A Fat4-Dchs1 signal between stromal and cap mesenchyme cells influences nephrogenesis and ureteric bud branching

Formation of the kidney requires reciprocal signaling among the ureteric tubules, cap mesenchyme and surrounding stromal mesenchyme to orchestrate complex morphogenetic events. The protocadherin Fat4 influences signaling from stromal to cap mesenchyme cells to influence their differentiation into nephrons. Here we characterize the role of a putative binding partner of Fat4, the protocadherin Dchs1. Mutation of Dchs1 leads to increased numbers of cap mesenchyme cells, which are abnormally arranged around the ureteric bud tips, and impairs nephron morphogenesis. Mutation of Dchs1 also reduces branching of the ureteric bud and impairs differentiation of ureteric bud tip cells into trunk cells. Genetically, Dchs1 is required specifically within cap mesenschyme cells. The similarity of Dchs1 phenotypes to stromal-less kidneys and to Fat4 mutants implicate Dchs1 in Fat4-dependent stroma-to-cap mesenchyme signaling. Antibody staining of genetic mosaics reveals that Dchs1 protein localization is polarized within cap mesenchyme cells, where it accumulates at the interface with stromal cells, implying that it interacts directly with a stromal protein. Our observations identify a role for Fat4-Dchs1 in signaling between cell layers, implicate Dchs1 as a Fat4 receptor for stromal signaling that is essential for kidney development, and establish that vertebrate Dchs1 can be molecularly polarized in vivo. D ev el op m en t A cc ep te d m an us cr ip t INTRODUCTION Metanephric kidney development requires interactions between three adjacent cell layers: the epithelial ureteric bud (UB), cap mesenchyme (CM), and stromal mesenchyme (stroma) (Fig. 1A) (Costantini and Kopan, 2010; Kopan et al., 2014). The UB forms a tubule that undergoes elongation and reiterative branching to form the collecting ducts. Growth and branching of the UB depends upon reciprocal cell signaling interactions with the neighboring CM, which coalesces around the tips of the branching epithelium. CM cells are also the progenitor cells for nephrons, which are created through a mesenchymal-to-epithelial transition to form a renal vesicle, which then undergoes further growth and morphogenesis (Costantini and Kopan, 2010; Kopan et al., 2014). The CM is surrounded by stroma. Genetic ablation of stromal cells or mutation of stromal-expressed genes including Ecm1, Foxd1, or Fat4 has revealed that stroma influences the development of the CM and UB (Das et al., 2013; Hatini et al., 1996; Hum et al., 2014; Levinson et al., 2005; Paroly et al., 2013). Nonetheless, there remain substantial gaps in our understanding of the cellular and molecular mechanisms that orchestrate the communication between stromal cells and other cell layers required for kidney morphogenesis. Here, we examine the role of Dchs1-Fat4 signaling in this process. Much of our understanding of Dchs1 and Fat4 comes from studies of their Drosophila homologues, Dachsous (Ds) and Fat. Ds and Fat are large cadherin family transmembrane proteins that bind to each other to regulate both Hippo signaling and planar cell polarity (PCP) (Matis and Axelrod, 2013; Reddy and Irvine, 2008; Staley and Irvine, 2012; Thomas and Strutt, 2012). Hippo signaling is a conserved signal transduction pathway best known for its influence on organ growth, which it controls by regulating a transcriptional co-activator protein called Yorkie (Yki), or in vertebrates D ev el op m en t A cc ep te d m an us cr ip t the Yki homologues Yap and Taz (Pan, 2010; Staley and Irvine, 2012). PCP is the polarization of cell morphology and cell behavior within the plane of a tissue (Goodrich and Strutt, 2011; Wansleeben and Meijlink, 2011). PCP signaling is intrinsically bidirectional, as it polarizes each pair of juxtaposed cells. Conversely in Fat-Hippo signaling Ds acts as a ligand that activates Fat, which functions as a receptor for Hippo signaling (Reddy and Irvine, 2008; Staley and Irvine, 2012), but there is also some evidence for a reciprocal Fat-to-Ds signal (Degoutin et al., 2013). Analysis of Fat4 and Dchs1 mutant mice has revealed that Dchs1-Fat4 signaling is essential for the morphogenesis of multiple mammalian organs, including the kidney (Mao et al., 2011; Saburi et al., 2008; Saburi et al., 2012; Zakaria et al., 2014). Requirements for DCHS1 and FAT4 in humans have been revealed by the linkage of mutations in these genes to Van Maldergem syndrome (Cappello et al., 2013). Mice mutant for Dchs1 or Fat4 have smaller kidneys, with fewer ureteric branches and a modest accumulation of small cysts (Mao et al., 2011; Saburi et al., 2008); hypoplastic kidneys have also been reported in Van Maldergem patients (Mansour et al., 2012). Differences between murine wild-type and Dchs1 or Fat4 mutant kidneys appear as early as E11.5, when the growth and branching of the UB in mutants lags behind that in wild-type embryos (Mao et al., 2011). Differentiation of nephron progenitor cells (CM) into nephrons was reported to be defective in Fat4 mutants (Das et al., 2013), reminiscent of the effect of stromal cell ablation on CM differentiation (Das et al., 2013; Hum et al., 2014), and it was suggested that Fat4 participates in stromal-to-CM signaling. The inhibition of nephron progenitor cell differentiation in Fat4 mutants was attributed to increased Yap activity (Das et al., 2013), although how this might be achieved is unclear, as the molecular pathway linking Fat to Yap identified in Drosophila does not appear to be conserved in mammals D ev el op m en t A cc ep te d m an us cr ip t (Bossuyt et al., 2014; Pan et al., 2013). Conversely, there is growing evidence that Ds-Fat PCP signaling mechanisms are conserved between insects and vertebrates, including the ability of human FAT4 to rescue PCP phenotypes in flies (Pan et al., 2013), and observations of abnormal cellular polarization in Dchs1 or Fat4 mutant mice (Mao et al., 2011; Saburi et al., 2008; Zakaria et al., 2014). Here, we focus on the role of Dchs1 in kidney development. We report that Dchs1 mutants share the expansion of CM identified in Fat4 mutants, consistent with their acting as a signaling pair. We also further characterize Dchs1 phenotypes in other cell types within the kidney, and show through conditional deletion that Dchs1 is specifically required within CM for the normal development of CM, UB, and stroma. Analysis of genetic mosaics establishes that the subcellular localization of Dchs1 is polarized within CM cells, where it accumulates on surfaces contacting stromal cells. Our observations suggest that Dchs1 functions as a receptor for a Fat4 signal from stromal cells that influences the behavior of CM, and indirectly, the UB. RESULTS Dchs1 functions in CM to influence kidney size and shape Immunolocalization and RNA in situ studies of E12.5 kidneys revealed that Fat4 and Dchs1 are expressed predominantly within the CM and stroma, rather than in the UB (Mao et al., 2011). Similarly, Fat4 and Dchs1 protein expression could be readily detected within the CM and stroma of E14.5 kidneys (Fig. 1B,D), whereas the UB appears to have levels of staining close to background (ie, as observed in mutant kidneys, Fig. 1C,E). These observations raise the question of how Fat4 and Dchs1 influence UB branching and growth. To determine where Dchs1 is genetically required for kidney development, we selectively removed Dchs1 from each of the three main cell D ev el op m en t A cc ep te d m an us cr ip t types within the developing kidney by using a Dchs1 conditional allele (Dchs1f) combined with either Hoxb7-Cre (expressed in UB)(Yu et al., 2002), Foxd1-Cre (expressed in stroma)(Humphreys et al., 2010), or Six2-Cre (expressed in CM and immediate derivatives)(Kobayashi et al., 2008). The efficacy and specificity of these Cre lines was confirmed using a conditional Rosa-26 lacZ reporter (Soriano, 1999)(Supplemental Fig. S1), and by Dchs1 antibody staining (see below). When kidneys from P0 mice with conditional deletion of Dchs1 in stromal cells or in the UB were examined, they appeared morphologically normal in terms of overall kidney size and shape (Fig. 1F-I, L,M,O,P). Conversely, conditional deletion of Dchs1 from CM resulted in smaller, rounder kidneys, reminiscent of those observed in Dchs1 mutant mice (Fig. 1J,K,N,Q) (Mao et al., 2011). Thus, Dchs1 is required in CM or its derivatives for overall kidney growth, and not in UB or stroma. Influence of Dchs1 on CM In developing wild-type kidneys, mesenchymal cells condense to form an ~2-3 cell wide cap around the UB tips; this cap is marked by strong expression of Six2 (Fig. 2A,C,E,G)(Kobayashi et al., 2008). Mutation of Fat4 results in increased numbers of CM cells around each ureteric bud tip (Fig. 2B,F)(Das et al., 2013). Dchs1 mutants exhibit a similar expansion of CM (Fig. 2D,H), consistent with inferences that Dchs1 and Fat4 function as signaling partners (Mao et al., 2011; Zakaria et al., 2014). In addition to the increase in thickness of the CM, the CM also appears disorganized in both Dchs1 and Fat4, i.e. the width and shape of the cap appears more irregular than in wild-type (Fig. 2A-H). Moreover, some CM cells appear mis-localized. In wild-type they are restricted to the distal side of the ureteric tips, near the surface of the kidney, but in mutants they can accumulate proximal to the ureteric tips (Fig. 2A-D). Increased thickness and D ev el op m en t A cc ep te d m an us cr ip t disorganization of CM has also been noted in kidneys with stromal deletion, or mutation of Ecm1, Foxd1, or Fat4 (Das et al., 2013; Hatini et al., 1996; Hum et al., 2014; Levinson et al., 2005; Paroly et al., 2013). To investigate the origin of these defects in CM thickness and organization, we examined CM cells in Fat4 mutants throughout kidney development, beginning at E10.5 when the ureteric epithelium first invades the metanephric mesenchyme. At E11.5, the thickness of cap mesenchyme cells appears similar between wild type and mutant kidneys (Fig. 2I,J), whereas by E12.5 the CM of Fat4 mutants is clearly abnormal (Fig. 2K,L). Abnormalities in kidney morphogenesis nonetheless appear by E11.5 (Mao et al., 2011), and one intriguing difference revealed by Six2 staining is that in Fat4 mutants the end of the UB remains completely surrounded by CM, whereas in wild-type, as the UB elongates, regions of the UB not at the tip (the UB stalk) are no longer directly in contact with Six2-expressing CM (Fig. 2I,J). To quantify the local increase in CM, we compared the volume of Cited1expressing cells per UB tip between Fat4 mutant and wild-type kidneys at E14.5; a three-fold increase was observed (Fig. 2Q). This was not associated with a detectable increase in CM cell proliferation (Fig. 2R-T). We also note that the local increase in CM around each UB tip does not correspond to an increase in total CM within the kidney, because there are fewer, more widely spaced, UB tips. This is illustrated by the observation that total Six2 expression, as measured by quantitative reversetranscription PCR (Q-PCR), normalized to kidney size (by using a ubiquitously expressed gene, GAPDH, as a Q-PCR standard) was similar between wild-type and Fat4 mutant kidneys (110% of wild-type, Fig. 3A). The observation that the total amount of CM is similar between wild-type and mutant kidneys (when normalized for kidney size) raises the question of whether Dchs1-Fat4 signaling has a specific affect on CM, or if the D ev el op m en t A cc ep te d m an us cr ip t increased CM at UB tips might simply reflect a similar amount of CM distributed amongst fewer tips? We do not favor this latter possibility, because the CM and UB tips normally engage in reciprocal signaling to maintain their fates (Kopan et al., 2014). Additionally, we note that Vangl2Lp mutants also have smaller kidneys with reduced branching (Yates et al., 2010), but they do not exhibit the expansion of CM observed in Dchs1 or Fat4 mutants (Fig. S2E,F). To determine where Dchs1 functions to influence CM cells, we examined kidneys with conditional deletion of Dchs1 in each of the three main cell types present within the early kidney. The expansion and irregular shape of CM was visible in kidneys with conditional deletion of Dchs1 in CM (Six2-Cre Dchs1f/-) (Fig. 2M-P). The phenotype appears less severe than in Dchs1 null mutants, but this likely reflects perdurance of Dchs1 mRNA and protein after conditional deletion of the gene, as Six2 expression in the kidney only begins around E10.5 (Oliver et al., 1995). No effect on CM thickness or organization could be detected in kidneys with conditional deletion of Dchs1 in stroma or UB (Fig. S2A-D). Thus, Dchs1 is required within the CM to influence CM thickness and organization. Influence of Dchs1-Fat4 signaling on UB development The smaller size of Dchs1 and Fat4 mutant kidneys correlates with reduced branching of the UB (Mao et al., 2011). Based on the density of UB tips in E14 to E15 kidneys, branching is reduced by deletion of Dchs1 in CM, but not UB or stroma (Figs 2P, S2A-D). Reduced branching was confirmed by counting all tips in E12.5 kidneys from mice with conditional deletion of Dchs1 in Six2–expressing cells (Six2-Cre Dchs1f/-), as compared to heterozygous controls (Six2-Cre Dchs1f/+) (Fig. 3B,D,E). This reduction is less than observed when comparing null animals to wild-type siblings (Mao et al., 2011), D ev el op m en t A cc ep te d m an us cr ip t but this could stem from a lag in loss of Dchs1 after conditional deletion, and as deletion of Dchs1 within UB or stroma had no detectable effect on kidney development, we conclude that Dchs1 function within CM influences the growth and branching of the neighboring UB. The observations that Dchs1 and Fat4 are predominantly expressed in CM and stroma, and that Dchs1 is genetically required in CM, raises the question of how they influence branching of the UB. The UB can be subdivided into distinct cell types based on cell behavior and molecular markers. One fundamental subdivision is between tip cells and non-tip (trunk) cells. Tip cells engage in reciprocal signaling with cap mesenchyme cells to maintain their respective fates (Costantini and Kopan, 2010; Dressler, 2009; Kopan et al., 2014). For example, tip cells express Wnt ligands, including Wnt9b and Wnt11, that signal to CM, whereas CM cells express regulators of Ret and FGF signaling, including GDNF and FGFs, that signal to tip cells. Dchs1-Fat4 signaling is not required for this feedback loop per se, because Gdnf, Ret, and Wnt 11 expression levels are all similar between wild-type and mutant kidneys (Fig. 3A)(Mao et al., 2011). However, the local expansion of CM in Dchs1 and Fat4 mutants could potentially increase the number of UB cells exposed to signals from CM, thereby increasing the population of ureteric cells specified as tip rather than trunk. To evaluate this hypothesis, we examined a tip cell marker, Sox9, in Fat4 mutant kidneys versus wildtype kidneys. Indeed, an increase in Sox9-expressing cells could be observed at ureteric bud tips (Fig. 3C,F-I). This expansion in Sox9-expressing cells correlates with loss of a trunk cell marker, the lectin DBA (Fig. 3J,K). We also quantified the expression levels of genes expressed in tip and trunk cells by Q-PCR (Fig. 3A). Two tip genes, Six2 and Ret, were expressed at similar levels between wild-type and mutant kidneys (Fig. 3A) (Mao et al., 2011). However, E-cad levels were reduced (to 70% of wild-type) in Fat4 mutant D ev el op m en t A cc ep te d m an us cr ip t kidneys (Fig. 3A). We interpret this as stemming from the reduction in the fraction of the kidney that is comprised by the UB (E-cad expressing cells), consistent with the reduced branching and more widely spaced UB tips observed by confocal microscopy. Taken together then, this Q-PCR analysis implies that the fraction of the UB system comprising tip cells (ie Sox9 and Ret expressing) is increased in mutants. Direct examination of a trunk marker (Wnt7b) revealed a modest reduction in RNA levels (to 88% of wild-type, Fig. 3A). Altogether, these observations are consistent with the hypothesis that the branching defects in Dchs1 and Fat4 could arise as a secondary consequence of the expansion of CM and a promotion of tip cell fate in neighboring UB cells, which could then delay the formation of new branches. Influence of Dchs1 and Fat4 on stromal cells Despite the increased thickness of the CM, the space between clusters of CM is increased in Dchs1 and Fat4 mutants (Fig. 2E-H), concomitant with the decreased branching of the UB. The space between CM is normally occupied by Foxd1-expressing stromal cells. To assess the identity of intervening cells in Dchs1 and Fat4 mutant embryos, a transgene expressing GFP under FoxD1 control was examined in E14.5 Dchs1 or Fat4 mutant kidneys. This revealed that the intervening space between CM is filled with FoxD1-expressing stromal cells in both wild-type and mutants (Fig. 3L,M, S2G,H). As the space between clusters of CM remains as narrow as in wild-type after conditional deletion of Dchs1 in the UB or stroma (Fig. S2A-D), but not after deletion of Dchs1 in CM (Fig. 2P), we infer that this stromal cell phenotype also stems from the requirement for Dchs1 in CM. This local expansion in stroma might simply reflect a similar number of FoxD1-expressing cells filling an expanded space between CM within a smaller mutant kidney. D ev el op m en t A cc ep te d m an us cr ip t Alternatively, it could be that Dchs1-Fat4-dependent signals specifically influence stromal cells. The continued expression of Foxd1 implies that normal stromal cell fate does not require Dchs1-Fat4 signaling. We also examined the expression of two genes that act in stromal cells to influence kidney development. Extracellular matrix 1 (Ecm1) is expressed by stromal cells in response to retinoic acid signaling, and influences UB branching at least in part by restricting Ret expression to UB tips (Paroly et al., 2013). Decorin (Dcn) expression is normally repressed by Foxd1 in stromal cells, and mis-expression of Dcn1 contributes to the Foxd1 mutant kidney phenotype (Fetting et al., 2014). However, Q-PCR analysis revealed that both of these genes are expressed at similar levels between wild-type and mutant kidneys (Ecm1 at 84% of wild-type, Dcn at 107% of wild-type, Fig. 3A). Abnormal nephrogenesis in Dchs1 and Fat4 mutants During early stages of nephrogenesis, CM cells condense to form a pre-cellular aggregate (PA), undergo a mesenchymal-to-epithelial transition to form a renal vesicle (RV), which as it begins to elongate into the nephron tubules transitions through distinctive comma-shaped body (CB) and S-shaped body (SB) stages (Fig. S3A). Further elongation and morphogenesis establishes the nephron tubules and glomeruli. Das et al (2013) reported that nephron differentiation was severely impaired in stromaless kidneys or Fat4 mutants. By contrast, Hum et al (2014) reported that stromaless kidneys had a similar extent of nephron differentiation as in controls, but differentiated nephron structures were mis-positioned. Using WT1 as a marker, which stains CM, distal regions of the RV, CB, and SB, and podocytes, we observed that nephron differentiation still occurs in Fat4 mutants, as visualized by the appearance of WT1expressing podocytes (Fig. 4A,B). However, there are abnormalities in early nephron D ev el op m en t A cc ep te d m an us cr ip t morphogenesis. The shape of the WT1-expressing portion of the CB and SB is more variable, and often differs from wild-type (Fig. 4C,D). Conditional deletion of Dchs1 within CM also resulted in abnormal of morphogenesis, including heterogeneity in the size and shape of the forming PA, RV, and CB (Fig. S3). In addition to strong expression in UB tips, Sox9 is also expressed during early stages of nephrogenesis, where it is visible within connecting tubules and in proximal regions of the RV, CB, or SB (Fig. 4G) (Reginensi et al., 2011). Using Sox9 as a marker, in wild type kidneys Sox9-expressing tubules that extend away from the kidney surface can be observed connecting the UB to the SSB (Fig. 4G, arrow). By contrast, in Dchs1 or Fat4 mutant kidneys, the initial orientation of these connecting tubules is frequently (23/48 scored) abnormal, extending parallel to the surface of kidney (Fig. 4H,I). The ends of these Sox9-expressing tubules are often associated with cysts (Fig. 4I), which become even more evident at later stages (Fig. 4B,F). Accumulation of small cysts in P0 kidneys of Dchs1 and Fat4 mutants has been reported previously (Mao et al., 2011; Saburi et al., 2008); these cysts were identified in Aquaporin2-expressing (marks collecting duct and connecting tubule) and Tamm-Horsfall Protein (THP) expressing tubules (marks nephron distal tubule and loop of Henle). We have extended cyst characterization here by identifying cysts in the outer (cortical) region associated with abnormal initiation of nephrogenesis; these cysts can express Sox9 or Cytokeratin 8 (CK8, a marker of the UB) (Fig. 4B,D,F,I). We also note that we detect cysts not only at P0, but also at E17.5 and E16.5. This is potentially significant as it was proposed that tubule dilation could be ascribed to defects in oriented cell division (Saburi et al., 2008), but the existence of oriented cell divisions earlier than P0 has been controversial (Karner et al., 2009; Yu et al., 2009). Our observations emphasize that nephrogenesis D ev el op m en t A cc ep te d m an us cr ip t occurs in Dchs1 and Fat4 mutants, but it is morphologically abnormal, and this abnormal nephron morphogenesis can be associated with cyst formation. Examination of Yap and Taz in Dchs1 and Fat4 mutants Das et al (2013) concluded that the local increase in CM thickness in Fat4 mutants stems from impaired differentiation of these nephron progenitor cells, and linked this impaired differentiation to activation of the Hippo pathway transcription factors Yap and Taz. This increased Yap/Taz activity was identified through increased nuclear localization of Yap in CM (Das et al., 2013). When we stained control kidneys, we observed a complex Yap distribution in the kidney, which appears to be consistent with prior studies (Das et al., 2013; Reginensi et al., 2013), and includes cells with predominantly nuclear (which can be dispersed or concentrated in sub-nuclear foci), predominantly cytoplasmic, and mixed distributions of Yap (Fig. 5A, S4A,C). The specificity of the antisera was confirmed by staining Yap mutant tissues (Fig. S4D). However, when we examined Yap localization in either Dchs1 or Fat4 mutants, we were unable to detect an obvious difference in Yap localization between wild-type and mutant CM (Fig. 5B, S4B). We are uncertain of the reason for this discrepancy with Das et al. (2013), but examined Yap localization using both conventional antigen retrieval methods (Fig. 5A,B), as well as using the tyramide signal amplification (TSA) approach employed by Das et al. (2013) (Fig. S4A,B). To further evaluate the potential relationship between Fat4-Dchs1 signaling and Yap/Taz activity, we examined genetic interactions. In Drosophila, where Fat regulates Yki activity, over-growth phenotypes of fat mutants are suppressed in yki heterozygotes (Reddy and Irvine, 2008). To assess whether a similar genetic interaction could be detected in murine kidneys, we compared Dchs1-/or Fat4-/kidneys to Dchs1-/Yap-/+ or D ev el op m en t A cc ep te d m an us cr ip t Fat4-/Yap-/+ kidneys. However, neither overall kidney morphology nor the expansion of CM were visibly altered by loss of one copy of Yap (Fig. 5C-E, S4E,F). Homozygous Yap mutants die at E8.5, but homozygous Taz mutants can survive. However, mutation of Taz did not suppress Fat4 kidney phenotypes, including smaller kidneys, reduced branching, and local expansion of CM (Fig. 5F-M, S4G). Instead, we found that Taz mutants, which have a cystic kidney phenotype on their own (Hossain et al., 2007; Makita et al., 2008; Tian et al., 2007), and Fat4 mutants, exhibit an enhanced cyst phenotype, such that Fat4 Taz double mutant kidneys were almost entirely filled with large cysts (Fig. 5F-I, S4G). Genetic enhancement between Fat4 and Taz is also reflected in the fact that while we could readily obtain P0 Fat4 or Taz single mutants, Fat4 Taz double mutants did not survive past E18.5. Thus, while earlier studies have revealed that Yap and Taz do play essential roles in kidney development (Das et al., 2013; Hossain et al., 2007; Makita et al., 2008; Reginensi et al., 2013; Tian et al., 2007), we did not obtain evidence supporting a role for Dchs1-Fat4 signaling in repressing Yap/Taz