Retinoic acid-dependent mechanisms patterning the cardiac progenitor fields

项目总结/摘要 先天性心脏缺陷（CHD）是最常见的出生缺陷类型，发生在1%的新生儿中。CHD 可导致婴儿患病、死亡，并可对成人造成更高的心血管并发症风险， 在矫正手术后。尽管冠心病的发病率很高，但其分子病因学尚不清楚， 明白要为冠心病患者开发靶向治疗，关键是要了解 引导心脏祖细胞进入特定心脏区域的机制。我们的长期目标是了解 调节正常心脏发育的保守机制。早期分化的心脏祖细胞 前侧板中胚层（ALPM）产生第一心野（FHF）， 祖细胞包括第二心脏区域（SHF）。已知体内视黄酸（RA）信号传导限制 通过间接抑制发育过程中的FGF信号传导， 胚胎前后轴的模式。对小鼠的研究表明， (TFs)在哺乳动物心血管发育过程中与TBX 1相互作用以调节FGF 8信号传导。的 RA调节ALPM内FHF和SHF规格的机制尚不清楚。的 本建议的具体目标是阐明RA调节FHF的分子机制 通过早期tbx 1-six 2a-Fgf信号传导级联来表达特异性。严格的RA调节对脊椎动物心脏至关重要 发育和六个TF在脊椎动物中进化保守，斑马鱼Six 2a是 与哺乳动物Six 2同源。有趣的是，我们对斑马鱼的初步研究表明， RA主要导致ALPM内FHF祖细胞的增加。我们的Bulk RNA-seq分析 早期斑马鱼胚胎揭示了在RA缺乏条件下six 2a的上调，并提示早期胚胎发育的可能性。 心脏发育对六个TF的需求比以前认为的要多。Six 2a的过表达导致 前化和更大的斑马鱼胚胎头，类似于在RA耗尽突变体中观察到的。 然而，我们目前还不了解RA信令下游的信令网络功能 调节ALPM内FHF祖细胞的规格。在目标1中，我们将使用新颖的six 2a-报告子 转基因系和six 2a突变体，以确定过量的Six 2a是否有助于扩大的FHF祖细胞 在RA信号缺陷的胚胎中。在目标2中，我们将进行遗传上位性实验， 确定Six 2a是否在RA信令下游的Tbx 1-FGF信令网络内起作用以限制CM ALPM中的祖先规范。由于RA在脊椎动物心脏发育中的作用， 这些研究将极大地提高我们对通路中的破坏如何的理解， 控制心脏祖细胞群分化的基因可能导致人类CHD。结果这些 研究将允许开发更好的治疗方法来治疗儿童和成人的CHD， 冠心病引起的心血管并发症。

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.