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Figure 6


Fig. 6. Ectopic neurogenesis in Hes-mutant boundaries. (A-I) Whole-mount in situ hybridization at E9.5. Mash1, Ngn2 and Math1 are not expressed in the ZLI or the isthmus of Hes3;Hes5 double-null embryos (A,D,G, arrowheads). In Hes1;Hes5 double-null embryos, Mash1 and Ngn2 are ectopically expressed in the ventral regions of the ZLI (B,E, asterisks) and the isthmus whereas Math1 expression is extended rostrally into the midbrain without interruption (H, arrow). In Hes1;Hes3;Hes5 triple-null embryos, ectopic expression of Mash1 and Ngn2 in the ZLI (C,F, asterisks) and the isthmus (C,F, arrowheads) is further enhanced. Ectopic Math1 expression in the midbrain is also enhanced (I, arrow). Note that embryos in H,I have an open brain with open isthmus (arrowheads). (J-L) Dll1 is not expressed in the ZLI or the isthmus of Hes3;Hes5 double-null embryos at E9.5 (J, arrowheads). Dll1 is ectopically expressed in the ZLI and the isthmus of Hes1;Hes5 double-null (K, asterisks) and Hes1;Hes3;Hes5 triple-null embryos (L, asterisks). (M-O) Flat-mount in situ hybridization of Ngn2 in the hindbrain. The hindbrain was cut along the ventral midline. Top is rostral. Ngn2 is not expressed in the interrhombomeric boundaries of Hes3;Hes5 double-null embryos (M, arrowheads). The interrhombomeric Ngn2-negative regions become obscure in Hes1;Hes5 double-null embryos (N, arrowheads) and are lacking in Hes1;Hes3;Hes5 triple-null embryos (O, bracket). (P-X) In situ hybridization of the spinal cord at E9.5. Math1 is ectopically expressed in the roof plate of Hes1;Hes3;Hes5 triple-null embryos (R, asterisk) but not of Hes3;Hes5 and Hes1;Hes5 double-null embryos (P,Q, arrowheads). Wnt1 expression is almost lost in the roof plate of Hes1;Hes3;Hes5 triple-null embryos (U). Ngn2 is ectopically expressed in the floor plate of Hes1;Hes3;Hes5 triple-null embryos (X, asterisk). (Y-Aa') Immunostaining (Y-Aa) and in situ hybridization (Y'-Aa') for Shh at E9.5. Shh is normally expressed in the floor plate (arrowheads) and the notochord (N) in Hes3;Hes5 double-null embryos (Y,Y'). Shh expression in the floor plate is downregulated in Hes1;Hes5 double-null embryos (Z,Z', arrowheads) and mostly lost in Hes1;Hes3;Hes5 triple-null embryos (Aa,Aa', asterisks) whereas that in the notochord (N) is not affected. Scale bars: 100 µm.





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