by the downregulation of
Gli expression.
There is probably a cell intrinsic program that regulates the response of
optic disc cells to Hh signaling. In contrast to astrocytes in the optic nerve
that continue to respond to Hh signaling into the postnatal period
(Wallace and Raff, 1999), the
response of optic disc astrocyte precursor cells is transient, averaging 4-5
days. One possible explanation for this difference is that optic disc
astrocyte precursors are responding to Shh released from the growth cones of
RGCs, such that once RGC axons have finished invading the optic disc, Shh
becomes limiting to optic disc cells. But this explanation is unlikely given
that astrocytes in the optic nerve are able to receive axon-associated Hh
signals right into the perinatal period
(Wallace and Raff, 1999
), and
that the addition of recombinant Shh-N to retinal explants in vitro did not
restore Gli expression in cells at the optic disc. Another
explanation for the downregulation of Hh responsiveness in optic disc
astrocyte precursor cells could be an alteration in the levels of their
extra-cellular matrix components in this region of the retina, as
extracellular matrix has been shown to have positive and negative influences
on Hh signaling (Pons et al.,
2001
).
Regulation of gliogenesis by Shh signaling
Glial cell specification from uncommitted neuroepithelial cells requires
Shh signaling (Pringle et al.,
1996). For example, oligodendrocytes are generated in specific
domains of the ventral neuroepithelium under the influence of Shh signaling
(reviewed by Bongarzone, 2002
).
However, in the optic stalk, which is part of the ventral neuroepithelium, Shh
signaling rather induces specification of astroglial lineage cells from
uncommitted neural stem cells. Upon induction by Shh in the ventral forebrain,
oligodendrocyte precursor cells migrate into the perinatal optic nerve where
they differentiate into mature oligodendrocytes. How neuroepithelial cells
interpret Shh signals appears to depend on the history and spatial location of
a given group of cells. For example, in the ventral spinal cord
(Orentas et al., 1999
;
Lu et al., 2000
),
metencephalon (Davies and Miller,
2001
) and telencephalon (Nery
et al., 2001
; Tekki-Kessaris et al., 2001), Shh signaling has been
shown to regulate the development of oligodendrocytes by inducing the
expression of oligodendrocyte fate specification genes such as Olig1
and Olig2 in neuroepithelial cells
(Sussman et al., 2002
).
However, in the optic stalk, Shh signaling rather induces and maintains the
expression of Pax2 that is required for astrocyte, but not
oligodendrocyte, lineage commitment.
Shh may also be involved in the development and maintenance of the
integrity of glial cells in other regions of the adult CNS. For instance, the
proliferation and organization of Müller glia in the developing and adult
retina (Jensen and Wallace,
1997; Wang et al.,
2002
) require Shh signaling, whereas in the adult cerebellum,
Bergman glia respond to Shh signaling from cerebellar Purkinje cells
(Traiffort et al., 1998
).
Misguidance of retinal axons in ThyCreShhn/c embryos is
due to the loss of Ntn1-expressing optic disc astrocyte precursor cells
Ntn1 from optic disc cells is required for the proper guidance of RGC axons
into the optic stalk (Deiner et al.,
1997; Shewan et al.,
2002
). The identical retinal axon guidance defects of
ThyCreShhn/c- and Ntn1-/--mutant mice
suggests that Shh is required for the development of the Ntn1-positive cells,
or for the induction of Ntn1 expression in optic disc astrocyte
precursor cells or both. However, the failure to observe any Pax2 or
Pdgfra expression at the optic disc in
ThyCreShhn/c mice implies these cells were unspecified in
the mutant mice. It is likely that the loss of optic disc astrocyte precursors
that ultimately results in a failure of Ntn1 expression at the optic
disc could have resulted in the axon guidance problems. However, it should
also be noted that the loss of optic disc cells in
ThyCreShhn/c mice could lead to abnormal optic disc
formation, as well as exposure of the potential subretinal space to exiting
RGC axons, both of which could exacerbate the axon misguidance.
Unlike Ntn1-/- mice, the eyes of
ThyCreShhn/c embryos are small and the retinal layers are
disrupted (Wang et al., 2002),
which could have caused the axon misrouting. However, two lines of evidence
indicate that disorganized retinal morphology was not responsible for the axon
misrouting. First, retinal rosettes were not observed until E17, but RGC axon
coiling in subretinal spaces at the optic disc was evident as early as E12.
Second, misguided RGC axons were not restricted to spaces in between retinal
rosettes, as axons were seen misrouted into peripheral retina in the absence
of lamination defects, and some misguided RGC axons were observed going
through rosettes. The smaller eye size of ThyCreShhn/c
mutants is consistent with the role of Shh in ocular growth and proliferation.
However, it is unlikely that the micropthalmia is responsible for the axon
misrouting, as micropthalmia per se does not result in RGC axon misguidance in
the retina. Conceivably, the misrouted axons in between the retinal pigment
epithelium and the neuroretina could obstruct communication between these
retinal layers thereby leading to the rosettes observed in
ThyCreShhn/c-mutant retina; however, evidence from our
studies and others do not support this conclusion. We observed regions of
normal retinal layering with misrouted axons in subretinal spaces and,
moreover, the retinal rosettes we observed in
ThyCreShhn/c-mutant retina were largely not associated
with misrouted axons in the subretinal space. Furthermore, the axon misrouting
in Ntn1-/--mutant mice did not result in any retinal
lamination abnormalities (Deiner et al.,
1997
).
As in Ntn1-/--mutant mice, RGC axons were still able to
exit the retina of ThyCreShhn/c mice. The earliest age at
which RGC-derived Shh signaling appears to act on optic disc astrocyte
precursor cells is E12, but Ntn1 is expressed in the optic fissure
and stalk prior to this age (Deiner et
al., 1997). As RGC differentiation begins some hours earlier than
E12, it is possible normal axon guidance could have occurred at the optic disc
prior to the loss of Ntn1-expressing cells. Thus, some of the later
generated RGC axons would then respond to guidance cues, such as N-CAM
(Brittis et al., 1995
), from
the early axons and follow them into the optic nerve.
RGC-derived Shh signaling in eye development
Shh signaling from RGCs plays an important role in the development of the
vertebrate visual system. RGC-derived Shh regulates the proliferation and
differentiation of retinal neuroblasts, thereby controlling the number and
organization of their synaptic connections
(Jensen and Wallace, 1997;
Levine et al., 1997
;
Stenkamp et al., 2000
;
Neumann and Nuesslein-Volhard,
2000
; Zhang and Yang,
2001
). By promoting the organization and structural integrity of
Müller glia in the retina, RGC-derived Shh signaling also helps maintain
the normal layering of the retina (Wang et
al., 2002
). In addition, the proliferation of perinatal optic
nerve astrocytes is regulated in part by RGC axon-associated Shh signaling
(Wallace and Raff, 1999
). We
now provide evidence that Shh from early-born retinal neurons is required for
optic disc and stalk neuroepithelial cell development.
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ACKNOWLEDGMENTS |
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