Modeling Gene Regulatory Networks for Early Cardiopharyngeal Development

先天性心脏缺陷影响~1%的新生儿，通常与颅面畸形有关。 DGS/CVFS（DiGeorge/VandeloFacial Syndrome）后者是 通常由自发性22q11.2从头缺失引起，导致TBX 1 单倍性不足是导致这种疾病的主要原因。TBX 1被认为在多能细胞中起作用。 早期第二心脏区和鳃头/咽头肌的祖细胞 胚胎，因此合并心脏和颅面畸形出现的早期缺陷 在心咽中胚层。多个相互作用的遗传修饰剂的存在， DGS突变暗示了复杂的基因调控网络（GRN）控制着早期 心咽发育尽管在确定关键遗传决定因素方面取得了进展， 脊椎动物胚胎的相对复杂性阻碍了心咽模型的进展， 网络.被囊玻璃海鞘是一个易于处理的模型， 可以在脊索动物中以前所未有的时空分辨率进行研究， 功能基因组学方法。在以前的研究中，全面的基因表达和 通过谱系特异性全基因组测定获得染色质可及性谱， 包括单细胞RNA-seq（scRNA-seq）和ATAC-seq。一种结合样本 开发了条形码、CRISPR/Cas9介导的诱变和scRNA-seq， 系统地询问编码和非编码遗传元件的功能， 高含量scRNA-seq测定。首先，这种方法将用于分析功能丧失 干扰在心咽谱系中表达的候选转录调节因子。 Ciona和可用的小鼠数据集将被整合到跨物种模型中， 心咽GRNs的保守和发散特征。其次，可达的扰动 选择的转录调节因子的非编码元件（加上高含量的scRNA-seq测定 和新的计算方法）将允许显式整合非编码元素， 我们的GRN模型的背景。最后，干扰心咽特异性 染色质可及性随后是谱系特异性ATAC-seq将进一步解开 转录调节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 心咽发育和先天性心颅面综合征的病因。

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.