PMC

(A) Representative staining of a wild type niche showing only a few old cells in direct contact with co-injected young cells (arrowheads). (B) Representative staining of Fgf20^−/− niche injected with wild type Six2⁺ P0 (red) and E12.5 (blue) progenitors. Old (red) Six2⁺ cells in contact with young (blue) co-injected cells marked with arrowheads. Scale bars, 30um. (C) Quantification of percentage (y-axis) of old cells in contact with the wild type co-injected cells in the wild type or FGF20−/− niches (x-axis) (p<0.001).

(A-D) A 1:1 mixture of FACS sorted E12.5 and P0 Six2⁺ cells imaged live with a fluorescent stereoscope immediately after plating on top of a filter (A) and 48hr culture with heparin, FGF9 and BMP7 (B-D). (E-H) High magnification image of fixed heterochronic co-cultures. Antibody staining of Six2 marked in green. Note that the initial culture was a monolayer of evenly distributed cells (E-E’”). By the end of 48hrs, all cells formed aggregates (F-F’” to H-H’”). Scale bar, 30μm.

(A) Antibody staining of Cited1-driven GFP (green), endogenous Cited1 protein (red) and Cyto8 (white, marking the UB). GFP overlaps with the endogenous protein (arrowhead). (B) Quantification of niche engraftment of E14.5 Cited1⁺ and Six2⁺ cells 4 days after co-injection. Cited1+ progenitors engraft better (69% vs. 31%, stdev=8%, p<10-25). (C) Left-Distribution of E12.5 Six2+ cells and E14.5, E18.5 and P0 Cited1+ progenitors in the CM niche 4 days after co-injection (E14.5: 65% vs. 35%, stdev=6%, n=24; E18.5: 45% vs. 55%, stdev=7%, n=40; P0: 27% vs. 73%, n=37). Right- Distribution of E14.5 vs. E18.5 and P0 Cited1+ progenitors in the CM niche 4 days after co-injection (E18.5 vs. E14.5: 67% vs. 33%, stdev=6%, n=15; P0 vs. E14.5: 79% vs. 21%, stdev=6%, n=15). See also .

(A-C) A representative example of the Imaris software analysis pipeline. Antibody staining labels CM (Six2, green) and UB (Cyto8, white); injected E12.5 tdTomato⁺ cells are red and P0 CFP⁺ cells are blue. Six2⁺ nuclei that are positive for either tdTomato (B) or CFP (C) are marked with bright green dots. (D) Criteria for segregating clusters; see text for detail. (E-F) Counting the number of “single” cells in this niche. 16 out of 28 young cells were single or in small (>4 cell) clusters, whereas 5 out of 5 old cells were single. (G) Quantification of the percentage of “single” Six2⁺ and Cited1⁺ cells to total cells by the end of 4-day culture in 37 injected niches (distance to closest cells ≤12μm). See also and .

(A) Schematic diagram of the experimental design. Lack of intrinsic differences predicts an even contribution from old and young cells to the CM 4-days after injection (left), whereas variation in intrinsic properties would predict exit of most old cells (right). (B-C) Representative Pax2-eYFP metanephroi injected with a 1:1 mixture of FACS sorted tdTomato or CFP positive Six2⁺ cells. (B’-C’) Immunostaining of the explants after 4 days in culture. Six2 (green) marks the CM and Cyto8 (white) labels the UB. Both young and old Six2⁺ cells contributed to the differentiated nephron epithelia (arrows), whereas the majority of cells remaining in the CM are young, regardless of the color. Only a few old cells could be found in each niche (arrowheads). (D) Quantification of injected cells remaining in individual niche after 4 days as the percentage of total injected cells remaining in that niche. On average, 5.7-fold more young cells remained in the niche compared to old cells in a total of 37 niches (stdev= ±8%, p<−52). Scale bars in (B-C) 1mm and (B’-C’) 30μm. See also .

(A) A heat map depicting the expression of genes in Cited1+ progenitors (E12.5, E14.5, E18.5 and P0), stromal cells (FoxD1+, E12.5) and RV cells. Unsupervised clustering visualize the expression patterns of genes (E12.5 magenta; E14.5 red; E18.5 blue; P0 brown; Foxd1+ stroma green; RV grey). Red, blue and yellow intensities indicate high, low and intermediate expression levels, respectively. (B) Analysis of single-cell RNA-Seq data using principle component analysis (PCA) (C) The algorithm Monocle was utilized to organize the cells using “pseudo-time analysis” of E14.5, E18.5 and P0 Cited1+ cells. (D) Gene expression profile from K-means cluster #1 showing the increase of ribosomal biosynthesis genes during progenitors aging. A dashed line represents the concomitant increase in expression of mTOR mRNA. (E) Graphic illustration showing the top five GO terms from K-means cluster 1. The X-axis displays the number of genes in the specified GO term, the Y-axis plots the p-value as a blue line. (F) Hierarchical clustering of cells based on genes in K-means cluster #1 shows the segregation of progenitors into three major clusters: one containing only E12.5 progenitors, a cluster containing E12.5, E14.5 and a few E18.5 progenitors and a large cluster containing only E18.5 and P0 progenitors. See also -.

(A) A representative E12.5 Pax2-eYFP kidney injected with Six2 ^{TGC +/tg}; Rosa ^+/tdTomato nephron progenitors at two different sites. Dotted line marks the branching UB. (B). A recipient kidney 96hrs after injection. Six2 antibody (green) labels the progenitor cells in the periphery. Cytokeratine-8 antibody (Cyto8, white) labels the collecting duct. (C). Enlarged image of one injection site (yellow frame in B). Arrow indicating injected progenitors in the CM and arrowheads pointing at differentiated descendants in the nephron epithelia. (D). 3hr pulse labeling of injected kidneys with EdU by the end of 48hrs (white). Magenta arrows point at triple positive (Six2⁺; tdTom⁺; EdU+) cells. (E, F) TUNEL staining of a kidney explant 24hrs (E) and 48hrs (F) after transplantation. Six2 antibody stains all CM (white), tdTomato (Red) labels injected cells, and TUNEL (green) labels apoptotic cells. (G). tdTomato⁺ progenitors differentiated into podocytes precursor [green, Wilm's tumor 1 (Wt1)], proximal tubule [white, Lotus Tetragonolobus Lectin (LTL)], (H) distal tubule (white, ECad^LO) and (I) thick ascending limb of Loop of Henle [green, Tamm-Horsfall Protein (THP)] but not UB (white, ECad^HI and Cyto8) or (J) stroma (green, PDGFRβ). Scale bar, 1mm in A, 200μm in B, 30 μm in C and D, 20μm in E and F and 30μm in G-I. See also .