The transcriptional, regulatory, and spatially defined changes from mid to late gestation in a non-human primate model

Project Summary Preterm birth is associated with decreased nephron endowment resulting in increased risk of chronic kidney disease and end-stage kidney disease. Human nephrogenesis is completed prior to birth at 32-36 weeks gestation. The majority of nephrons are formed through the poorly understood process, lateral branch nephrogenesis (LBN). Nephron acquisition by LBN is unique to primates and occurs after 22 weeks gestation in the human. During LBN, an elongating, non-branching ureteric bud (UB) tip induces nephron development with each nephron directly connected to the ureteric stalk. In contrast, mice do not undergo LBN. Instead, mice generate nephrons exclusively through branch phase nephrogenesis (BpN) wherein the UB undergoes branching morphogenesis with each branch generating approximately two nephrons. The rhesus macaque is the only known model that undergoes LBN like the human and is thus the sole model in which to define signaling pathways controlling LBN. The central hypothesis is that the cell composition and spatial cell-cell signaling in the LBN niche are distinct from BpN and negatively altered by preterm birth. The long-term goals are to leverage the molecular mechanisms enabling LBN to develop strategies to maintain and/or extend LBN-supporting signaling pathways in preterm infants. The objective of this proposal is to identify the progressive compositional, regulatory, and spatially defined signaling environments in mid to late-gestation primate nephrogenesis. This will enable the development of novel approaches to maintain or extend the LBN process postnatally in preterm infants. The rationale is that the unique rhesus tissues already available and to be obtained through this proposal provide the optimal resources to define critical cellular and signaling changes that occur during human kidney development at single-cell and spatial resolution. The first aim is to determine compositional signaling pathway shifts and regulatory relationships from mid to late-gestation primate kidneys. Single-cell multimodal transcription and ATAC-seq analyses will be performed from replicate rhesus fetal kidneys in conjunction with powerful new integrative regulatory prediction analyses. The second aim will be to elucidate spatially defined differences in WNT signaling in mid to late-gestation primate kidneys. A key benefit of using rhesus tissue for these mechanistic studies is that the tissue will be handled and fixed through a consistent protocol, thus minimizing variation in RNA degradation that may falsely result in differences between gestational periods. Spatial expression of key genes within the WNT pathway, including WNT ligands, receptors, inhibitors, and targets will be determined in mid to late-gestation kidneys by RNAScope Hiplex assay and again validated in high-quality human archival tissues to ensure conservation. Expected outcomes include a comprehensive cellular and transcriptional atlas of primate kidney nephrogenesis determination of cell-cell signaling and spatial interactions that drive primate nephron endowment.