Figure 4. Nephron progenitors intermix with interstitial progenitors but no significant changes in gene expression are observed.

(A) E15.5 kidneys were sectioned and immunostained for the tip marker Etv4 (red) and cytokeratin (cyan). Distinct ureteric branch tip domains are still present in Wnt11 mutants as indicated (arrowheads). (B) E15.5 sections were stained for the matrix protein fibronectin (Fn1; red) and cytokeratin (cyan). Note the exclusion of fibronectin from the nephron progenitor niche in wild type animals (line marks boundary between the nephron progenitors and interstitial progenitors). In Wnt11^tm1a/tm1a kidneys the fibronectin boundary is disrupted and staining observed in the nephron progenitor niche (arrowheads). Insets show zoomed view of the progenitor niche. (C) Immunolocalization of Foxd1 +interstitial progenitors (blue) in conjunction with Six2 +nephron progenitors (red) at E15.5 reveals mixing of the two cell populations in Wnt11 mutant kidneys (insets show zoomed view of cell mixing). Foxd1 +cells can infiltrate (arrowheads) the nephron progenitor niche and are found near the ureteric branch tips (cyan). (D) Quantitation of tissue sections immunostained for both Six2, Foxd1, and cytokeratin. There is an increase in the number of Foxd1 +cells touching the ureteric branch tips in Wnt11 mutants. 30 ureteric tip domains from n = 3 biological replicates were quantified. (E) Quantitation of Six2 cell neighbors. The number of Foxd1 +cells touching a Six2 +cell is divided by the number of Six2 +cells touching the same cell. In Wnt11 mutants a Six2 +cell is just as likely to have as many Foxd1 +neighbors as Six2 +neighbors. Three biological replicates were quantified, 10 Six2 +cells per sample. (F) Fold-changes associated with RNA-seq of either whole kidneys or Six2 +cells from wild type and Wnt11 mutant kidneys. The fold-change was calculated from the average of n = 6 for each genotype in whole kidney analysis and n = 3 for each genotype in the nephron progenitor analysis. Example genes which define each progenitor population (ureteric branch tip, nephron progenitor, and interstitial progenitor) are shown. No significant changes (>1.5 fold change) are observed. All error bars represent SEM. All significance values were determined by t-test. ns = p > 0.05, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 4—figure supplement 1. Several Wnt receptors are expressed in the nephron progenitors and display weakly penetrant phenotypes upon deletion.

(A) RNA-seq from E16.5 Six2 +nephron progenitors reveals the expression of several Wnt pathway receptors and co-receptors. RPKM values are listed for each gene. Only genes with an RPKM >1 are shown. (B) β-galactosidase stains of Fzd2^+/- and Fzd7^+/- kidneys at E15.5. Alleles have a lacZ reporter inserted into the coding region. (C) Wholemount in situ hybridization for Fzd2 and Fzd7 on E15.5 urogenital systems show that they largely recapitulate the β-gal expression patterns. (D) Immunostaining for Six2 (red), β-galactosidase (green), and cytokeratin (blue) on E15.5 kidney sections from Fzd2 and Fzd7 heterozygous animals to show the expression domains of each receptor. Both are found within the nephron progenitors, but Fzd7 to a lesser extent. (E) Wholemount immunostains for Six2 (green) and cytokeratin (red) were performed on mutants and appropriate controls at E15.5 for each receptor listed (Wnt11 is included for comparison). For Ror2, the conditional line was crossed to the Six2TGC^tg line to specifically remove Ror2 in the nephron progenitors (Ror2^c/c;Six2TGC^tg/+). No receptor mutant completely recapitulates the Wnt11 mutant phenotype. Insets show 2X magnification. (F) High resolution view shows the slightly disorganized Six2 +nephron progenitors (green) in E15.5 Ptk7^-/- kidneys although cap boundaries are still visible and the phenotype only occurred in one of two mutants analyzed. (G) Wholemount immunostains for Six2 (green) on E15.5 kidneys from Fzd2^-/- and Fzd2^-/-;Fzd7^+/- ±. Slightly disorganized caps were observed in one of three kidneys examined.