The roles of histone methyl‐transferase kmt2d in vasculogenesis
Authors: Serrano Angie , Demarest Bradley L. , Tristani‐Firouzi Martin , Yost H. Joseph 
Published: June 2021

INTRODUCTION

Congenital heart defects (CHD) arise from abnormal development of the heart and major blood vessels. An estimated 40,000 infants are affected by CHD each year in the United States. Despite its high incidence, our understanding of the complex genetic anomalies leading these defects is limited. Recent studies linked mutations in the ubiquitous histone methyl‐transferase KMT2D with congenital heart defects and Kabuki Syndrome, a condition with a spectrum of CHD and other congenital disorders. While KMT2D has well‐described roles in myocardium development during cardiogenesis, it is unknown whether KMT2D has regulatory functions during vasculogenesis, which could contribute to the spectrum of heart and other defects in Kabuki Syndrome. KMTD2 has recently been shown to interact with Notch signaling in Drosophila. While Notch pathway has well‐established roles in vasculogenesis and angiogenesis, its regulation by KMT2D during cardiovascular development has not been explored. Zebrafish provide a strong model for the analysis of vasculogenesis defects, since it can survive early development in absence of blood circulation.

OBJECTIVE

The primary objective of this study is to describe the roles of Kmt2d and its regulatory link with Notch signaling during zebrafish vasculogenesis.

METHODS

Using the CRISPR/Cas9 technology, we generated Zebrafish kmt2d null mutants that recapitulate the phenotypic features of human Kabuki Syndrome. In order to study endocardial/endothelial development, the stable mutant kmt2dzy59 was crossed with the transgenic line tg(kdrl:GFP). kmt2dzy59;tg(kdrl:GFP) cardiovascular phenotype was characterized with super resolution confocal microscopy and fluorescent‐activated cell sorting (FACS).

Kmt2d and Notch pathway interaction was assessed by G0 knockdown analysis in the Notch reporter line tg(TP1:EGFP). Results were confirmed and expanded by RNAseq of single embryos from genetic crosses at 24hpf. Single embryo transcriptome analysis allows us to capture transcriptomes in clutches of embryos before the appearance of phenotypes, and to post‐facto genotype sibling embryos by transcriptome analysis with Kallisto and SNP mapping.

RESULTS

In this study we generated Zebrafish kmt2d null mutants that recapitulate the phenotypic features of Kabuki Syndrome. We characterized for the first time a cardiovascular developmental defect in kmt2dzy59 embryos, including hypoplastic ventricle, aortic arch disruption, and altered cell cycle profiles. kmt2d CRISPR/Cas9 injection in a Notch reporter line showed expanded Notch signaling in endocardial/endothelial cells. RNAseq analysis of kmt2dzy59 embryos showed a substantial upregulation of Notch signaling components.

CONCLUSION

Our results demonstrate that Kmt2d regulates zebrafish vasculogenesis. We also provide evidence of regulatory interactions between Kmt2d and Notch signaling which modify endothelial/endocardial cell behavior.

Support or Funding Information

This work in HJY laboratory was supported by NHLBI CvDC U01HL0981

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.