Tulp3 knockout results in decrease of urp1 expression and scoliosis during zebrafish development. (A) Representative confocal images of MZtulp3 embryos (without curvature phenotype (normal), mild and severe ventral body curvature) and respective control embryos at 2dpf immunostained with anti-RF and anti-acetylated Tubulin as a ciliary marker. Loss of Tulp3 results in no obvious defects in RF formation compared to the control. Numbers represent embryos displaying RF disorganization and embryos that have been analysed in total from 3 independent experiments (control: n = 33, n = 42 and n = 26; MZtulp3 (normal): n = 11, n = 38 and n = 24; MZtulp3 (mild): n = 30, n = 28 and n = 36; MZtulp3 (severe): n = 22, n = 28 and n = 25). Scale bar: 10 μm. (B) WISH analysis reveals reduced urp1 expression in ventral CSF-cNs (black arrowheads) of MZtulp3 embryos (presenting with a ventral curvature phenotype) compared to the control at 28hpf. Expression levels of urp2 (black arrow) and pkd2l1 (black arrowheads; serves as a control as its expression in CSF-cNs has been shown to be unaffected in ciliary mutants42,47) are comparable between MZtulp3 and control embryos at 28hpf. (C) qPCR analysis reveals unaltered expression of urp2 and pkd2l1 while urp1 expression was significantly reduced in MZtulp3 embryos compared to the respective control at 2dpf. (D, E) Representative images (lateral and dorsal views) and quantification of adult (18 months) tulp3 mutant zebrafish displaying scoliosis phenotypes (black arrows) in comparison to the respective control analysed from 3 independent breedings (control: n = 15, n = 13 and n = 0; heterozygous tulp3 mutant: n = 29, n = 6 and n = 8; homozygous tulp3: n = 15, n = 40 and n = 7); total number of adult fish used for analyses are shown above respective bar.
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