Variable phenotypes and penetrance between and within different zebrafish transition zone mutants

Meckel Syndrome, Nephronophthisis, Joubert Syndrome, and Bardet-Biedl Syndrome have mutations in proteins that localize to the ciliary transition zone (TZ). The phenotypically distinct syndromes suggest these TZ proteins have differing functions. However, mutations in a single TZ gene can result in multiple syndromes suggesting the phenotype is influenced by modifier genes. We performed a comprehensive analysis of ten zebrafish TZ mutants including mks1, tmem216, tmem67, rpgrip1l, cc2d2a, b9d2, cep290, tctn1, nphp1, and nphp4, as well as mutants in ift88 and ift172. Our data indicate variations in phenotypes exists between different TZ mutants, supporting different tissue specific functions of these TZ genes. Further we observed phenotypic variations within progeny of a single TZ mutant, reminiscent of multiple disease syndromes being associated with mutations in one gene. In some mutants the dynamics of the phenotype became complex with transitory phenotypes that are corrected over time. We have also demonstrated that multiple-guide derived CRISPR/Cas9 F0 “Crispant” embryos recapitulate zygotic null phenotypes, and rapidly identified ciliary phenotypes in 11 cilia-associated gene candidates (ankfn1, ccdc65, cfap57, fhad1, nme7, pacrg, saxo2, c1orf194, ttc26, zmynd12, and cfap52). Overall design: The homozygous mutant (including ift172, ift88, b9d2, mks1, cc2d2a, tmem216, cep290, tmem67) embryos were obtained from multiple pairs of heterozygous mutant crosses. The rpgrip1l-/- embryos were obtained from multiple pairs of homozygous crosses. The wild-type embryos were obtained from multiple pairs of AB-strain wild-type crosses (the zebrafish strain used for generating all cilia-related mutants). To obtain embryos from same developmental stage for each allele, 20-min timed mating were performed. For 5 days post fertilization (dpf) analysis, RNA was harvested from, ift172, ift88, b9d2, tmem216, cep290, tmem67 mutant embryos (~30-35 mutant embryos/sample) sorted at 2 dpf based on the curled-down tail (CDT) phenotype. b9d2 and mks1 mutant embryos were identified by genotyping DNA extracted from the 5-dpf embryo tail. For 2 dpf analysis, RNA was harvested from rpgrip1l-/- embryos (~30-35 embryos/sample) that were sorted into two groups based on the CDT and normal-looking (normal) phenotype. RNA samples were prepared with Qiagen RNeasy Plus Mini Kit. RNA library was prepared with Illumina RNA with PolyA selection package and sequenced with Illumina HiSeq 2x150bp, single index.