Retinoic acid-dependent mechanisms patterning the cardiac progenitor fields

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Project Summary/Abstract Congenital heart defects (CHDs) are the most common type of birth defects, occurring in 1% of newborns. CHDs can lead to infant illness, mortality and can pose a higher risk for cardiovascular complication in adults, even after corrective surgery. Despite their high prevalence, the molecular etiology underlying CHDs are not well understood. To develop targeted therapies for patients with CHDs, it is crucial to understand the fundamental mechanisms that direct cardiac progenitors into the specific heart fields. Our long-term goal is to understand the conserved mechanisms that regulate normal heart development. Early differentiating cardiac progenitors within the anterior lateral plate mesoderm (ALPM) give rise to the first heart field (FHF), while later differentiating progenitors comprise the second heart field (SHF). It is known that retinoic acid (RA) signaling in vivo restricts the cardiac progenitor fields within the ALPM by indirectly repressing FGF signaling during developmental patterning of the embryonic anterior-posterior axis. Studies in mice have indicated that Six transcription factors (TFs) interact with TBX1 to regulate FGF8 signaling during mammalian cardiovascular development. The mechanism by which RA regulates specification of the FHF and SHF within the ALPM is not understood. The specific aims of this proposal are to elucidate the molecular mechanisms by which RA regulates FHF specification through an early tbx1-six2a-Fgf signaling cascade. Tight RA regulation is critical for vertebrate heart development and Six TFs are evolutionarily conserved among vertebrates, with zebrafish Six2a being homologous to mammalian Six2. Interestingly, our preliminary studies in zebrafish indicate that a deficiency in RA predominantly leads to an increase in FHF progenitors cells within the ALPM. Our Bulk RNA-seq analysis of early stage zebrafish embryos revealed an upregulation of six2a in RA-deficient conditions and suggest an earlier requirement for Six TFs in heart development than previously thought. Overexpression of six2a results in anteriorization and larger zebrafish embryo heads, similar to what is observed in the RA-depleted mutants. However, we currently have no understanding of the signaling network functioning downstream of RA signaling to regulate the specification of FHF progenitors within the ALPM. In Aim 1, we will use novel six2a-reporter transgenic lines and six2a mutants to determine if excess Six2a contributes to the enlarged FHF progenitor population in RA signaling deficient embryos. In Aim 2, we will perform genetic epistasis experiments to determine if Six2a functions within a Tbx1-FGF signaling network downstream of RA signaling to limit CM progenitor specification within the ALPM. Due to the role of RA in vertebrate heart development and the conservation of Six TFs, these studies will greatly improve our understanding on how disruption in the pathways that control the differentiation of cardiac progenitor populations can lead to CHDs in humans. As a result, these studies will allow for the development of better therapeutics to treat CHDs in children as well as adults with cardiovascular complications resulting from CHDs.