Intro/background: Mammalian kidney development is driven by reciprocal signalling interactions within a tissue microenvironment known as the nephrogenic niche. Traditionally, kidney development was thought to be driven primarily by interactions between the epithelial ureteric tip and mesenchymal nephron progenitor populations, with the nephrons that form in this environment considered passive products. Challenging this view, we discovered that Wnt4-mediated BMP-Smad signaling from the developing nephron is essential for maintaining nephron progenitor cell state and sustaining kidney development. Single-cell RNA sequencing (scRNA-seq) of global and conditional Wnt4 knockout mice was instrumental in this discovery. However, key questions remained unresolved due to low recovery of target cell types, a lack of change in expected Wnt4 target genes, and the loss of spatial context in scRNAseq.
Methods/Results: To further interrogate the molecular mechanisms of this novel cellular interaction and its effect on the tissue microenvironment, we profiled five Wnt4 knockout (KO) and three wild type (WT) kidneys using the 10x Genomics Xenium Mouse Tissue Atlassing panel (X genes), supplemented with 50 custom probes targeting Wnt4-regulated genes, markers and regulators of progenitor self-renewal and differentiation. Quality control was performed on the data, demonstrating high data integrity, consistency, and reliability, with key metrics indicating excellent quality. Independent clustering and manual annotation of KO and WT samples at multiple resolutions revealed reduced nephron differentiation and altered stromal cell populations in KO kidneys. Differential expression analysis and marker plots revealed impaired nephron and ureteric progenitor cell maintenance, and an expanded stromal progenitor cell domain in the KO samples. Analysis of Wnt4 targets and signalling pathway gene sets affirmed expected changes in BMP-SMAD signalling, and identified additional changes in the FGF and retinoic acid signalling pathways that were not evident in our prior scRNAseq analysis.
Conclusion: Our findings reinforce the crucial role of Wnt4-mediated BMP-Smad signaling in maintaining nephron progenitor cell state and driving kidney development. By leveraging spatial transcriptomics, we identified disruptions in nephron differentiation, progenitor maintenance, and stromal composition in Wnt4 KO kidneys. These insights illustrate the potential of spatial transcriptomics to evaluate interdependent signalling interactions within complex tissues. Future investigations will further elucidate these pathways and their relevance to WNT4-related congenital kidney abnormalities.