Loss of function of Kmt2d, a gene mutated in Kabuki syndrome, affects heart development in Xenopus laevis

Abstract

Background Kabuki syndrome is a haploinsufficient congenital multi‐organ malformation syndrome, which frequently includes severe heart defects. Mutations in the histone H3K4 methyltransferase KMT2D have been identified as the main cause of Kabuki syndrome, however, the role of KMT2D in heart development remains to be characterized. Results Here we analyze the function of Kmt2d at different stages of Xenopus heart development. Xenopus Kmt2d is ubiquitously expressed at early stages of cardiogenesis, with enrichment in the anterior region including the cardiac precursor cells. Morpholino‐mediated knockdown of Kmt2d led to hypoplastic hearts lacking the three‐chambered structure. Analyzing different stages of cardiogenesis revealed that development of the first and second heart fields as well as cardiac differentiation were severely affected by loss of Kmt2d function. Conclusion Kmt2d loss of function in Xenopus recapitulates the hypoplastic heart defects observed in Kabuki syndrome patients and shows that Kmt2d function is required for the establishment of the primary and secondary heart fields. Thus, Xenopus Kmt2d morphants can be a valuable tool to elucidate the etiology of the congenital heart defects associated with Kabuki syndrome.

The role of Kabuki Syndrome genesKMT2DandKDM6Ain development: Analysis in Human sequencing data and compared to mice and zebrafish

“…kmt2d mutations in zebrafish cause defects in cardiovascular development, angiogenesis and aortic arch formation, as well as cardiac hypoplasticity (Serrano et al, 2019) while Kmt2d associates with genes in cardiomyocytes, and myocardial deletion of Kmt2d reduces cardiac gene expression during murine heart development (Ang et al, 2016). In Xenopus , morpholino-based knockdown of kmt2d causes cardiac hypoplasticity, reduced expression of early cardiac developmental genes tbx20 , isl1 , nkx2.5 (Schwenty-Lara, Nurnberger, et al, 2019), and impaired formation and migration of NCCs by reducing the expression of early NCC-specific markers like pax3 , foxd3 , twist , and slug (Schwenty-Lara, Nehl, et al, 2019). Interestingly we did not observe these same defects in single zebrafish Kabuki Syndrome mutants.…”

“…Recent studies have significantly contributed to our understanding of cardiac development and function as regulated by KMT2D (Ang et al, 2016; Schwenty-Lara, Nurnberger, et al, 2019; Serrano et al, 2019), and KDM6A (Lee et al, 2012). The PHD domain of zebrafish kmt2d , which shares 86.1% identity with human KMT2D , was targeted by CRISPR-mediated gene disruption in a recent study on kmt2d in heart development (Serrano et al, 2019).…”

“…They report a robust Notch signaling hyperactivation in endocardial and endothelial cells that affect vasculogenesis and angiogenesis, and a rescue of cardiovascular phenotype by pharmacological inhibition of Notch signaling (Serrano et al, 2019). Another recent study on kmt2d showed morpholino-based knockdown of kmt2d in Xenopus causes heart defects (Schwenty-Lara, Nurnberger, et al, 2019), and impairs NCC development (Schwenty-Lara, Nehl, & Borchers, 2019). In mouse studies, development of limbs, palate, central nervous system, and heart are impaired upon mutating Kmt2d , with hypoplasia in frontonasal bone, fully penetrant cleft palate, mandible hypoplasia, deficits in palatal shelf elevation and cranial base ossification (Fahrner et al, 2019; Shpargel, Mangini, Xie, Ge, & Magnuson, 2020).…”

Using Xenopus to analyze neurocristopathies like Kabuki syndrome

“…Whether the NC induction and migration defects observed in Kmt2d‐deficient Xenopus embryos are due to species‐dependent differences or caused by divergent experimental approaches remains to be investigated (Schwenty‐Lara et al, 2020). Similarly, while cardiac defects have been carefully examined in conditional Kmt2d knockout mice from a morphological and epigenetic perspective (Ang et al, 2016), Xenopus loss‐of‐function experiments have dissected to what extent the different steps of heart development are affected by Kmt2d depletion (Schwenty‐Lara et al, 2019). These findings showed that Kmt2d knockdown has only minor effects on cardiac mesoderm induction, while cardiac differentiation was severely affected (Schwenty‐Lara et al, 2019).…”

“…Similarly, while cardiac defects have been carefully examined in conditional Kmt2d knockout mice from a morphological and epigenetic perspective (Ang et al, 2016), Xenopus loss‐of‐function experiments have dissected to what extent the different steps of heart development are affected by Kmt2d depletion (Schwenty‐Lara et al, 2019). These findings showed that Kmt2d knockdown has only minor effects on cardiac mesoderm induction, while cardiac differentiation was severely affected (Schwenty‐Lara et al, 2019). With the analysis of craniofacial and cardiac malformations two major Kabuki patient symptoms have been systematically addressed using the Xenopus system.…”