Fig. 5. The roof plate is expanded and Grem1 expression is reduced in mes/r1-S2GOF;F8 embryos. (A-D') Transverse sections through r1 of a control and a mes/r1-S2GOF;F8 embryo at 48 somites (E11.75), stained with Hematoxylin and Eosin. Every third or fourth section in the series was assayed by RNA in situ hybridization with a probe for Otx2, to locate the posterior limit of the mesencephalon (not shown). The broken lines in the inset in A illustrate the approximate levels at which the anterior (A) and posterior (P) sections were cut, i.e. within 40-100 and 130-250 µm of the posterior limit of the Otx2 expression domain (indicated in blue), respectively. The sections in all panels are shown at the same magnification. The regions demarcated by broken boxes in A-D are shown at fourfold higher magnification in A'-D', respectively. The mediolateral width of the dorsal midline domain in which the neuroepithelium is only two or three cell layers thick is indicated by red brackets (A'-D'). (E-L,O,P) RNA in situ hybridization assays in whole mount using the probes indicated. Dorsal views of embryos of the genotypes indicated, collected at the stages denoted. Anterior is towards the top, posterior towards the bottom. (E-I) The roof plate that bisects the Fgf8 expression domain is indicated by arrowheads. In control and mes/r1-S2GOF embryos, the Fgf8-negative roof plate is difficult to discern; it is considerably expanded mediolaterally in the mes/r1-S2GOF;F8 embryo (open arrowhead in G). The lateral and ventral aspect of the Fgf8 expression domain in r1 is visible in E and G on the left side, through the roof plate. (J-L) A broken red line is drawn at the same distance posterior to the isthmic constriction at the mes/r1 boundary (black arrow). Bmp7 expression marks the roof plate, and the roof plate widens closer to the isthmic constriction in the mes/r1-S2GOF;F8 embryo than in the control and mes/r1-S2GOF embryos. (M,N) Transverse sections through anterior r1 at approximately the same AP level as in A and B, hybridized with a probe for Msx1. The mediolateral extent of the Msx1 expression domain from the center of the roof plate (open circle) is indicated by a broken red arrow. (O,P) Grem1 expression is readily detected at the dorsal midline of r1 in the control embryo, but is barely detected in r1 of the mes/r1-S2GOF;F8 embryo (red asterisk).