Hypoxia impedes differentiation of cranial neural crest cells into derivatives relevant for palatogenesis

Orofacial clefts (OFC) are clefts of the lip and/or palate. They are the second-most prevalent congenital malformation. OFC result from failures in fusion processes of facial structures during embryonic development. Craniofacial structures are derived from ectodermal epithelial cells and cranial neural crest cells (CNCCs). In fact, the ectomesenchymal CNCCs are the source of most midface structures. They proliferate heavily during development and differentiate into a variety of tissues such as bone, cartilage and smooth muscle. Risk factors for OFC are multifactorial and include genetic components but also environmental factors. One environmental factor is hypoxia that can be caused by several reasons during pregnancy, such as tobacco smoking, medication or living at high altitudes. Knowledge about the molecular mechanisms of how hypoxia evokes OFC is at large. We here show that hypoxia has only modest effects on proliferating CNCCs, but dramatically influences their differentiation potential. We detected massive perturbations of differentiation in all of our tested differentiation paradigms, i.e. chondrocytes, osteoblasts and smooth muscle cells. The transcriptional induction of the majority of regulated genes during each of these processes was grossly impaired by hypoxic conditions, as evidenced by genome-wide transcriptomic analyses. Bioinformatic analyses pointed to a central role of cytoskeletal organization and amino acid metabolism being compromised during all three differentiation processes. We could identify several OFC risk genes amongst the genes with constrained induction during hypoxia. Our analyses reveal a drastic influence of hypoxia on the differentiation potential of CNCCs as a possible source for the occurrence of OFC. To assess the differentiation potential of CNCCs under normoxic and hypoxic conditions, we differentiated the cells under 21% and 0.5% O2 into chondrocyte, osteoblast or smooth muscle cell lineages and compared them to proliferating cells. We used four replicates for normoxic and three replicates for hypoxic conditions. We then performed gene expression analysis using RNA sequencing.