PROJECT SUMMARY/ABSTRACT Salivation is a critical physiological activity that aids digestion, maintains oral health, and supports functions such as speech, swallowing and taste sensation. Salivary gland dysfunction results from ageing, diseases such as Sjögren syndrome and from radiotherapy for head and neck cancers. Therapeutic irradiation causes permanent damage to salivary glands, highlighting their poor regenerative ability. One potential obstacle to recovery of salivation may be that damage, particularly during radiation therapy, is inflicted not only on saliva-generating epithelial cells but also on supporting mesenchymal cells. Indeed, preservation or restoration of mesenchymal cell function may constitute an ideal therapeutic target, as an optimized mesenchymal microenvironment may augment the function and regenerative capacity of residual salivary gland epithelial cells and their progenitors. A knowledge of how mesenchymal cells function during homeostasis and contribute to regeneration after injury thus may provide a new approach to activate mechanisms that protect salivary glands and enhance their repair. In many organs the mesenchymal expression of signals that provide regenerative feedback to the epithelium during homeostasis and injury repair is induced by expression of a Hedgehog (Hh) protein signal from the epithelium. Using mouse genetic models, cell lineage tracing, and single-cell transcriptomics, we have discovered that Desert hedgehog (DHH), the least studied of the three mammalian Hh family members, drives an epithelial-mesenchymal feedback (EMF) circuit in the major adult salivary glands, and that activity of this circuit is crucial for salivary gland maintenance and for regeneration after radiation injury. Importantly, although DHH expression in cells of the salivary gland epithelium is essential for regeneration, our findings also highlight a vital role for mesenchymal response to this signal for execution of the regenerative program. Here we propose to elucidate the role of Hh signaling in salivary gland homeostasis and regeneration by characterizing at a single cell level the transcriptomic and epigenetic consequences of EMF circuit activity, and to assess the conservation of DHH-driven EMF circuitry in human salivary glands. With the goal of manipulating Hh pathway activity for protection from or enhancement of tissue repair after radiation injury, we have developed a conformation-specific nanobody against the Hh receptor Patched1 that activates Hh pathway response. This nanobody can be targeted to specific cell and tissue types, thus mitigating potential adverse effects arising from systemic Hh pathway activation. With this agent, we will test the possibility that precise, tissue-targeted activation of the Hh pathway can effectively enhance endogenous reparative mechanisms for salivary gland protection from and restoration after injury from irradiation like that administered in head and neck cancer therapy.