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.