Fig. 7.

Lhx9 can directly induce hcrt expression. (A) Ectopic lhx9-expressing neurons also express hcrt in embryos injected with an hs:lhx9 plasmid, fixed 1 h after heat shock at 24 hpf, and analyzed using double fluorescent ISH with hcrt-specific and lhx9-specific probes. Arrowhead indicates endogenous Hcrt neurons. A single 1.5 μm confocal section is shown. Supplementary material Movie 4 contains the complete confocal image stack. The bright field (BF) overlay and dashed white circles show the position of the eye. The boxed region is shown at higher magnification in the inset. (B) Schematic of the zebrafish 1 kb hcrt promoter, including putative Lhx9 binding sites A and B. (C) Sequence of a previously characterized mammalian Lhx9 binding site compared with sites A and B in the zebrafish hcrt promoter and in mutated constructs. Gray shading, dashes and red boxes indicate conserved nucleotides, deleted nucleotides and mutated nucleotides, respectively. (D-I) Embryos were injected with a plasmid containing both hcrt:EGFP and hs:lhx9, and some injected embryos were heat-shocked at 24 hpf. Deletion or scrambling of putative Lhx9 binding sites reduced the number (D,F-I) and intensity (F-I) of EGFP-expressing cells. Cell counts indicate Hcrt cells per brain with (endogenous and ectopic Hcrt cells) and without (endogenous Hcrt cells only) heat shock. Mean±s.e.m. is shown. n, number of brains analyzed. ***P<0.001 compared with the wild-type promoter (one-way ANOVA followed by Bonferroni's correction for multiple comparisons). (E) Yellow box indicates area shown in F-I. (J) EMSA showing that the Lhx9 homeodomain (HD) binds to the wild-type (WT) site A (arrow), but not the scrambled site A, in vitro. Scale bars: 10 μm.