Hedgehog signaling controls a gene regulatory network necessary for directing early cardiovasculogenesis

摘要 先天性心脏病(CHD)是最常见的出生缺陷--每100人中就有3人患有先天性心脏病 出生。尽管CHD的发生频率很高，但由于普遍存在的 非编码变异和遗传修饰。作为一个实验室，我们试图通过理解来提炼这种复杂性 涉及CHD的潜在基因调控网络。在这项研究中，我们发现了一个基因调控网络 对于Hedgehog(HH)信号下游心血管谱系的正确发展是必不可少的，a 掌握细胞命运决定的调节器。我们之前的工作已经表明，Hedgehog信号是必需的 用于第二个心场衍生的心脏结构。利用遗传诱导命运图谱，我们观察到晚期 标记心脏祖细胞(E8.5和更高版本)只标记第二个心区结构，而早期标记 心脏祖细胞(E6.5)还标记了第一心场来源的结构。所有HH的生殖系移除 信号，通过消融一个关键的膜结合受体，平滑(SMO)，导致发育不全 心管呈线形，心腔扩张失败，早期胚胎死亡。有条件删除SMO 使用Mesp1-Cre概括了生殖系表型，而使用Nxk2-5-Cre支持去除SMO 小室的形成，限制了HH信号对早期中胚层的要求。Mesp1+细胞的RNA序列 过表达Gli3R，主要的HH信号转录抑制因子，揭示了谱系的上调。 在胚胎7.5天以下确定区分心血管血统的转录程序- 调节与维持祖细胞身份有关的因素。我们假设刺猬发出的信号 通过以下方式控制正确建立心血管谱系所需的基因调控网络 控制中胚祖细胞的分化时间。为达致这个目标，我们建议 1)验证和研究转录干扰细胞命运决定的发育机制 心源性中胚层2)研究严重发育不良心脏缺陷的细胞和机制基础 由早期HH信令中断引起。这些数据将被整合为一种手段，不仅可以理解 HH控制早期中胚层图案化的机制，但提供基础知识 通过为心脏发育不良表型提供一种新的机制来阐明冠心病的病因。

ABSTRACT Congenital heart disease (CHD) is the most common birth defect—occurring in as many as 3 in every 100 live births. Despite its frequency, the genetic etiologies of CHD remain elusive due to prevalent contribution of noncoding variation and genetic modifiers. As a lab we attempt to distill this complexity though understanding the underlying gene regulatory networks involved in CHD. In this study, we uncover a gene regulatory network essential for the proper development of cardiovascular lineages downstream of the hedgehog (hh) signaling, a master regulator of cell fate determination. Our previous work has shown that Hedgehog signaling is required for second heart field-derived cardiac structures. Using Genetic Inducible Fate Mapping, we observed that late labeling of cardiac progenitors (E8.5 and later) labeled only second heart field structures while early labeling of cardiac progenitors (E6.5) additionally labeled first heart field derived structures. Germline removal of all hh signaling, through ablation of a key membrane-bound receptor, smoothened (smo), caused hypoplasia of the linear heart tube, a failure of chamber expansion, and early embryonic lethality. Conditional smo removal using Mesp1-Cre recapitulated the germline phenotype whereas smo removal using Nxk2-5-Cre supported chamber formation, restricting the requirement for hh signaling to early mesoderm. RNA-seq of Mesp1+ cells overexpressing Gli3R, the primary hh-signaling transcriptional repressor revealed upregulation of lineage- determining transcriptional programs for differentiating cardiovascular lineages at embryonic day 7.5 but down- regulation of factors involved with maintenance of progenitor identity. We hypothesize that hedgehog signaling controls a gene regulatory network required for the proper establishment of cardiovascular lineages by controlling the differentiation timing of mesodermal progenitors. As a means to accomplish this, we propose to 1) Validate and study the developmental mechanisms of transcriptional disruption of cell fate decisions in the cardiogenic mesoderm 2) Study the cellular and mechanistic basis for the severe hypoplastic cardiac defects caused by disruption in early hh singaling. These data will be integrated as a means to understand not only the mechanism by which hh controls early mesodermal patterning, but to contribute foundational knowledge towards the elucidation of CHD etiology by providing a novel mechanism for hypoplastic cardiac phenotypes.