Project Summary/Abstract The kidney is patterned along a cortical to medullary axis with specific segments of the nephron, collecting duct and vasculature all lying adjacent to each other in histologically distinct domains. In order for a kidney to function, different cell types from different cellular lineages must form at the same anatomical location. Although there has been some insight into how the individual lineages become patterned (such as proximal distal patterning of the nephron), how the different cell types/lineages coordinate their development resulting in the global patterning of the organ is unknown. We have recently found that the renal interstitial cells show extensive heterogeneity and patterning along the cortical/medullary axis of a newborn mouse kidney[1]. The patterned domains of the renal interstitium precisely map to the different anatomical domains within the kidney. How the different interstitial cell types arise and what role they play in kidney development/function are unknown. We hypothesize that the interstitium functions to relay and integrate signals from the different lineages and in turn, reinfources and integrates the differentiation of the renal parenchyma along the cortical/ medullary axis. Using bioinformatic analysis of single cell RNA-Seq data, we have identified unique transcriptional signatures for the different interstitial cell types. This information will allow us to understand how the pattern is established as well as its function. In this proposal, we will focus on the specification and function of a sub-population of interstitial cells we will refer to as the proximal tubule (PT) interstitium. In this proposal, we will investigate the mechanisms underlying specification of a subpopulation of renal fibroblasts we refer to as the proximal tubules interstitium (PT interstitium). Notch/Rbpj and Yap/Taz transcription factors are both active within this population and ablation of either pathway using Foxd1Cre has revealed overlapping roles in the specification of this cell type. Our preliminary analysis indicates that the PT interstitium is necessary for the maturation of the adjacent proximal tubules. We hypothesize that the PTs and/or endothelia produce Notch/Rbpj and Yap/Taz activators and that cells with overlapping pathway activation become PT intersitium. The PT interstitium produces signals that promote the differentiation/maturation of the PTs. This crosstalk allows the co-maturation and integration of the proximal tubules and other cortical cell types. We further hypothesize that disruption of normal cortical-medullary pattern in renal organoids leads to defects in Yap/Taz and/or Notch/Rbpj signaling and contributes to the lack of nephron maturation in these tissues. These hypotheses will be tested here. Completion of these aims will open up an entirely new field of kidney interstitial biology that will have a long and lasting impact on the multiple fields including kidney development, kidney ...