Fig. 4. Her5 activity can inhibit ngn1 until 24 hpf, in a dose-dependent manner, but does not affect HuC expression. Whole-mount in situ hybridization for ngn1 (blue) and her5 (red), with immunocytochemistry for HuC protein expression (brown), at the stages indicated (bottom left) following a two-hour heat-shock (hs) between the 8- and 15-somite stages (A-G), the 15- and 20-somite stages (H,I) and at 24 hpf (J,K) in hsp-her5 transgenic heterozygotes (+/tg), homozygotes (tg/tg) or their non-transgenic siblings (wt). Dorsal views of flat-mounted anterior neural tubes (A-E,H-K) or tails (F,G), anterior to the top; bracket indicates the IZ, vertical black bar indicates the MH domain. ngn1 expression is downregulated in a dose-dependent manner immediately after heat-shock at any of these stages (B,C,I). This phenotype is stable in the MH domain (blue arrows to restored ngn1 expression in the fore- and hindbrain in E). In contrast, neuronal precursors already expressing HuC are only moderately affected (brown arrows in F-I) [HuC is first detectable in vcc neurons at about the 20-somite stage (H,I), thus at the 15-somite stage we focused on HuC expression in the tail (F,G)[. At 24 hpf, only the MH domain is sensitive to ectopic Her5 activity (blue arrows to unaffected ngn1 expression in the fore- and hindbrain in K). IZ, intervening zone; hs, heat-shock; som, somites.

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