Project Summary/Abstract Three-quarters of a million Americans have End Stage Renal Disease (ESRD). Their only treatment options are dialysis, with poor long term survival prospects, or a curative kidney transplant, with too few kidneys to meet patient need. One way to address the shortage of transplantable kidneys is to develop a thorough insight into essential cell types and how they are generated, and then to use this insight to generate functional, cell-based kidney replacements. The physiologically relevant epithelial networks of the kidney arise from two distinct progenitor populations. Nephron progenitors generate the nephrons, of which there are approximately one million in the human kidney, and adjacent ureteric progenitors generate the branched network to the collecting system, to which nephrons connect. Homeostasis is maintained by specialized cell types in both the nephron and the collecting duct. Notably, water and salt, and pH balance, are regulated by principal cells (PCs) and intercalated cells (ICs), respectively. There are three subtypes of intercalated cells: type A intercalated cells (A-ICs), type B intercalated cells (B-ICs) and non-A-non-B intercalated cells that perform distinct functions in the regulation of pH homeostasis. Interestingly, the McMahon group recently demonstrated both PCs and ICs have dual origins from both nephron and ureteric progenitor cells, an unusual developmental process. Here, I aim to identify the mechanisms generating similar, but not identical, IC subtypes from distinct kidney progenitor populations. In Specific Aim 1, I will focus on identifying the targets of transcription factors that are known to be essential for the development of ICs: Tfcp2l1 and Foxi1. I will also profile the targets of transcription factors that have not yet been described in ICs: Dmrt2 distinguishes both nephron and ureteric epithelial derived IC-A cell types from Hmx2/Hmx3 expressing IC-B cell types. Differential Hmx gene expression sub-divides the IC-B populations: Hmx2+ B-ICs and non-A-nonB ICs are nephron-derived and Hmx2+/Hmx3+ B-ICs are ureteric lineage derived. We hypothesize that Tfcp2l1 and Foxi1 initiate a general IC developmental program, while Dmrt2, Hmx2, and Hmx3 have mutually repressive actions in the generation of IC subtypes. I will use biochemical approaches and generate tagged alleles to identify regulatory interactions for these transcription factors within ICs of both lineages. In Specific Aim 2, I will use genetic approaches in vivo and a ureteric organoid model to determine the requirement of these factors in the functional transcriptional interplay generating mammalian ICs. Taken together, this proposed work will define a regulatory framework for the development of intercalated cells within the ureteric and nephron lineages in the kidney and provide new insights into the regulatory programs governing cell types involved in mammalian kidney function.