Fig. 2. Her5 as a candidate to control IZ formation. (A-C) Whole-mount in situ hybridization at the 3-somite stage (A, dorsal view, anterior to the top) and at 24 hpf (B,C, sagittal views, anterior to the left) with the markers indicated (bottom left, color-coded in A). Note that her5 expression in wild-type embryos delineates the IZ (bracket) from the onset of neurogenesis (A) until at least 24 hpf (IZ identified by the gap in zcoe2 staining in B). (D) Structures of the wild-type and mutant forms of her5 cDNA and their encoded proteins used for functional assays. Top: full-length her5 cDNA as determined from our genomic analyses (A.T. and L.B.-C., unpublished), which starts at ATG1 and encodes nine additional N-terminal amino acids compared to the published sequence (Müller et al., 1996) (see box for protein sequence). Bottom: hsp-her5 construct used to generate transgenic lines for conditional misexpression; this construct is built from the clone of Müller et al. (Müller et al., 1996) such that the first ATG is deleted and the second ATG is used for the generation of an otherwise fully functional Her5 protein (see Materials and Methods). As a control, a morpholino directed against ATG2 (MO^tg, inset) inhibits translation of the transgene mRNA but not that of the endogenous her5 (data not shown, see Materials and Methods). For loss-of-function experiments, a morpholino directed against ATG1 (MO^her5, inset) was used, which inhibits the function of the endogenous Her5 mRNA. Abbreviations as Fig. 1 plus, b, basic DNA-binding motif; HLH, helix-loop-helix dimerization motif; IZ, intervening zone; mes, mesencephalon; r1, rhombomere 1.

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