Fig. 2

her5 expression and activity at the MH junction are independent of Notch signalling. (A-C) Expression of notch1a (Bierkamp and Campos-Ortega, 1993) revealed by whole-mount in situ hybridisation (blue staining) in wild-type embryos at the stages indicated (bottom right). (A,B) Flat-mounted views of the MH area, anterior towards the top; (C) lateral view, anterior leftwards. In B, double staining for krox20 expression (red) identifies rhombomeres 3 and 5. Note the faint expression of notch1a (arrow) at the ventral midline of the IZ (bracket) at all stages. (D-S) Expression of her5 and ngn1 as indicated (bottom line) in three- to five-somite wild-type (D,F,H,J,P,R) or mock-treated embryos (L,N) versus: (E,G) embryos injected at the two-cell stage with nicd-myc RNA; (I,K) deadly-seven (des) notch1a-deficient mutants; (M,O) embryos treated with the gamma-secretase inhibitor DAPT; and (Q,S) embryos injected in at the two-cell stage with Delta^Stu mRNA. All views are dorsal, anterior towards the top; in D-G and Q lineage tracers (Myc and β-galactosidase, respectively) are revealed in brown by immunocytochemistry. NICD inhibits her5 expression and decreases the number of neurons per proneural cluster (arrows in G). All other manipulated or mutant contexts increase this number (e.g. compare the intensity of ngn1 staining between control and experimental embryo in the vcc in K,O,S with J,N,R, arrows); however, none of these manipulations affects her5 expression or the presence and size of the IZ (I,M,Q). des, homozygote deadly-seven embryos; IZ, intervening zone; NICD, Notch intracellular domain; som, somite stage.