Highlights d Increased lymphatic specification in zebrafish mutants for splicing factor Nova2 d Vascular Nova2 regulates splicing of pre-mRNAs encoding signaling molecules d Nova2-regulated splicing limits MAPK/ERK signaling in developing vessels d Nova2 limits the Vegfc-Flt4-Mapk-Erk-Prox1 axis to restrict lymphatic specification
The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites) and CRISPR/Cas9 complexes that perfectly complement one strain injected. Sequencing of targeted sites showed biased, allele-specific editing for the perfectly complementary sequence in the majority of cases (14/19). To test utility, we examined whether phenotypes generated by F0 injection could be internally controlled with such polymorphisms. Targeting of genes bmp7a and chordin showed reduction in the frequency of phenotypes in injected ‘heterozygotes’ compared with injecting the strain with perfect complementarity. Next, injecting CRISPR/Cas9 complexes targeting two separate sites created deletions, but deletions were biased to selected chromosomes when one CRISPR/Cas9 target contained a polymorphism. Finally, integration of loxP sequences occurred preferentially in alleles with perfect complementarity. These experiments demonstrate that single nucleotide polymorphisms (SNPs) present throughout the genome can be utilised to increase the efficiency of in cis genome editing using CRISPR/Cas9 in the zebrafish model.
The endocardium plays important roles in the development and function of the vertebrate heart; however, few molecular markers of this tissue have been identified and little is known about what regulates its differentiation. Here, we describe the Gt(SAGFF27C); Tg(4xUAS:egfp) line as a marker of endocardial development in zebrafish. Transcriptomic comparison between endocardium and pan-endothelium confirms molecular distinction between these populations and time-course analysis suggests differentiation as early as eight somites. To investigate what regulates endocardial identity, we employed npas4l, etv2 and scl loss-of-function models. Endocardial expression is lost in npas4l mutants, significantly reduced in etv2 mutants and only modestly affected upon scl loss-of-function. Bmp signalling was also examined: overactivation of Bmp signalling increased endocardial expression, whereas Bmp inhibition decreased expression. Finally, epistasis experiments showed that overactivation of Bmp signalling was incapable of restoring endocardial expression in etv2 mutants. By contrast, overexpression of either npas4l or etv2 was sufficient to rescue endocardial expression upon Bmp inhibition. Together, these results describe the differentiation of the endocardium, distinct from vasculature, and place npas4l and etv2 downstream of Bmp signalling in regulating its differentiation.
Twenty unrelated Russian ARVC patients were examined by collection of personal and family history, physical examination, ECG, Echo-CG, myocardial biopsy and cardiac MRI. Analysis of PKP2 and DSG2 genes was performed by Sanger sequencing. Results: We found three rare genetic variants in PKP2 gene: two truncating mutations (D.W538X and F.1523_1538del) and one missense variant, p.S140F. Patients had manifestation at third-fourth decade of life with high-grade ventricular arrhythmia; ICDs were implanted in two cases. We found three rare genetic variants in DSG2 gene: p. S194L, p.N245H, p.R49H, and two missense VUCSes (p. V533I and p.V158G). Variants p.S194L, p.N245H and p. R49H are «probably damaging» by PolyPhen2. Four patients carrying 5 genetic variants had a manifestation at third decade of life with ventricular tachycardia, RV hypertrophy, and indications for ICD. Conclusion: We identified eight genetic variants in 20 Russian ARVC index patients. Three probands (15%) were PKP2-positive. Four probands (25%) carry mutations in DSG2 gene. This prevalence matches with the prevalence of ARVD9 and ARVD10 in European populations. Truncated mutations and probably damaging variants account for 25% mutations. This ratio of positive DNA-diagnostics tests looks reasonable to be introduced into routine clinical practice in our Centre.
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