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First published online 29 August 2007
doi: 10.1242/dev.02884
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Max-Planck-Institute for Brain Research, Department of Neurochemistry, Deutschordenstr. 46, 60528 Frankfurt/M., Germany.
Author for correspondence (e-mail:
veithopker{at}aol.com)
Accepted 9 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: BMP, Eye development, Retinal pigment epithelium, RPE specification
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Martinez-Morales et al.,
2004). Enlargement of the distal portion of the optic vesicle and
dorsal expansion divides the optic vesicle into three territories
(Hilfer, 1983): the narrow
optic stalk (proximal), the neural retina (NR) and the retinal pigment
epithelium (RPE). Formation of the lens vesicle from the surface ectoderm
induces the distal region of the optic vesicle to invaginate and this process
results in the development of the bilayered optic cup. The inner layer
develops into the multilayered NR, whereas the outer layer develops into the
single-layered, pigmented RPE (reviewed by
Chow and Lang, 2001).
In vertebrates, optic vesicle cells initially co-express a number of
transcription factors (TFs) that become restricted to NR, RPE and optic nerve
later on, implicating that these cells are competent to develop into these
tissues (reviewed by Martinez-Morales et
al., 2004). Extrinsic signals emanating from the surface ectoderm
and ocular mesenchyme appear to induce and repress specific TFs, which
subsequently pattern the optic vesicle into NR and RPE domains (for reviews,
see Chow and Lang, 2001;
Martinez-Morales et al.,
2004). For example, fibroblast growth factors (FGFs) expressed in
the surface ectoderm and/or distal optic vesicle appear to be involved in NR
induction and differentiation (Pittack et
al., 1997; Hyer et al.,
1998; Nguyen and Arnheiter,
2000; Vogel-Höpker et
al., 2000; Martinez-Morales et
al., 2005). Embryonic transplantations and in ovo explant cultures
of the chick optic vesicle have shown that the dorsoventral polarity of the
eye is already specified by stage 10
(Uemonsa et al., 2002;
Kagiyama et al., 2005). At
this time point, the dorsal half of the optic vesicle is fated to develop
mainly into RPE, whereas the ventral portion develops mainly into NR
(Kagiyama et al., 2005).
Little is known about the molecular mechanisms that specify the RPE
(reviewed by Martinez-Morales et al.,
2004). The mesenchyme adjacent to the optic vesicle appears to be
crucial for RPE development, but the molecular nature of the signal(s) is
still unclear (reviewed by Chow and Lang,
2001; Martinez-Morales et al.,
2004). Activin, a member of the transforming growth factor-ß
(TGF-ß) superfamily, or a related growth factor appears to be released
from the mesenchyme to induce RPE development
(Fuhrmann et al., 2000).
Cell-intrinsic TFs mediate the effect of mesenchymal signalling molecules on
RPE development (reviewed by Chow and Lang,
2001). The best-studied example is the microphthalmia-associated
transcription factor (MITF), a basic helix-loop-helix leucine zipper TF that
is crucial for the acquisition and maintenance of RPE cell identity (reviewed
by Martinez-Morales et al.,
2004). Ectopic Mitf expression in cultured avian neural
retina cells results in the induction of pigmentation by initiating the
expression of two markers of differentiated pigment cells: melanosomal matrix
protein 115 (MMP115) and tyrosinase
(Mochii et al., 1998;
Planque et al., 1999). By
contrast, inhibition of Mitf by small interfering RNAs (siRNA)
decreases MMP115 expression and promotes de-differentiation of the
RPE (Iwakiri et al., 2005). In
Mitf mutants, the RPE remains unpigmented and displays areas
developing into a second NR (Bumsted and
Barnstable, 2000; Nguyen and
Arnheiter, 2000). Members of the orthodenticle-related family of
TFs, Otx1 and Otx2, are also required for RPE specification
during vertebrate eye development
(Martinez-Morales et al.,
2001; Martinez-Morales et al.,
2003). In Otx1/Otx2 mutants, RPE development is
disturbed and instead the outer layer of the optic cup develops NR-like
features. Similar to Mitf, Otx2 overexpression induces a pigmented
phenotype in cultured NR cells. Otx1 and Otx2 are initially
expressed in the entire optic vesicle. Subsequently, Otx2 expression
is maintained in the presumptive RPE and expression persists in the adult RPE
(reviewed by Martinez-Morales et al.,
2004).
There appear to be differences in the Mitf expression pattern
between chick and mouse (Mochii et al.,
1998; Fuhrmann et al.,
2000; Nguyen and Arnheiter,
2000). In chick, Mitf expression seems to be restricted
to the dorsal region of the optic vesicle, the presumptive RPE, and this
region is covered by the surrounding mesenchyme. By contrast, the entire mouse
optic vesicle is initially covered by a small amount of mesenchyme and here
Mitf expression is observed throughout the optic vesicle. Once the
mesenchyme is displaced at the distal part of the optic vesicle at the time
this region contacts the FGF-expressing surface ectoderm, Mitf
expression is inhibited and instead NR induction occurs in the mouse
(Bora et al., 1998;
Nakayama et al., 1998;
Nguyen and Arnheiter, 2000).
The paired-like homeobox gene Chx10 is a specific marker of retinal
progenitor cells and functions to repress Mitf expression in the
distal optic vesicle (Rowan et al.,
2004; Horsford et al.,
2005). Moreover, overexpression of Chx10 in the chick RPE
causes downregulation of Mitf expression and other pigment markers,
leading to a nonpigmented RPE (Rowan et
al., 2004). Thus, the current model is that the ocular mesenchyme
is necessary to induce the RPE domain during vertebrate eye development,
whereas FGFs released from the surface ectoderm ensure that the NR develops at
the distal part of the optic vesicle (reviewed by
Chow and Lang, 2001).
Like activin, BMPs belong to the TGF-ß superfamily and several BMP
ligands and their receptors are expressed in the developing chick and mouse
eye and surrounding tissues (reviewed by
Chow and Lang, 2001;
Martinez-Morales et al.,
2004). BMPs are involved in several aspects of vertebrate eye
development. For example, BMP signalling is required for patterning the eye
primordia during blastula and gastrula stages in zebrafish
(Hammerschmidt et al., 2003),
whereas later on BMPs function in both dorsal and ventral patterning of the
vertebrate eye (Koshiba-Takeuchi et al.,
2000; Sakuta et al.,
2001; Adler and Belecky-Adams,
2002; Sasagawa et al.,
2002; Murali et al.,
2005). In addition, the generation of retina-specific BMP type 1
receptor mutant mice has shown that different threshold levels of BMP
signalling regulate distinct developmental processes such as dorsoventral
patterning of the NR, as well as NR growth and differentiation
(Murali et al., 2005). At
present, however, the possible involvement of BMP signalling in RPE
development during optic vesicle stages has not been established (for a
review, see Martinez-Morales et al.,
2004).
In this study, we show that BMP family members are expressed at the right time and place to be involved in inducing Mitf expression in the chick optic vesicle. Mitf expression is first observed at optic vesicle stages, being strongest in the distal optic vesicle that is covered by the BMP-expressing surface ectoderm. Gain-of-function experiments show that BMPs are sufficient to elicit RPE development in vivo. BMP treatment converts cells of the presumptive optic stalk and NR region into RPE. By contrast, interfering with BMP signalling at optic vesicle stages inhibits RPE formation and induces NR-specific gene expression in the outer optic cup. Thus, we provide evidence that during optic vesicle stages, BMPs are necessary and sufficient for RPE development in vivo.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and Henrique
et al. (Henrique et al., 1995)
and on cryostat sections using the technique described by Reissmann et al.
(Reissmann et al., 1996). In
some cases, we enhanced the signal by staining whole-mount embryos two to
three times or by leaving the colour reaction on sections overnight. Antisense
RNA probes specific for chicken Bmp2, Bmp4 and Bmp7
(Reissmann et al., 1996;
Vogel-Höpker and Rohrer,
2002), Bmp5 (Oh et
al., 1996), Bmpr1b (L. Niswander, Sloan-Kettering
Institute, NY), Rx and Fgf8 (T. Ogura, Tohoku University,
Aoba, Japan), Mitf (Mochii et
al., 1998), MMP115
(Rowan et al., 2004),
Chx10 (D. Schulte, MPI Brain Research, Frankfurt/M, Germany),
Wnt2b (H. Roelink, University of Washington, Seattle, WA) and
Sox10 (M. Wegner, University of Erlangen, Germany) were used.
In vivo manipulations of the developing chick embryo
Gain-of-function experiments
A 2 µl drop of recombinant mouse BMP5 or BMP4 (0.7 mg/ml or 1 mg/ml;
R&D Systems) was placed in a Petri dish and about eight drops (10 µl
each) of distilled water were placed around it to keep it from evaporating.
Ten to fifteen agarose beads (Affi-Gel blue beads, Biorad) were added to the
BMP solution, taking care to avoid transferring any fluid with the beads.
These beads were incubated in the BMP4 or BMP5 solution for a minimum of 1
hour at room temperature.
Fertile white leghorn chicken eggs were incubated at 37.8°C until they
reached the desired stages (stages 8-12) according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951). The embryonic membranes were removed and a small incision
was made either temporal (posterior) to the optic vesicle/cup or into the
midline of the forebrain. One BMP-soaked bead was transferred to the egg,
inserted through the slit in the membranes and placed either temporal to the
optic vesicle/cup into the mesenchyme or placed into the forebrain/optic
vesicle region. The embryos were left to develop at 37.8°C until they
reached the desired stages (stages 13-26). At this point, the embryos were
fixed in 4% paraformaldehyde in PBS (PFA) at 4°C for 24-48 hours. Embryos
to be used for whole-mount ISH were dehydrated and stored in 100% methanol.
Those intended for ISH on sections were cryoprotected overnight in 15% sucrose
in PBS at 4°C; consecutive 12-16 µm sections were then cut and analysed
by ISH. For control experiments, beads were soaked in PBS and implanted
according to the same protocol.
Loss-of-function experiments
Noggin-expressing Chinese hamster ovary (CHO B3A4) cells were cultured and
implanted as described (Vogel-Höpker
and Rohrer, 2002). Briefly, for implantation, a 90% confluent
culture was harvested and centrifuged to form a pellet for implantation. The
embryonic membranes of stage 8-12 chick embryos were removed and
noggin-expressing CHO cells implanted/injected into the mesenchyme temporal to
the optic vesicle or into the optic vesicle using fine glass micropipettes.
For control experiments, CHO cells were cultured, harvested and implanted
according to the same protocol. After incubation for a further 1-6 days, the
embryos were fixed and sectioned as described above.
Replication-competent RCAS (B) retroviruses engineered to express the
dominant-negative BMPR1B (referred to here as dnBmpR1b) were kindly provided
by L. Niswander. Retroviral stocks were prepared as described previously
(Vogel et al., 1995;
Vogel et al., 1996). For the
infection of embryos with dnBmpr1b-RCAS (B), or with RCAS (B) as control,
retroviral stock was injected either into the optic vesicle or into the
mesenchyme temporal to the optic vesicle at stages 6-11, using fine glass
micropipettes. The embryos were incubated for a further 3-8 days and analysed
as described above.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the chick, the separation of the optic vesicle into NR and RPE domains
is initiated in the distal region of the optic vesicle at stage 10 (see
below). Initially, Otx2 transcripts are detected throughout the optic
vesicle (data not shown) (Bovolenta et al.,
1997). At stage 10, Otx2 expression weakens in the distal
portion of the optic vesicle (Fig.
1A) and, by stage 13, Otx2 transcripts are abundant in
the dorsal part of the optic vesicle, the cells that will give rise to the RPE
(Fig. 1B). Otx2
expression is maintained in the RPE thereafter and, from about stage 23
onwards, Otx2 expression is also detected in NR cells (unoperated eye
in Fig. 5B)
(Bovolenta et al., 1997).
|
;
Mochii et al., 1998). At
stages 13-18, Wnt2b expression is detected in the presumptive RPE and
no transcripts are detected within the NR
(Fig. 3B)
(Jasoni et al., 1999).
Next, we investigated the time point at which the NR domain is established
during chick eye development. The retinal homeobox-containing gene Rx
is initially expressed throughout the optic vesicle (data not shown)
(Mathers et al., 1997). At
stage 10, Rx expression was seen to be downregulated in the
presumptive RPE (Fig. 1G) and,
by stage 13, expression was restricted to cells in the distal portion of the
optic vesicle (Fig. 1H).
Chx10 is a NR-specific gene expressed in progenitor cells of the NR.
At stage 10, Chx10 expression is detected distally in the temporal
region of the optic vesicle (Fig.
1I) (Fuhrmann et al.,
2000), the region where Mitf transcripts are first
downregulated (compare Fig. 1C
or Fig. 2T with
Fig. 1I).
A second NR-specific marker is FGF8, which appears to be involved in NR
induction and differentiation
(Vogel-Höpker et al.,
2000; Martinez-Morales et al.,
2005). At stage 10, Fgf8 transcripts were not detected in
the distal neuroepithelium of the chick optic vesicle
(Fig. 1K). Fgf8
transcripts in the presumptive NR are first observed at stage 11/12 (13-16
somites) (Vogel-Höpker et al.,
2000; Crossley et al.,
2001) and expression persists in the central region of the chick
NR at optic cup stages (Fig.
1L) (Vogel-Höpker et al.,
2000).
Thus, in the chick, the subdivision of the optic vesicle into NR and RPE is observed at stage 10.
BMP expression during the initial stages of chick eye development
A signal released from the mesenchyme is thought to be the primary inducer
of Mitf expression in the chick and mouse optic vesicle
(Fuhrmann et al., 2000;
Kagiyama et al., 2005). At
stage 9, Mitf expression was seen to be strongest in the distal part
of the optic vesicle that is covered by the surface ectoderm
(Fig. 2G). The first
mesenchymal cells that surround the dorsal region of the optic vesicle are of
neural crest origin (Johnston et al.,
1979; Hilfer,
1983), suggesting that initially a signal released from the
surface ectoderm induces Mitf expression within the optic vesicle,
rather than a signal released from the adjacent mesenchyme. To document the
presence of neural crest-derived mesenchyme in more detail, we next compared
the expression of the neural crest marker gene Sox10 with the
Mitf expression pattern during the initial stages of chick eye
development. At stage 8+, the optic primordia are first visible. At this
stage, Sox10 expression was detected in migrating neural crest cells
in the dorsal neural folds (Fig.
2C). At stages 9 and 10, Sox10 expression was restricted
to migrating neural crest cells that overlie the dorso-temporal part of the
optic vesicle and no transcripts were observed in the distal region
(Fig. 2D,E) where Mitf
expression is strongest (Fig.
2G,H,R).
|
;
Furuta and Hogan, 1998). In
the chick, Trousse et al. (Trousse et al.,
2001) did not detect Bmp7 transcripts in the
neuroepithelium of the optic vesicle or overlying ectoderm until stage 13,
whereas we previously observed Bmp7 transcripts in the presumptive
RPE at stages 11-16 (Vogel-Höpker et
al., 2000). Therefore, we investigated the expression pattern of
BMP family members and their receptors in comparison with the Mitf
expression pattern during the initial stages of chick eye development (stages
8-14). By stage 8+ (6 somites) the neural folds have closed and the
neuroepithelium of the optic primordia are first visible. At this time point,
the neuroepithelium appeared to be close to the surface ectoderm
(Fig. 2) and transcripts of
Bmp4, Bmp5 and Bmp7 were detected in the dorsal neural
folds. Within the neural folds, Bmp7 expression was diffuse
(Fig. 2I), whereas there
appeared to be a regional restriction of Bmp4 and Bmp5
transcripts (Fig. 2L,O). Both
Bmp4 and Bmp7 transcripts, but not Bmp5
transcripts, were observed in the overlying ectoderm between stages 8 and 11
(Fig. 2B,I-Q). Both
Bmp4 and Bmp7 transcripts were also detected in the ectoderm
overlying the ocular mesenchyme at stage 10
(Fig. 2K,N). At optic cup
stages (stages 13-16), Bmp4 transcripts are present in the dorsal NR,
whereas Bmp5 and Bmp7 transcripts are detected in the
presumptive RPE and surrounding mesenchyme (data not shown)
(Vogel-Höpker et al.,
2000; Trousse et al.,
2001).
BMP receptor type 1a and type 1b are expressed in the eye field during the
initial stages of vertebrate eye development
(Furuta and Hogan, 1998;
Trousse et al., 2001;
Hyer et al., 2003). Consistent
with these observations, we detected Bmpr1b transcripts in the
neuroepithelium of the optic vesicle, the overlying ectoderm and surrounding
mesenchyme at stages 8-10 (data not shown).
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|
BMP application induces RPE development in the presumptive optic stalk and NR
If BMP levels determine whether the cells of the neuroepithelium of the
optic vesicle acquire a RPE instead of a NR phenotype, overexpression of BMPs
should result in ectopic generation of RPE from cells of the optic vesicle
(presumptive NR/optic stalk region). We implanted BMP-soaked beads into the
head mesenchyme or optic vesicle at stages 8-12 and analysed these embryos for
changes in NR and RPE gene expression patterns. Vertebrate BMPs have been
divided into two subgroups, suggesting that different ligands might have
different functions during embryogenesis. In our experiments, we implanted
BMP4 and BMP5, which belong to different BMP subclasses. BMP4 and BMP2 belong
to the Dpp family, whereas both BMP5 and BMP7 belong to the 60A family
(Zhao, 2002).
As described above, Otx2 and Mitf expression is
downregulated in cells that develop into NR, but maintained in cells that will
develop into RPE during vertebrate eye development. By contrast, the
RPE-specific marker MMP115, which is involved in melanin pigment
production, is first detected in the presumptive RPE at stage 13
(Fig. 1F). Similarly,
Wnt2b expression is detected in the presumptive RPE at early optic
cup stages (Fig. 3B)
(Jasoni et al., 1999;
Fuhrmann et al., 2000).
Application of BMP4 or BMP5 at stages 8-12 induced (MMP115, Wnt2b)
and maintained (Otx2, Mitf) RPE genes in both the distal and proximal
region of the optic vesicle in 43% of the embryos (n=19/44). In some
cases in which the bead had been placed close to the eye region, optic cup and
nerve formation was not observed, so that the BMP-treated eyes had still optic
vesicle-like morphology (Fig.
3E; Fig. 4E-G). The
distal and proximal regions of these BMP-treated eyes had lost the
characteristic morphology of the multilayered NR and optic stalk/nerve,
respectively. Instead, these regions developed RPE-like features, including
the appearance of pigment granules (Fig.
5I,J). In these embryos, the expression of Mitf, Otx2,
MMP115 and Wnt2b was maintained or induced in the proximal
region that normally develops into the optic nerve
(Fig. 4F;
Fig. 5C,F), and/or in the
distal region that normally gives rise to the NR
(Fig. 3E;
Fig. 4E,F,I;
Fig. 5D,G,N,O). In BMP-treated
embryos that developed RPE-like features, expression of NR-specific genes such
as Rx, Chx10 and Fgf8 was downregulated or absent in the
distal optic vesicle (Fig.
4G,H; Fig. 5J,K).
Three BMP-treated embryos that were left to develop until stage 25/26
developed a single-layered pigmented region within the neuroepithelium of the
forebrain, expressing both Otx2 and MMP115 (data not
shown).
|
). These results show that BMPs are sufficient to induce RPE
development in vivo.
|
).
Noggin-expressing CHO cells were injected either into the head mesenchyme
or into the ventricle of developing chick embryos at stages 8-11. At several
time points after the injection, the embryos were analysed for the expression
of RPE- and NR-specific markers. In general, the noggin-treated eyes were
smaller than the contralateral unoperated eye and displayed aberrant
development of the optic stalk/nerve (coloboma), NR, RPE and lens as
previously reported by Adler and Belecky-Adams
(Adler and Belecky-Adams,
2002). We therefore implanted the cells slightly further away, at
the level of the midbrain. In 32% of the embryos, parts of the outer optic cup
no longer had a single-layered morphology and instead a region developed with
NR-like morphology (Fig. 3F,
arrowheads; Fig. 6B,D,
arrowheads; Fig. 6E-J, arrows).
In these regions, the pigment marker MMP115, Wnt2b and Mitf
expression was downregulated (n=7/22;
Fig. 3F;
Fig. 6B,D,J) and pigment
granules were not observed (Fig.
6E,F,J, arrow). Instead, we observed expression of the retinal
markers Rx and Chx10 in these regions
(Fig. 6G; data not shown).
Pax6 is initially expressed throughout the optic vesicle, but
expression is lost from the proximal RPE at late optic cup stages
(Fig. 6I, arrowhead) (reviewed
by Martinez-Morales et al.,
2004). At stage 25, Pax6 expression is strong within the
chick NR and no Pax6 transcripts are observed within the
single-layered RPE (Fig. 6H,I).
Overexpression of Pax6 in the chick RPE induces transdifferentiation
of the RPE into NR (Azuma et al.,
2005). Following noggin treatment, we observed strong
Pax6 expression in a small, multilayered region of the RPE
(Fig. 6H,I), whereas expression
of the RPE-specific gene MMP115 was downregulated and pigment
granules were absent (Fig. 6J,
arrow). However, weak induction of Pax6 expression within the RPE
(Fig. 6H, arrowhead) did not
result in the downregulation of MMP115 (data not shown). In control
experiments, CHO cells were grafted into the mesenchyme temporal to the optic
vesicle of stage 10-11 chick embryos. In these cases, eye morphology was
normal and MMP115 expression and pigment granules were restricted to
the RPE (n=12; data not shown).
In a second set of experiments, we blocked BMP signalling within the RPE by viral overexpression of a dnBmpr1b construct. Injection of dnBmpR1b-RCAS (B) into the eye field at stages 6-11 resulted in partial loss of RPE development in 21% (7/33) of cases. The most dramatic effects were observed when the operation was carried out at stage 6/7. The outer layer of the optic cup was no longer single-layered and instead developed a NR-like morphology (3/4 cases). Expression of both Otx2 and MMP115 was downregulated in the outer optic cup (Fig. 7F,G,J). By contrast, the NR marker Chx10 was now detected in the outer layer of the optic cup (Fig. 7H). Thickening of the outer layer was not as prominent when the operation was carried out at stages 8-11 (observed in 4/29 cases; Fig. 7K,L). Injection of control RCAS (B) retrovirus at the same stages of development did not result in any alterations in gene expression, and pigment granules were still observed in the outer layer of the optic cup (n=8; Fig. 7A-D).
Taken together, the data suggest that BMP signalling is required during the initial stages of chick eye development for proper development of the RPE.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vertebrates, the neuroepithelium of the optic vesicle initially
co-expresses several TFs that are involved in RPE and NR development. For
example, Mitf and Otx2 are initially expressed in the entire
optic vesicle, but expression is subsequently maintained only in the
presumptive RPE. MITF and OTX2 are key signals involved in initiating and
maintaining pigmentation in the RPE of vertebrates (for reviews, see
Chow and Lang, 2001;
Martinez-Morales et al.,
2004). The retinal homeobox-containing gene Rx, which is
also initially expressed throughout the optic vesicle, becomes downregulated
in the presumptive RPE, whereas expression is maintained in the presumptive NR
(Mathers et al., 1997). In
this study, we show that in the chick optic vesicle, RPE development is
initiated first and that induction of NR development, marked by Chx10
expression, leads to the separation of the chick optic vesicle into NR and
RPE. Expression of Chx10, a marker of retinal progenitor cells, is
detected at stage 10 in the distal region of the chick optic vesicle (this
study) (Fuhrmann et al., 2000)
(for a review, see Chow and Lang,
2001) at the time when Mitf expression is downregulated
in this region (this study). Members of the FGF family - Fgf1, Fgf2
and Fgf19 - are expressed in the surface ectoderm overlying the
distal portion of the chick optic vesicle (reviewed by
Chow and Lang, 2001;
Martinez-Morales et al., 2004;
Kurose et al., 2004). The
separation of the optic vesicle into NR and RPE domains is initiated through
FGF-mediated induction of Chx10, which subsequently leads to the
repression of Mitf (Horsford et
al., 2005) and possibly also of Otx2 in the presumptive
NR. An antagonistic interaction between Chx10 and Mitf
regulates retinal cell identity. CHX10 negatively regulates Mitf
expression by binding to its promoter, thereby ensuring NR development in the
distal portion of the optic vesicle (Rowan
et al., 2004; Horsford et al.,
2005). Thus, it appears that, similar to the situation in mouse,
RPE development is the fate of the neuroepithelium of the optic vesicle in the
absence of NR-inducing signals. Removal of the ectoderm after BMP-mediated RPE
induction and before FGF production should thus lead to RPE development.
Indeed, surface ectoderm removal at stage 10 prevents the separation of the
optic vesicle into NR and RPE, and instead a pigmented vesicle develops
(Hyer et al., 1998;
Nguyen and Arnheiter, 2000).
At stage 10, Mitf expression is mainly observed in the distal optic
vesicle, whereas at this time only a few cells express Chx10 (this
study). Thus, in the absence of FGF-induced Chx10 expression, the
neuroepithelial cells will mainly develop into RPE and only a few neuronal
cells are observed (Hyer et al.,
1998). FGF application to the distal optic vesicle restores proper
separation of the NR and RPE domains in the absence of the surface ectoderm
(reviewed by Martinez-Morales et al.,
2004). FGF family members are also expressed in the presumptive NR
(reviewed by Chow and Lang,
2001; Martinez-Morales et al.,
2004; Kurose et al.,
2004). For example, in the chick, Fgf8 and Fgf19
transcripts are observed in the distal optic vesicle at about the time when
Chx10 expression is first detected in this region
(Vogel-Höpker et al.,
2000; Crossley et al.,
2001; Kurose et al.,
2004). Indeed, FGF8 application into the chick ocular mesenchyme
inhibits Mitf, Otx2 and Bmp7 expression in the presumptive
RPE and Bmp7 expression in the surrounding mesenchyme, and this
allows NR development to occur in the outer optic cup
(Vogel-Höpker et al.,
2000; Martinez-Morales et al.,
2005). On the other hand, BMP application leads to a
downregulation of Fgf8 expression within the NR, and this allows RPE
development to occur in the distal region of the neuroepithelium (this study,
see below).
|
;
Kagiyama et al., 2005).
Previous studies considered the mesenchyme as a source of RPE-inducing signals
for three reasons. First, Mitf expression was first detected in the
presumptive chick RPE at stage 12/13
(Mochii et al., 1998;
Fuhrmann et al., 2000), at the
time the presumptive RPE is surrounded by mesenchyme. Second, in the mouse,
the initial expression of Mitf throughout the optic vesicle coincides
with the time when it is entirely covered by a small amount of mesenchyme
(Bora et al., 1998;
Nguyen and Arnheiter, 2000).
Third, embryonic transplantations and explant studies supported the idea that
the mesenchyme induces RPE-specific gene expression within the neuroepithelium
of the optic vesicle (Fuhrmann et al.,
2000; Kagiyama et al.,
2005). In the chick, Mitf expression is induced in the
distal optic vesicle before mesenchymal cells are present (this study)
(Hilfer, 1983;
Johnston et al., 1979;
Sullivan et al., 2004;
Kagiyama et al., 2005), and
here expression is strongest in the distal optic vesicle that is covered by
the Bmp4- and Bmp7-expressing surface ectoderm. Our
gain-of-function experiments show that ectopic BMP application at optic
vesicle stages can induce the development of a single-layered RPE, including
the appearance of pigment granules, by inducing the pigment cell marker
MMP115 and/or maintaining the expression of Otx2 and
Mitf in cells that would normally have developed into the optic nerve
or NR. In vitro studies have shown that optic vesicles isolated at stages
11-15 and cultured in the presence of mesenchyme express Mitf, Wnt2b
and MMP115 (Fuhrmann et al.,
2000). The results of these co-culture experiments may be
explained by BMP-producing/-containing mesenchyme that has a maintenance
function at this later stage (see below). Fuhrmann et al.
(Fuhrmann et al., 2000)
reported that activin, but not BMPs, can substitute for the mesenchyme to
induce RPE development in optic vesicle explants. The discrepancy with the
present in vivo data might be best explained by the fact that BMPs specify
different cell fates in a concentration-dependent manner and that the BMP
concentrations used were either too low or too high to elicit RPE induction
(Wilson et al., 1997;
Simeoni and Gurdon, 2007). BMP
beads applied close to the optic vesicle induce the development of a
single-layered RPE in cells that would have normally developed into NR or
optic stalk. By contrast, following exposure to a lower BMP concentration
owing to a different position of the BMP-soaked bead, only single cells within
the NR itself express the RPE-specific marker MMP115.
What is the cellular mechanism that is responsible for the generation of
RPE instead of a two-layered optic cup with NR? BMP treatment does not lead to
increased apoptosis, excluding the possibility of selective death of
presumptive NR (Ohkubo et al.,
2002). The significant defects in eye vesicle morphogenesis upon
BMP overexpression raised the question of whether the effect of BMPs is direct
or, alternatively, is secondary to an invagination defect. Optic vesicle
invagination fails when the NR domain has not been correctly specified
(Uemonsa et al., 2002). The
finding that lower BMP levels do not interfere with optic cup formation and
lead to RPE-specific gene expression in single cells within the NR argues in
favour of a direct BMP-induced differentiation process (e.g.
Fig. 5O).
In the chick and mouse, several BMP family members and relevant receptors
are expressed at the right time and place to play a role in inducing and
maintaining RPE development (Lyons et al.,
1995; Dudley and Robertson,
1997; Furuta et al.,
1997; Furuta and Hogan,
1998; Wawersik et al.,
1999; Fuhrmann et al.,
2000; Vogel-Höpker et
al., 2000; Crossley et al.,
2001; Trousse et al.,
2001; Belecky-Adams et al.,
2002; Müller and Rohrer,
2002; Hyer et al.,
2003; Liu et al.,
2003). BMPs mainly signal via complexes composed of type 1 and
type 2 transmembrane serine/threonine kinase receptors, which are both
required for signal transduction (Mishina,
2003). Activated type 1 receptor kinases subsequently
phosphorylate intracellular mediators known as Smad proteins. The type 1
receptors, also known as activin receptor-like kinases (ALKs), ALK1 (ACVRL1),
ALK2 (ACTR1; ACVR1), ALK3 (BMPR1A) and ALK6 (BMPR1B) phosphorylate SMAD1,
SMAD5 and SMAD8 (also known as SMAD9 in mouse), which transduce the
extracellular signal to the nucleus. Activin receptor type 2 mediates BMP
signalling when bound to BMPR1A or BMPR1B (for reviews, see
Balemans and Van Hul, 2002;
Larsson and Karlsson, 2005).
In the chick, Bmpr1a, Bmpr1b and activin type 2a and type 2b
receptors are expressed in the neuroepithelium of the optic vesicle and/or
surrounding tissues at optic vesicle stages (data not shown)
(Stern et al., 1995;
Fuhrmann et al., 2000;
Hyer et al., 2003) and ACTR1
is present in the optic primordia of the developing mouse embryo
(Yoshikawa et al., 2000).
Interestingly, neither the ßA nor ßB activin subunit has been
detected at optic vesicle stages in the developing chick embryo
(Fuhrmann et al., 2000),
whereas phosphorylated SMAD1 was observed in both the neuroepithelium of the
optic vesicle and in the surface ectoderm
(Belecky-Adams et al., 2002;
Faure et al., 2002;
Sakai et al., 2005). We
finally demonstrate the physiological importance of BMPs in RPE development by
interfering with BMP signalling at optic vesicle stages. Application of the
BMP-inhibitor noggin or of the dnBmpR1b construct downregulated MMP115,
Mitf and Otx2 expression in the RPE and instead induced the
expression of the NR marker genes (e.g. Chx10, Rx). FGF8 application
into the mesenchyme near to the optic vesicle/cup induces the development of a
second NR in the outer optic cup
(Vogel-Höpker et al.,
2000; Martinez-Morales et al.,
2005). However, during chick eye development, Fgf8 and
Fgf19 are expressed within the NR, but NR induction in the outer
optic cup does not occur. If BMPs within the RPE and FGFs within the NR act
antagonistically, the absence of BMPs within the RPE should allow the
development of NR-like features in the outer optic cup (see Figs
6 and
7). BMP inhibition at optic cup
stages results in the upregulation of Fgf8 expression within the NR
itself (Adler and Belecky-Adams,
2002).
BMPs have multiple functions during early and late stages of vertebrate eye
development (Koshiba-Takeuchi et al.,
2000; Sakuta et al.,
2001; Adler and Belecky-Adams,
2002; Sasagawa et al.,
2002; Hammerschmidt et al.,
2003; Murali et al.,
2005). For example, deletion of the BMPR1A/B function specifically
within the mouse retina leads to reduced growth of the NR and failure of
retinal neurogenesis (Murali et al.,
2005). We show that at optic vesicle stages, BMPs are involved in
patterning the vertebrate eye by regulating RPE gene expression within the
neuroepithelium of the optic vesicle. On the basis of our results, we propose
the following model (Fig. 8).
Within a short period of time, both RPE and NR specification are induced by
signals released from the overlying ectoderm. Initially, the BMP-expressing
surface ectoderm is involved in inducing and maintaining Mitf
expression in the neuroepithelium of the chick optic vesicle. At this time,
the optic vesicle is in direct contact with the surface ectoderm (this study)
(Johnston et al., 1979;
Hilfer, 1983;
Sullivan et al., 2004;
Kagiyama et al., 2005). The
subdivision of the optic vesicle into NR and RPE domains is initiated by FGFs
(e.g. FGF1, 2 and/or 19) released from the surface ectoderm a few hours later
at stage 9/10. FGF-mediated induction of Chx10 expression in the
distal portion of the optic vesicle downregulates genes involved in RPE
development (e.g. Mitf). Subsequently, during early optic cup stages,
BMPs (e.g. BMP5 and BMP7) in the presumptive RPE itself, the mesenchyme and/or
released from the surrounding tissues (dorsal surface ectoderm, diencephalon)
into the mesenchyme, are involved in stabilising the RPE domain in the outer
optic cup. FGF family members (e.g. FGF3, 8, 15 and 19), being now expressed
in the NR itself, maintain Chx10 expression and allow NR development
to occur adjacent to the RPE. Thus, at the early optic cup stages when the NR
and RPE are in close contact, BMPs/MITF within the RPE and FGFs/CHX10 within
the NR, act antagonistically to ensure vertebrate eye development.
BMP ligands are expressed in overlapping domains and genetic studies
strongly argue that BMP family members are functionally redundant in vivo
(Solloway et al., 1998;
Solloway and Robertson, 1999;
Kim et al., 2001). It is
possible that cooperative signalling of different BMP family members, which
may also involve BMP heterodimers (Butler
and Dodd, 2003), might be involved in regulating RPE development
at optic vesicle stages. However, which specific BMP family members are
involved in RPE specification, differentiation and maintenance remains to be
elucidated.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adler, R. and Belecky-Adams, T. L. (2002). The
role of bone morphogenetic proteins in the differentiation of the ventral
optic cup. Development
129,3161
-3171.
Azuma, N., Tadokoro, K., Asaka, A., Yamada, M., Yamaguchi, M.,
Handa, H., Matsushima, S., Watanabe, T., Kida, Y., Ogura, T. et al.
(2005). Transdifferentiation of the retinal pigment epithelium to
the neural retina by transfer of the Pax6 transcriptional factor.
Hum. Mol. Genet. 14,1059
-1068.
Balemans, W. and Van Hul, W. (2002). Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev. Biol. 250,231 -250.[CrossRef][Medline]
Belecky-Adams, T. L., Adler, R. and Beebe, D. C. (2002). Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation. Development 129,3795 -3802.[Medline]
Bora, N., Conway, S. J., Liang, H. and Smith, S. B. (1998). Transient overexpression of the Microphthalmia gene in the eyes of Microphthalmia vitiligo mutant mice. Dev. Dyn. 213,283 -292.[CrossRef][Medline]
Bovolenta, P., Mallamaci, A., Briata, P., Corte, G. and
Boncinelli, E. (1997). Implication of OTX2 in pigment
epithelium determination and neural retina differentiation. J.
Neurosci. 17,4243
-4252.
Bumsted, K. M. and Barnstable, C. J. (2000).
Dorsal retinal pigment epithelium differentiates as neural retina in the
microthalmia (mi/mi) mouse. Invest. Ophthalmol. Vis.
Sci. 41,903
-908.
Butler, S. J. and Dodd, J. (2003). A role for BMP heterodimers in roof plate-mediated repulsion of commissural axons. Neuron 38,389 -401.[CrossRef][Medline]
Chow, R. L. and Lang, R. A. (2001). Early eye development in vertebrates. Annu. Rev. Cell Dev. Biol. 17,255 -296.[CrossRef][Medline]
Crossley, P. H., Martinez, S., Ohkubo, Y. and Rubenstein, J. L. (2001). Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108,183 -206.[CrossRef][Medline]
Dudley, A. T. and Robertson, E. J. (1997). Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient mice. Dev. Dyn. 208,349 -362.[CrossRef][Medline]
Faure, S., de Santa Barbara, P., Roberts, D. J. and Whitman, M. (2002). Endogenous patterns of BMP signaling during early chick development. Dev. Biol. 244, 44-65.[CrossRef][Medline]
Fuhrmann, S., Levine, E. M. and Reh, T. (2000). Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127,4599 -4609.[Abstract]
Furuta, Y. and Hogan, B. L. (1998). BMP4 is
essential for lens induction in the mouse embryo. Genes
Dev. 12,3764
-3775.
Furuta, Y., Piston, D. W. and Hogan, B. L. M. (1997). Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development 124,2203 -2212.[Abstract]
Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.[CrossRef]
Hammerschmidt, M., Kramer, C., Nowak, M., Herzog, W. and Wittbrodt, J. (2003). Loss of maternal Smad5 in zebrafish embryos affects patterning and morphogenesis of optic primordia. Dev. Dyn. 227,128 -133.[CrossRef][Medline]
Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D. (1995). Expression of Delta homologue in prospective neurons in chick. Nature 375,787 -790.[CrossRef][Medline]
Hilfer, S. R. (1983). Development of the eye of the chick embryo. Scan. Electron Microsc. III,13
