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Patterning the optic neuroepithelium by FGF signaling and Ras activation

Shulei Zhao¹, Fang-Cheng Hung², Jennifer S. Colvin³, Andrew White³, Weilie Dai¹, Frank J. Lovicu^1,*, David M. Ornitz³ and Paul A. Overbeek^1,2,

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
³ Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA
^* Present address: Department of Anatomy and Histology, Institute for Biomedical Research, University of Sydney, NSW 2006, Australia

View larger version (100K): [in a new window]   Fig. 1. Altered RPE differentiation in family OVE1070. Embryos, transgenic (A,C,E) and non-transgenic (B,D,F) for CPV2-FGF9 (OVE1070), were assayed for ocular pigmentation (A,B), for expression of the TRP2-lacZ transgene (C,D), and by in situ hybridization for expression of Cdh3 (P-cadherin) (E, F). OVE1070 mice were mated to a C57BL/6 partner to produce pigmented offspring. In E13.5 transgenic embryos, the dorsal RPE (rNR) lost its pigmentation and became a neural retina-like tissue (A). By contrast, the entire RPE in non-transgenic embryos was pigmented (B). When OVE1070 mice were mated with TRP2-lacZ transgenic mice, the offspring carrying both CPV2-FGF9 and TRP2-lacZ lost ß-gal activity in the dorsal presumptive RPE (rNR) at E10.5 (C). In offspring carrying TRP2-lacZ only, the entire prospective RPE expressed lacZ (D). In E12.5 OVE1070 embryos, Cdh3 (P-cadherin) expression (red) was attenuated in the dorsal presumptive RPE (rNR) (E), while in wild-type embryos, Cdh3 was expressed in the entire RPE (F). Cdh3 (P-cadherin) expression was also detected in the corneal and conjunctival epithelia in both types of embryos. D-V, dorsal-ventral orientation; L, lens; LP, lens pit; NR, neural retina; rNR, RPE-derived neural retina. Scale bars: 50 µm in C,D; 100 µm in A,B,E,F.

View larger version (155K): [in a new window]   Fig. 2. Expression of transgenic and endogenous FGF9. In family OVE1070, transgene expression was first detected in the dorsal presumptive RPE (rNR) at E10.5 by in situ hybridization using an SV40 riboprobe (A). At E11.5, the transgene remained expressed in the dorsal rNR and was also expressed in the posterior of the lens vesicle (LV) (B). By E12.5, transgene expression was turned off in the rNR and became restricted to the lens (C). In chimeric embryos (OVE1070: ROSA26) (D), clusters of ß-gal-positive cells (arrows) were present in the rNR, indicating that cells from ROSA26 mice (blue cells) were induced to become neural retinal cells by the transgenic FGF9 secreted from neighboring cells. During normal eye development (E,F), expression of endogenous Fgf9 was detected in the prospective neural retina but not in the prospective RPE at E10.0 (E) and E11.5 (F). Fgf9 is also expressed in the ventral (yellow arrow in E) but not dorsal (yellow arrowhead) region of the optic stalk. Abbreviations: D-V, dorsal-ventral orientation; L, lens; LP, lens pit; LV, lens vesicle; NR, neural retina; rNR, RPE-derived neural retina. Scale bars: 50 µm in A,B,D-F; 100 µm in C.

View larger version (123K): [in a new window]   Fig. 3. Neural patterning in OVE1070 transgenic eyes. In E15.5 OVE1070 embryos, BrdU incorporation was detected in a high percentage of the cells in the RPE-derived neural retina (rNR), analogous to the original neural retina (NR) (A). Very few cells in the unaffected ventral RPE were actively proliferating (A). Immunohistochemistry with antibody to neuron-specific ß-tubulin III showed that retinal ganglion cells (g) had been generated in both the NR and rNR at E15.5 (B). The tissue morphology and the absence of ganglion cells in the anterior margin between NR and rNR indicate that this tissue was not converted to neural retina. At postnatal day 6 (C), both the NR and rNR became laminated and formed three distinct cellular layers referred to as the ganglion cell layer (g), the inner nuclear layer (i) and the outer nuclear layer (o). Immunohistochemistry with antibody to rhodopsin showed that photoreceptors were born and located basally in both retinae (C). Abbreviations: L, lens; Scale bars: 50 µm in C; 100 µm in A,B.

View larger version (97K): [in a new window]   Fig. 4. Expression of retinal transcription factors in wild-type (top panels) and in OVE1070 transgenic (bottom panels) embryos. In situ hybridization was performed to assay for expression of Rax (Rx) at E10.5 (A,B), Chx10 at E11.5 (C,D), Pax6 (E, F) and Atoh7 (Math5) (G,H) at E13.5, and Mitf at E11.5 (I,J). Expression of neuronal genes (Rx, Chx10, Pax6 and Math5) was activated in the RPE-derived neural retina (rNR), while Mitf expression was downregulated. In wild-type eyes (E), Pax6 was expressed in the lens epithelium and neural retina, and in the anterior (white arrowheads) but not in the posterior region (yellow arrowheads) of the RPE. D-V, dorsal-ventral orientation; L, lens; LP, lens pit; L, lens vesicle. Scale bars: 50 µm in A-D,I,J; 100 µm in E-H.

View larger version (110K): [in a new window]   Fig. 5. Induction of neural retina from the presumptive RPE by TRP2-rasT24. Hematoxylin/Eosin staining of an E16.5 transgenic embryo showed that nearly the entire presumptive RPE was converted to a second neural retina (rNR) (A). Antibody labeling (green fluorescence) using anti-ß-tubulin III indicated that ganglion cells had differentiated in both the endogenous neural retina (NR) and rNR (B, boxed region in A). BrdU incorporation showed that numerous cells in the rNR were actively proliferating, analogous to the cells in the original neural retina (NR) (C). Postmitotic ganglion cells (g) were readily visible in the NR, but not as prevalent in the rNR (red arrowhead). Blood cells (arrows in B,C) showed autofluorescence. L, lens. Scale bars: 50 µm in B,C; 100 µm in A.

View larger version (122K): [in a new window]   Fig. 6. Expansion of RPE into the presumptive neural retina in Fgf9-/- embryos. In wild-type E13.5 eyes (A,C), the neural retina (NR) is morphologically distinguished from the RPE. The anterior boundary of the RPE (arrow) is clearly defined by the shape of the cells and the presence of pigment granules. In E13.5 Fgf9-/- embryos, the anterior border of the ventral neural retina is consistently affected (B,D). The RPE extends around the anterior margin and occupies a small zone on the inner layer of the optic cup (arrows in B,D). High-magnification images of the boxed regions in A,B are shown in C,D, respectively. Dark-field images of the wild-type (E) and mutant (F) eyes clearly show extension of pigmentation around the anterior margin in Fgf9-/- embryos. Scale bars: 60 µm in A,B; 20 µm in C,D; 40 µm in E,F.