Regulatory mechanisms that dictate stem and differentiated cell populations of the salivary gland

PROJECT SUMMARY Salivary gland (SG) morphogenesis, homeostasis and regeneration requires programs of self-renewal, proliferation and differentiation of stem/progenitor epithelial cells. Identification of the transcriptional and signaling networks that govern these cellular processes is critical to better understand how they impact normal SG development and disease. However, the gene-regulatory mechanisms that control cell fate and lineage choices in the SG are not well understood, particularly in the broader context of the chromatin environment. To address this knowledge gap, we have focused our studies on the master Transcriptional Factor (TF) p63, especially the epithelial enriched DNp63 isoform which plays an indispensable role in the SG. We have shown that during SG development, DNp63+ cells are enriched in the epithelial cells of the developing placode and initial bud. Utilizing genetic lineage tracing, we have demonstrated that p63+ multipotent precursor cells give rise to all epithelial cell lineages of the SG during development and adult gland maintenance. Additionally, we have shown DNp63 maintains the adult SG basal and myoepithelial (MEC) stem/progenitor cell populations through modulating TGF- b/Activin signaling. Moreover, DNp63 directs MEC cellular plasticity during homeostasis and in response to injury and regeneration. Our comprehensive analysis and preliminary results from genetic and genomic datasets have led us to a broad hypothesis that p63 serves as a linchpin regulator in both normal and injured states of the SG and asserts its molecular effects by direct regulation of target genes, including key transcription and chromatin modifying factors, and by an integrated modulation of the higher-order chromatin and the epigenetic landscape. This hypothesis will be tested in two aims using well-characterized p63 mouse knockouts for SG development and regeneration and ex-vivo explants. Our studies will be powered by cutting- edge techniques of single cell genomics and epigenomics and chromatin based protein interaction. In Aim 1, we will evaluate the role of p63 during the early stages of salivary gland development and branching morphogenesis with a focus on Midkine (Mdk), a member of the pleiotrophin (PTN)/MK family of secreted heparin-binding growth factors. In Aim 2, we will determine the role of p63 in submandibular gland (SMG) tissue repair and regeneration using well established regeneration and injury induced mouse model systems and define the transcriptomic, chromatin landscape of these cells at single cell resolution. Finally, we will perform proteomic and integrative analyses to identify and examine the hierarchy of p63 and its targets and co-regulators in the regulatory network in SG. This work is innovative and significant because our proposed use of sophisticated technologies to examine transcriptional and epigenetic mechanisms of gene regulation will lead to new discoveries into SG organogenesis and regeneration. Long term, such knowledge will enrich existing molecular paradigms of cell-specific regulatory networks and signaling pathways and guide therapeutic interventions in human patients with SG dysfunction.