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

Hedgehog 信号传导控制指导早期心血管发生所需的基因调控网络

• 批准号: 9769123
• 负责人: Alexander Guzzetta
• 金额: $ 5万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2021-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769123
• 关键词: Ablation; Biological Assay; Cardiac; Cardiac development; Cardiovascular system; Cell Death; Cell Proliferation; Cells; Complex; Congenital Abnormality; Data; Defect; Development; Diagnosis; Down-Regulation; Embryo; Erinaceidae; Etiology; Excision; Failure; Foundations; Frequencies; Gene Expression; Gene Order; Genes; Genetic; Genetic Predisposition to Disease; Genetic Transcription; Heart; Heart Abnormalities; Heart Atrium; Investigation; Knowledge; Label; Life; Ligands; Live Birth; Maintenance; Mammals; Membrane; Mesoderm; Molecular; Morphogenesis; Mus; Mutation; Neural tube; Pathway interactions; Patients; Pattern; Phenotype; Process; Public Health; Regulator Genes; Resolution; Role; Signal Transduction; Structure; Testing; Transcription Coactivator; Transcription Repressor/Corepressor; Tube; Untranslated RNA; Up-Regulation; Variant; Work; cardiogenesis; congenital heart disorder; developmental genetics; differential expression; hedgehog signal transduction; mutant; novel; overexpression; premature; prevent; progenitor; programs; receptor; receptor binding; response; smoothened signaling pathway; spatiotemporal; transcription factor; transcriptome; transcriptome sequencing

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.

摘要 先天性心脏病（CHD）是最常见的出生缺陷，每100个婴儿中就有3个患有先天性心脏病 出生尽管其频率，冠心病的遗传病因仍然难以捉摸，由于普遍的贡献， 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 冠心病相关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， hedgehog（hh）信号下游的心血管谱系正常发育所必需的， 决定细胞命运的主要调节器。我们以前的工作表明，刺猬信号是必需的， 用于第二心脏场导出的心脏结构。使用遗传诱导命运图谱，我们观察到， 标记心脏祖细胞（E8.5和更晚）仅标记第二心脏区域结构，而早期标记心脏祖细胞（E8.5和更晚）仅标记第二心脏区域结构。 心脏祖细胞（E6.5）另外标记第一心野衍生的结构。所有hh的生殖细胞去除 信号，通过消融一个关键的膜结合受体，smoothened（smo），引起发育不全， 直线形心管、腔室扩张失败和早期胚胎死亡。条件性smo去除 使用Mesp 1-Cre重现了种系表型，而使用Nxk 2 -5-Cre去除smo支持了 室形成，限制了对早期中胚层的HH信号传导的需求。Mesp 1+细胞的RNA-seq 过表达Gli 3R，主要的hh信号转录抑制子显示了谱系的上调， 在胚胎第7.5天确定用于区分心血管谱系的转录程序， 调节与维持祖细胞身份有关的因子。我们假设刺猬信号 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： 控制中胚层祖细胞的分化时间。为了实现这一目标，我们建议： 1)研究转录破坏细胞命运决定的发育机制， 心源性中胚层2）研究严重发育不良性心脏缺损的细胞学和机制基础 是由早期HH信号中断引起的。这些数据将被整合为一种手段，不仅可以了解 hh控制早期中胚层模式的机制，但要贡献基础知识 通过为发育不全的心脏表型提供一种新的机制来阐明CHD的病因。