Forming Nephrons Promote Nephron Progenitor Maintenance via Paracrine BMP4 Signaling

Background: Current models propose that interactions between stromal, ureteric, and nephron progenitors are sufficient to drive kidney development. In this study, we aimed to investigate whether the forming nephron is a passive product of the niche, or whether it is an active partner in the signalling interactions required for ongoing progenitor maintenance and branching morphogenesis. Methods: Kidney size and morphology were assessed in global Wnt4 knockout (KO, Wnt4GCE/GCE), and conditional Wnt4 knockout (cKO, Six2TGC-Wnt4fl/fl) mice using brightfield imaging, histology and immunofluorescence at embryonic day (E) 17.5. Nephron progenitor cell number, dispersal, proliferation and death were examined through wholemount and section immunofluorescence at E14.5 and E17.5. Conditional deletion of Wnt4 was assessed by ddPCR in sorted Six2GFP+ and LTL+ cells. Transcriptional changes were assessed with bulk and single cell RNAseq in KO and cKO kidneys; select results were validated by quantitative hybridisation chain reaction (HCR). Nephron progenitor cell dispersal from the tip was assessed by confocal microscopy in wholemount wildtype and Wnt4 KO kidneys after culture with or without 100ng/mL BMP4. Results: Kidney size, branching, and nephron progenitor cell number were severely reduced in Wnt4 KO kidneys. Conditional deletion of Wnt4 with Six2Cre was incomplete but led to decreased kidney size, lower nephron progenitor cell number, increased cell death and dispersal from the tip, and reduced branching morphogenesis. Single cell RNAseq revealed a downregulation of BMP signalling effectors Id1 and Id3 in nephron progenitor cells, implicating Wnt4 target BMP4 as a paracrine signal mediating feedback from the committing nephron. Reduced expression of Bmp4, Id1, and Id3 was validated by HCR; Fgf8 was unchanged. Recombinant BMP4 restored nephron progenitor compaction in cultured Wnt4 mutant kidneys and blocked differentiation in wildtype controls, mirroring the role of BMP7-MAPK signalling in progenitor self-renewal. Conclusion: This study illustrates a crucial role for developing nephrons in maintaining the nephron progenitor population, partly via paracrine BMP4 signalling. These results suggest an indirect relationship between nephron formation and branching morphogenesis and shed light on the mechanisms of renal hypodysplasia associated with human WNT4 mutations. Funding: Government Support – Non-U.S.