Multimodal transcriptomic profiling of the embryonic palate enables novel in utero cleft therapy [bulk RNA-Seq]

Cleft palate is a common disorder of development resulting from failure of growth, migration, elevation, and osteogenic fusion of embryonic cranial neural crest-derived palatal shelves. Despite progress in recent decades, the molecular pathways involved in this failure are not well understood. Here, we present a multimodal, spatiotemporal transcriptomic profiling of the developing palate through integrated, unbiased single-cell and bulk RNA-sequencing and multiplexed in situ mRNA mapping of osteogenic cell lineages. We then show that loss of Pax9, a critical transcription factor orchestrator of Wnt signaling in palate development, results in increased expression of sclerostin (Sost), a known antagonist of Wnt signaling. Finally, we reveal that a single dose of sclerostin-neutralizing monoclonal antibody restores Wnt signaling and corrects cleft palate defects in utero in Pax9-/- mouse embryos. Overall design: Herein, we begin to unveil the complex transcriptomic milieu driving morphogenetic gradients of expression and development of the secondary palate. The primary aims were three-fold: (1) define the temporospatial transcriptomic profile of the embryonic secondary palate; (2) assess the Wnt-dependent mechanisms driving palate morphogenesis and failure of palate fusion in Pax9-/- embryos; and (3) explore the potential role of a well-known Wnt antagonist protein, sclerostin, in the pathogenesis of cleft palate through the testing of a novel drug delivery approach targeting this protein for the treatment of cleft palate in utero.