PROJECT SUMMARY Human cleft lip with or without cleft palate (CL/P), the most common craniofacial birth defect, is caused by im- paired fusion of the facial prominences. During normal morphogenesis, the frontonasal and maxillary promi- nences fuse at a three-way seam, the lambdoidal junction (l), to form the upper lip/primary palate. If the epithe- lium that covers the prominences persists at the l, orofacial clefting ensues, as we previously reported in mouse embryos deficient for PBX transcription factors (TFs). We described that: 1) prominence fusion requires coordi- nation of two distinct cellular behaviors, apoptosis and epithelial-to-mesenchymal transition (EMT), respectively, in two discrete subpopulations within the l epithelium; 2) these two subpopulations are characterized by different molecular signatures; and 3) mice deficient for PBX TFs with CL/P lack both of these epithelial subpopulations. Our preliminary evidence in the mouse indicates that: I) the λ epithelium is heterogeneous and comprises 6 main cell subpopulations prior to prominence fusion, as demonstrated by single cell RNA sequencing; II) one of the subpopulations, that we termed “l fusion effectors”, is enriched for genes that have been associated with human or mouse orofacial clefting, for genes encoding pro-apoptotic factors, and for inhibitors of cell cycle progression; III) “l fusion effector” cells are located at the tip of the prominences; and IV) the proportion of “l fusion effectors” is perturbed in compound Pbx1/2 mutant embryos with CL/P compared to controls. Based on these results, we posit that the “l fusion effector” cluster comprises cells that are prime executors of prominence fusion; is halted in the cell cycle, a prerequisite for subsequent cellular changes, like apoptosis and EMT, to achieve fusion; and is quantitatively and/or qualitatively perturbed in mouse models of CL/P. We will test this hypothesis via the following Specific Aims: 1) Determine the spatiotemporal dynamics of the epithelial “l fusion effector” subpopulation throughout midface prominence fusion. We will establish whether the “l fusion effector” clus- ter is transient or if it persists after fusion, as well as track the progenitors and descendants of “l fusion effector” cells in vivo across space and developmental time. 2) Establish the molecular mechanisms underlying cell cycle arrest in “l fusion effector” cells during upper lip/primary palate fusion. We will uncover in vivo cell cycle dynamics of “l fusion effector” cells and assess whether PBX-dependent regulation of cell cycle genes mediates cell cycle control in this cluster. 3) Determine whether the perturbations of the “λ fusion effector” cluster in Pbx1/2 mutants are recapitulated in p63- or Bmpr1a-deficient mice with CL/P. We will establish whether the cellular and transcriptional changes of the l epithelium resulting in CL/P in Pbx1/2 mutants are recapitulated in all three mouse models, or if they are distinct. This research...