Fusion of the facial prominences to form the upper lip and primary palate is a critical part of normal human development and failure of this process results in a type of orofacial clefting affecting ~1.5 in 1,000 human births termed cleft lip with or without cleft palate (CL+/-P). Despite the importance of this process, we only have limited knowledge of the genes and cell types that are responsible for carrying out successful fusion of the facial prominences at a critical region termed the lambdoid junction, where the medial nasal process meets with the lateral nasal process and maxillary prominence. Its small size and complex organization have made this fusion zone difficult to isolate and study using conventional gene expression analyses. With single cell RNA sequencing (scRNA-seq), we assessed the cell types, cell behaviors, and gene expression profiles for the wild-type lambdoid junction. We identified particular cell types associated with fusion and specific gene signatures expressed within these populations. Importantly, many of these genes have been relatively understudied in model organisms. Building on these preliminary data, the goal of this proposal is to investigate if and how these cell populations and their gene signatures are altered in mouse models of human clefting. First, I will study if and how the expression of the marker genes of the cell populations at the fusion zone is altered in a small set of mouse models of human orofacial clefting. I will use models that are: (a) of full penetrance and invariable expressivity; (b) clearly involving the l junction in any pathology; (c) relevant to human orofacial clefting; and (d) that represent different scenarios of l junction fusion failure. Specifically, I will use Trp63, Bmpr1a, Tfap2a, and Aldh1a3 models of clefting and craniofacial dysmorphology. For each of these models, I will assess the expression of the marker genes by both in situ hybridization and by qRT-PCR, and assess if there are similarities and differences in how these genes and their associated cell populations are altered compared to control embryos. Secondly, I will perform lambdoid junction scRNA-seq on one of the models to obtain a more in-depth analysis of cell populations and gene expression. The region corresponding to the lambdoid junction will be isolated from stage-matched wild-type and mutant embryos, and dissociated into single cells for sequencing in a routine and reproducible procedure. Subsequently, standard bioinformatics procedures will be employed to assess if and how the cell populations and gene expression profiles of the relative cell populations have been altered in the cleft lip model. Finally, any differences will be verified and extended to the other models using in situ hybridization and qRT-PCR to determine if similar or different mechanisms underlie these various mouse clefting models. The work in this proposal will provide a crucial understanding for how orofacial clefting occurs at single cell resolution, which could ultimately be used to prevent and treat human orofacial clefting.
Orofacial clefting, one of the most common human birth defects, occurs in ~1.5 in 1000 newborns and its presence requires multiple operations to correct the defect and this frequently reduces the quality of life for both the child and the parents. Insufficient information exists concerning the mechanisms of orofacial clefting to enable the majority of these defects to be detected or prevented prenatally. We are using animal model systems and state of art single cell technology to determine how orofacial clefting proceeds and to identify new mechanisms that mediate normal facial fusion so that we may apply this knowledge to understand and ultimately treat the origins of human orofacial clefting.
