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Published on: September 1, 2017
Variable phenotypes and penetrance between and within different zebrafish ciliary transition zone mutants
Jun Wang1, Holly R Thomas1, Robert G Thompson1
1Department of Pharmacology and Toxicology, University of Alabama at Birmingham School of Medicine, Birmingham, AL 35294, USA.
Abstract:
Meckel syndrome, nephronophthisis, Joubert syndrome and Bardet-Biedl syndrome are caused by mutations in proteins that localize to the ciliary transition zone (TZ). The phenotypically distinct syndromes suggest that these TZ proteins have differing functions. However, mutations in a single TZ gene can result in multiple syndromes, suggesting that 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 that variations in phenotypes exist 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 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).
Insights
Mutations in transition zone (TZ) proteins cause distinct ciliopathies, but modifier genes influence phenotypes. Zebrafish models reveal tissue-specific TZ gene functions and complex, variable disease presentations, aiding identification of new cilia-associated genes.
Area of Science:
- Genetics
- Cell Biology
- Developmental Biology
Background:
- Ciliary transition zone (TZ) proteins are crucial for ciliopathies like Meckel, nephronophthisis, Joubert, and Bardet-Biedl syndromes.
- Distinct syndromes suggest varied TZ protein functions, yet single gene mutations can cause multiple syndromes, implying genetic modifiers.
Purpose of the Study:
- To investigate tissue-specific functions of TZ genes using zebrafish models.
- To explore the genetic basis of phenotypic variability in ciliopathies.
- To rapidly identify novel cilia-associated genes using CRISPR/Cas9 technology.
Main Methods:
- Comprehensive analysis of ten zebrafish TZ mutants (mks1, tmem216, tmem67, rpgrip1l, cc2d2a, b9d2, cep290, tctn1, nphp1, nphp4) and additional mutants (ift88, ift172).
- Phenotypic characterization of TZ mutants, including observation of variations within and across mutants.
- Application of multiple-guide CRISPR/Cas9 in F0 'crispant' embryos to identify ciliary phenotypes in candidate genes.
Main Results:
- Phenotypic variations among different TZ mutants support tissue-specific roles for these genes.
- Intra-mutant phenotypic variability and transient phenotypes were observed, mirroring complex human ciliopathies.
- CRISPR/Cas9 identified ciliary phenotypes in 11 novel cilia-associated gene candidates.
Conclusions:
- Zebrafish TZ mutants reveal complex gene functions and phenotypic variability in ciliopathies.
- Modifier genes likely influence ciliopathy phenotypes.
- CRISPR/Cas9 is an effective tool for rapid identification of genes involved in ciliary function.
