Fig. 4. The retinal pigmented epithelium includes several cell populations. Double in situ hybridisation was performed with X-Smo in blue (arrows in A) and X-bhh in red (weak in bright field, A; strong under fluorescence, arrows in B). (C) Overlapping staining shows that some cells are negative for expression of both genes in between the two expression domains (white and black arrows give the approximate limits of X-bhh and X-Smo expression domains, respectively). (D,E) Immunostaining with the antibody XAR1 on retinal cross-section of stage 36 and 42, respectively. Staining is detected in the RPE except in the peripheral pigmented epithelium (bars in E). (F) In situ hybridisation on stages 38 embryos with a Xotx5 probe. Xotx5 is expressed in the photoreceptor layer (arrowhead) and strongly in the RPE (arrows), excluding the peripheral pigmented epithelium (bars). (G-I) Staining for BrdU uptake in retina cross-sections of stage 42 embryos. (G) The retina in bright field; (H) BrdU immunostaining. (I) An overlap of G and H shows that some BrdU-positive cells are present in the peripheral pigmented epithelium (arrowheads). Arrows indicate some BrdU-positive cells in the CMZ. (J-L) Double staining for BrdU uptake and Gli3 expression. Gli3 staining is shown in J, BrdU immunostaining in K. Double staining in L shows that some BrdU-positive cells in the peripheral pigmented epithelium are also Gli3 positive (arrow). le, lens; CMZ, ciliary marginal zone. Scale bar: 45 µm in A-F; 15 µm in G-L.