Abstract 19803: TMEM88: A Novel Regulator of Cardiac Development Identified Through Chromatin Dynamics

Abstract

Cardiac development is dependent on the biphasic activities of Wnt/β-catenin signaling where it is required for mesoderm development but must be blocked to specify the cardiogenic mesoderm. The mechanism mediating this inhibition of Wnt/β-catenin signaling in cardiac progenitor cells is not known. Methods and Results. RNA seq was used to analyze genes most highly induced in the cardiac progenitor cell during cardiac differentiation of human ES cells. Transmembrane protein 88 (TMEM88) was identified for further study. In line with previous work showing TMEM88 binds Disheveled by a C-terminal PDZ binding motif, we used transgenic hES cells to show that TMEM88 blocks Wnt/β-catenin signaling during cardiac differentiation. Chromatin dynamics showed marked suppression (H3K27me3) of this locus in the transition through mesoderm with a switch to an activated state (H3K4me3) in the cardiac progenitor; a signature resembling other genes involved in mediating cell fate changes such as transcription factors. By qPCR, TMEM88 is highly up-regulated prior to Nkx2.5 and Isl1 expression. In situ hybridization of developing zebrafish embryos showed expression of TMEM88 specifically in the cardiogenic ventro-lateral mesoderm overlapping the expression profile of cardiac transcription factors such as Nkx2.5 and MEF2c. Short hairpin RNA targeting the TMEM88 transcript was used to determine its requirement for cardiac differentiation of hES cells. Suppression of TMEM88 resulted in a failure to specify the cardiac lineage based on significantly reduced Nkx2.5, Tbx5, and cTnT expression. Linear regression analysis revealed a dose dependent effect of increased TMEM88 significantly correlating with higher cTnT expression. In vivo, morpholino knockdown of TMEM88 in zebrafish markedly alters heart development with some embryos failing to generate a heart. Phylogenetic analysis of TMEM88 orthologs identifies the expression of this gene only in vertebrate chordates. Conclusion. These data show that TMEM88 is required for cardiac development in vitro and in vivo acting mechanistically in a cell autonomous manner to inhibit Wnt/β-catenin signaling during the specification of the cardiac lineage in progenitor cells of the primordial heart.