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. 2020 May 1;9:e53895. doi: 10.7554/eLife.53895

Figure 4. Absence of R-spondins from progenitors causes lack of MET and downregulation of β-catenin target genes.

(A) Schematic outline of the strategy used for progenitor-specific deletion of Rspo3. Six2:Cre-DM stands for (Six2:Cre, Rspo1-/-, Rspo3fl/fl). (B) Macroscopic view reveals smaller kidneys (K) in mutant E18.5 embryos. H and E staining reveals a complete absence of glomeruli (compare iii and iv). (C) Immunolabelling for SIX2 (red), WT1 (green) and JAG1 (white) revealed a mild reduction of progenitors and confirmed the lack of nephrons on the molecular level (D). In situ hybridization performed on E14.5 embryos revealed persistence of Wnt9b expression, but dramatic reduction of class I (Wnt4) and class II (Bmp7, Tfrc, Slc12a) β-catenin target genes in the nephrogenic lineage. Dotted white lines highlight the ureter and orange lines outline the CM compartment. (E) Quantification with RNA-Scope technology combined with Halo software analysis shows a reduction of Axin2 expression by 51% in the nephrogenic zone of mutant kidneys compared to control (p<0.0001). (n = 4 embryos isolated from four litters for control genotype, and n = 7 embryos isolated from six litters for mutant genotype). Each dot or square represents the total RNAScope signal detected per nephrogenic area normalised to the total number of cells present in this field. See Figure 4—source data 1. Columns are means ± SEM with p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).

Figure 4—source data 1. Source data for Figure 4E: Quantification of Axin2 RNA-Scope signal.

Figure 4.

Figure 4—figure supplement 1. Analysis of progenitor-specific deletion reveals a requirement of R-Spondins for MET.

Figure 4—figure supplement 1.

(A) Schematic outline of the experimental strategy. (B) H and E staining of E14.5 kidney sections reveals an absence of forming nephrons. (C) Wnt9b Class II target genes are reduced upon nephron progenitor specific depletion of Rspo3. Ureteric tips are outlined with a dashed white line, the cap mesenchyme with an orange line. (D) RNA-Scope analysis demonstrates reduced Axin2 expression in the nephrogenic zone of developing kidney Six2:Cre-DM mutants (E14.5). Axin2 probe was detected with a chromogenic substrate (pink colour) that fluorescences (red signal). Nuclei were counterstained with Hematoxylin and with Hoechst (blue).