Stromal–epithelial crosstalk regulates kidney progenitor cell differentiation

Journal name:
Nature Cell Biology
Volume:
15,
Pages:
1035–1044
Year published:
DOI:
doi:10.1038/ncb2828
Received
Accepted
Published online
Corrected online

Abstract

Present models suggest that the fate of the kidney epithelial progenitors is solely regulated by signals from the adjacent ureteric bud. The bud provides signals that regulate the survival, renewal and differentiation of these cells. Recent data suggest that Wnt9b, a ureteric-bud-derived factor, is sufficient for both progenitor cell renewal and differentiation. How the same molecule induces two seemingly contradictory processes is unknown. Here, we show that signals from the stromal fibroblasts cooperate with Wnt9b to promote differentiation of the progenitors. The atypical cadherin Fat4 encodes at least part of this stromal signal. Our data support a model whereby proper kidney size and function is regulated by balancing opposing signals from the ureteric bud and stroma to promote renewal and differentiation of the nephron progenitors.

At a glance

Figures

  1. The cortical stroma regulates Wnt9b target activation and differentiation of the nephron progenitors.
    Figure 1: The cortical stroma regulates Wnt9b target activation and differentiation of the nephron progenitors.

    (a) Schematic representation of the embryonic kidney indicating the Wnt9b-expressing ureteric bud epithelium in red, the renewing nephron progenitors that express Wnt9b Class II targets in light grey, the differentiating nephron progenitors or pre-tubular aggregates (PTAs) that express Wnt9b Class I targets in dark grey and the Foxd1-positive stromal fibroblasts in blue. (b,cHaematoxylineosin-stained P1 wild-type (b) and Foxd1cre;Rosa26DTA mutant (c) kidney sections. Arrows indicate nephron progenitor cells. (di) Expression of Class II/progenitor gene Cited1 (d,e), Amphiphysin (f,g) and Class I/PTA gene Lef1 (h,i) in wild-type (d,f,h) and mutant (e,g and i) kidneys at E15.5. All sections have been co-stained with antibodies against the Wnt9b-independent progenitor marker Six2 (in red) and the ureteric bud epithelium marker cytokeratin (CK; in blue). Quantification of the Six2 expression domain showed that it was significantly expanded from 2.1 cell layers in the wild type to 5.3 layers in mutants (n = 4, P<0.000001). Statistics source data can be found in Supplementary Table S1. The ureteric bud in bi is outlined with a white dashed line. di are captured at ×63 magnification. Scale bars, 100 μm (b,c) and 50 μm (all other panels).

  2. Expression of Class II targets is independent of a Wnt ligand but dependent on
β-catenin in stromaless and Fat4 mutants.
    Figure 2: Expression of Class II targets is independent of a Wnt ligand but dependent on β-catenin in stromaless and Fat4 mutants.

    (ai) Whole-mount images of wild-type (ac), Foxd1cre;Rosa26DTA (df) and Fat4 null (gi) kidneys cultured from E11.5 for 48 h in the presence of dimethylsulphoxide (DMSO; a,d,g), 5 μM IWP2 (inhibitor of Wnt production; b,e,h) or the β-catenin destabilizer IWR1 (c,f,i). All tissues were hybridized with an antisense probe against Pla2g7. Scale bars, 1 mm. (j,k) Quantitative analysis of gene expression by quantitative PCR with reverse transcription (qRT–PCR). cDNA levels of Class II/progenitor targets Tafa5 and Pla2g7 and Class I/PTA target C1qdc2 were assessed after treating wild-type or Fat4 mutant kidneys with IWP2 (j) or IWR1 (k) or dimethylsulphoxide. Dimethylsulphoxide-treated wild-type levels are considered baseline. Shown is the mean value of qRT–PCR data from three different experiments. Error bars indicate s.e.m. *P<0.05; #P<0.01.

  3. Nephron progenitors of stromal mutants show increased nuclear Yap and reduced pYap.
    Figure 3: Nephron progenitors of stromal mutants show increased nuclear Yap and reduced pYap.

    (al) P1 wild-type (ad), Foxd1cre;Rosa26DTA (eh) and Fat4 null (il) kidneys were subjected to immunostaining using either an antibody against Yap or a pYap-specific antibody (green in merged images), the progenitor marker Six2 (in red) and the ureteric bud epithelium marker cytokeratin (CK; in blue). Single channel images for pYap (b,f and j) and Yap (d,h and l) are shown beneath each merged image. The asterisks indicate the progenitor domain in mutants with significantly reduced pYap expression. All images were captured at ×63 magnification. Scale bars, 20 μm.

  4. Nuclear Yap/Taz drives Wnt9b Class II/progenitor and represses Class I/PTA target gene expression in isolated progenitor cell cultures.
    Figure 4: Nuclear Yap/Taz drives Wnt9b Class II/progenitor and represses Class I/PTA target gene expression in isolated progenitor cell cultures.

    (a) Western blot on whole-cell lysates from isolated mesenchymal cells transfected with scrambled, Yap- or Taz-specific siRNA for 72 h. Blots were probed with the following antibodies: Yap, Taz, Class II target genes (Cited, Tafa5, Pla2g7, Amphiphysin), Class I target gene (Pax8), Wnt9b-independent progenitor markers (Six2 and Sall1) and a marker of differentiation, E-cadherin. (b) qRT–PCR on cDNA from cells transfected with a scrambled siRNA or siRNAs against Taz and Yap using primers for Yap, Taz, Class II Class I targets and Wnt9b-independent progenitor genes. (c) Western blot of cells 72 h after transfection with vector, full-length or Fat4-ECD plasmids. (d) Metanephric mesenchyme (MM) cells (stained with CellTracker, red) were co-cultured with cells co-expressing empty vector, full-length Fat4 or Fat4-ECD and GFP (green) then stained with anti-Yap antibody (teal) and DAPI (blue). Arrows indicate the nucleus. Note the lack of Yap in the nucleus of mesenchymal cells adjacent to Fat4-expressing cells. Scale bars, 20 μm. In all in vitro experiments, cultured cells were isolated from 20 embryos from 3 pregnant females. The qRT–PCR data shown are 1 representative example from 3 individual experiments. Uncropped images of blots are shown in Supplementary Fig. S9.

  5. Ablation of Yap/Taz from the progenitors alters expression of Class I and Class II target genes in the nephron progenitors.
    Figure 5: Ablation of Yap/Taz from the progenitors alters expression of Class I and Class II target genes in the nephron progenitors.

    (ar) Sections from E18.5 wild type (a,c,e,g,i,k,m,o,q) or Six2cre; Yapflox/flox/Tazflox/flox mutant (b,d,f,h,j,l,n,p,r) kidneys were stained with antibodies against pYap (green), the progenitor marker Six2 (red) and the ureteric bud epithelium gene, cytokeratin (CK in blue; a,b; single-channel images for pYap are shown in a’,b’), haematoxylin and eosin (H&E; c,d); sections were stained with antibodies against LTL (red), the epithelial marker E-cadherin (green) and DAPI (blue; e,f), the Wnt9b/Class II target Cited1 (in green), the ureteric bud epithelium marker dolichos biflorus agglutinin (DBA, in red) and DAPI (blue; g,h; g’,h’ show a higher magnification with the ureteric bud outlined in white), the antisense Wnt9b/Class II target Pla2g7 (i,j), the antisense Wnt9b/Class II target Tafa5 (k,l), the antisense Wnt9b/Class I target Pax8 (m,n; m’,n’ show the higher magnification with the ureteric bud outlined in white), the antisense Wnt9b/Class I target Wnt4 (o,p), antibodies against the Class II target Amphiphysin (in red), β-catenin (in green) and E-cadherin (blue; q,r; the corresponding single channels for Amphiphysin and β-catenin are shown in q’,r’ and q”,r”, respectively). (s,t) Model showing the expression of progenitors in wild type and Yap/Taz double mutants. The self-renewing progenitor cells (light grey) are significantly reduced or lost in the mutants and are replaced by the differentiating progenitors (dark grey). Dashed lines indicate the ureteric bud in all panels. Arrows in a,b and a’,b’ indicate reduced Six2-positive cells and the asterisks indicate the PTAs; arrows in c,d indicate epithelial structures lost in the mutants; arrow in h indicates loss of Cited1 expression from ureteric bud tips; and arrows in n and p indicate ectopic expression of target genes. Scale bars, 0.1 mm (a,b), 10 μm (cp) and 50 μm (q,r).

  6. Fat4 mutants exhibit an expansion of the progenitor domain and reduced differentiation.
    Figure 6: Fat4 mutants exhibit an expansion of the progenitor domain and reduced differentiation.

    (ap) Wild-type (a,c,e,g,i,k,m,o) and Fat4 null (b,d,f,h,j,l,n,p E18.5) kidneys stained with haematoxylin and eosin staining (a,b), antisense probes against Six2 (c,d) and Eya1 (e,f) and the Wnt9b/Class II target genes Cited1 (g,h) and Tafa5 (i,j), or antibodies against Amphiphysin (green), the ureteric bud epithelium marker cytokeratin (CK in red) and DAPI (k,l), Pax2 (red), E-cadherin (green) and β-catenin (blue; m,n), Six2 (red), the Wnt9b/Class I target gene Lef1 (green) and cytokeratin (blue; o,p). Quantification of Six2-positive cells revealed that the progenitor domain was on average 4 cell layers thick in mutants as compared with 2.1 cell layers in wild-type kidneys (n = 4, P<0.000001). Wild-type kidneys had an average of 51.5 Lef1-positive structures per section whereas Fat4 mutants had only 28 (n = 4, P = 0.007). Statistics source data can be found in Supplementary Table S1. All images are taken at ×20 magnification. Scale bars, 100 μm.

  7. Fat4 mutants express Class II/progenitor genes in the absence of Wnt9b.
    Figure 7: Fat4 mutants express Class II/progenitor genes in the absence of Wnt9b.

    (ax) Wild-type (a,e,i,m,q,u,a’), Fat4 mutant (b,f,j,n,r,v,b’), Wnt9b mutant (c,g,k,o,s,w,c’) and Wnt9b/Fat4 double mutant (d,h,l,p,t,x,d’) kidneys at P1 (ad and a’–d’), E11.5 (et) or E15.5 (ux). Sections were stained with haematoxylin and eosin (ad), anti-Six2 (green), DBA (red) and DAPI (blue; a’–d’), anti-Six2 (red), the Wnt9b/Class II target gene Cited1 (green) and cytokeratin (blue, CK in eh), antisense probes against Pla2g7 (il), Tafa5 (mp) and the Wnt9b/Class I target genes C1qdc2 (qt) and Wnt4 (ux). Dashed lines indicate the ureteric bud. The asterisks in ad indicate expanded progenitor cell domains. The asterisks in wx indicate medullary stromal expression of Wnt4. The letter a in w,x indicates the adrenal gland. In ad images are captured at ×40 magnification and scale bars, 20 μm. All other images are at ×20 magnification and scale bars are 100 μm.

  8. Model for the interaction between the stroma and the ureteric bud on the progenitor cells.
    Figure 8: Model for the interaction between the stroma and the ureteric bud on the progenitor cells.

    The outlined area of the nascent kidney is magnified on the right. Wnt9b, produced by the ureteric bud (red), signals canonically to both the renewing progenitors (grey cells) and to the differentiating progenitors (green cells). Fat4, produced by the stroma, promotes differentiation by enhancing the nuclear export of Yap/Taz within the progenitors (green cells), which allows expression of Class I β-catenin targets. In the renewing progenitors, levels of pYap are low. Unphosphorylated Yap is localized predominantly in the nucleus and activates the expression of Class II β-catenin targets and promotes progenitor renewal. We propose that cells induced to differentiate by Fat4 are only transiently located next to the stroma and then migrate to the stalk side of the ureteric bud where they initiate MET.

  9. Characterization of the stromaless mutants
    Supplementary Fig. 1: Characterization of the stromaless mutants

    Panel A: Ablation of the stroma in the Foxd1cre; Rosa26DTA mutant kidneys. Comparison of stromal markers in wildtype (A, C, E) and Foxd1Cre;Rosa26DTA (B, D, F) kidneys at e15.5 (A-D) and 18.5 (E, F). At E15.5, the stromal expression of Foxd1 (green in A and B) cortical to the progenitor cells is lost (arrows in A and B.). Meis1/2 (green in C–F) is decreased in mutants at E15.5 (arrow in D) and completely lost by E18.5 (arrow in F). All sections are co-stained with the progenitor marker Six2 (red) and the UB marker cytokeratin (blue in A-D) or the lectin Dolichous biflorus agglutinin (DBA. White in E, F). In A and B, the cortex of the kidney is up and the medulla is down. In C–F, cortex is to the left, the medulla is to the right. Scale bar  =  50 microns. Panel B: Ablation of the stroma affects the activation of distinct classes of Wnt9b target genes in the nephron progenitors. In situ hybridization on sections of E15.5 wildtype (A, C, E, G) and FoxdCre;Rosa26DTA (B, D, F, H) kidneys hybridized with antisense probes to Class II/progenitor targets Tafa5 (A, B) and Pla2g7 (C,D) and Class I/PTA targets Pax8 (E,F) and C1qdc2 (G,H). Note: Expanded domain of Class II targets and significant reduction in Class I. All images captured at 20x magnification. Scale bar  =  100 microns..

  10. Localization of YAP in vivo and in vitro.
    Supplementary Fig. 2: Localization of YAP in vivo and in vitro.

    Left panel: Low mag localization of total and phosphorylated Yap: E18.5 wild type (A, B) and Fat4 null (C) kidneys were subjected to immunostaining using YAP and pYAP antibodies (in green). All slides were co-stained with Six2 (red) and Cytokeratin (blue). Single channel images for pYap or Yap are shown in black and white below the corresponding color panel. In all panels, the cortex is up and medullary region is down. Arrowheads in A’ and B’ indicates cortical zone while the arrows indicate the medullary region. Scale bar  =  100 microns. Right panel: Expression of YAP in cell cultures: Immunostaining of MM progenitor cells with anti-Yap antibody (green). YAP predominantly exhibits nuclear expression at low density and diffuse expression throughout the cell at higher density. Nuclei are co-stained with Dapi (blue). Scale bar: 20 microns.

  11. Kinetics of TAZ/YAP ablation from the progenitors.
    Supplementary Fig. 3: Kinetics of TAZ/YAP ablation from the progenitors.

    Wildtype (A, C, E, G) and Six2Cre; Yapflox/flox;Tazflox/flox (B, D, F, H) kidneys from E11.5 (A, B), 12.5 (C,D), 13.5 (E, F) and 18.5 (G, H) embryos stained with antibodies to pYAP (green), Six2 (red) and Cytokeratin (CK, blue). Single channel images for pYap are shown in black and white to the right of the corresponding color panel. Note efficient ablation of pYap from the Six2 expressing domain by E13.5. Scale bars  =  100 microns.

  12. Response of cultured progenitor cells to Wnt and Yap signaling.
    Supplementary Fig. 4: Response of cultured progenitor cells to Wnt and Yap signaling.

    Mesenchymal cells isolated from 20 embryos from 3 distinct mothers were cultured and subjected to QrtPCR to analyze Wnt9b target gene expression and Six2 after the following treatments: (A) transfection with an empty plasmid or a Lef1/beta-catenin fusion construct; (B) addition of IWP2 or IWR1 to the media for 48 hrs. Shown is one representative experiment analysed in triplicate PCR reactions. .

  13. Percent proliferation of Six2 positive progenitor cells in wild type, Fat4 and Six2Cre; Tazflox/flox;Yapflox/flox mutants.
    Supplementary Fig. 5: Percent proliferation of Six2 positive progenitor cells in wild type, Fat4 and Six2Cre; Tazflox/flox;Yapflox/flox mutants.

    Kidneys were co-stained with Six2, cytokeratin and pHH3. The number of Six2/pHH3 double positive cells was quantified for wildtype TAZ/YAP and Fat4 mutants. 10.5% (p = 0.004) of Six2 positive cells were PHH3 positive in the Six2cre; Tazflox/flox; YAPflox/flox mutants, 16% (p = 0.02) in Fat4 mutants and 13% in wild type. Data is the mean from 5 different kidneys for TAZ/YAP mutants (compared to 5 wildtype littermates) and 4 kidneys from Fat4 mutants (compared to 4 wildtype littermates). Statistics source data can be found in Table1. Error bars indicate SEM.

  14. Expression of Class I targets requires a Wnt ligand in wildtype, stromaless and Fat4 mutants.
    Supplementary Fig. 6: Expression of Class I targets requires a Wnt ligand in wildtype, stromaless and Fat4 mutants.

    A–F: In situ hybridization analyzing the expression of a Class I/PTA target C1qdc2 in kidney explants isolated from E11.5 wild type (A-B), Foxd1cre; Rosa26DTA (C-D) and Fat4 null (E-F) kidneys treated either with DMSO (A, C, E) or a Wnt ligand production inhibitor IWP2 (B, D, F).

  15. Expression of stromal and progenitor targets in various mutants.
    Supplementary Fig. 7: Expression of stromal and progenitor targets in various mutants.

    Panel A: The stroma develops normally in Fat4 mutants. E13.5 (A, B, E, F, I, J, M, N) and 18.5 (C, D, G, H, K, L, O, P) wild type (A, C, E, G, I, K, M, O) and Fat4 null (B, D, F, H, J, L, N, P) kidney sections stained with the progenitor marker Six2 (red), the UB/collecting duct marker CK (blue) and the cortical stroma markers Foxd1 (green in A-D) and Meis 1/2 (green in E-H) and the medullary stroma markers Slug (green in I-L) and Lef1 (green in M-P). Panel B: Characterization of Class II/progenitor targets in Wnt9b/Vangl2 double mutants: E15.5 (A-D) and 18.5 (E-H) kidney sections from wild type (A,E), Wnt9b null (B), Wnt9b hypomorph (neo) (F), Vangl2lp/lp (C, G) and Wnt9b−/−; Vangl2lp/lp and Wnt9bneo/neo;Vangl2lp/lp mutants were subjected to immunostaining for progenitor genes Cited1 (green), Six2 (red) and the UB marker cytokeratin (blue). Panel C: pSmad1/5/8 expression remains unaltered in wild type and mutant kidneys: E18.5 wildtype (A,B), Six2Cre;Yapflox/flox;Tazflox/flox (C,D) and Fat4−/− (E, F) kidneys stained with antibodies to pSmad1/5/8 (green) and the collecting duct marker DBA (red). Images show the cortical regions (A, C, E) and the medullary region (B, D, F). There is no change in pSmad activity in Fat4 or Yap/Taz mutants relative to wildtype. Scale bar  =  100 microns.

  16. Characterization of wildtype, Wnt9b, Fat4 and YAP/TAZ mutant kidneys.
    Supplementary Fig. 8: Characterization of wildtype, Wnt9b, Fat4 and YAP/TAZ mutant kidneys.

    A: Fate mapping of the nephron progenitors in Wnt9b null kidneys. The Six2 expressing progenitor cells were lineage traced in SixCre;RosaYap (a,c,e,g) and SixCre;RosaYap;Wnt9b−/− (b,d,f,h) kidneys by co-staining with antibodies to GFP (green) and the stromal proteins (in red) Foxd1 (a,b), Meis1/2 (c,d), Slug (e,f) and Lef1 (g,h). The ureteric bud epithelia was stained with anti-cytokeratin (blue) B: In situ hybridization using antisense riboprobes for Taz and Yap on Wildtype and Fat4 null kidneys at E18.5. C: Wildtype (A, E, I), Taz single (B, F, J), YAP single (C, G, K) and Taz and YAP double mutants (D, H, L) were analyzed for the expression of the following targets. ClassII/progenitor target Amphiphysin (in red), pYAP (in green) (A-D); Class II target Cited1 (in red) and GFP (in green, indicating Six2cre activity) (E-H); Class I/PTA target Pax8 (in purple) (I-L). The ureteric bud epithelia was stained with DBA (blue in A-H). Scale bar 100 microns.

  17. Un-cropped images showing molecular weight markers for all Western Blots included in the main text.
    Supplementary Fig. 9: Un-cropped images showing molecular weight markers for all Western Blots included in the main text.

Change history

Corrected online 29 August 2013
In the version of this Article originally published, there was an error in Fig. 4a. The labels "Yap siRNA" and "Taz siRNA" were switched. This has been corrected in the PDF and HTML versions of the Article.

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Author information

Affiliations

  1. Department of Internal Medicine (Nephrology), University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA

    • Amrita Das,
    • Courtney M. Karner &
    • Thomas J. Carroll
  2. Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA

    • Amrita Das,
    • Courtney M. Karner,
    • Mei Xin,
    • Eric N. Olson &
    • Thomas J. Carroll
  3. Cancer and Developmental Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • Shunsuke Tanigawa &
    • Alan O. Perantoni
  4. Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA

    • Lawrence Lum
  5. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9148, USA

    • Chuo Chen
  6. Present address: Department of Internal Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA

    • Courtney M. Karner

Contributions

Experiments were designed by A.D., S.T., C.M.K., A.O.P. and T.J.C. Experiments were performed by A.D., S.T. and C.M.K. Data were interpreted by A.D., S.T., C.M.K., A.O.P. and T.J.C. M.X., L.L., C.C. and E.N.O. provided mice or other reagents. The paper was written by A.D., S.T., A.O.P. and T.J.C.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Characterization of the stromaless mutants (898 KB)

    Panel A: Ablation of the stroma in the Foxd1cre; Rosa26DTA mutant kidneys. Comparison of stromal markers in wildtype (A, C, E) and Foxd1Cre;Rosa26DTA (B, D, F) kidneys at e15.5 (A-D) and 18.5 (E, F). At E15.5, the stromal expression of Foxd1 (green in A and B) cortical to the progenitor cells is lost (arrows in A and B.). Meis1/2 (green in C–F) is decreased in mutants at E15.5 (arrow in D) and completely lost by E18.5 (arrow in F). All sections are co-stained with the progenitor marker Six2 (red) and the UB marker cytokeratin (blue in A-D) or the lectin Dolichous biflorus agglutinin (DBA. White in E, F). In A and B, the cortex of the kidney is up and the medulla is down. In C–F, cortex is to the left, the medulla is to the right. Scale bar  =  50 microns. Panel B: Ablation of the stroma affects the activation of distinct classes of Wnt9b target genes in the nephron progenitors. In situ hybridization on sections of E15.5 wildtype (A, C, E, G) and FoxdCre;Rosa26DTA (B, D, F, H) kidneys hybridized with antisense probes to Class II/progenitor targets Tafa5 (A, B) and Pla2g7 (C,D) and Class I/PTA targets Pax8 (E,F) and C1qdc2 (G,H). Note: Expanded domain of Class II targets and significant reduction in Class I. All images captured at 20x magnification. Scale bar  =  100 microns..

  2. Supplementary Figure 2: Localization of YAP in vivo and in vitro. (489 KB)

    Left panel: Low mag localization of total and phosphorylated Yap: E18.5 wild type (A, B) and Fat4 null (C) kidneys were subjected to immunostaining using YAP and pYAP antibodies (in green). All slides were co-stained with Six2 (red) and Cytokeratin (blue). Single channel images for pYap or Yap are shown in black and white below the corresponding color panel. In all panels, the cortex is up and medullary region is down. Arrowheads in A’ and B’ indicates cortical zone while the arrows indicate the medullary region. Scale bar  =  100 microns. Right panel: Expression of YAP in cell cultures: Immunostaining of MM progenitor cells with anti-Yap antibody (green). YAP predominantly exhibits nuclear expression at low density and diffuse expression throughout the cell at higher density. Nuclei are co-stained with Dapi (blue). Scale bar: 20 microns.

  3. Supplementary Figure 3: Kinetics of TAZ/YAP ablation from the progenitors. (413 KB)

    Wildtype (A, C, E, G) and Six2Cre; Yapflox/flox;Tazflox/flox (B, D, F, H) kidneys from E11.5 (A, B), 12.5 (C,D), 13.5 (E, F) and 18.5 (G, H) embryos stained with antibodies to pYAP (green), Six2 (red) and Cytokeratin (CK, blue). Single channel images for pYap are shown in black and white to the right of the corresponding color panel. Note efficient ablation of pYap from the Six2 expressing domain by E13.5. Scale bars  =  100 microns.

  4. Supplementary Figure 4: Response of cultured progenitor cells to Wnt and Yap signaling. (134 KB)

    Mesenchymal cells isolated from 20 embryos from 3 distinct mothers were cultured and subjected to QrtPCR to analyze Wnt9b target gene expression and Six2 after the following treatments: (A) transfection with an empty plasmid or a Lef1/beta-catenin fusion construct; (B) addition of IWP2 or IWR1 to the media for 48 hrs. Shown is one representative experiment analysed in triplicate PCR reactions. .

  5. Supplementary Figure 5: Percent proliferation of Six2 positive progenitor cells in wild type, Fat4 and Six2Cre; Tazflox/flox;Yapflox/flox mutants. (317 KB)

    Kidneys were co-stained with Six2, cytokeratin and pHH3. The number of Six2/pHH3 double positive cells was quantified for wildtype TAZ/YAP and Fat4 mutants. 10.5% (p = 0.004) of Six2 positive cells were PHH3 positive in the Six2cre; Tazflox/flox; YAPflox/flox mutants, 16% (p = 0.02) in Fat4 mutants and 13% in wild type. Data is the mean from 5 different kidneys for TAZ/YAP mutants (compared to 5 wildtype littermates) and 4 kidneys from Fat4 mutants (compared to 4 wildtype littermates). Statistics source data can be found in Table1. Error bars indicate SEM.

  6. Supplementary Figure 6: Expression of Class I targets requires a Wnt ligand in wildtype, stromaless and Fat4 mutants. (225 KB)

    A–F: In situ hybridization analyzing the expression of a Class I/PTA target C1qdc2 in kidney explants isolated from E11.5 wild type (A-B), Foxd1cre; Rosa26DTA (C-D) and Fat4 null (E-F) kidneys treated either with DMSO (A, C, E) or a Wnt ligand production inhibitor IWP2 (B, D, F).

  7. Supplementary Figure 7: Expression of stromal and progenitor targets in various mutants. (651 KB)

    Panel A: The stroma develops normally in Fat4 mutants. E13.5 (A, B, E, F, I, J, M, N) and 18.5 (C, D, G, H, K, L, O, P) wild type (A, C, E, G, I, K, M, O) and Fat4 null (B, D, F, H, J, L, N, P) kidney sections stained with the progenitor marker Six2 (red), the UB/collecting duct marker CK (blue) and the cortical stroma markers Foxd1 (green in A-D) and Meis 1/2 (green in E-H) and the medullary stroma markers Slug (green in I-L) and Lef1 (green in M-P). Panel B: Characterization of Class II/progenitor targets in Wnt9b/Vangl2 double mutants: E15.5 (A-D) and 18.5 (E-H) kidney sections from wild type (A,E), Wnt9b null (B), Wnt9b hypomorph (neo) (F), Vangl2lp/lp (C, G) and Wnt9b−/−; Vangl2lp/lp and Wnt9bneo/neo;Vangl2lp/lp mutants were subjected to immunostaining for progenitor genes Cited1 (green), Six2 (red) and the UB marker cytokeratin (blue). Panel C: pSmad1/5/8 expression remains unaltered in wild type and mutant kidneys: E18.5 wildtype (A,B), Six2Cre;Yapflox/flox;Tazflox/flox (C,D) and Fat4−/− (E, F) kidneys stained with antibodies to pSmad1/5/8 (green) and the collecting duct marker DBA (red). Images show the cortical regions (A, C, E) and the medullary region (B, D, F). There is no change in pSmad activity in Fat4 or Yap/Taz mutants relative to wildtype. Scale bar  =  100 microns.

  8. Supplementary Figure 8: Characterization of wildtype, Wnt9b, Fat4 and YAP/TAZ mutant kidneys. (739 KB)

    A: Fate mapping of the nephron progenitors in Wnt9b null kidneys. The Six2 expressing progenitor cells were lineage traced in SixCre;RosaYap (a,c,e,g) and SixCre;RosaYap;Wnt9b−/− (b,d,f,h) kidneys by co-staining with antibodies to GFP (green) and the stromal proteins (in red) Foxd1 (a,b), Meis1/2 (c,d), Slug (e,f) and Lef1 (g,h). The ureteric bud epithelia was stained with anti-cytokeratin (blue) B: In situ hybridization using antisense riboprobes for Taz and Yap on Wildtype and Fat4 null kidneys at E18.5. C: Wildtype (A, E, I), Taz single (B, F, J), YAP single (C, G, K) and Taz and YAP double mutants (D, H, L) were analyzed for the expression of the following targets. ClassII/progenitor target Amphiphysin (in red), pYAP (in green) (A-D); Class II target Cited1 (in red) and GFP (in green, indicating Six2cre activity) (E-H); Class I/PTA target Pax8 (in purple) (I-L). The ureteric bud epithelia was stained with DBA (blue in A-H). Scale bar 100 microns.

  9. Supplementary Figure 9: Un-cropped images showing molecular weight markers for all Western Blots included in the main text. (243 KB)

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