Jomok, Beninio

The present forward mutagenesis screen strategies in vertebrates are difficult to identify genes and mutants affecting a specific organ of interest. Here, we report a strategy to enrich cardiac mutants by leveraging the transparency of zebrafish embryos and the high efficiency of pGBT-RP2 (RP2), a gene-breaking transposon-based in vivo protein trap cassette. By selectively raising up founder fish with Red Fluorescence Protein (RFP) reporter expression in the heart, we successfully generated 15 cardiac mutant lines. All tagged loci exhibit cardiac expression and the overall knockdown efficiency at transcript level is greater than 95%. One of the cardiac lines, RP2_ #135, is a homozygous embryonic lethal line that is caused by a RP2 insertion in methionine adenosyltransferase 2a (mat2a) . Taking advantage of the LoxP sites inside the RP2 vector, we further demonstrated that the cardiac phenotypes in RP2_ #135 is due to a disrupted expression of mat2a in cardiomyocytes, as indicated by a conditional rescue assay using Tg(cmlc2:Cre-YFP) , a myocardium specific Cre transgenic line. Finally, we show that the reduced cardiomyocyte number, but not size, in the mat2a mutant is conveyed by the p53-mediated apoptosis pathway. Together, our data prove the feasibility of a facile approach to generate a vertebrate cardiac mutant collection with the following two unique features. First, the expression dynamics of each tagged gene is reported by RFP. Second, the causality between the tissue-specific gene expression and the resulted cardiac phenotypes can be attested genetically.

Rationale: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. Objective: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.