Fig. 5. Changing requirement of Gli2A- and Gli3R-mediated Shh signaling in maintaining gene expression domains in ventral and dorsal mes/r1. RNA in situ hybridization with Shh, Gli1, Gli3 and Pax7 probes. The analysis was performed on representative sections along the AP axis of the mes/r1; sections shown are at the level of the posterior mes (see Fig. 3G). V, ventral; D, dorsal. The mes is outlined where necessary. (A-E) Shh is induced and maintained in the mes ventral midline (VM) of Smo cko and Gli2-En1 cko mutants (B,D,E, arrowheads) and comparable with controls (A, inset in E), but is absent from the mes VM in Gli2-null mutants (C, red arrowhead). Inset in C shows that Shh is induced in the midline of the ventral diencephalon (di) (arrow) similar to control embryos (A). (F-J) The ventrolateral expression of Gli1 observed in control sections (F, inset in J) is lost in Smo cko mutants (G,J, red arrowheads), but is induced weakly in Gli2-null and Gli2-En1 cko mutants (H,I, arrowheads). Gli1 is expressed in mesenchyme (m) in E9.5 control and mutants. (K-T) Gli3 and Pax7 are expressed in dorsolateral mes in control (K,P) but are extended ventrally to the VM in Smo-En1 cko mes (L,Q, arrows). Expression of both markers remains dorsally restricted in Smo-Nes cko mutants (O,T, arrows). In Gli2-null and Gli2-En1 cko mutant mes, Pax7 expression remains restricted (R,S, arrows), but Gli3 expression is expanded ventrally, albeit in a dorsal-to-ventral gradient (M,N). This results in overlapping Gli3 and Gli1 expression domains (H,I,M,N, arrowheads). Scale bars: 25 µm.