Molecular pathogenesis of congenital heart disease mediated by neural crest and second heart field cells

摘要 心脏流出道(OFT)与毗邻的主要动脉易受发育的影响 胚胎发育过程中的侮辱会导致先天性心脏病。他们的部分原因 易损性是因为OFT的形态发生需要两个神经元的相互作用 冠状细胞(NCC)和邻近的第二心野(SHF)中胚层细胞。这件事发生在 在胚胎咽器的动态扩张过程中。要理解这两个 在并列的群体中，我们在谱系后进行单细胞RNA测序(scRNA-seq) 净化，主要集中在NCC。心脏NCC(CNCC)传统上被定义为 通过细胞命运进程的特定基因标记物进行定位而不是分子上，因为 它们的多能性和不断变化的基因图谱。使用scRNA-seq，我们能够识别 咽部器官中可能存在CNCC。这是通过使用早期的 血管平滑肌基因，如Acta2，作为指导。从这一点上，我们确定了三个 小鼠胚胎日的CNCC数量，E10.5。我们将它们称为Tbx2和Tbx3 (Tbx2/3)、Isl1和Acta2群体，基于这些基因的不同表达。我们 提示Tbx2/3和Isl1谱系细胞可能独立进化并对 咽弓动脉和OFT的平滑肌细胞。这项建议已经确定了 为了通过双重血统追踪来探索这三个群体的起源和命运轨迹， 确定内部基因的功能，并建立控制其基因调控网络 发展。我们不仅将检查NCC的变化，还将评估周围环境 SHF中胚层细胞，因为改变CNCC可能也会影响这些祖细胞。我们 也有从小鼠胚胎中纯化的SHF中胚层细胞的scRNA-seq数据。论 另一方面，SHF中所需基因的改变可能会扰乱CNCC 发展。作为一个具体的例子，TBX1，22q11.2缺失综合征的基因是 在SHF中表达，但不在CNCC中表达，但它对其功能有很大影响。我们将测试这一想法 在TBX1缺失突变胚胎中，CNCC不能从表达Sox10的祖细胞进化而来 并且不能进入OFT。在这个计划中要研究的许多基因是 与人类患者的先天性心脏病有关。总体而言，这一计划将有所帮助 确定CNCC的分子方面和CNCC-SHF细胞命运的编排 胚胎发生。

ABSTRACT The cardiac outflow tract (OFT) with adjoining major arteries is susceptible to developmental insults during embryogenesis that leads to congenital heart disease. Part of the reason for their vulnerability is because the morphogenesis of the OFT requires the interaction of both neural crest cells (NCCs) and adjacent second heart field (SHF) mesoderm cells. This takes place during dynamic expansion of the embryonic pharyngeal apparatus. To understand these two juxtaposed populations, we performed single cell RNA-sequencing (scRNA-seq) after lineage purification, focusing mainly on NCCs. Cardiac NCCs (CNCCs) have traditionally been defined positionally rather than molecularly by specific gene markers of cell fate progression because of their multipotency and changing genetic profiles. Using scRNA-seq, we were able to identify putative CNCCs in the pharyngeal apparatus. This was achieved by using expression of early vascular smooth muscle genes, such as Acta2, as a guide. From this, we identified three CNCC populations at mouse embryonic day, E10.5. We refer to them as the Tbx2 and Tbx3 (Tbx2/3), Isl1 and Acta2 populations, based upon distinct expression of these genes. We suggest that the Tbx2/3 and Isl1 lineage cells may independently evolve and contribute to smooth muscle cells of the pharyngeal arch arteries and OFT, respectively. This proposal is set to explore the origin and fate trajectories of these three populations by dual lineage tracing, by determining the function of genes within, and to build gene regulatory networks controlling their development. Not only will we examine changes in NCCs but also will evaluate surrounding SHF mesoderm cells because altering CNCCs might affect these progenitor cells as well. We also have scRNA-seq data on the SHF mesoderm cells purified from mouse embryos. On the other hand, it is possible that alteration of genes required in the SHF can disrupt CNCC development. As one specific example, Tbx1, the gene for 22q11.2 deletion syndrome is expressed in the SHF but not CNCCs, but it greatly affects their function. We will test the idea that in Tbx1 null mutant embryos, CNCCs fail to progress from Sox10 expressing progenitors and are not able to enter the OFT. Many of the genes to be investigated in this program are associated with congenital heart disease in human patients. Overall, this program will help identify molecular aspects of CNCCs and the orchestration of CNCC-SHF cell fates in embryogenesis.