Fig 2: Nephron progenitor polarity is disrupted in Wnt11 mutants.(A) E15.5 kidney sections were immunostained for Six2 (red), GM130 (Golgi; green) and DAPI (grey). The Golgi (white arrowheads) show a polarization to the distal end of nephron progenitors in wild type kidneys and this polarization is disrupted in Wnt11 mutants where they are localized closer to the ureteric branch tip (white outline). (B) Staining for GFP (green), GM130 (red), and cytokeratin (cyan) highlighting the normal polarization of Golgi within the nephron progenitor at E15.5. The letter ‘a’ marks the distance from the cell center to the foot of the cell (contact with tip) and ‘b’ marks the distance from the Golgi to the foot. (C) Ratio of the distances described in B) for wild type and mutant cells. Wild type cells show a smaller ratio indicating the Golgi lie farther from the ureteric tip than Wnt11 mutant cells, supporting their polarized nature and the loss in Wnt11 mutants. Three biological replicates and ~20 cells from each replicate were quantified. (D) E15.5 kidney sections immunostained for GFP (green), integrin a8 (Itga8; red) and cytokeratin (CK, cyan). Image shows the overlap of GFP +signal with Itga8 in cells close to the tip (white arrowhead), suggesting polarization of Itga8. (E) E15.5 kidney sections immunostained for Six2 (red), Itga8 (green), and cytokeratin (cyan). Arrowhead points to Itga8 polarization towards the ureteric branch tip in wild type kidneys, which is lost in Wnt11 mutants. (F) E15.5 wholemount immunostains for Six2 (red), desmin (Des; green), and cadherin 1 (Cdh1; blue) show the polarization of desmin foci (Des-foci) toward the ureteric branch tip which is disrupted in Wnt11 mutant kidneys. Since the desmin stain appears aster-like with wispy projections, the central focal point (foci) were identified for ease of distance quantitation. Foci were automatically located by Imaris imaging software as the most intense focal point of the desmin stain. (G) Quantitation of the percentage of desmin foci located greater than 5 µm from the ureteric tip in each genotype showing the greater dispersion from the tip in Wnt11 mutants. 385 foci from five control tips and 353 foci from four tips were analyzed. 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.

Fig 3: (A) Cre- animal stained for ITGA8 (red) at 20X. Magnified images of ITGA8 (B) and PDGFRß (C) staining are shown in panels to the right. (D) Merged image shows co-staining of ITGA8 in PDGFRß+ lung cells. (E) Cre+ animal showed markedly reduced ITGA8 staining (red). Magnified images of ITGA8 (F) and PDGFRß+ (G) staining in Cre+ animals are shown in panels to the right. (H) Merged image shows reduced ITGA8 staining in PDGFRß+ cells. (I-K) Analysis of BALF from Cre+ and Cre- mice revealed no differences in WBC, RBC, and total protein (mean±SEM, n = 3). (L,M) Baseline mechanics showed no difference in airway resistance and elastance (mean±SEM, n = 6).

Fig 4: In vitro characterization of PDGFRß-selected cells isolated from lungs of Pdgfrb-Cre+;Itga8flox/- (Cre+) and WT or Itga8flox/flox (Cre-) mice.(A) Western blot showing the absence of ITGA8 expression in Cre+ cells compared to Cre- cells. (B) Adhesion assay showing decreased binding to the substrates nephronectin (NPNT) and fibronectin (FN) by Cre+ cells. (C-G) PDGFRß-selected cells were treated with TGFß (10 ng/ml) or vehicle. mRNA expression is presented as fold change relative to Cre- in the untreated group (vehicle). Genes include: Itga8 (C), Col1a1 (D), Acta2 (E), Ctgf (F), and Fn1 (G). (*p<0.05, mean±SEM, n = 4).

Fig 5: Expression of ITGA8 in the CFK. A) Whole kidney extracts were prepared from CFK and subjected to Immunoblot analysis with antiserum against ITGA8 antiserum. B-E) Representative co-immunofluorescence images of CFK sections with isotype control antibodies (B) or ITGA8 (red) and ECAD (green) antisera; nuclei were stained with DAPI; a strong ITGA8 signal (red) (arrow) (C) was observed on surface of NP population that did not express ECAD (green) (arrow) (D); also shown on the merged image (arrow) (E); ITGA8 expression (arrowhead) (C) in the ECAD-positive renal vesicle (arrowhead) (D); also shown on the merged image (arrowhead) (E); a weaker ITGA8 signal (concave arrowhead) (C) was detected in the distal segment of the ECAD-positive S-shaped body (concave arrowhead) (D); also shown on the merged image (concave arrowhead) (E). F-H) Representative co-immunofluorescence images of CFK sections with ITGA8 (red) and NCAM1 (green) antisera; a subpopulation of ITGA8-positive (red) (arrow) (F) expressed strong NCAM1 signal (green) (arrow) (G); also shown on the merged image (arrow) (H); ITGA8 signal (arrowhead) (F) in the NCAM1-positive renal vesicle (arrowhead) (G); also shown on the merged image (arrowhead) (H). ITGA8 expression (concave arrowhead) (F) in the distal segment of the S-shaped body identified by NCAM1 expression (concave arrowhead) (G); also shown on the merged image (arrowhead) (H). Scale bars: 20 µm.