Fig. 5. Boundary defects in the developing nervous system. (A-H) SEM analysis of wild-type (A,B,E,F) and Hes1;Hes5 double-null (C,D,G,H) embryos. Parts of A and C are enlarged in B and D, respectively. At E10.5, the ZLI (arrows) and the isthmus (arrowhead) are clearly formed in the wild type (A,B). By contrast, in Hes1;Hes5 double-null embryo (C,D), the ZLI is lacking (asterisks) and the isthmus is only partially formed (arrowheads). The interrhombomeric boundaries are clearly formed in the wild type at E9.5 (E, arrowheads) and E10.5 (F, arrowheads). They are not significantly affected at E9.5 (G, bracket) but become ambiguous at E10.5 (H, bracket) in Hes1;Hes5 double-null embryos. (I,J) In situ hybridization for Shh at E10.5. Shh is expressed in the wild-type ZLI (I, arrowhead) but not in Hes1;Hes5 double-null ZLI (J, asterisk). (K-N) In situ hybridization for Lfng at E9.5 (K,L) and Pax6 at E10.5 (M,N). In the wild-type ZLI (K,M), Lfng and Pax6 are not expressed (arrowheads). In contrast, in Hes1;Hes5 double-null embryos (L,N), the Lfng- and Pax6-negative regions are lacking (asterisks). (O-Cc) In situ hybridization for Wnt1 (O-Q), Fgf8 (R-T), En1 (U-W), Otx2 (X-Z) and Gbx2 (Aa-Cc) at E9.5 and E8.5 (insets of X-Cc). Wnt1, Fgf8 and En1 are normally expressed in the isthmus of Hes3;Hes5 double-null embryos (O,R,U, arrowheads). This expression is downregulated in Hes1;Hes5 double-null embryos (P,S,V, arrowheads) and more severely impaired in Hes1;Hes3;Hes5 triple-null embryos (Q,T,W, asterisks). In Hes3;Hes5 double-null embryos, Otx2 is expressed rostrally whereas Gbx2 is expressed caudally to the isthmus (X,Aa, arrowheads). The Otx2 and Gbx2 expression domains are less clearly separated in Hes1;Hes5 double-null embryos at E9.5 (Y,Bb, asterisks), and their borders become even more ambiguous in Hes1;Hes3;Hes5 triple-null embryos at E8.5 and E9.5 (Z,Cc, asterisks).