ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland.

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

Transcriptomic and Single-Cell Analysis Reveals Regulatory Networks and Cellular Heterogeneity in Mouse Primary Sjögren's Syndrome Salivary Glands.

Sjögren's Syndrome (SS) is a chronic autoimmune disease of unknown etiology which primarily affects the salivary and lacrimal glands resulting in the loss of secretory function. Treatment options for SS have been hampered due to the lack of a better understanding of the underlying gene regulatory circuitry and the interplay between the myriad pathological cellular states that contribute to salivary gland dysfunction. To better elucidate the molecular nature of SS, we have performed RNA-sequencing analysis of the submandibular glands (SMG) of a well-established primary Sjögren's Syndrome (pSS) mouse model. Our comprehensive examination of global gene expression and comparative analyses with additional SS mouse models and human datasets, have identified a number of important pathways and regulatory networks that are relevant in SS pathobiology. To complement these studies, we have performed single-cell RNA sequencing to examine and identify the molecular and cellular heterogeneity of the diseased cell populations of the mouse SMG. Interrogation of the single-cell transcriptomes has shed light on the diversity of immune cells that are dysregulated in SS and importantly, revealed an activated state of the salivary gland epithelial cells that contribute to the global immune mediated responses. Overall, our broad studies have not only revealed key pathways, mediators and new biomarkers, but have also uncovered the complex nature of the cellular populations in the SMG that are likely to drive the progression of SS. These newly discovered insights into the underlying molecular mechanisms and cellular states of SS will better inform targeted therapeutic discoveries.

p63 and Its Target Follistatin Maintain Salivary Gland Stem/Progenitor Cell Function through TGF-ß/Activin Signaling.

Multipotent ΔNp63-positive cells maintain all epithelial cell lineages of the embryonic and adult salivary gland (SG). However, the molecular mechanisms by which ΔNp63 regulates stem/progenitor (SP) cell populations in the SG remains elusive. To understand the role of ΔNp63 in directing cell fate choices in this gland, we have generated ΔNp63-deleted adult mice and primary salivary cell cultures to probe alterations in SP cell differentiation and function. In parallel, we have leveraged RNA-seq and ChIP-seq-based characterization of the ΔNp63-driven cistrome and scRNA-seq analysis to molecularly interrogate altered SG cellular identities and differentiation states dependent on ΔNp63. Our studies reveal that ablation of ΔNp63 results in a loss of the SP cell population and skewed differentiation that is mediated by Follistatin-dependent dysregulated TGF-ß/Activin signaling. These findings offer new revelations into the SP cell gene regulatory networks that are likely to be relevant for normal or diseased SG states.

Transcriptomic and Network Analysis of Minor Salivary Glands of Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized primarily by immune-mediated destruction of exocrine tissues, such as those of the salivary and lacrimal glands, resulting in the loss of saliva and tear production, respectively. This disease predominantly affects middle-aged women, often in an insidious manner with the accumulation of subtle changes in glandular function occurring over many years. Patients commonly suffer from pSS symptoms for years before receiving a diagnosis. Currently, there is no effective cure for pSS and treatment options and targeted therapy approaches are limited due to a lack of our overall understanding of the disease etiology and its underlying pathology. To better elucidate the underlying molecular nature of this disease, we have performed RNA-sequencing to generate a comprehensive global gene expression profile of minor salivary glands from an ethnically diverse cohort of patients with pSS. Gene expression analysis has identified a number of pathways and networks that are relevant in pSS pathogenesis. Moreover, our detailed integrative analysis has revealed a primary Sjögren's syndrome molecular signature that may represent important players acting as potential drivers of this disease. Finally, we have established that the global transcriptomic changes in pSS are likely to be attributed not only to various immune cell types within the salivary gland but also epithelial cells which are likely playing a contributing role. Overall, our comprehensive studies provide a database-enriched framework and resource for the identification and examination of key pathways, mediators, and new biomarkers important in the pathogenesis of this disease with the long-term goals of facilitating earlier diagnosis of pSS and to mitigate or abrogate the progression of this debilitating disease.