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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. |
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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. |
