RESEARCH ARTICLE 3483
Development 134, 3483-3493 (2007) doi:10.1242/dev.02884
Bone morphogenetic proteins specify the retinal pigment
epithelium in the chick embryo
Frank Müller*, Hermann Rohrer and Astrid Vogel-Höpker†
In vertebrates, the neuroepithelium of the optic vesicle is initially multipotential, co-expressing a number of transcription factors
that are involved in retinal pigment epithelium (RPE) and neural retina (NR) development. Subsequently, extrinsic signals
emanating from the surrounding tissues induce the separation of the optic vesicle into three domains: the optic stalk/nerve, the NR
and the RPE. Here, we show that bone morphogenetic proteins (BMPs) are sufficient and essential for RPE development in vivo.
Bmp4 and Bmp7 are expressed in the surface ectoderm overlying the optic vesicle, the surrounding mesenchyme and/or
presumptive RPE during the initial stages of eye development. During the initial stages of chick eye development the
microphthalmia-associated transcription factor (Mitf), important for RPE development, is expressed in the optic primordium that is
covered by the BMP-expressing surface ectoderm. Following BMP application, the optic neuroepithelium, including the
presumptive optic stalk/nerve and NR domain, develop into RPE as assessed by the expression of Otx2, Mitf, Wnt2b and the
pigmented cell marker MMP115. By contrast, interfering with BMP signalling prevents RPE development in the outer layer of the
optic cup and induces NR-specific gene expression (e.g. Chx10). Our results show that BMPs are sufficient and essential for RPE
development during optic vesicle stages. We propose a model in which the BMP-expressing surface ectoderm initiates RPE
specification by inducing Mitf expression in the underlying neuroepithelium of the optic vesicle.
INTRODUCTION
The vertebrate eye primordia are first visible as an outgrowth of the
prosencephalic neuroepithelium (reviewed by Chow and Lang,
2001; 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
Max-Planck-Institute for Brain Research, Department of Neurochemistry,
Deutschordenstr. 46, 60528 Frankfurt/M., Germany.
*Present address: Institute for Physiological Chemistry, Martin-Luther-University,
Hollystr. 1, D-06097 Halle, Germany
†
Author for correspondence (e-mail: [email protected])
Accepted 9 July 2007I
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-␤ (TGF-␤)
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 microphthalmiaassociated 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 dedifferentiation 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
DEVELOPMENT
KEY WORDS: BMP, Eye development, Retinal pigment epithelium, RPE specification
3484 RESEARCH ARTICLE
MATERIALS AND METHODS
Assaying gene expression in chick embryos by in situ
hybridisation (ISH)
ISH was performed on whole embryos according to Wilkinson (Wilkinson,
1993) 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 ␮l 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 ␮l 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
␮m 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.
RESULTS
Gene expression in the neuroepithelium of the
optic vesicle during the initial stages of chick eye
development
At the beginning of eye development, the entire optic vesicle coexpresses several genes known to be involved in RPE and NR
development. To determine the time point when RPE and NR
development is initiated in the chick, we analysed and compared the
distribution of transcripts of genes known to be involved in RPE and
NR development in vertebrates.
DEVELOPMENT
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-␤ 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 retinaspecific 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.
Development 134 (19)
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).
No Mitf transcripts were observed in the eye primordia of the
chick at stage 8 (Fig. 2F). However, Mitf expression was observed
in the optic vesicle at stage 9, where expression is strongest in the
distal region (Fig. 2G) that is covered by the overlying surface
ectoderm (Fig. 2, compare G with J). In the temporal part of the optic
vesicle, downregulation of Mitf expression was observed in the distal
portion at stage 10 (Fig. 1C; Fig. 2S,T), while expression is still
observed in the distal optic vesicle more nasally (Fig. 2R).
Subsequently, at around stage 12/13, Mitf transcripts were restricted
to the presumptive RPE (Fig. 1D). A marker of differentiated
pigment cells is melanosomal matrix glycoprotein 115 (MMP115),
which is involved in melanin production. Unlike Otx2 and Mitf, we
did not observe MMP115 expression at the initial stages of chick eye
development (Fig. 1E). The first MMP115 transcripts were detected
in the presumptive RPE from stage 13 onwards (Fig. 1F; Fig. 4C).
This is about five stages earlier than previously reported (Mochii et
al., 1988; 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 homeoboxcontaining 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
RESEARCH ARTICLE 3485
Fig. 1. Division of the chick optic vesicle into a NR and RPE
domain at stage 10. Schematic (above) illustrates the location of the
sections in the temporal part of the optic vesicle at stage 10/11.
(A-F) RPE development. (A) At stage 10, Otx2 expression weakens in
the distal region of the optic vesicle, the presumptive NR (arrowheads).
(B) At stage 13, Otx2 transcripts are abundant in the presumptive RPE
(arrows). (C) At stage 10, Mitf expression is downregulated in the distal
region of the optic vesicle (arrowheads). (D) Mitf expression is observed
in the presumptive RPE at stage 13 (arrows). (E) MMP115 expression is
not observed in the optic vesicle at stage 11 (arrows). (F) The first
MMP115 transcripts appear in the presumptive RPE at stage 13
(arrows). (G-L) NR development. (G) Rx expression is downregulated in
the presumptive RPE at stage 10 (arrows). (H) Rx expression is restricted
to the distal region of the optic vesicle, the presumptive NR at stage 13
(arrowheads). (I) Parallel section of the optic vesicle shown in C. At
stage 10, Chx10 expression is detected in the distal region of the optic
vesicle (arrowheads), in the region where Mitf expression weakens.
(J) Strong Chx10 expression is detected in the distal region, the
presumptive NR at stage 13 (arrowheads). (K) Fgf8 expression is not
detected in the optic vesicle at stage 10 (arrowheads). (L) At stage 13,
Fgf8 expression is observed in the distal region of the optic vesicle, just
beneath the forming lens placode. Note that in all panels at stage
10/11, the temporal region of the optic vesicle is shown. LP, lens
placode.
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
DEVELOPMENT
BMPs in RPE development
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).
Two candidate genes that are detected in the presumptive lens
ectoderm in the mouse are Bmp4 and Bmp7 (Furuta et al., 1997;
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
Development 134 (19)
Fig. 2. Comparison of the Sox10, Mitf and Bmp expression
patterns during the initial stages of chick eye development.
(A) Schematic illustrating the location of the section in H, showing the
nasal part of the optic vesicle at stage 10/11. (B) Bmp4 expression in
the surface ectoderm overlying the optic vesicle following whole-mount
in situ hybridisation (13 somites). The optic primordium is shown at
stage 8 (6 somites; C,F,I,L,O), at stage 9 (7-9 somites; D,G,J,M,P) and
stage 10 (10-12 somites; E,H,K,N,Q). (C) At stage 8, Sox10-positive
neural crest cells are observed in the dorsal neural folds of the
prosencephalon (arrows). (D) Sox10-expressing neural crest cells are
detected in the dorsal-most region of the prosencephalon (arrows) and
no transcripts are detected distally. (E) Sox10 expression is detected in
neural crest cells overlying the dorsal part of the optic vesicle (arrows).
(F) At stage 8, Mitf expression is not observed in the optic primordium
(arrow). (G) Mitf expression is strongest in the distal part of the optic
vesicle at stage 9 (arrows). The arrowheads indicate neural crest cells
dorsally. (H) At stage 10, Mitf expression is observed in the presumptive
RPE (dorsal optic vesicle) and in the distal part (arrows). See A for the
location of this section; see also R,S,T. (I) Bmp7 expression is observed
in the ectoderm overlying the optic primordium at stage 8 (arrows), and
diffuse expression is detected in the neural folds (arrowheads).
(J) Parallel section of the embryo shown in G. At stage 9, strong Bmp7
expression is observed in the overlying ectoderm (arrows). Bmp7
transcripts are also detected in the dorsal ectoderm that covers the
neural crest cells (arrowheads). (K) Bmp7 expression in the ectoderm
overlying the distal region of the optic vesicle at stage 10 (arrows).
Transcripts are still observed in the ectoderm overlying the mesenchyme
(arrowhead). (L) At stage 8, Bmp4 transcripts are detected in the
overlying ectoderm (arrow) and in the neural folds (arrowheads).
(M) Strong Bmp4 expression is detected in the ectoderm overlying the
distal portion of the optic vesicle at stage 9 (arrows). Weak or no
expression is observed in the dorsal-most ectoderm overlying the
mesenchymal cells (arrowheads). (N) At stage 10, Bmp4 expression is
still strong in the ectoderm overlying the distal portion of the optic
vesicle (arrows), whereas weak expression is observed in the ectoderm
overlying the surrounding mesenchyme (arrowhead). Note that Bmp4
expression appears to be stronger in the ectoderm overlying the dorsal
portion of the optic vesicle. (O) At stage 8, Bmp5 expression is strong in
the dorsal midline, the neural folds (arrowheads). Transcripts appear to
be absent from the overlying ectoderm. (P) Bmp5 expression weakens
in the dorsal midline at stage 9 (arrowhead). (Q) No Bmp5 transcripts
are detected in the neuroepithelium of the chick optic vesicle and
surrounding tissues (arrow) at stage 10. (R) Nasal region of the optic
vesicle at stage 10. Mitf expression in the distal and dorsal part of the
optic vesicle (arrowheads). (S) Higher magnification of the Mitf
expression pattern in the more-temporal region of the optic vesicle
shown in H. Mitf expression is detected in both the dorsal and distal
region of the optic vesicle (arrowheads), although expression weakens
ventrally (arrow). (T) In the most-temporal region of the optic vesicle,
Mitf expression is downregulated in the disto-ventral region (arrow).
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
DEVELOPMENT
3486 RESEARCH ARTICLE
Fig. 3. Effects of BMP and noggin application on the Wnt2b
expression pattern during early stages of eye development.
(A) Schematic of a stage 10/11 chick embryo showing the implantation
site of the BMP5-soaked bead in E. (B) At stage 13, Wnt2b transcripts
are restricted to the presumptive RPE (arrow). The arrowhead indicates
Wnt2b expression in the ectoderm. (C) Wnt2b transcripts are detected
in the RPE (arrows) and surface ectoderm (arrowhead) on the
contralateral side of the BMP5-treated eye shown in E. (D) Wnt2b
expression in the contralateral, untreated eye following implantation of
noggin-expressing cells. Wnt2b transcripts are restricted to the RPE
(arrowhead) and no transcripts are detected within the NR. The arrow
shows Wnt2b expression within the ectoderm and anterior lens.
(E) Following BMP5 application (asterisk), Wnt2b expression is also
detected in the distal region of the optic vesicle, the presumptive NR
(arrows). The arrowhead indicates Wnt2b expression in the ectoderm.
(F) Parallel section of the noggin-treated eye shown in Fig. 5B,D. Wnt2b
expression is downregulated in the entire outer optic cup (arrowheads).
In the surface ectoderm (arrow) and anterior lens, Wnt2b expression is
still detected.
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).
In summary, BMP family members are expressed at the right time
(stage 8/9) and place (surface ectoderm) to be involved in RPE
specification by inducing Mitf expression in the neuroepithelium of
the chick optic vesicle.
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
RESEARCH ARTICLE 3487
Fig. 4. Effects of BMP5 application on the distribution of genes
expressed within the NR and RPE at optic cup stages. (A)
Schematic illustrating the location of the BMP5-soaked bead following
implantation at stage 10/11 as shown in E-I; B-D are PBS-soaked bead
controls (B) In control embryos, Mitf expression is weakly detected in
the RPE at stage 15 (arrowheads). (C) The RPE-specific marker MMP115
is restricted to the RPE at this stage (arrowheads). (D) Strong Rx
expression is detected in the NR at this stage (arrowhead). (E) Following
implantation of a BMP5-soaked bead (asterisk), optic cup formation is
not observed and Mitf expression is detected in the distal optic vesicle
(arrowheads). (F) Parallel section of the embryo shown in E and G.
MMP115 expression is induced in the presumptive NR (arrowhead) and
the optic stalk region following BMP5 exposure. (G) Implantation of a
BMP5-soaked bead (asterisk) leads to downregulation of Rx expression
in the presumptive NR. (H) BMP5 application downregulates Chx10
expression in the distal optic vesicle/cup, the presumptive NR (right,
arrowheads). By contrast, Chx10 expression is strongly observed within
the presumptive NR of the contralateral eye (left, arrow). (I) Parallel
section of the embryo shown in H. MMP115 expression is induced by
BMP5 in the presumptive NR (arrowheads), whereas in the
contralateral, unoperated eye, MMP115 transcripts are absent from the
NR (left, arrow). L, Lens.
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
DEVELOPMENT
BMPs in RPE development
3488 RESEARCH ARTICLE
Development 134 (19)
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).
BMP beads placed into the mesenchyme lying more temporal to
the optic vesicle did not prevent optic cup formation, and eye
morphology, including lens development, appeared normal (Fig.
4H,I; Fig. 5L-O). In 44% of these cases, the RPE-specific marker
MMP115 was expressed in single cells within the NR (n=12/27; Fig.
4I; Fig. 5N,O). Application of PBS-soaked beads into the optic
vesicle or into the mesenchyme temporal to the optic vesicle did not
DEVELOPMENT
Fig. 5. Effects of BMP4 application at the optic
vesicle stage (stage 8/9) on the distribution of
genes expressed within the NR and RPE at
stage 24. (A) Schematic illustrating the
implantation site of the BMP4-soaked bead
following the operations at stage 8/9. (B) Following
BMP4 application into the optic vesicle, optic cup
formation is not observed. Instead, a huge vesicle
with RPE-like morphology, expressing Otx2 in both
the proximal (C) and distal (D) regions developed.
The arrowhead indicates Otx2 expression in the RPE
of the contralateral, unoperated eye. Note that at
this stage (stage 24), Otx2 transcripts are also
observed in cells of the native NR (Bovolenta et al.,
1997). This expression pattern is also seen in a
small ventral region of the BMP-treated eye that
has still NR-like morphology (arrow). (C) The entire
optic stalk region has a single-layered RPE-like
morphology expressing Otx2 (arrows) following
BMP4 exposure. (D) Following BMP4 application,
the region that normally develops into the NR has a
RPE-like morphology and expresses Otx2 (arrows).
(E) BMP4 application into the optic vesicle inhibits
NR and optic stalk/nerve development and induces
Mitf expression in the entire optic vesicle. Note that
the entire eye has RPE-like morphology (arrow). In
the unoperated, contralateral eye, Mitf expression
is restricted to the RPE (arrowhead) and no
transcripts are observed in the multilayered NR.
(F) Mitf expression (arrows) in the optic stalk/nerve
region that developed a single-layered morphology
following BMP4 application. (G) Following BMP
treatment, Mitf expression is observed in the region
that normally develops into the multilayered NR
(arrows). (H) Parallel section of the embryo shown
in B. Chx10 expression is only detected in a small
ventral portion of the BMP-treated eye (arrow). In
the contralateral eye, strong Chx10 expression is
restricted to cells of the NR (arrowhead). (I) The
optic stalk/nerve region of the BMP-treated eye has
RPE-like morphology and pigment granules
(arrows) are observed. (J) Following BMP4
application, expression of the NR-specific marker
Chx10 is not observed in the region that normally
develops into the NR. Instead, this region is pigmented (arrows) and has RPE-like morphology. (K) Fgf8 expression is not observed in the distal
region of the BMP-treated eye (arrows). (L) Following BMP4 application more temporal to the optic vesicle, eye morphology including lens
development appears to be normal although the operated eye is slightly smaller. (M) MMP115 expression is restricted to the outer optic cup, the
developing RPE and no transcripts are observed within the NR (arrows). (N,O) BMP4 application induced MMP115 expression in single cells of the
NR (arrows). The asterisk indicates the neurepithelium of the diencephalon. L, Lens.
BMPs in RPE development
RESEARCH ARTICLE 3489
convert NR into RPE (n=12; data not shown) (Vogel-Höpker et al.,
2000). These results show that BMPs are sufficient to induce RPE
development in vivo.
BMP signalling is required for RPE development
To address the functional relevance of BMPs by loss-of-function
experiments during the initial stages of eye development, we
interfered with BMP signalling using the protein noggin. Noggin
specifically inhibits BMP signalling by binding to BMP dimers,
thereby preventing their interaction with cell surface receptors
(reviewed by Balemans and Van Hul, 2002).
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 singlelayered 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).
DEVELOPMENT
Fig. 6. Effects of interfering with BMP signalling
on the distribution of transcripts known to be
involved in NR and RPE development. (A) Mitf
expression in the contralateral, untreated eye
following implantation of noggin-expressing CHO
cells. Mitf expression is restricted to the RPE
(arrowhead). (B) Inhibition of BMP signalling
downregulates Mitf expression in the outer optic cup
(arrowheads) and only a small single-layered region
still expresses Mitf (arrows). (C) MMP115 expression in
the contralateral, untreated eye following
implantation of noggin-expressing cells. MMP115
transcripts are restricted to the RPE. (D) Parallel
section of the noggin-treated eye shown in B.
MMP115 expression is downregulated in the outer
optic cup (arrowheads), although MMP115 expression
is still maintained in a small region that still has RPElike morphology (arrow). (E) Following implantation of
noggin-expressing CHO cells, the single-layered RPE
suddenly thickens in the proximal region of the eye
(arrow). Pigmentation is still observed in the
unaffected areas of the RPE (arrowheads). (F) Higher
magnification of the eye shown in E. The RPE is
pigmented (arrowhead), whereas the adjacent multilayered area is unpigmented (arrow). (G) Parallel section of the noggin-treated eye shown in E
and F. Rx expression is observed in the native NR and in the outer optic cup that has NR-like morphology (arrow). Expression is absent in the singlelayered RPE (arrowhead). (H) Proximal region of a noggin-treated eye at stage 25. At this stage, Pax6 expression is strongly detected in the native
NR, whereas transcripts are absent from the pigmented single-layered RPE (see also arrowhead in I). Following noggin treatment, strong Pax6
expression is induced in a small, multilayered region of the outer layer of the optic cup (box). The arrowhead indicates weak Pax6 expression in the
RPE. Note that Pax6 expression is also observed in the brain on the right side. (I) Higher magnification of the multilayered region in the outer optic
cup shown in H. This region strongly expresses Pax6 (arrow), whereas expression is not observed in the single-layered, pigmented RPE (arrowhead).
(J) Parallel section of the eye shown in H and I. MMP115 expression is downregulated in the Pax6-expressing region that is multilayered (arrow).
3490 RESEARCH ARTICLE
Development 134 (19)
Taken together, the data suggest that BMP signalling is required
during the initial stages of chick eye development for proper
development of the RPE.
DISCUSSION
In this study, we have determined the time point at which the optic
vesicle is subdivided into NR and RPE domains. We present
evidence from gain- and loss-of-function studies that BMPs are
necessary and sufficient for RPE development during optic vesicle
stages in the chick. In addition, the BMP expression pattern in
comparison to the expression of RPE and/or NR marker genes
suggests that the BMP-expressing surface ectoderm, rather than the
mesenchyme, is involved in RPE specification by inducing Mitf
expression in the underlying optic vesicle.
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; MartinezMorales 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; MartinezMorales 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 (VogelHöpker et al., 2000; Martinez-Morales et al., 2005). On the other
DEVELOPMENT
Fig. 7. Effects of interfering with BMP
signalling on the distribution of transcripts
known to be involved in NR and RPE
development. (A-D) Control experiments
overexpressing a viral RCAS (B) (labelled RCAS-B)
construct only (parallel sections). (A) Eye
morphology is normal following viral infection
with RCAS (B). Expression of the viral reverse
transcriptase gene (RT) indicates infected areas of
the RPE (arrows). (B) In the eyes infected with
RCAS (B), Otx2 expression is still restricted to the
RPE (arrowheads) and no transcripts are observed
in the NR at stage 20. (C) Following viral injection
of RCAS (B), MMP115 expression is unchanged
and is restricted to the RPE (arrowheads).
(D) Expression of the NR-specific marker Chx10 is
not observed in the outer layer of the optic cup
following injection of RCAS (B) (arrowheads).
(E-H) The effects following injection of the viral
dnBmpR1b-RCAS (B) construct at stage 6. (E) Following injection of dnBmpR1b, a large region of the outer layer of the optic cup is thickened
(arrowheads). The infection of the RPE is shown by the expression of RT. (F) Otx2 expression weakens in the outer layer of the optic cup following
injection of dnBmpR1b (arrowheads). (G) MMP115 expression is downregulated following inhibition of BMP signalling (arrowheads). (H) Expression
of the NR-specific gene Chx10 is induced in the outer layer of the optic cup following viral overexpression of the dnBmpR1b construct (arrowheads).
(I) RT expression in the outer layer of the optic cup (arrowheads) following viral infection of the dnBmpR1b construct at stage 7. (J) MMP115
expression is downregulated (arrowheads) in the infected region of the outer layer of the optic cup. Strong MMP115 expression is still observed in
the single-layered portion (arrow). (K) RT expression within the proximal RPE (arrowheads) following infection at stage 10. (L) The arrowheads
indicate the expression of the NR marker gene Chx10 in the RPE in the parallel section. L, lens; NR, neural retina.
BMPs in RPE development
RESEARCH ARTICLE 3491
(A) RPE SPECIFICATION
(B) NR SPECIFICATION
(C) MAINTENANCE
stage 8/9
stage 10
stage 11 - 17
SURFACE ECTODERM
BMPs
OPTIC VESICLE
SURFACE ECTODERM
DISTAL
OPTIC VESICLE
MESENCHYME
BMPs
Mitf
BMPs
FGFs
Chx10
RPE
Mitf
NR
Chx10
Mitf
BMPs
FGFs
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).
It has been suggested that RPE development is initiated by signals
released from the ocular mesenchyme (for a review, see MartinezMorales et al., 2004; 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
DEVELOPMENT
Fig. 8. Proposed model for the regulation of the RPE and NR domain in the developing chick eye. (A) The RPE is specified first at stage 9.
BMP genes expressed in the surface ectoderm at stage 8 (e.g. Bmp4 and Bmp7) induce Mitf expression in the underlying optic vesicle. Mitf
expression is strongest in the region of the optic vesicle that directly contacts the overlying ectoderm. (B) At stage 10, NR specification is initiated by
signals released from the FGF-expressing surface ectoderm. FGF-mediated (e.g. FGF1, 2 and 19) induction of Chx10 in the distal portion of the optic
vesicle inhibits Mitf and Otx2 expression in this region, which results in the subdivision of the optic vesicle into a RPE and NR domain. (C) At early
optic cup stages, Mitf expression is still maintained in the presumptive RPE by BMPs expressed within the RPE itself and in the surrounding
mesenchyme (e.g. BMP5, BMP7). At these stages, BMP family members expressed in the dorsal surface ectoderm and adjacent diencephalon could
also be emanating into the underlying mesenchyme to maintain Mitf expression and hence RPE development. On the other hand, FGFs present in
the NR itself (e.g. FGF3, 8, 15 and 19) maintain Chx10 expression, which allows NR development in the adjacent inner layer of the optic cup. The
antagonistic interaction between BMPs/MITF within the RPE and FGFs/CHX10 within the NR ensures the development of the vertebrate eye at early
optic cup stages.
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 ␤A nor ␤B
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.
Development 134 (19)
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.
We thank Sabine Richter for excellent technical assistance; R. Harland, B.
Houston, R. Johnson, M. Mochii, L. Niswander, H. Roelink, T. Ogura for
providing reagents; and Sabine Fuhrmann, Juan Ramon Martinez-Morales,
Dorothea Schulte and Jochen Wittbrodt for critical reading of the manuscript.
F.M. and H.R. were supported from the SFB 269. A.V.-H. was supported by the
Deutsche Forschungsgemeinschaft.
References
Adler, R. and Belecky-Adams, T. L. (2002). The role of bone morphogenetic
proteins in the differentiation of the ventral optic cup. Development 129, 31613171.
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.
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.
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.
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 platemediated repulsion of commissural axons. Neuron 38, 389-401.
Chow, R. L. and Lang, R. A. (2001). Early eye development in vertebrates. Annu.
Rev. Cell Dev. Biol. 17, 255-296.
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.
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.
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.
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.
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.
Hamburger, V. and Hamilton, H. (1951). A series of normal stages in the
development of the chick embryo. J. Morphol. 88, 49-92.
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.
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.
Hilfer, S. R. (1983). Development of the eye of the chick embryo. Scan. Electron
Microsc. III, 1353-1369.
Horsford, C. J., Nguyen, M. T., Sellar, G. C., Kothary, R., Arnheiter, H. and
McInnes, R. R. (2005). Chx10 repression of Mitf is required for the maintenance
of mammalian neuroretinal identity. Development 132, 177-187.
Hyer, J., Mima, T. and Mikawa, T. (1998). FGF-1 patterns the optic vesicle by
directing the placement of the neural retina domain. Development 125, 869877.
DEVELOPMENT
3492 RESEARCH ARTICLE
Hyer, J., Kuhlman, J., Afif, E. and Mikawa, T. (2003). Optic cup morphogenesis
requires pre-lens ectoderm but not lens differentiation. Dev. Biol. 259, 351-363.
Iwakiri, R., Kobayashi, K., Okinami, S. and Kobayashi, H. (2005). Suppression
of Mitf by small interfering RNA induces transdifferentiation of chick embryonic
retinal pigment epithelium. Exp. Eye Res. 81, 15-21.
Jasoni, C., Hendrickson, A. and Roelink, H. (1999). Analysis of chicken Wnt-13
expression demonstrates coincidence with cell division in the developing eye and
is consistent with a role in induction. Dev. Dyn. 215, 215-224.
Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L. and
Coulombre, A. J. (1979). Origins of avian ocular and periocular tissues. Exp. Eye
Res. 29, 27-43.
Kagiyama, Y., Gotouda, N., Sakagami, K., Yasuda, K., Mochii, M. and Araki,
M. (2005). Extraocular dorsal signal affects the developmental fate of the optic
vesicle and patterns the optic neuroepithelium. Dev. Growth Differ. 47, 523536.
Kim, R. Y., Robertson, E. J. and Solloway, M. J. (2001). Bmp6 and Bmp7 are
required for cushion formation and septation in the developing mouse heart.
Dev. Biol. 235, 449-466.
Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose, T., Uno, K.,
Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K. et al.
(2000). Tbx5 and the retinotectum projection. Science 287, 134-137.
Kurose, H., Bito, T., Adachi, T., Shimizu, M., Noji, S. and Ohuchi, H. (2004).
Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis
and eye development, compared with Fgf15 expression in the mouse. Gene
Expr. Patterns 4, 687-693.
Larsson, J. and Karlsson, S. (2005). The role of Smad signaling in hematopoiesis.
Oncogene 24, 5676-5692.
Liu, J., Wilson, S. and Reh, T. (2003). BMP receptor 1b is required for axon
guidance and cell survival in the developing retina. Dev. Biol. 256, 34-48.
Lyons, K. M., Hogan, B. L. and Robertson, E. J. (1995). Colocalization of BMP 7
and BMP 2 RNAs suggests that these factors cooperatively mediate tissue
interactions during murine development. Mech. Dev. 50, 71-83.
Martinez-Morales, J. R., Signore, M., Acampora, D., Simeone, A. and
Bovolenta, P. (2001). Otx genes are required for tissue specification in the
developing eye. Development 128, 2019-2030.
Martinez-Morales, J. R., Dolez, V., Rodrigo, I., Zaccarini, R., Leconte, L.,
Bovolenta, P. and Saule, S. (2003). OTX2 activates the molecular network
underlying retina pigment epithelium differentiation. J. Biol. Chem. 278, 2172121731.
Martinez-Morales, J. R., Rodrigo, I. and Bovolenta, P. (2004). Eye
development: a view from the retina pigmented epithelium. BioEssays 26, 766777.
Martinez-Morales, J. R., Del Bene, F., Nica, G., Hammerschmidt, M.,
Bovolenta, P. and Wittbrodt, J. (2005). Differentiation of the vertebrate retina
is coordinated by an FGF signaling center. Dev. Cell 8, 565-574.
Mathers, P. H., Grinberg, A., Mahon, K. A. and Jamrich, M. (1997). The Rx
homeobox gene is essential for vertebrate eye development. Nature 387, 603607.
Mishina, Y. (2003). Function of bone morphogenetic protein signaling during
mouse development. Front. Biosci. 8, d855-d869.
Mochii, M., Agata, K., Kobayashi, H., Yamamoto, T. S. and Eguchi, G. (1988).
Expression of gene coding for a melanosomal matrix protein transcriptionally
regulated in the transdifferentiation of chick embryo pigmented epithelial cells.
Cell Differ. 24, 67-74.
Mochii, M., Mazaki, Y., Mizuno, N., Hayashi, H. and Eguchi, G. (1998). Role of
Mitf in differentiation and transdifferentiation of chicken pigmented epithelial
cell. Dev. Biol. 193, 47-62.
Müller, F. and Rohrer, H. (2002). Molecular control of ciliary neuron
development: BMPs and downstream transcriptional control in the
parasympathetic lineage. Development 129, 5707-5717.
Murali, D., Yoshikawa, S., Corrigan, R. R., Plas, D. J., Crair, M. C., Oliver, G.,
Lyons, K. M., Mishina, Y. and Furuta, Y. (2005). Distinct developmental
programs require different levels of Bmp signaling during mouse retinal
development. Development 132, 913-923.
Nakayama, A., Nguyen, M. T., Chen, C. C., Opdecamp, K., Hodgkinson, C. A.
and Arnheiter, H. (1998). Mutations in microphthalmia, the mouse homolog of
the human deafness gene MITF, affect neuroepithelial and neural crest-derived
melanocytes differently. Mech. Dev. 70, 155-166.
Nguyen, M.-T. and Arnheiter, H. (2000). Signaling and transcriptional regulation
in early mammalian eye development: a link between FGF and MITF.
Development 127, 3581-3591.
Oh, S. H., Johnson, R. and Wu, D. K. (1996). Differential expression of bone
morphogenetic proteins in the developing vestibular and auditory sensory
organs. J. Neurosci. 16, 6463-6475.
Ohkubo, Y., Chiang, C. and Rubenstein, J. L. R. (2002). Coordinate regulation
RESEARCH ARTICLE 3493
and synergistic actions of BMP4, SHH and FGF8 in the rostral prosencephalon
regulate morphogenesis of the telencephalic and optic vesicles. Neuroscience
111, 1-17.
Pittack, C., Grunwald, G. B. and Reh, T. A. (1997). Fibroblast growth factors are
necessary for neural retina but not pigmented epithelium differentiation in chick
embryos. Development 124, 805-816.
Planque, N., Turque, N., Opdecamp, K., Bailly, M., Martin, P. and Saule, S.
(1999). Expression of the Microthalmia-associated basic helix-loop-helix leucine
zipper transcription factor Mi in avian neuroretina cells induces a pigmented
phenotype. Cell Growth Differ. 10, 525-536.
Reissmann, E., Ernsberger, U., Francis-West, P. H., Rueger, D., Brickell, P. M.
and Rohrer, H. (1996). Involvement of bone morphogenetic protein-4 and
bone morphogenetic protein-7 in the differentiation of the adrenergic
phenotype in developing sympathetic neurons. Development 122, 2079-2088.
Rowan, S., Chen, A., Young, T. L., Fisher, D. E. and Cepko, C. L. (2004).
Transdifferentiation of the retina into pigmented cells in ocular retardation mice
defines a new function of the homeodomain gene Chx10. Development 131,
5139-5152.
Sakai, D., Tanaka, Y., Endo, Y., Osumi, N., Okamoto, H. and Wakamatsu, Y.
(2005). Regulation of Slug transcription in embryonic ectoderm by ß-cateninLef/Tcf and BMP-Smad signaling. Dev. Growth Differ. 47, 471-482.
Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani, T., Iemura, S.,
Yamamoto, T. S., Ueno, N. and Noda, M. (2001). Ventroptin: a BMP-4
antagonist expressed in a double-gradient pattern in the retina. Science 293,
111-115.
Sasagawa, S., Takabatake, T., Takabatake, Y., Muramatsu, T. and Takeshima,
K. (2002). Axes establishment during eye morphogenesis in Xenopus by
coordinate and antagonistic actions of BMP4, Shh, and RA. Genesis, 33, 86-96.
Simeoni, I. and Gurdon, J. B. (2007). Interpretation of BMP signaling in early
Xenopus development. Dev. Biol. 308, 82-92.
Solloway, M. J. and Robertson, E. J. (1999). Early embryonic lethality in
Bmp5;Bmp7 double mutant mice suggests functional redundancy within the
60A subgroup. Development 126, 1753-1768.
Solloway, M. J., Dudley, A. T., Bikoff, E. K., Lyons, K. M., Hogan, B. L. and
Robertson, E. J. (1998). Mice lacking Bmp6 function. Dev. Genet. 22, 321-339.
Stern, C. D., Yu, R. T., Kakizuka, A., Kintner, C. R., Methews, L. S., Vale, W.
W., Evans, R. M. and Umesono, K. (1995). Activin and its receptors during
gastrulation and the later phases of mesoderm development in the chick
embryo. Dev. Biol. 172, 192-205.
Sullivan, C. H., Braunstein, L., Hazard-Leonards, R. M., Holen, A. L., Samaha,
F., Stephens, L. and Grainger, R. M. (2004). A re-examination of lens
induction in chicken embryos: in vitro studies of early tissue interactions. Int. J.
Dev. Biol. 48, 771-782.
Trousse, F., Esteve, P. and Bovolenta, P. (2001). Bmp4 mediates apoptotic cell
death in the developing chick eye. J. Neurosci. 21, 1292-1301.
Uemonsa, T., Sakagami, K., Yasuda, K. and Araki, M. (2002). Development of
dorsal-ventral polarity in the optic vesicle and its presumptive role in eye
morphogenesis as shown by embryonic transplantation and in ovo explant
culturing. Dev. Biol. 248, 319-330.
Vogel, A., Rodriguez, C., Warnken, W. and Izpisua-Belmonte, J. C. (1995).
Dorsal cell fate specified by chick Lmx-1 during vertebrate limb development.
Nature 378, 716-720.
Vogel, A., Rodriguez, C. and Izpisua-Belmonte, J.-C. (1996). Involvement of
FGF-8 in initiation, outgrowth and patterning of the vertebrate limb.
Development 122, 1737-1750.
Vogel-Höpker, A. and Rohrer, H. (2002). The specification of noradrenergic locus
coeruleus (LC) neurones depends on bone morphogenetic proteins (BMPs).
Development 129, 983-991.
Vogel-Höpker, A., Momose, T., Rohrer, H., Yasuda, K., Ishihara, L. and
Rapaport, D. H. (2000). Multiple functions of fibroblast growth factor-8 (FGF-8)
in chick eye development. Mech. Dev. 94, 25-36.
Wawersik, S., Purcell, P., Rauchman, M., Dudley, A. T., Robertson, E. J. and
Maas, R. (1999). BMP7 acts in murine lens placode development. Dev. Biol.
207, 176-188.
Wilkinson, D. G. (1993). Whole mount in situ hybridisation of vertebrate
embryos. In In Situ Hybridisation (ed. D. G. Wilkinson), pp. 75-83. Oxford:
Oxford University Press.
Wilson, P. A., Lagne, G., Suzuki, A. and Hemmati-Brivanlou, A. (1997).
Concentration-dependent patterning of the Xenopus ectoderm by BMP4 and its
signal transducer Smad1. Development 124, 3177-3184.
Yoshikawa, S., Aota, S., Shirayoshi, Y. and Okazaki, K. (2000). The ActR-I
activin receptor protein is expressed in notochord, lens placode and pituitary
primordium cells in the mouse embryo. Mech. Dev. 91, 439-444.
Zhao, G.-Q. (2002). Consequences of knocking out BMP signaling in the mouse.
Genesis 35, 43-56.
DEVELOPMENT
BMPs in RPE development