Otpb is a critical downstream effector of Fezl in DA progenitors. (A-F) Embryos were injected with either a buffer solution (A,B,E) or DNA constructs containing a heat shock element (HSE) expression cassette that drives a GFP tracer together with either otpb (C,F; at 10-20 pg per embryo) or the constitutively active pac1* (D) cDNAs and were grown at 28°C. At 7 hours post fertilization (hpf) embryos were transferred to a permissive temperature of 38°C for a period of 45 minutes and were shifted back to a 28°C incubator. Mosaic embryos expressing the GFP tracer in the ventral diencephalon were selected for further analysis and were thereafter harvested at 52 hpf. DA neurons were scored by either an RNA probe directed to dopamine transporter (dat; A-D) or an anti-tyrosine hydroxylase (TH) antibody (E,F). A-D are siblings derived from a cross between tofm808-/- and tofm808-/+ fish. Subsequent to the phenotypic analysis, embryos were separated into a multiwell dish and were genotyped by sequencing. (G) Bar chart showing the relative changes in DA cell number following conditional activation of Otpb in WT embryos that were injected with a plasmid harboring a heat shock element-driven otpb/gfp (HSE-otpb/gfp) expression cassette. At the indicated times after fertilization, embryos were subjected to a 45 minute temperature shock (38°C) and the number of dat+ hypothalamic DA neurons in each embryo was scored at 52 hpf. Scale bar: 50 μm.
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