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) tofm808 mutants. (C,F) tofm808 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 tofm808 embryos were scored by TH staining followed by sequencing. Black arrowheads mark deficient otpb expression domains. (G) Delayed IT development in tofm808. Relative number of IT/OT neurons in too few (tofm808) embryos, toffezlMO and their WT siblings at different developmental stages. toffezlMO indicate embryos, in which fezl-directed antisense morpholino was injected into tofm808. Embryos were scored by co-staining of IT and TH followed by sequencing of the tofm808 mutation. WT embryos display 4-7 IT cells (IT>3) on each side of the brain whereas tofm808 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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