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First published online 29 August 2007
doi: 10.1242/dev.02884

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Bone morphogenetic proteins specify the retinal pigment epithelium in the chick embryo

Frank Müller^*, Hermann Rohrer and Astrid Vogel-Höpker

Max-Planck-Institute for Brain Research, Department of Neurochemistry, Deutschordenstr. 46, 60528 Frankfurt/M., Germany.

View larger version (61K): [in this window] [in a new window]   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.

View larger version (57K): [in this window] [in a new window]   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).

View larger version (70K): [in this window] [in a new window]   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.

View larger version (70K): [in this window] [in a new window]   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.

View larger version (79K): [in this window] [in a new window]   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.

View larger version (106K): [in this window] [in a new window]   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 RPE-like 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 single-layered 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).

View larger version (84K): [in this window] [in a new window]   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.

View larger version (9K): [in this window] [in a new window]   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.