Poster #1: Alexandra Mantel PhD

Gene expression dynamics of human and mouse craniofacial development at the single-cell level

Alexandra Manchel1*, Nagham Khouri-Farah2*, Emma Wentworth Winchester2, Brian M. Schilder3,4, Kelsey Robinson5, Sarah W. Curtis5, Nathan G. Skene3,4, Elizabeth J. Leslie-Clarkson5, Justin Cotney1,6 Affiliations 1 Department of Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA 2 Graduate Program in Genetics and Developmental Biology, UConn Health 3 Department of Brain Sciences, Faculty of Medicine, Imperial College London, London, W12 0BZ, UK 4 UK Dementia Research Institute at Imperial College London, London, W12 0BZ, UK 5 Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA 6 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA * These authors contributed equally to this work.

Craniofacial development gives rise to the face’s complex structure and involves the interplay of diverse and transient cell types. However, our understanding of human-specific craniofacial developmental mechanisms and genetics remains limited. We present a single-nucleus RNA sequencing (snRNA-seq) atlas of human craniofacial development from craniofacial tissues of 24 embryos, spanning 6 key time points during embryogenesis (4–8 post-conception weeks). This resource reveals 8 major cell types from 95,892 nuclei, including muscle progenitors, 19 cranial neural crest cell (CNCC) subtypes, 22 ectodermal subtypes, and 25 mesenchymal subtypes. We performed comparisons to snRNA-seq of mouse E10.5 to E15.5 craniofacial tissue, which reflect major morphological landmarks of the human tissues profiled. Integration with human and mouse spatial transcriptomics data facilitated annotation of cell subtype clusters that correspond to anatomical features such as the palate and the mandible. This enabled linking of common variants associated with facial morphology and risk for orofacial clefts to anatomically distinct cellular subtypes. We additionally examined the spatial mapping of the risk associations of orofacial clefts and identified significant associations in the pharyngeal regions of the human spatial transcriptomics. Surprisingly, we found enrichment of rare de novo protein-altering variants from orofacial cleft patients in marker genes that specify epithelial subtypes from the palate and palate surface. This finding challenges the current paradigm that CNCCs are likely driving non-syndromic orofacial clefting. This atlas offers unprecedented insights into the cellular and genetic mechanisms shaping the human face and pinpoints specific, spatially restricted cell subtypes as contributors to orofacial clefts. Funding sources: EJLC (R01-DE030342, X01-HG010835, X01-HD100701, X01-HL132363) and JC (NIDCR 1R01DE028945, NIDCR 1R03DE028588, NIGMS 5R35GM119465).