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.
Kidney development depends on reciprocal interactions between two tissues, the ureteric bud and the metanephric mesenchyme1,2. Signals produced by the mesenchyme promote reiterative branching morphogenesis of the ureteric bud, and signals from the bud support survival and proliferation of nephron progenitor cells within the mesenchyme. In addition, the bud produces a signal(s) that induces a subset of the progenitors to undergo a mesenchymal-to-epithelial (MET) transition to form an intermediate condensed structure known as the pre-tubular aggregate (PTA), which proceeds to an epithelial structure referred as the renal vesicle. The renal vesicle will undergo morphogenesis to form the nephron, a structure consisting of the renal corpuscle, the proximal tubule, the loop of Henle, the distal tubule and the connecting tubule. The ureteric bud will give rise to the collecting ducts and ureter.
A significant portion of the inductive activity attributed to the ureteric bud can be assigned to Wnt9b (ref. 3). Previous studies have shown that Wnt9b signals to the nephron progenitor cells and activates at least two molecularly and spatially distinct programs; one that promotes progenitor cell proliferation/renewal (referred to as the Class II/progenitor signature) and another that induces their differentiation (referred to as the Class I/pre-tubular aggregate or PTA signature)4. In the absence of Wnt9b, the progenitor domain is specified correctly but does not expand and the PTAs and renal vesicles do not form3,4. Both programs are activated by the transcription factor β-catenin4. A question that arises is how the same molecule promotes two seemingly contradictory, program-specific responses.
The nephron progenitors are encapsulated by a population of fibroblasts known as the stroma (Fig. 1a). These cells are ideally positioned to influence the fate of the nephron progenitors. Indeed, ablation of the transcription factor Foxd1 from the stroma results in expansion of nephron progenitor cells and a severe deficit in MET/differentiation5,6. However, the precise mechanism underlying this phenotype is unclear.
Here, we show that the stromal cells produce a signal(s) that regulates progenitor cell renewal. This signal is at least in part encoded by the atypical cadherin Fat4. Fat4 normally functions to modify β-catenin activity, promoting the differentiation program and repressing the renewal program. We propose that Fat4 accomplishes this role by modulating the activity of Yap and Taz within the nephron progenitors. By providing opposing progenitor renewal and differentiation signals, the ureteric bud and the stroma provide a niche that ensures proper nephron endowment and optimal kidney function.
The stroma promotes differentiation
To test a potential role for the stroma in nephron progenitor fate, these cells were ablated by generating pups carrying both a Cre inducible form of the diphtheria toxin A chain (RosaDTA; ref. 7) and Foxd1Cre (ref. 8).
Foxd1Cre;RosaDTA pups died within 24 h of birth. As expected, examination of embryonic day (E)18.5 kidneys revealed a complete absence of the cortical stromal cells and their derivatives (Supplementary Fig. S1A). Foxd1-positive stromal cells were absent at E15.5 although there were still some Foxd1-derived medullary stromal cells (Supplementary Fig. S1A). Thus, the Foxd1Cre;RosaDTA mouse results in deletion of the cortical stroma by at least E15.5.
Kidneys of P1 Foxd1Cre;RosaDTA pups (which we will refer to as stromaless) were smaller than wild-type kidneys and were fused to the body wall. Haematoxylin–eosin-stained sections revealed an expanded zone of mesenchymal cells capping the ureteric buds (Fig. 1c). Staining with an antibody against Six2 demonstrated that the nephron progenitor domain of mutants was significantly expanded in the Foxd1cre;RosaDTA mutants (Fig. 1e,g,i).
To determine whether stromaless kidneys had a normal Wnt9b response, we assessed the expression of both Class I/PTA and Class II/progenitor target genes at E15.5. Class I targets include Pax8, Wnt4, Lef1 and C1qdc2 (ref. 4). Class II targets include Cited1, phospholipase A2 group 7 (Pla2g7), Amphiphysin (Amph) and expressed sequence AW049604 (Tafa5/Fam19a5; ref. 4). At E15.5, all Class II/progenitor Wnt9b targets examined were expressed throughout the expanded progenitor domain of the stromaless mutants (Fig. 1e,g and Supplementary Fig. S1B). However, the expression of Class I/PTA targets was significantly reduced or absent (Fig. 1i and Supplementary Fig. S1B). Although most kidneys were largely devoid of PTAs and renal vesicles, a very low number of PTAs and renal vesicles did form, most likely corresponding to regions of retained stroma. These data suggest that a signal(s) from the cortical stroma suppresses renewal and/or promotes differentiation of the nephron progenitor cells.
Previous studies have found that the stroma produces secreted frizzleds and it has been suggested that these signals affect the activity of Wnt ligands produced by the ureteric bud and/or renal vesicles6,9,10. To determine whether ablation of the stroma could affect the strength of ureteric-bud-derived Wnt9b signalling, we cultured stromaless mutants with a small molecule that inhibits Wnt production (IWP2) by repressing the fatty acyl transferase porcupine11,12.
Unexpectedly, Foxd1Cre;RosaDTA kidneys treated with the highest dosage of IWP2 still maintained the expression of Wnt9b Class II/progenitor targets (although at slightly reduced levels) after 48 h of culture (Fig. 2e). Interestingly, IWP2 treatment blocked expression of class I/PTA targets in both wild-type and stromaless kidneys (Supplementary Fig. S6). These data suggest that in the absence of stroma, Class II target expression is less dependent on a Wnt ligand.
To determine whether β-catenin was still necessary for the expression of Wnt9b targets in stromaless kidneys, Foxd1Cre;RosaDTA kidneys were cultured with a compound that destabilizes β-catenin (inhibitor of Wnt response 1 or IWR1; Fig. 2c,f; refs 11, 12). Both wild-type and Foxd1Cre;RosaDTA mutant kidneys lost expression of Wnt9b/β-catenin Class I and II targets in the presence of IWR1. These data indicate that removal of the stroma makes Wnt9b Class II target gene expression less dependent on a Wnt ligand, but does not lessen dependence on β-catenin.
Stromal signals affect Yap activation
Recent studies have shown that β-catenin activity can be regulated by crosstalk with the Hippo/Warts pathway13,14,15,16. Hippo and Warts (Mst1/2 and Lats1/2 in mice) are serine/threonine kinases that regulate cell proliferation and differentiation by activating the phosphorylation and nuclear exclusion of the transcriptional regulator Yorkie. Loss of Hippo/Warts signalling in the heart results in increased levels of nuclear Yap (a mouse orthologue of Yorkie) and inappropriate activation of the β-catenin pathway16. Given the observed effect on β-catenin activity, we examined the localization and activation status of Yap in stromaless kidneys using antibodies that recognize either total or phosphorylated forms of the protein17,18,19,20.
In wild-type kidneys, we found that total Yap protein was ubiquitously expressed. However, Yap showed differences in its sub-cellular localization with a higher nuclear to cytoplasmic ratio in differentiated cells in the more central or medullary region of the kidney (Fig. 3c,d and Supplementary Fig. S2A). As expected, phospho- (p-) Yap expression and nuclear Yap were largely exclusive of each other. pYap expression was strong in the cortical stromal, ureteric bud and nephron progenitor cells (Supplementary Fig. S2A’). Levels of pYap abruptly decreased in all three lineages as differentiation proceeded. The medullary region was largely devoid of pYap expression (Supplementary Fig. S2A’).
Yap protein levels were next assessed in Foxd1Cre;RosaDTA kidneys. Although total Yap levels did not change significantly in mutants, there was a striking increase in the levels of nuclear Yap in all three lineages within the nephrogenic zone of mutant kidneys (Fig. 3g,h). Conversely, phosphorylation of Yap was significantly reduced in the Six2-positive cells of Foxd1Cre;RosaDTA kidneys (Fig. 3e,f). These data suggest that signals from the cortical stroma regulate Yap phosphorylation and localization within the nephron progenitors.
Yap/Taz regulates Wnt9b activity
Our data suggest that nuclear Yap (and Taz) stimulate the expression of Wnt9b target genes. To test this hypothesis, nephron progenitors were isolated and cultured21,22. As in the stromaless mice, Yap protein was predominantly nuclear in the cultured cells although, as previously shown, the levels of nuclear Yap were inversely correlated to cell density (Supplementary Fig. S2B).
Cited1, Pla2g7, Tafa5 and Amphiphysin are all expressed in the isolated progenitors in the absence of Wnt9b (Fig. 4a) and all tested were positively affected by Lef1/β-catenin activity (Supplementary Fig. S4A). We next investigated whether the expression of these genes was dependent on Wnt production. Consistent with observations made in Foxd1Cre;RosaDTA kidneys, treatment of isolated progenitor cells with IWP2 had no significant effect on the expression of these progenitor target genes (Supplementary Fig. S4B).
We next investigated whether Wnt9b/Class II target expression was dependent on Yap/Taz activity. Cells in which Yap/Taz mRNA/protein levels were knocked-down using short interfering RNA (siRNA)-mediated silencing showed a significant repression of the Class II/progenitor targets Cited1, Tafa5, Pla2g7 and Amphiphysin (Fig. 4a,b) whereas the levels of Wnt9b-independent progenitor targets such as Sall1 and Six2 were only moderately reduced (Fig. 4a,b). Expression of the class I/PTA targets Pax8 and E-cadherin was significantly increased in these cells (Fig. 4a,b). These findings suggest that nuclear Yap/Taz enhances the expression of Wnt9b Class II/progenitor target genes whereas it represses the expression of Class I/PTA targets.
To determine whether this role for Yap/Taz was conserved in vivo, we ablated both genes from the kidney nephron progenitors using Six2Cre. Both Yap and Taz are expressed in largely overlapping domains in the kidney progenitor cells (Supplementary Fig. S8B) and single ablation of either gene had little effect on Wnt9b target gene expression (with the exception of Cited1, which was lost in both single mutants Supplementary Fig. S8C). Although Six2Cre; Yapflox/flox; Tazflox/flox mutants maintained Yap protein to E13.5, levels were greatly decreased at E15.5 and by birth, most of the progenitor cells and their derivatives showed no detectable levels of Yap (Supplementary Fig. S3 and Fig. 5a,b). As expected, given the kinetics of Yap ablation, the first several days of kidney development did not seem to be affected in these mutants (Supplementary Fig. S3). However, Six2Cre; Yapflox/flox; Tazflox/flox mutants died within 24 h of birth.
At E18.5, Yap/Taz double mutant kidneys were smaller than those of wild-type littermates and exhibited a greatly reduced nephrogenic zone and decreased density of epithelia in the more central regions of the kidney indicative of a reduction in nephron number (Fig. 5d). Staining of mutant kidneys with Lotus tetragonolobus lectin (LTL), a marker of proximal tubules, revealed a severe decrease in the number and length of these structures in mutants (Fig. 5e,f). Further, P1 double mutants had significantly fewer glomeruli than wild-type kidneys (Supplementary Table S1). However, the collecting duct epithelium, which is derived from the ureteric bud lineage, looked relatively normal.
The paucity in progenitor-cell-derived structures in mutants could be caused by a deficit in progenitor cell number or progenitor cell differentiation. To examine the role of Yap/Taz in progenitor cell maintenance/proliferation, we stained kidneys with an antibody against the Wnt9b Class II/progenitor target Cited1 as well as the Wnt9b-independent progenitor marker Six2. Most ureteric bud tips lacked a Cited1-positive mesenchymal cap (arrows in Fig. 5h), even though a single layer of Six2-positive cells (as opposed to 2–3 cell layers in the wild type) still surrounded the ureteric bud of some mutants (Fig. 5 and Supplementary Fig. S3). Many tips completely lacked progenitor cells. Transcripts of Class II targets Pla2g7, Tafa5 and Amphiphysin showed similar reductions in double mutants (Fig. 5j,l and r respectively). Importantly, β-catenin protein was still expressed in mutants (Fig. 5r–r”). Thus, consistent with what was observed in vitro, Yap/Taz activity is required for normal β-catenin Class II/progenitor target gene expression in vivo.
Loss of Class II target expression correlates with low rates of progenitor cell proliferation4. Therefore, we compared the percentage of pHH3/Six2-positive cells in the Yap/Taz mutants with the wild type. Mutants showed a significant decrease in cell proliferation rates (Supplementary Fig. S5).
To determine whether Yap/Taz activity affected the expression of the Class I/PTA targets, we assessed the expression of Pax8 and Wnt4 in P1 kidneys. Both genes showed expanded/ectopic expression in Yap/Taz mutants. Pax8 and Wnt4 showed strong ectopic expression in the cortical mesenchyme, expanding into what would normally be the expression domain of Class II/progenitor targets (Fig. 6d,n’ and p).
These data suggest that Yap/Taz normally represses the expression of Class I/PTA markers in the nephron progenitors. Although we observed ectopic expression of Class I/PTA targets, the formation of renal vesicles seemed to be delayed in mutants suggesting that Yap/Taz activity is required for proper differentiation. Thus, the reduced size of the kidney and the reduction of nephron number observed in Yap/Taz mutants are most likely caused by a combination of reduced progenitor cell renewal as well as defects in differentiation.
Fat4 regulates Yap/Taz activity in the progenitors
Our data suggest that a signal(s) from the stroma regulates Yap/Taz activity within the progenitors. In flies, members of the Fat and Ds family of atypical cadherins have been shown to mediate the activity of the Yap/Taz homologue Yorkie23. Intriguingly, one member of this family, Fat4, is expressed predominantly in the stroma of the embryonic kidney24,25.
To determine whether Fat4 regulates the phosphorylation status of Yap/Taz, we analysed the expression of the Yap protein in E15.5 and P1 Fat4−/− kidneys. Consistent with the observations in the stromaless kidneys, phosphorylation of Yap was normal in the stroma and the ureteric bud epithelium, but significantly reduced in most Six2-positive progenitor cells in Fat4 mutant kidneys (Fig. 3i,j). Further, total Yap protein was predominantly localized in the nucleus of Fat4 mutant progenitor cells (Fig. 3k,l). These results suggest that Fat4 regulates the activity/localization of Yap in the mammalian kidney.
Fat4 nulls exhibit an expanded progenitor domain
We next sought to determine whether ablation of Fat4 had an impact on the nephron progenitor population. Fat4 null kidneys are smaller than their wild-type littermates at birth but seem to have an expanded progenitor domain (Fig. 6b). The expanded progenitor cells expressed Wnt9b nephron progenitor (Class II) targets as well as other progenitor markers (Fig. 6d,f,h,j,l,n,p). However, the number of Lef1-positive structures suggested that Fat4 mutants had significantly fewer nephrons than the wild type (Fig. 6p and Supplementary Table S1).
To determine whether the expansion of the nephron progenitor population was the result of an increased rate of proliferation or simply a failure to differentiate, we quantified the percentage of pHH3-positive progenitor cells in the wild type and Fat4 mutants. Fat4 mutants showed a mild but significant increase in the percentage of pHH3-positive progenitor cells relative to the wild type (Supplementary Fig. S5 and Table S1).
We next determined whether Fat4 mutant kidneys continued to express Wnt9b/Class II target genes after treatment with IWP2 (similar to stromaless kidneys). Indeed, the low level expression of Wnt9b Class II target genes was maintained, whereas Class I/PTA targets were lost (Fig. 2h,j,k and Supplementary Fig. S6). Fat4 mutants lost expression of both classes of targets after treatment with IWR1 (Fig. 2i,j,k and Supplementary Fig. S6). Thus, ablation of Fat4 renders expression of Wnt9b Class II/progenitor targets less dependent on a Wnt ligand but does not relieve dependence on β-catenin.
To determine the effect of Fat4 ablation on Wnt9b activity in vivo, we generated Fat4/Wnt9b double mutants. Fat4/Wnt9b double mutant kidneys were hypoplastic with a moderately branched ureteric bud system. Unlike Wnt9b null kidneys, double mutant buds were capped with progenitor cells although they still lacked renal vesicles and their derivatives (Fig. 7d). Consistent with the results observed on culture of Fat4 mutants with IWP2, we found that expression of Wnt9b Class II/progenitor target genes Cited1, Pla2g7 and Tafa5 was rescued in Wnt9b mutants on co-ablation of Fat4 (Fig. 7h,l and p). However, class I/PTA target expression was not rescued (Fig. 7t,x). These data suggest that Fat4 inhibits the expression of Wnt9b Class II/progenitor targets and promotes the expression of Class I/PTA targets in vivo.
Previous studies suggest that Fat4 interacts with Vangl2 to regulate PCP during tubule diameter maintenance in the kidney26. However, removal of Vangl2 from mice carrying either a null or hypomorphic allele of Wnt9b (ref. 27; Wnt9b−/−; Vangl2lp/lp and Wnt9bneo/neo; Vangl2lp/lp) did not rescue the progenitor domain or expression of Wnt9b Class II/progenitor targets, suggesting that this phenotype is independent of the Vangl2/PCP pathway (Supplementary Fig. S7B).
The cortical stroma and mural cells of Fat4 mutant kidneys were molecularly and phenotypically indistinguishable from the wild type (Supplementary Fig. S7A). Further, we did not observe ectopic expression of progenitor markers within the stromal compartment of Fat4 mutants (Supplementary Fig. S7A). These data suggest that the expansion in progenitors was not caused by a transformation of the stroma into progenitors.
Reciprocally, lineage tracing of progenitor cells in kidneys revealed that the Six2Cre-expressing derivatives were present in Wnt9b mutants until E15.5 (Supplementary Fig. S8A). Co-labelling of Six2Cre;RosaYFP; Wnt9b−/− with antibodies against GFP and stromal markers Foxd1, Meis1/2 or Slug demonstrated that the progenitors of Wnt9b mutants were not mis-specified or trans-fated towards a stromal fate (Supplementary Fig. S8A). These findings rule out the possibility that the phenotypes observed in Wnt9b, Fat4 or Wnt9b/Fat4 double mutants were the results of defects in nephron progenitor or stromal cell specification or fate.
Fat4 acts non-autonomously
Our model suggests that stromal Fat4 acts on the adjacent progenitors. To determine whether Fat4 can act non-autonomously, we co-cultured isolated progenitors with cells transfected with full-length Fat4. Yap localization shifted from predominantly nuclear to cytoplasmic when progenitor cells were cultured adjacent to Fat4-expressing cells (Fig. 4d). A construct lacking the cytoplasmic domain of Fat4 (Fat4-ECD) had the same effect (Fig. 4c). Both constructs resulted in increased levels of phosphorylated Yap. All results were independent of cell density, indicating that Fat4 is capable of activating Yap/Taz in a non-autonomous manner.
We next assessed the effect of Fat4 on progenitor cell differentiation. Both full-length and the extra-cellular domain of Fat4 had a similar effect on gene expression as knockdown of Yap/Taz, repressing class II/progenitor gene expression (with the exception of Pla2g7) and enhancing differentiation/MET as assessed by E-cadherin (Fig. 4c). In sum, these data suggest that stromally derived Fat4 non-autonomously regulates Yap/Taz activity within a subset of the nephron progenitors, which promotes their differentiation.
The embryological studies performed by Clifford Grobstein during the 1950s established one of the central tenets of metanephric kidney development: nephron progenitor maintenance and differentiation rely on signals produced by the ureteric bud epithelium1,2. However, there is growing evidence that final kidney form relies on inductive and inhibitory crosstalk between multiple cell types present in the organ anlagen5,6,28,29,30,31. In this study, we provide evidence that the cortical stroma inhibits nephron progenitor cell expansion and promotes its differentiation. We propose that Fat4, produced by the stroma, provides a signal that antagonizes nephron progenitor renewal and promotes differentiation by modulating response to Wnt9b.
We suggest that Yap/Taz plays a crucial role in regulating Wnt9b signature activation. Precisely how this is accomplished is not clear. Several recent studies have shown direct interaction between β-catenin and Yap/Taz signalling13,14,15,16. Our data are consistent with the idea that Fat4 signalling promotes the expression of Wnt9b/β-catenin differentiation targets, and inhibits the expression of the progenitor renewal targets. We propose that nuclear Yap/Taz and β-catenin cooperate to activate the nephron progenitor signature and loss of nuclear Yap/Taz (and possibly gain of cytoplasmic pYap/Taz) is necessary for the expression of the differentiation signature (Fig. 8). As we have not identified conserved Tead- (the DNA binding co-factor for Yap and Taz) binding sites within either class of target gene, the effect of Yap/Taz on β-catenin targets may not be direct. Nevertheless, Yap/Taz must impinge on Wnt9b/β-catenin activity at some point.
Although Fat4 is expressed in the stroma (refs 24, 25 and this study), we cannot rule out the possibility that low levels of Fat4 present in the progenitors constitute the active pool of protein. However, the observation that DTA deletion of the stromal cells results in a similar phenotype to Fat4 ablation would seem to support a stromal source. We propose that Fat4 binds to another factor produced by the progenitors to activate Yap/Taz in the progenitors. The mouse orthologue of Dachsous and Fat3, a paralogue of Fat4, are both expressed in the progenitor cells where they could be acting as receptors for stromal Fat4 (refs 24, 25, 31). It will be interesting to determine whether either of these factors mediates Yap/Taz activity.
The Foxd1Cre;RosaDTA kidneys elicit a more severe phenotype than mutation of Fat4 alone. Inappropriate production of other signals (such as Bmps, ref. 6) produced by the ectopic endothelial or capsular cells observed in Foxd1 (ref. 6) mutants may lead to the more severe phenotype observed in these mutants. In support of this model, we did not detect ectopic pSmad1/5/8 activity in Fat4 or SixCre; Yapflox/flox; Tazflox/flox mutants (Supplementary Fig. S7C), although they are detectable in Foxd1 mutants6. These data suggest that the phenotypes resulting from ablation of Fat4 are a subset of those observed in Foxd1 and stromaless kidneys.
In summary, we have revealed that ureteric-bud-derived Wnt9b and stromal-derived Fat4 provide opposing signals that regulate kidney progenitor cell maintenance and differentiation. Although this interaction is clearly essential for kidney development, we feel that the crosstalk between the Wnt and Fat/Yap pathways may be representative of a more general mechanism underlying stromal/epithelial interactions in multiple tissues. Given the increasing evidence of stromal involvement in numerous human pathological conditions, these findings are likely to have a significant impact on our understanding of human disease.
All animals were housed, maintained and used according to protocols approved by the Institutional Animal Care and Use Committees at the University of Texas Southwestern Medical Center and following the guidelines from the NCI-Frederick Animal Care and Use Committee. For each experiment, female mice of 7–8 weeks of age were crossed with a male of 9–10 weeks of age. Plugs were checked and the embryos were collected at the desired time points for further analysis. The number of mice used for each experiment was as follows: Wnt9b−/− (5–6 pregnant females at each time point), Wnt9bneo/neo (5 pregnant females), Fat4−/− (6–8 pregnant females for each time point), Rosa26DTA (6–8 pregnant females for each experiment), Taz/Yap (3 females at the early time point and 6–7 to collect E18.5 and P1), Vangl2 and Ror (2–3 pregnant females for each), Six2 Cre, Foxd1Cre and KspCre (1 male for each). Mice of the desired genotype were randomly selected and the investigator was blinded to allocation.
Creation of a conditional Yap and Taz mutant allele.
The Yap and Taz targeting vector was constructed using pGKneoF2L2DTA harbouring two loxP sites encompassing a neomycin resistance cassette flanked by two FRT sites32. Targeting arm sequences were isolated from 129SvEv genomic DNA by PCR. The Yap 5′ targeting arm (5 kb), Yap 3′ targeting arm (4 kb), Yap knockout arm (2 kb), Taz 5′ targeting arm (5 kb), Taz 3’ targeting arm (4 kb) and Taz knockout arm (2 kb) were cloned into the vector using NotI, XmaI, EcoRV, EcoRV, EcoRV and XmaI respectively. The sequence-verified targeting vector was linearized and electroporated into 129SvEv-derived embryonic stem cells. Targeting of the mutant allele was screened through Southern blot analysis. The Yap 5′ probe detects a 6 kb DNA fragment in addition to the wild-type 9 kb fragment after EcoRI digestion and the Yap 3′ probe detects a 10 kb DNA fragment plus the wild-type 8 kb fragment after PstI digestion in the presence of the Yap-targeted allele. Similarly, the Taz 5′ probe detects a 10 kb fragment in addition to the wild-type 12 kb fragment on StuI digestion and the Taz 3′ probe detects an 8 kb fragment in addition to the wild-type 10 kb fragment on BamHI digestion if homologous recombination occurred. Targeted ES cells were injected into blastocysts to generate chimaeric mice. High-percentage chimaeric males were bred with mice expressing FLPe recombinase to remove the neomycin resistance cassette, producing YaploxP and TazloxP alleles33. PCR genotyping using a forward primer upstream of the 5′ loxP site, a first reverse primer located in the knockout arm, and a second reverse primer downstream of the 3′ loxP site produces 600 base pair (bp), 457 bp, 338 bp, 655 bp, 496 bp and 704 bp bands for YaploxP, YapWT, Yapfloxed, TazloxP, TazWT and Tazfloxed alleles, respectively. Primer sequences for PCR genotyping are as follows: YF: 5′-ACATGTAGGTCTGCATGCCAGAGGAGG-3′; YR1, 5′-AGGCTGAGACAGGAGGATCTCTGTGAG-3′; YR2, 5′-TGGTTGAGACAGCGTGCACTATGGAGC-3′; TF, 5′-GGCTTGTGACAAAGAACCTGGGGCTATCTGAG-3′; TR1, 5′-CCCACAGTTAAATGCTTCTCCCAAGACTGGG-3′; TR2, 5′-AACTGCTAACGTCTCCTG CCCCTGACCTCTC-3′.
Ex vivo organ culture.
Isolated kidney explants were cultured as previously described4. Briefly, E11.5 kidneys were cultured at the air/medium interface for 48 h. Small-molecule Wnt antagonists (5 μM of IWP2 and 100 μM of IWR1) were added to the medium and replaced every 12 h. After 48 h of culture the kidneys were fixed in ice-cold 4% paraformaldehyde and washed thoroughly in PBS before subjecting them to further analysis. All analyses were repeated at least three times, with a minimum of four different kidneys in each experiment.
Tissues and primary metanephric mesenchyme cell cultures.
Metanephric mesenchymes were enzymatically separated from the T-shaped ureteric buds from E13.5 rat embryos and cultured on laminin-coated BD-Biocoat 6-cm dishes (BD Biosciences) in serum-free Ham’s F12:DMEM 1:1 (Gibco, BRL) medium with supplements as described previously (ref. 21) and with the addition of 10 ng ml−1 human TGF- α and 50 ng ml−1 human FGF2. Tissues were grown for 10 days, resulting in a 50–100-fold increase in cell numbers. Cells were removed from plates with trypsin–EDTA solution (Gibco-Invitrogen).
Plasmids and electroporation.
Lef-1/β-catenin fusion complementary DNA was generously provided by W. Bichmeier and J. L. Gordon (ref. 3), and cloned into pCMV–tag3–myc plasmid (Stratagene). Primary metanephric mesenchyme cells were transfected with the Amaxa Nucleofector 96-well Shuttle System (Lonza Group). Briefly, 1 million cells per transfection were resuspended in 20 μl of P3 Primary Cell 96-well Nucleofector Kit (Lonza). pCMV–tag3–myc or pCMV–tag3–myc–Lef-1/β-catenin plasmid (2 μg) was mixed with cell suspensions and transferred to the well of a 96-well Nucleoplate module. For Fat4 experiments, empty vector or Fat4 plasmids (4 μg) were mixed with 1 million cells in 20 μl of 96-well Nucleofector solution. Nucleofections were performed according to the manufacturer’s instructions using program DN-100, and then cells were seed on laminin-coated 24 well. At 24–72 h post transfection, cells were collected by TRIZOL (Invitrogen) for RNA isolation or RIPA buffer for protein analysis.
Scrambled siRNA (#4390843, Ambion), Yap siRNA (sense: 5′-GUUUACUACAUAAACCAUAtt-3′, anti-sense: 5′-UAUGGUUUAUGUAGUAAACtt-3′, #4390771, siRNA ID: s170198, Ambion) or ON-TARGETplus SMART pool Rat Wwtr1 (Taz) siRNA (#1: 5′-GAGAUGACCUUCACGGCCA-3′; #2: 5′-AGUCCUAUCACGUGACCGA-3′; #3: 5′-CUUACGUUACACACAAAUA-3′; #4: 5′-GGUGAAAAUUCGGGUCAGA-3′, siRNA ID: L-088521-02-0005, Dharmacon) was transfected into rat metanephric mesenchyme cells by Lipofectamine RNAi MAX (Invitrogen) transfection reagent following the manufacturer’s protocol. Culture medium was changed 24 h post transfection and transfection efficiency was analysed by qRT–PCR or immunoblotting.
In situ hybridization.
For section in situ hybridization, kidneys isolated at specific stages were fixed overnight in 4% PFA (in PBS) at 4 °C and cryopreserved in 30% sucrose. Tissues were frozen in OCT (Tissue Tek) and sectioned at 10 μm. Sections were subjected to in situ hybridization as previously described4. The following probes were used: Fat4 (linearized with BamHI and transcribed with T3 polymerase), Yap (linearized with BamHI and transcribed with T3 polymerase), Taz (linearized with EcoRI and transcribed with T3 polymerase), Cited, Six2, SalI Pla2g7, C1qdc2 and Wnt4 (ref. 4).
Histology, immunohistochemistry and immunocytochemistry.
Kidneys isolated at birth were formaldehyde fixed and paraffin embedded. Sections (5 μm) from paraffin-embedded kidneys were subjected to haematoxylin and eosin staining. For immunohistochemistry, fixed kidneys were embedded in OCT and sectioned on a cryostat. Frozen sections were washed with PBS and blocked with 5% serum for an hour at room temperature and incubated with different primary antibodies at 4 °C overnight. The following antibodies were used: Cited1 (Neo Markers, Cat. RB1219, dilution 1:500), Six2 and Amphiphysin (ProteinTech, Cat. 11562-1, 13379-1; dilution 1:500), DBA (dolichos biflorus agglutinin) and LTL (lotus tetragonolobus lectin; VectorLabs, Cat. B1035, B1325, 1:500), E-cadherin (Invitrogen, Cat. 131900, dilution 1:500), cytokeratin (CK; Sigma, Cat. C2562, clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2,1:500), Foxd1 (Santa Cruz, Cat. Sc-47585 dilution 1:500 with tyramide amplification), Meis1/2 (Santa Cruz, Cat. Sc-10599, dilution 1:1,000 with tyramide amplification), Lef1 and Slug (Cell Signaling, Cat. 2230, 9585; 1:250). For staining with Yap (Cell Signaling, Cat. 4911, 4912; 1:500, tyramide amplified) and pYap (Cell Signalling 1:500, tyramide amplified) antibodies, washes were carried out in 1×TBS with 0.1% Tween 20 instead of PBS, and sections were antigen retrieved using citrate buffer at pH 6.0 and blocked with 10% normal goat serum. Slides were incubated overnight with primary antibodies. After primary incubation, sections were washed and incubated with HRP-tagged secondary antibodies for 1 h at room temperature. Further, signal was detected with tyramide amplification. Slides were washed and re-stained with additional markers according to the above-mentioned immunohistochemistry protocol. Slides were then mounted with Vectashield and images were captured with a Zeis LSM500 microscope. For pSmad1/5 stains, frozen sections were fixed with 4% PFA, permeabilized with 1% SDS at 37 °C for 10 min, blocked for an hour at room temperature (with 1% BSA + 0.1% fish gelatin + 0.1% Tween 20) primary antibody (Cell Signaling, Cat. 9516; diluted in block 1:300) was incubated overnight. The following day the staining was developed using biotinylated secondary antibody followed by a streptavidin reaction.
All histological analyses were performed on at least six different kidney samples from at least three different embryos. Images shown are representative from at least six different kidneys. One E18.5 kidney from an animal genotyped as being a Fat4 mutant was identified that showed no difference in pYap staining in Six2-positive cells. pYap was downregulated in Six2-positive cells in all other (greater than 6) kidneys examined.
Primary metanephric mesenchyme cells were cultured (low density: 0.1 million, high density: 1 million cells) on 4-well chamber slides (Thermo, #155382), which were manually coated with laminin (5 μg per well, BD Biosciences). Cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min and further washed 3 times with PBS, 5 min each. The cells were then blocked for 1 h at room temperature with 10 % goat serum in PBS, and incubated with Yap mouse monoclonal antibodies (1:50) overnight. The cells were then washed with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody for 1 h. The cells were treated with DAPI, mounted on slides with Vectashield (Vector Laboratories), and visualized using a Zeiss LSM710 confocal microscope (Carl Zeiss) at ×40 magnification. Confocal images were captured and analysed by the ZEN image program (Zeiss).
Metanephric mesenchyme cells were lysed with modified RIPA buffer containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1% deoxycholate, 50 mM sodium fluoride, 50 mM sodium orthovanadate, 1 mM phenylmethyl sulphonyl fluoride, and proteinase inhibitor cocktail (Roche). Cell lysates were homogenized in an ultrasonicator for 15 s four times on ice. The protein concentration was determined by the Rc-protein assay kit with a BSA standard (Bio-Rad Laboratories). Protein samples (10 μg each) were denatured by 4× LDS buffer at 75 °C for 10 min and resolved in 4–2% Bis-Tris gels with MOPS buffer (Invitrogen). Proteins were electrotransferred to PVDF membranes (Bio-Rad, Hercules). Membranes were blocked with 5% non-fat dry milk in TBST and incubated overnight at 4 °C with primary antibodies. Membranes were washed with TBST, followed by species-specific HRP-conjugated secondary antibodies. Bound antibodies were visualized using the SuperSignal West Pico Chemiluminescent Substrate system (ThermoFisher Scientific) according to the manufacturer’s instructions. Antibodies were obtained as follows: against Yap/Taz (#8418), pYap (#4911), Yap (#4912), phospho-MST (#3681) and Pax8 (#9857) from Cell Signaling Technology; against Six2 (#11562), Pla2g7 (#15526) and Amphiphysin (#13379) from Proteintech; against β-Actin (A5441) from Sigma; against E-cadherin (#610181) from BD Biosciences; against CITED1 (#RB-9219) from Lab Vision; against Sall1 (#ab31526) from Abcam; against Tafa5 (#F5148) from R&D systems; Yap monoclonal antibody (M01; #H00010413) from Abnova.
In vitro co-culture.
To distinguish between the Fat4-expressing effector cells and the wild-type progenitor cells, we cultured cells on laminin-coated 4-well coverslips after co-transfection of Fat4 plasmids (4 μg per 1 million cells) and GFP plasmids (0.5 μg per 1 million cells) by electroporation as described above. Twenty-four hours later wild-type metanephric mesenchyme cells (progenitor cells stained with CellTracker Red CMTPX, Invitrogen) were added to the effector cells (0.5 million cells per well). Cultured metanephric mesenchyme cells were fixed with 4% PFA for immunofluorescence microscopy after 24 h culture as described above.
Progenitor cell layer counts.
Kidneys were sectioned and stained for ureteric bud (cytokeratin or DBA) and progenitor cell markers (Cited1 and/or Six2). Sections that passed through the lumen of the ureteric bud were subsequently used to count progenitor cells. The progenitor cell layer atop the ureteric bud was counted from 3–4 different kidneys and an average was calculated per ureteric bud tip for each kidney.
Kidneys isolated at birth were paraffin embedded and sectioned at 5 μm thickness. Every fifth section was collected throughout the entire kidney and non-overlapping images were taken at ×10 magnification. The number of glomeruli was counted from each image and an average was calculated from 4 different mice. Note, P1 kidneys have been previously shown to have more than 10,000 glomeruli; however, the sampling method was significantly different from ours.
Cultured kidneys treated with various compounds were cultured for 48 h and stored in RNA-later. A minimum of 9 kidneys for each treatment and genotype were pooled together to isolate RNA using the Qiagen mini kit. cDNA was made using 1.5–2 μg of RNA for all samples using iScript reagents from BioRad. Real-time analysis was performed and amplification was calculated comparing the CT values of target genes to cyclophilin (used as an internal control) and the fold change by comparing the CT values of untreated versus treated samples. The experiment was carried out three times and the error bar represents the s.e.m.
For rat metanephric mesenchyme qRT–PCR, the following primers were used: Rattus_Six2F: 5′-CAAGAATGAAAGCGTGCTCA-3′; Rattus_Six2R: 5′-CTTCTCCGCCTCGATGTAGT-3′; Rattus_Cited1F: 5′-ATGCCAACCAGGAGATGAAC-3′; Rattus_Cited1R: 5′-TGGCAGTAGGAGAGCCTGTT-3′; Rattus_Pax8F: 5′-AACTCGATCAGATCCGGCCA-3′; Rattus_Pax8R: 5′-CCAAGTCCACAATGCGTTGC-3′; Rattus_SalIF: 5′-ATCAGCGGTGTGAAGCAGCT-3′; Rattus_SalIR: 5′-TGCTGTCACCATGCTCACGT-3′; Rattus_YAPF: 5′-TGCTCAACATCTCAGACAGT-3′; Rattus_YAPR: 5′-TGTTGTCTGATCATTGTGATTTAAG-3′; Rattus_Wnt4F: 5′-AGTGACAAGAGCATGCAGCAGT-3′; Rattus_Wnt4R: 5′-ATAGGCGATGTTGTCCGAGCAT-3′; Rattus_E−cadherinF: 5′-TTGCCACAGATGATGGTTCACCC-3′; Rattus_E−cadherinR: 5′-TGGTGATGACATGGGGCTTCGG-3′; Rattus_C1qdc2F: 5′-GGCACGACAGGAACTCAAGAA-3′; Rattus_C1qdc2R: 5′-AGAGCTCAGGTCCAGAGGCTT-3′; Rattus_Pla2g7F: 5′-TTGCGCCTCTTATATGGTTC-3′; Rattus_Pla2g7R: 5′-TAAATTGTCCTGAAGGCTCCG-3′; Rattus_Tafa5F: 5′-GGAGTTGGAGGACCAGAACAA-3′; Rattus_Tafa5R: 5′-ACAGAGGAGGTGGTGGCTT-3′; Rattus_GAPDHF: 5′-GCTCTCTGCTCCTCCCTGTTCTA-3′; Rattus_GAPDHR: 5′-CTTGTCTATGAGACGAGGCTGGC -3′; Rattus_TAZF: 5′-GGACACAGGTGAAAATTCGG-3′; Rattus_TAZR- 5′-AAGTCCCGAGGTCAACATTT-3′.
For mouse qRT–PCR analysis, the following primers were used: Mus_Pla2g7F: 5′-ACA ACT CCT GCA AGC TGG AAT-3′; Mus_Pla2g7R: 5′-AAG TAA GTT GCC GAT GCA GA-3′; Mus_Tafa5F: 5′-AAC CTG TGA GAT TGT GAC CCT-3′; Mus_Tafa5R: 5′-TCG AGT GGT GCC TGC TAT C-3′; Mus_C1qdc2F: 5′-ACA TGA CGT GGT TGA ACT TTG-3′; Mus_C1qdc2R: 5′-CAG TAA GGC CTC TGG GGT AA-3′; Mus_CyclophilinF: 5′-GGA GAT GGC ACA GGA GGA A-3′; Mus_CyclophilinR: 5′-GCC CGT AGT GCT TCA GCT T-3′.
Statistical analysis was performed and P values were determined for all quantitative studies using Student’s t-test with equal variance.
We thank Y. Yang (NIH, USA) for providing the Vangl2 mutant mice, M. Takeichi (RIKEN Center for Developmental Biology, Japan) for providing the Fat4 full-length and ECD plasmids, J. Cabera for artwork and O. Cleaver, Q. Li and D. Marciano for reading and commenting on the manuscript. This work was supported by a post-doctoral fellowship from the A.H.A. to A.D. and from the Japanese Society for the Promotion of Science to S.T., the NIH (R01DK080004 and R01DK095057 to T.C.) and National Cancer Institute’s Center for Cancer Research (A.P.). This work was supported by the George O’Brien Kidney Research Center at UTSW.
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Pediatric Nephrology (2017)