Dissecting the Spatial Multiome of the Embryonic Palate

Objectives: Cleft palate (CP) is among the most common developmental anomalies in humans. While it is known that CP often results from failed osteogenic differentiation of cranial neural crest-derived progenitor cells, there is a disparity in understanding the molecular mechanisms governing osteogenesis during palate development. Thus, we lack the ability to develop novel treatments to prevent or correct these anomalies in patients. The aim of this study was to increase our fundamental understanding of these molecular mechanisms - creating the first multi-omic atlas of palate development.
Methods: In this multimodal, unbiased multi-omic dissection of the embryonic palate, we employed next-generation sequencing approaches (bulk RNA-seq, single-cell RNA-seq, 3D spatial RNA-seq, and ATAC-seq) and in vivo highly multiplexed spatial validation of identified effector molecules across developmental stages (E13.5-P0). Murine embryonic palatal shelves were microdissected for tissue (RNA and protein), single-cell, and single-nuclei-level processing. Littermates were formalin-fixed and paraffin-embedded for histological processing and in vivo validation in biological triplicate. All bioinformatic data were integrated and statistically assessed through Seurat.
Results: Unique morphogenetic transcriptomic and epigenetic signatures were identified in pre-fusion (E13.5-E14.5) and post-fusion (E15.5-P0) clusters. Staged differential analyses and functional enrichment pathway profiling revealed novel temporal gene expression and epigenetic patterns associated with specific developmental events. RNA velocity and pseudotime trajectory analyses revealed highly dynamic transcriptional regulation of osteogenic cell clusters. In situ spatial resolution of coronal mid-palatal sections revealed novel marker genes specific to palatal mesenchyme through development, offering novel insight into key molecular signaling programs driving palatal fusion.
Conclusions: This integrated spatiotemporal analysis provides the first unbiased, multi-omic (gene expression, epigenetic, and protein-level) developmental template of palate development. Such knowledge will increase our ability to develop and translate safe and effective therapeutic solutions to prevent or correct CP in patients.