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Fig. 3
Transient lhx9 overexpression induces ectopic Hcrt neurons. (A) Zebrafish embryos were injected with tol2 transposase mRNA and a plasmid in which a heat shock-inducible promoter regulates the expression of a candidate gene (hs:gene x). (B,C) Anterior views of 26hpf embryos after ISH showing endogenous hcrt expression (B) and mosaic expression of gene x 1 h after heat shock (C). Approximately 10-20% of cells overexpress gene x. White box in B,C indicates the hypothalamus. (D) Control embryos injected with an hs:EGFP plasmid exhibit normal hcrt expression at 120hpf. (E) Embryos overexpressing lhx9 contain ectopic Hcrt cells that are dorsal and caudal relative to endogenous Hcrt neurons. (F) Ectopic Hcrt cells exhibit neuronal morphology, as visualized using Tg(hcrt:EGFP) larvae. White and yellow boxes in D-F indicate endogenous and ectopic Hcrt cells, respectively.