Although kidneys of equal size can vary 10-fold in nephron number at birth, discovering what regulates such variation has been hampered by a lack of quantitative parameters defining kidney development. Here we report a comprehensive, quantitative, multiscale analysis of mammalian kidney development in which we measure changes in cell number, compartment volumes, and cellular dynamics across the entirety of organogenesis, focusing on two key nephrogenic progenitor populations: the ureteric epithelium and the cap mesenchyme. In doing so, we describe a discontinuous developmental program governed by dynamic changes in interactions between these key cellular populations occurring within a previously unappreciated structurally stereotypic organ architecture. We also illustrate the application of this approach to the detection of a subtle mutant phenotype. This baseline program of kidney morphogenesis provides a framework for assessing genetic and environmental developmental perturbation and will serve as a gold standard for the analysis of other organs.

We have raised an antibody specifically recognizing endogenous mouse SRY protein and used it to investigate the molecular and cellular mode of action of SRY in testis determination. We find that expression of SRY protein closely mirrors the expression of Sry mRNA in mouse genital ridges and is detectable for 6 to 8 h after the mRNA ceases to be detectable. The subset of somatic cells that expresses SRY begins to express SOX9 almost immediately. Since these SOX9-positive cells go on to develop as Sertoli cells, it appears that SRY expression marks the pre-Sertoli cell lineage and leads to up-regulation of Sox9 expression cell-autonomously. However, a small proportion of SOX9-positive cells did not appear to express SRY, possibly reflecting the additional involvement of paracrine signaling in activating Sox9 transcription in these cells. We confirmed by ex vivo cell mixing experiments that SRY is able to engage receptor-mediated signaling to up-regulate Sox9 expression. Finally, we showed by employing specific inhibitors that the causative signaling molecule is prostaglandin D2 (PGD2) and that PGD2 can induce Sox9 transcription in cultured XX gonads. Our data indicate a mechanism whereby Sry uses both a cell-autonomous mechanism and a PGD2-mediated signaling mechanism to stimulate expression of Sox9 and induce the differentiation of Sertoli cells in vivo.

While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.

While the molecular cues initiating testis determination have been identified in mammals, the cellular interactions involved in generating a functional testis with cord and interstitial compartments remain poorly understood. Previous studies have shown that testis cord formation relies on cell migration from the adjacent mesonephros, and have implicated immigrant peritubular myoid cells in this process. Here, we used recombinant organ culture experiments to show that immigrant cells are endothelial, not peritubular myoid or other interstitial cells. Inhibition of endothelial cell migration and vascular organisation using a blocking antibody to VE-cadherin, also disrupted the development of testis cords. Our data reveal that migration of endothelial cells is required for testis cord formation, consistent with increasing evidence of a broader role for endothelial cells in establishing tissue architecture during organogenesis.

Background: Human kidney organoids hold promise for studying development, disease modelling and drug screening. However, the utility of stem cell-derived kidney tissues will depend on how faithfully these replicate normal fetal development at the level of cellular identity and complexity. Methods: Here, we present an integrated analysis of single cell datasets from human kidney organoids and human fetal kidney to assess similarities and differences between the component cell types.Results: Clusters in the combined dataset contained cells from both organoid and fetal kidney with transcriptional congruence for key stromal, endothelial and nephron cell type-specific markers. Organoid enriched neural, glial and muscle progenitor populations were also evident. Major transcriptional differences between organoid and human tissue were likely related to technical artefacts. Cell type-specific comparisons revealed differences in stromal, endothelial and nephron progenitor cell types including expression of WNT2B in the human fetal kidney stroma.Conclusions: This study supports the fidelity of kidney organoids as models of the developing kidney and affirms their potential in disease modelling and drug screening.