Project description:FGF signaling has been implicated in the regulation of osteogenesis in both intramembranous and endochondral ossification. In the developing palate, the anterior bony palate forms by direct differentiation of cranial neural crest-derived mesenchymal cells, but the signals that regulate osteogenic cell fate remains unclear. In the present study, we present evidence that locally activated FGF8 signaling in the anterior palate leads to complete bone loss of the palatine process of the maxilla (ma) and ectopic cartilage formation. This aberrant developmental process was accompanied by significantly elevated level of cell proliferation, which contributes to abnormally thickened ma, and complete inhibition of Osterix expression, which accounts for the lack of bone formation. Consistent with the phenotype, RNA-Sequencing (RNA-Seq) analysis further demonstrated that augmented FGF8 signaling downregulated genes involved in ossification, biomineral tissue development, and bone mineralization, but upregulated genes involved in cell proliferation, cartilage development, and cell fate commitment. Expression validation of selected genes supported the RNA-Seq results. We conclude that FGF signaling functions as a negative regulator of osteogenic fate but promotes chondrogenesis of cranial neural crest cell-derived mesenchyme in the hard palate, which will have implication in directed differentiation of precursor cells for clinical application.
Project description:Being a transcription factor, Shox2 binds to specific DNA sites to regulate gene expression via modulating chromatin status. To investigate the impact of Shox2 on the chromatin accessible landscape in the anterior hard palate, we subjected FACS-sorted Shox2+ cells from the palate of E14.5 Shox2Cre/+;Nkx2.5F/F;R26RmTmG and Shox2Cre/-;Nkx2.5F/F;R26RmTmG mice to ATAC-Seq analysis.
Project description:Cellular binary fate decisions require the progeny to silence genes associated with the alternative fate. The major subsets of alpha:beta T cells have been extensively studied as a model system for fate decisions. While the transcription factor RUNX3 is required for the initiation of Cd4 silencing in CD8 T cell progenitors, it is not required to maintain the silencing of Cd4 and other helper T lineage genes. The other runt domain containing protein, RUNX1, silences Cd4 in an earlier T cell progenitor, but this silencing is reversed whereas the gene silencing after RUNX3 expression is not reverse. Therefore, we hypothesized that RUNX3 and not RUNX1 recruits other factors that maintains the silencing of helper T lineage genes in CD8 T cells. To this end, we performed a proteomics screen of RUNX1 and RUNX3 to determine candidate silencing factors.
Project description: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.
Project description: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.
Project description:To characterize the anterior palate-specific chromatin landscape of the distal Shox2-binding sites, we conducted Shox2 ChIP-Seq again in parallel with H3K27ac ChIP-Seq on the Shox2+ domain anterior palate and the Shox2- posterior palate. Integrative analysis of the Shox2 and H3K27ac ChIP-Seq datasets and aggregate plots of binding signals of H3K27ac in the anterior and posterior palate, respectively, versus the summits of Shox2 binding sites showed close association of Shox2 binding peak with enriched H3K27ac in the anterior palate but not the posterior palate, indicate that Shox2 occupancy is highly related to active enhancer elements.
