Modeling Gene Regulatory Networks for Early Cardiopharyngeal Development

先天性心脏病影响约1%的新生儿，并经常与颅面部相关 缺陷，如DiGeorge/心脏速度面部综合征(DGS/CVFS)。后者是 通常由自发的从头22q11.2缺失引起，这导致了tbx1 单倍体功能不全是这种疾病的主要原因。TBX1被认为在多能性中发挥作用 早期第二心野和分支/咽头肌的祖细胞 胚胎，因此心脏和颅面联合畸形出现在早期的缺陷中 在心咽中胚层。多个相互作用的遗传修饰物的存在 DGS外显率提示复杂的基因调控网络(GRN)控制早期 心咽发育。尽管在识别关键基因决定因素方面取得了进展，但 脊椎动物胚胎的相对复杂性阻碍了心咽建模的进展 网络。被囊状乔纳是一种易驯化的模型，在那里早期心咽发育 可以在脊索动物中以前所未有的空间和时间分辨率进行研究，使用 功能基因组学方法。在以前的研究中，全面的基因表达和 染色质可及性图谱通过谱系特异性全基因组分析获得， 包括单细胞RNA-seq(scRNA-seq)和ATAC-seq。一种组合样本的方法 条形码、CRISPR/Cas9介导的突变和scRNA-seq被开发为 系统地询问编码和非编码遗传元件的功能 高含量scRNA-seq分析。首先，这种方法将被用来描述功能丧失 在心咽谱系中表达的候选转录调节因子的扰动。 Ciona和可用的老鼠数据集将被集成到跨物种模型中，以共同学习 心咽GRN的保守和发散特征。接下来，可访问性的摄动 所选转录调节因子的非编码元件(加上高含量scRNA-seq分析 和新的计算方法)将允许显式集成 我们GRN模型的背景。最后，心咽特异的扰动调节器 染色质的可及性以及特定血统的atac-seq将进一步解开 转录调控因子对可及性与活性的影响。将这些数据集集成到 持久和不断发展的GRN模型将支持对早期 先天性心颅面部综合征的病因与心咽发育。

Congenital heart defects affect ~1% of newborn infants and are often associated with craniofacial defects as is the case in the DiGeorge/CardioVeloFacial syndrome (DGS/CVFS). The latter is often caused by spontaneous de novo 22q11.2 deletions, which result in TBX1 haploinsufficiency, a major cause of the disease. TBX1 is thought to function in multipotent progenitors for the second heart field and branchiomeric/pharyngeal head muscles in early embryos, and thus combined cardiac and craniofacial malformations emerge from early defects in the cardiopharyngeal mesoderm. The existence of multiple interacting genetic modifiers of DGS penetrance hints at the complex gene regulatory network (GRN) controlling early cardiopharyngeal development. Despite progress in identifying key genetic determinants, the relative complexity of vertebrate embryos has hindered progress in modeling cardiopharyngeal networks. The tunicate Ciona is a tractable model where early cardiopharyngeal development can be studied with unprecedented spatial and temporal resolution in chordates, using functional genomics methods. In previous studies, comprehensive gene expression and chromatin accessibility profiles were obtained by lineage-specific whole genome assays, including single cell RNA-seq (scRNA-seq) and ATAC-seq. A method combining sample barcoding, CRISPR/Cas9-mediated mutagenesis, and scRNA-seq was developed to systematically interrogate the function of coding and non-coding genetic elements using high-content scRNA-seq assays. First, this approach will be used to profile loss-of-function perturbations for candidate transcription regulators expressed in the cardiopharyngeal lineage. Ciona and available mouse datasets will be integrated into cross-species models to jointly learn conserved and divergent features of cardiopharyngeal GRNs. Next, perturbations of accessible non-coding elements for selected transcription regulators (plus high-content scRNA-seq assays and new computational methods) will permit the explicit integration of non-coding elements in the context of our GRN models. Finally, perturbing regulators of cardiopharyngeal-specific chromatin accessibility followed by lineage-specific ATAC-seq will further disentangle the impact of transcription regulators on accessibility vs. activity. Integrating these datasets into lasting and evolving GRN models will support comprehensive understanding of early cardiopharyngeal development and the etiology of congenital cardio-craniofacial syndromes.