Kics2 loss-of-function impacts on embryonic development and cilium functionality (A) Kics2 depletion results in edema formation, which is not rescued by variants identified in this study. Live zebrafish at 48 hpf. Arrow indicates edema. NI, non-injected; CTRL MO, control MO; + KICS2 WT, + K262?, + D296E, + Y393C, embryos co-injected with Kics2 splMO and capped RNA encoding human KICS2 WT or identified variants. Scale bar: 300 ?m. (B) Stacked bar graph of edema formation summarizing 7 experiments with 77?121 embryos in total. Two-tailed Fisher?s exact test. ????p < 0.0001, ?p = 0.035, and ns: p > 0.05. (C) Loss of Kics2 function leads to defective otolith seeding at 48 hpf. Scale bar: 100 ?m. (D) Stacked bar graph summarizing otolith defects in 7 experiments with 77?121 embryos in total. Two-tailed Fisher?s exact test. ????p < 0.0001, ??p = 0.0018, and ns: p > 0.05. (E) Kics2 depletion affects heart looping. Images of cmcl2 in situ hybridization at 48 hpf (scale bar: 100 ?m; A, atrium; V, ventricle). Graph displays correct (D), unlooped (N), and inversely (L) looped hearts. Two-tailed Fisher?s exact test. ????p < 0.0001. 8 experiments with 220?239 embryos. (F) Insulin (ins) in situ hybridization to detect pancreas localization. Dashed line: midline. Scale bar: 200 ?m. Graph displays correctly (R) and wrongly (L) placed pancreata. Two-tailed Fisher?s exact test. ????p < 0.0001. 8 experiments with 221?238 embryos. (G) Southpaw (spaw) expression is randomized upon kics2 knockdown but can be partially rescued by rapamycin treatment. Arrowheads: expression in the lateral plate mesoderm. Scale bar: 200 ?m. Two-tailed Fisher?s exact test. ????p < 0.0001 and ?p = 0.0341. 5 experiments with 109?120 embryos. (H) Confocal stacks of 6 somite stage Kupffer?s vesicles (KVs) stained for acetylated tubulin (acTub; magenta) and PKC? (green). Scale bar: 20 ?m. (I) Kics2 manipulation does not affect the number of cilia per KV. 2 experiments with 12?19 embryos. Kruskal-Wallis test with Dunn?s multiple comparison test. ns: p > 0.05. (J) KV cilia become elongated upon loss of Kics2 function. 2 experiments with 135?323 cilia. Kruskal-Wallis test with Dunn?s multiple comparison test. ????p < 0.0001 and ???p = 0.001.
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