Journal: eLife

Article Title: Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment

doi: 10.7554/eLife.40392

Figure Lengend Snippet: ( 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. 10.7554/eLife.40392.014 Figure 4—source data 1. Quantitation of nephron progenitor metrics.

Article Snippet: Antibodies utilized in this study include: Six2 (Proteintech, 11562–1-AP, RRID: AB_2189084 , 1:1000 for section, 1:250 for wholemount), pan-cytokeratin (Sigma, C2931, RRID: AB_258824 , 1:250), GFP (Aves Labs, GFP-1020, RRID: AB_10000240 , 1:500), integrin α8 (R and D Systems, AF4076, RRID: AB_2296280 , 1:500), Etv4 (Abgent, AP6642B, RRID: AB_1967633 , 1:500), Krt8/18 (Abcam, ab53280, RRID: AB_869901 , 1:500), Cdh1 (BD Transduction Laboratories, 610182, RRID: AB_397581 , 1:500), Foxd1 (Santa Cruz Biotechnology, sc-47585, RRID: AB_2105295 , 1:1000), fibronectin (Sigma, F3648, RRID: AB_476976 , 1:500), desmin (DAKO, M0760, RRID: AB_2335684 , 1:500), GM130 (BD Transduction Laboratories, G65120/610822, RRID: AB_398141 , 1:500), and β-galactosidase (Biogenesis, 4600–1409, RRID: AB_2314513 , 1:1000).

Techniques: Marker, Staining, Mutagenesis, Quantitation Assay, RNA Sequencing