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We analyze the timing of neural patterning in Xenopus and the mechanism by which the early pattern is generated. With regard to timing, we show that by early gastrula, two domains of the anteroposterior (A/P) pattern exist in the presumptive neurectoderm, since the opl gene is expressed throughout the future neural plate, while the fkh5 gene is expressed only in more posteriorectoderm. By mid-gastrula, this pattern has become more elaborate, with an anterior domain defined by expression of opl and otx2, a middle domain defined by expression of opl and fkh5, and a posterior domain defined by expression of opl, fkh5 and HoxD1. Explant assays indicate that the late blastula dorsal ectoderm is specified as the anterior domain, but is not yet specified as middle or posterior domains. With regard to the mechanism by which the A/P pattern is generated, gain and loss of function assays indicate that quantitatively and qualitatively different factors may be involved in inducing the early A/P neural pattern. These data show that neural patterning occurs early in Xenopus and suggest a molecular basis for initiating this pattern.
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Fig. 1. Expression of fkh5 begins at the onset of gastrulation in posterior dorsal ectoderm, and is subsequently expressed in diencephalon, midbrain, hindbrain, and spinal cord. (A) Northern analysis of fkh5. 1.25 embryo equivalents per lane were analyzed for fkh5 RNA (top row) at various embryonic stages shown. Ethidium stained 18S rRNA is the loading control (bottom row). (B) Whole-mount in situ analysis of fkh5. Embryos were analyzed by in situ hybridization and in some cases sectioned as described in Section 4. Embryo orientations are indicated by: A, anterior; P, posterior. Arrows in (a,c,e) indicate the dorsal lip of the blastopore. (a) Stage 10.5, dorsal view. (b) Stage 10.5, sagittal section, showing fkh5 expression is in non-involuted tissue only. The dotted line indicates the boundary between involuted and non-involuted tissue. (c) Stage 11.5, dorsal view. (d) Stage 11.5, sagittal section, showing fkh5 expression in the dorsal ectoderm only. (e) Stage 13, dorsal view. (f) Stage 13, sagittal section. Bracket indicates fkh5 expression. (g) Stage 19, dorsal view. Arrows in (g) indicate positions of sections in (h,i). (h,i) Coronal sections of stage 19. Brackets indicate fkh5 expression. (j) Stage 27, lateral view. (k–n) Double whole-mount in situ hybridizations (as described in Section 4). In all panels, embryos are stage 16–18 embryos, with fkh5 always in purple and opl, otx2 or en2/krox20 in blue. (k) fkh5 and otx2 double in situ, dorsal view. (l) fkh5 and otx2 double in situ, anterior view. Arrows in (k,l) indicate the overlap of fkh5 and otx2. (m) fkh5 and opl, dorsal view. (n) fkh5 and opl, anterior view. (o) fkh5 and en2/krox20 mixed probe, dorsal view. (p) fkh5 and en2/krox20 mixed probe, anterior view. Brackets in (o,p) indicate the partial overlap of fkh5 expression stripes with en2 in the midbrain. The arrowhead indicates partial overlap with the r5 stripe of krox20 expression in the midbrain.
Fig. 2. In situ hybridization reveals the A/P pattern in the presumptive forebrain at stage 10+, and a greater A/P pattern in the ectoderm at stage 11.5. Embryos were analyzed by in situ hybridization (as described in Section 4). All views are dorsal, with anterior to the left. A, anterior; P, posterior. (A) Stage 10+, opl expression. (B) Stage 10+, fkh5 expression. (C) Stage 10.5, opl expression. (D) Stage 10.5, fkh5 expression. (E) Stage 10.5, otx2 expression. (F) Stage 10.5, HoxD1 expression. (G) Stage 11.5, opl expression. (H) Stage 11.5, fkh5 expression. (I) Stage 11.5, otx2 expression. (J) Stage 11.5, HoxD1 expression. (K) Diagram of anterior (A), middle (M) and posterior (P) domains at mid-gastrula. (L) Diagram of gene expression corresponding to A, M, and P domains at mid-gastrula. opl is expressed in all three domains. otx2 expression is primarily restricted to the anterior domain, with lower expression in the midline of the middle domain. fkh5 expression is seen in middle and posterior domains. HoxD1 is expressed in the posterior domain only.
Fig. 5. Expression of opl in the late blastulaectoderm is uniform and UV-insensitive, while dorsal localization of opl and fkh5 in early gastrula is prevented by UV. Whole-mount in situ hybridization is shown for opl in normal embryos or embryos irradiated with UV light during the first cell cycle. In (c,e,g,i) dorsal is towards the top. All views are of the animal hemisphere for opl in situ hybridizations, and of the vegetal pole for fkh5 in situ hybridizations. (a) Expression of opl in control stage 9.5 (late blastula) embryos. (b) Expression of opl at stage 9.5 in embryos treated with UV light during the first cell cycle. (c) Expression of opl in control stage 10.5 (early gastrula) embryos. (d) Expression of opl at stage 10.5 in UV-treated embryos. (e) Expression of fkh5 in control stage 10.5 embryos. (f) Expression of fkh5 at stage 10.5 in UV-treated embryos. (g) Expression of opl in control stage 11.5 (mid-gastrula) embryos. (h) Expression of opl at stage 11.5 in UV-treated embryos. (i) Expression of fkh5 in control stage 11.5 embryos. (j) Expression of fkh5 at stage 11.5 in UV-treated embryos. Expression of opl at stage 9.5 is unaffected by UV treatment, but dorsal localization of opl and fkh5 expression at stage 10.5 and 11.5 is prevented by UV. Embryos harvested at stage 30 equivalent showed that the UV treatment was effective, with all embryos having a dorso-anterior index of 0 or 1 (data not shown). Embryos hybridized to an opl sense probe, as a control for non-specific staining, showed no staining (data not shown).
