Abstract
Kabuki Syndrome patients have a spectrum of congenital disorders, including congenital heart defects, the primary determinant of mortality. Seventy percent of Kabuki Syndrome patients have mutations in the histone methyl-transferase KMT2D. However, the underlying mechanisms that drive these congenital disorders are unknown. Here, we generated and characterized zebrafish kmt2d null mutants that recapitulate the cardinal phenotypic features of Kabuki Syndrome, including microcephaly, palate defects, abnormal ear development, and cardiac defects. The cardiac phenotype consists of a previously unknown vasculogenesis defect that affects endocardium patterning and, consequently, heart ventricle lumen formation. Additionally, zebrafish kmt2d null mutants have angiogenesis defects depicted by abnormal aortic arch development, hyperactive ectopic blood vessel sprouting, and aberrant patterning of the brain vascular plexus. We demonstrate that zebrafish kmt2d null mutants have robust Notch signaling hyperactivation in endocardial and endothelial cells, including increased protein levels of the Notch transcription factor Rbpj. Our zebrafish Kabuki Syndrome model reveals a regulatory link between the Notch pathway and Kmt2d during endothelium and endocardium patterning and shows that pharmacological inhibition of Notch signaling rebalances Rbpj protein levels and rescues the cardiovascular phenotype by enhancing endothelial and endocardial cell proliferation and stabilizing endocardial patterning. Taken together, these findings demonstrate that Kmt2d regulates vasculogenesis and angiogenesis, provide evidence for interactions between Kmt2d and Notch signaling in Kabuki Syndrome, and suggest future directions for clinical research.
Highlights
The zebrafish Kmt2d protein (UniProt E7F2F7) only has 44.3% amino acid identity with the human Histone-Lysine N-Methyltransferase 2D (KMT2D) (UniProt O14686), BLAST analysis of the individual protein domains showed that the Plant Homeo Domain (PHD) located at the N terminus of the zebrafish Kmt2d has 86.1% identity with the human PHD (UniProt O14686: aa 5030 to 5137 and 126 to 217) and the Su-Enhancer-of-zeste and Trithorax (SET) and Post-SET domains located at the C terminus of the zebrafish protein have 99.1% and 100% identity with the human SET (O14686: aa 5398 to 5513) and Post-Set domains (O14686: aa 5521 to 5537), respectively
Kabuki Syndrome is a rare multisystemic developmental disorder mainly characterized by postnatal growth deficit, distinct facial features, hearing defects, abnormal neurologic development, immune dysfunction, and Congenital heart defects (CHD), predominantly left-sided defects and coarctation of the aorta [1,3,62]
We developed a genetic zebrafish model for Kabuki Syndrome that recapitulated cardinal phenotypic traits of the human pathology but, most importantly, allowed us to uncover previously unknown cardiovascular defects precipitated by abnormal endothelial/endocardial cell patterning
Summary
Chemical inhibition of a downstream target of this pathway (v-raf murine sarcoma viral oncogene homolog B1 [BRAF] inhibitor), partially rescued the craniofacial and neuroanatomical phenotype of kmt2d-depleted zebrafish larvae in transient knockdown and kmt2d+/− heterozygous crosses [29]. These findings suggest a pathway involved in some aspects of KS neurological defects and establishes the utility of zebrafish for drugs screening in KS. MAPK, Mitogen-activated protein kinase; MFI, Median Fluorescence Intensity; MF20, Myosin Heavy Chain Antibody; MIP, maximum intensity projection; mo, mouth; MsigDB, Molecular Signatures Database; MsV, mesencephalic vein; NCA, nasal ciliary artery; NES, Normalized Enrichment Score; NICD, Notch intracellular domain; n.s., not significant; OMIM, Online Mendelian Inheritance in Man; ORA, opercular artery; OV, optic vein; oft, outflow tract; PHD, Plant Homeo Domain; pH3, phospho Histone 3; RAS, retrovirus-associated DNA sequence; RIN, RNA integrity number; RNA-seq, RNA sequencing; RTqPCR, reverse transcription-quantitative polymerase chain reaction; SET, Su-Enhancer-ofzeste and Trithorax; sgRNA, single-guide RNA; SR, superresolution; VA, ventral aorta; ventr/ven, ventricle
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