Genetic underpinnings of craniofacial disorders explored with spatial sequencing

口腔畸形，包括牙齿畸形(缺牙、多牙)、唇裂和/或唇裂 盘状畸形(口面部裂，OFC)和唾液腺异常(异位或发育不全)是最常见的出生方式 美国的缺陷。对于这些疾病的预防性治疗的设计将需要一个精确的理解 转录调控网络(TRN)控制相关组织的发育。研究项目： 模型生物是无价的，例如，揭示了这些结构中的间充质起源于 其中的神经脊和上皮细胞主要来源于口腔外胚层。然而，目前还不清楚这些TRN是如何 在发育时间内和口腔的空间域内部署。此外，这些TRN的一些方面 很可能是人类特有的，例如那些调节第二牙列发育的基因，它确实 不会出现在啮齿类动物身上。最后，上面提到的所有障碍都有遗传基础，因为没有一个没有所有的 遗传风险得到了充分的解释。了解人体组织中的TRNS是找到候选基因的最可靠的方法 隐藏着这种风险的基因。我们空间转录组的最新进展，以及获得捐赠的途径 人类胎儿组织，使得这些重要的问题能够以精确的时空方式得到解决。这里 在目标1中，我们建议在四个关键的发育时间点在整个人脸上进行SCI空间 (受孕后7-9、10-12、13-15和16-18周)。然后我们将重点放在第二味觉和 OFC的基因基础。我们将使用计算算法来推断成员资格和监管 调节腭上皮和腭间充质不同区域分化的TRNS等级； 这些TRN的排名成员是隐藏OFC缺失遗传能力的有力候选者。在目标2中，我们 将利用第一个目标的结果来开发将诱导多能干细胞(IPSC)转化为 腭部上皮和间充质细胞。我们将设计2个编码和2个非编码变体的iPSC 与OFC相关，将工程化IPSC分化为腭部细胞类型，并将分化后的细胞 单细胞RNA-seq.这将揭示特定的细胞类型，以及它们发育过程中受影响的步骤 通过突变，阐明OFC的发病机制。这些实验将确定强烈的 口裂缺失遗传性候选，改进与以下相关的DNA变异的功能测试 并为类似地分析其他遗传性颅面部疾病提供了数据集。

Malformations of the oral cavity, which include dental anomalies (hypodontia, hyperdontia), cleft lip and or cleft plate (orofacial cleft, OFC), and salivary gland anomalies (ectopic or aplasia), are among the most common birth defects in the US. The design of preventative therapies for these disorders will require a precise understanding of the transcriptional regulatory networks (TRNs) governing development of the relevant tissues. Studies in model organisms have been invaluable, for instance revealing that mesenchyme in these structures derives from neural crest and epithelia in them derives largely from oral ectoderm. However, it is unclear how these TRNs are deployed over developmental time and within spatial domains of the mouth. Moreover, aspects of these TRNs are likely to be human specific, for instance those regulating the development of secondary dentition, which does not occur in rodents. Finally, all of the disorders mentioned above have a genetic basis, in none has all of the heritable risk been fully explained. Knowledge of the TRNs in human tissue is the surest way to find candidate genes to harbor such risk. Recent advances in our spatial transcriptomics (sciSpace), and access to donated human fetal tissue, permit these important questions to be addressed in a precise spatio-temporal manner. Here we propose, in Aim 1, to conduct sciSpace over the entire human face at four critical developmental timepoints (7-9, 10-12, 13-15, and 16-18 weeks post conception). We will then focus on the secondary palate and the genetic underpinnings of OFC. We will use computational algorithms to deduce the membership and regulatory hierarchy of TRNs regulating differentiation of distinct domains of palate epithelium and palate mesenchyme; top ranking members of these TRNs are strong candidates to harbor the missing heritability for OFC. In Aim 2, we will use the results of the first aim to develop protocols for converting induced pluripotent stem cells (iPSC) into palate epithelium and mesenchymal cells. We will engineer iPSC with 2 coding and 2 non-coding variants associated with OFC, differentiate the engineered iPSC into palate cell types, and subject the differentiated cells to single cell RNA-seq. This will reveal the specific cell types, and the step in their development, that is affected by the variants, illuminating the pathogenic mechanisms of OFC. These experiments will identify strong candidates for the missing heritability for orofacial cleft, improve functional tests of DNA variants associated with it, and provide the datasets to similarly analyze other inherited craniofacial disorders.