Transcriptomic Atlas of Embryonic Human Craniofacial Development

A majority of craniofacial development occurs early in pregnancy and to fully understand how craniofacial defects arise, it is essential to observe gene expression during this critical time period. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from embryonic development -4 to 8 weeks post conception. To observe how genes are organized in this system, we have constructed co-expression networks from the bulk RNA-seq. Strong disease candidates are likely genes that are co-expressed with many other genes, serving as regulatory hubs within these networks. We have identified 29 modules of co-expressed genes and ~1800 hub genes and found enrichment of craniofacial relevant biology suggesting that these networks could reveal new disease genes and potential targets of previously identified disease genes. We leveraged large functional genomics databases including GTEx and GnomAD to reveal genes that are specifically expressed in craniofacial tissue. We integrate these data with our co-expression networks in order to prioritize the genes in this study. Our analysis revealed 268 novel or underappreciated disease candidate genes. RNA-seq performed on dissected human craniofacial tissue from three replicates of 4 time points and a culture model of cranial neural crest cells (CNCCs). Single-nuclei-RNA-seq was performed on two replicates of CS20 craniofacial primary tissue. ChIP-seq performed on CNCCs and chromatin states called to compare to previously published chromatin states from primary craniofacial tissue. **RAW data for Samples GSM5919238 and GSM5919239 are deposited in dbGAP**

颅面部发育的大部分过程发生于妊娠早期，若要全面阐明颅面部缺陷的发生机制，在这一关键时期观察基因表达情况至关重要。为此，我们对受孕后4至8周胚胎发育阶段的人类颅面部组织开展了批量RNA测序（bulk RNA-seq）与单细胞RNA测序（single-cell RNA-seq）。为解析该系统中基因的组织模式，我们基于批量RNA测序数据构建了共表达网络。与大量其他基因共表达的基因，极有可能是潜在的强疾病候选基因，可作为这些共表达网络中的调控枢纽。我们已鉴定出29个共表达基因模块和约1800个枢纽基因，且这些模块显著富集颅面部相关生物学功能，表明该网络可用于发现新的疾病基因，以及解析已鉴定疾病基因的潜在靶点。我们借助包含GTEx与GnomAD在内的大型功能基因组学数据库，筛选出了特异性表达于颅面部组织的基因。我们将这些数据与共表达网络进行整合，以对本研究中的基因进行优先级排序。本研究的分析结果鉴定出268个全新的或此前未被充分关注的疾病候选基因。我们对4个时间点、每个时间点3份重复的解剖人类颅面部组织，以及颅神经嵴细胞（cranial neural crest cells, CNCCs）的培养模型进行了RNA测序。我们对CS20颅面部原代组织的2份重复样本开展了单核RNA测序（single-nuclei RNA-seq）。我们对CNCCs进行了染色质免疫沉淀测序（ChIP-seq），并鉴定了染色质状态，以与此前已发表的颅面部原代组织的染色质状态进行对比。样本GSM5919238与GSM5919239的原始数据已提交至dbGAP数据库。