HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development.

Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular cardiomyocytes (VCs) and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5(+) progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5(+) progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors. Author summary Congenital heart defects are the most common malformations found in newborns, with many of these defects disrupting development of the outflow tract, the structure where blood is expelled from the heart. Despite their frequency, we do not have a grasp of the molecular and genetic mechanisms that underlie most congenital heart defects. Here, we show that zebrafish embryos containing a mutation in a gene called histone deacetylase 1 (hdac1) have smaller hearts with a reduction in the size of the ventricle and outflow tract. Hdac1 proteins limit accessibility to DNA and repress gene expression. We find that loss of Hdac1 in zebrafish embryos leads to increased expression of genes that are also induced by excess retinoic acid, a teratogen that induces similar outflow tract defects. Genetic loss-of-function studies support that ectopic expression of ripply3, a common target of both Hdac1 and retinoic acid signaling that is normally restricted to a subset of posterior pharyngeal cells, contributes to the smaller hearts found in zebrafish hdac1 mutants. Our study establishes a mechanism whereby the coordinated repression of genes downstream of Hdac1 and retinoic acid signaling is necessary for normal vertebrate outflow tract development.