PROJECT SUMMARY Eccrine sweat glands are the most abundant glands in human skin and are essential for thermoregulation and water balance. Patients with severe burn injuries and congenital sweat gland deficiencies cannot properly maintain body temperature, which can lead to heat stroke and organ failure. The recent identification of multipotent stem cells in sweat glands presents great potential to help these people to thermoregulate efficiently by opening the way for de novo sweat gland regeneration. Nonetheless, regeneration of fully functional sweat glands will require better understanding of the endogenous signals that normally regulate differentiation of sweat gland stem cells. Precise innervation and neuronal control are indispensable for sweat glad function since neuronal signals elicit sweating, and further, as we previously discovered in our lab, these signals are critical for sweat gland maturation. Sweat glands and neurons rely on each other for precise co-development. However, very little is known about how neuronal inputs influence sweat gland development, especially sweat gland stem cells. We hypothesize that neuronal signals are critical for the specification of sweat gland stem cells during development. Using mouse sweat glands as a model, we will investigate the molecular changes in sweat gland stem cells during development when lacking innervation, and further identify the downstream pathway within sweat gland stem cells that mediate the effect of neurotransmitters upon stem cell maintenance. Through combined use of mouse genetics, immunofluorescent confocal and light sheet imaging, single-cell RNA sequencing, fluorescence-activated cell sorting, and tissue explant and cell culture systems, we plan to: 1) investigate the effect of denervation during sweat gland development and homeostasis; 2) dissect the roles of specific neurotransmitters in sweat ducts and sweat gland development; and 3) understand the mechanism(s) by which calcium mediates neuronal control of sweat gland stem cell fate. Completion of our proposed studies will provide novel insights into molecular mechanisms by which nervous system influence sweat gland morphogenesis and cell fate determination at each developmental stage, as well as better understanding on how sweat glands and nerves co-develop into a functional unit. Ultimately, our proposed work will contribute to regenerative therapies for patients suffering from sweating deficiency.