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FIG. 2. Whole mount in situ hybridization showing expression of Xbr-1 during development. In panels D Xbr-1 expression in the eye anlage is marked with an arrowhead. (A, B) Xbr-1 expression of stages 10.5 and 11.5 gastrulae. Shown are dorsal views and the closing blastopore is at the top. Xbr-1 is not expressed on the dorsal side. (C) Lateral view of the embryo in (B), showing that Xbr-1 expression is strong on the ventral side of the gastrula. At mid-gastrulation (stage 11.5, B), the Xbr-1 negative area has a keyhole shape, i.e., it is narrow posteriorly and flares out anteriorly. Based on its location on the dorsal side we suggest that the area that does not express Xbr-1 at stage 10.5 corresponds to the prospective neural plate. However, the fate map of the early gastrula shows that the prospective neural plate is much larger and it extends well to the lateral sides of the gastrula (Keller et al., 1992). Therefore, it is likely that the Xbr-1-negative region covers only part of the neural plate, possibly the ventral neural tube and/or the floor plate posteriorly and the prospective forebrain anteriorly. (D) Dorsal-anterior view of Xbr-1 expression at late gastrula/early neurula stage (stage 13). The remnant of the blastopore is to the top and the prospective anterior neural plate to the bottom of the figure. The color reaction has been purposely overdeveloped to reveal the bilateral Xbr1 staining in the anterior neural plate, in the area of the prospective eyes. (E) Anterior view of Xbr-1 expression at the neural plate stage (stage 16). Xbr-1 is expressed in the dorsal eye anlage in the anterior-lateral neural plate. The dotted line defines the limits of the anterior neural plate. (F, G) Side views of Xbr-1 expression at stage 25 (F) and stage 26 (G). Arrow in F points to Xbr-1 expression in the tailbud, asterisk to expression in the pharynx. (H, I) Frontal (H) and dorsal (I) views of stage 27 embryos, showing strong expression in the eye. (J) High magnification of a stage 25/26 eye. Xbr-1 expression is restricted to the dorsal half. (K, L) Side views of stage 30 (K) and stage 33/34 (L) embryos. Arrow in L points to the tailbud. At stage 33/34 the hybridization signal in the eye is obscured by eye pigment. However, in high magnification (M) of the eye region the bluish hybridization signal of Xbr-1 expression (arrowhead) can be distinguished from the brown eye pigment (open arrow). The position of the lens is marked (l). Expression of Xbr-1 is still found only in the dorsal half of the eye but it is more restricted than earlier (compare with M). Abbreviations: cg, cement gland; l, lens.
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FIG. 3. Sections showing expression of Xbr-1, BMP-4, and X-Notch-1 during eye development, after whole-mount in situ hybridization. (A, B, C, D, E, H, K) Expression of Xbr-1 (I, L), expression of X-Notch-1 (G, J), and expression of BMP-4. (A, B, C) Transverse sections at the levels of the eyes at stage 25 (A), stage 26 (B), and stage 278 (C). Expression is found in the area between the prospective neural retina and the pigment epithelium (A) and ends up in the margin of the eye (C). Note that the pigment epithelium thins out during development (from A to C) and eventually envelops the back of the neural retina (arrows in J, K, L). (D) Posterior transverse section of a stage 25 embryo showing expression of Xbr-1 in the dorsal neural tube (dt) and the lateral mesoderm (lm). (E, F) Comparison of Xbr-1 and X-Notch-1 expression in the eyes at stage 25/26. The area between arrowheads expresses Xbr-1 (E) but not X-Notch-1 (G). Note that at this stage X-Notch-1 is expressed uniformly in the prospective neural retina. (G, H, I, J, K, L) High magnification of sections through the eyes, showing a comparison between expression of BMP-4 (G, J), Xbr-1 (H, K), and X-Notch-1 (I, L) at stage 31/32 (G, H, I) and stage 33/34 (J, K, L). The area of the eye that expresses Xbr-1 (H, K) and BMP-4 (G, J) is the part of the prospective dorsal ciliary margin and is included between arrowheads. These sections show that expression of BMP-4 and Xbr-1 in the eye is largely coincident at both stages. The same area of the eye appears negative for X-Notch-1 expression (area between arrowheads in I, L). Note that as the neural retina differentiates, between stages 312 (I) and 33/34 (L), X-Notch-1 expression becomes gradually restricted to the periphery of the eye, but is never observed at the very edge of the eye (arrowheads). In (G, H, I) the prospective pigment epithelium has thinned out and cannot be easily distinguished. In (J, K, L) arrows point to the pigment epithelium which is now easily recognizable by the brown pigment. In some sections the lens (l) has been detached. Abbreviations: di, diencephalon; dt, dorsal neural tube; l, lens; lm, lateral mesoderm; n, notochord; nr, prospective neural retina; pe, prospective pigment epithelium; s, somite.
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