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Fig. 3. Aberrant otpb expression in fezl-deficient embryos. Embryos (at 52 hpf, lateral view) were subjected to whole mount in situ hybridization with either antisense otpb (A-C) or pac1 (D-F) RNA probes followed by immunostaining with an antibody directed against tyrosine hydroxylase (TH; A-F) that detects DA neurons. (A,D) Wild type (WT). (B,E) tof^m808 mutants. (C,F) tof^m808 embryos were injected, at one-cell stage, with an antisense Fezl morpholino (fezlMO) that blocks proper splicing leading to retention of intron 2 and a shorter protein that lacks most of the zinc finger domain (sp3; see Fig. S6 in the supplementary material). WT heterozygous and tof^m808 embryos were scored by TH staining followed by sequencing. Black arrowheads mark deficient otpb expression domains. (G) Delayed IT development in tof^m808. Relative number of IT/OT neurons in too few (tof^m808) embryos, tof^fezlMO and their WT siblings at different developmental stages. tof^fezlMO indicate embryos, in which fezl-directed antisense morpholino was injected into tof^m808. Embryos were scored by co-staining of IT and TH followed by sequencing of the tof^m808 mutation. WT embryos display 4-7 IT cells (IT>3) on each side of the brain whereas tof^m808 display a deficit (IT<3) in IT cell number between 46 and 52 hours of development. The bars were normalized to 100%. The number of scored embryos is indicated on each bar. (H) A scheme depicting an overlay of TH, otpb and pac1 expression domains as they appear in WT embryos (at 52 hpf). HB, hindbrain; LC, locus coeruleus; NPO, neurosecretory preoptic area; PT, posterior tuberculum; Tel, telencephalon. Scale bar: 50 µm.

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