Loss of function of the TMEM67 transcripts containing missense mutations in a zebrafish phenotype rescue assay. (a) Bright-field microscopy of morphant zebrafish embryos stained with riboprobes against myod, pax2, and krox20 using in situ hybridization. Uninjected, uninjected embryos. MO, embryos injected with morpholino. WT + MO, embryos co-injected with OaMKS3 mRNA plus MO. MT + MO embryos co-injected with mutant OaMKS3(I681N;I687S) mRNA plus MO. (b) Zebrafish embryos at the 11–12-somite stage stained by in situ hybridization with riboprobes against myod, pax2, and krox20. The graded embryos were injected with 0.17–0.33 pmol mks3 MO, while normal embryos were injected with the solvent (Danieau buffer and phenol red dye mixture). The grade 1 morphant shows a shortened body axis, kinked notochords, and wide somites compared with the normal embryo, and exhibited one or two out of the three developmental aberrations (see Methods). Embryos with grade 2 phenotypes showed all three structural defects to a moderate degree. Grade 3 embryos showed more severe developmental aberrations. The embryos with grade 4 phenotype showed severe developmental malformations and died within or soon after 14 hpf. (c) Graph showing the phenotypic rescue of mks3 morphant embryos co-injected at the 11–12 somite stage of development with ovine OaMKS3 mRNA plus MO and scored using in situ hybridization (ISH). The percentages of the 11–12 somite rescued zebrafish embryos (with 50 ng mRNA) are shown in different morphological grades. Embryos at the 11–12-somite stage were assessed by myoD, pax2, and krox20 labeling using ISH and observed under a bright-field microscope. Co-injection of MO plus 50 ng ovine wild type OaMKS3 mRNA (MO + WT) resulted in a significant restoration of the normal phenotype in the morphant embryos compared with MO-injected embryos (**p < 0.0001, 2-tailed Fisher Exact test), whereas the co-injection of MO plus ovine mutant OaMKS3(I681N;I687S) mRNA (MO + MT) did not significantly restore the normal phenotype in the morphant embryos compared with morpholino-injected embryos (p = 0.2903). The number indicated at the top of each bar denotes the total number of embryos per injection condition. Abbreviations: MO, morpholino; WT, OaMKS3 mRNA; MT, OaMKS3(I681N;I687S) mRNA; ns, not significant.