Pronephric Tubulogenesis Requires Daam1-Mediated Planar Cell Polarity Signaling

BASIC RESEARCH www.jasn.org

Rachel K. Miller,* Sol Gomez de la Torre Canny,† Chuan-Wei Jang,‡ Kyucheol Cho,*§ ʈ Hong Ji,* Daniel S. Wagner,† Elizabeth A. Jones, Raymond Habas,¶ and Pierre D. McCrea*§

*Department of Biochemistry and Molecular Biology, the University of Texas M.D. Anderson Cancer Center, Houston, Texas; †Department of Biochemistry and Cell Biology, Rice University, Houston, Texas; ‡Program in Developmental Biology, Baylor College of Medicine, Houston, Texas; §Program in Genes and Development, ʈ University of Texas Graduate School of Biomedical Sciences, Houston, Texas; Department of Biological Sciences and Molecular Physiology, Warwick University, Coventry, United Kingdom; and ¶Department of Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania

ABSTRACT Canonical ␤-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway.

J Am Soc Nephrol 22: 1654–1667, 2011. doi: 10.1681/ASN.2010101086

Multiple organs including the kidney, lung, mam- lial transition to form the epithelial tubules of the mary gland, gut, heart, and ear are composed of tubule nephron, the basic unit of filtration.30 Such nephric networks, the formation of which requires Wnt sig- tubules serve not only as conduits for fluid flow within naling. Several of these systems utilize canonical Wnt/ the kidney but also as a filtration device for selective ␤-catenin signaling at early inductive stages followed absorption and excretion. by downregulation at later developmental time 1–7 points. Noncanonical Wnt signals in turn regulate Received October 20, 2010. Accepted April 23, 2011. later tubulogenesis processes, including the polarized Published online ahead of print. Publication date available at 8–16 migration of branching cells. As learned from www.jasn.org. studies in varied organ systems, the Wnt pathways ap- Correspondence: Dr. Pierre D. McCrea, The University of Texas 17–29 pear to exhibit key roles in tube formation. M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit The kidney is an essential organ that functions in 1000, Houston, TX 77030. Phone: 713-834-6277; Fax: 713-834- 6273; E-mail: [email protected]; or Dr. Rachel K. Miller, filtering wastes, absorbing nutrients and water, and The University of Texas M.D. Anderson Cancer Center, 1515 Hol- maintaining body fluid osmolarity. The kidney devel- combe Boulevard, Unit 1000, Houston, TX 77030. Phone: 713-834- ops from the intermediate mesoderm and undergoes 6307; Fax: 713-834-6273; E-mail: [email protected] inductive events promoting mesenchymal-to-epithe- Copyright © 2011 by the American Society of Nephrology

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Three progressive developmental stages characterize mamma- Wnt signaling.15 Mutations in seahorse, the zebrafish protein product lian kidney formation, resulting in the pronephros, mesonephros, of which interacts with Inversin and Dishevelled, likewise result in and ultimately the adult kidney—the metanephros.31 On the cystic kidneys,14 whereas Vangl2, homologous to Drosophila PCP other hand, amphibian and fish kidneys transition only once, component Van Gogh, regulates the posterior tilt of primary cilia from the pronephric (embryonic) to the mesonephric (adult) within the zebrafish pronephric duct.10 In nonkidney mucociliary stage. Formation of the mesonephros (and metanephros in mam- epithelia, Dishevelled’s DEP and PDZ domains, which each contrib- mals) requires the preceding formation of the pronephros,32,33 ute to noncanonical Wnt signaling, respectively participate in cilio- and nephrons within each distinct stage form as a consequence of genesis or cilia orientation.52 Finally, loss of the fat4 protocadherin in similar molecular events, thereby producing analogous tubular mice, which in Drosophila is needed to orient PCP in the wing,53 morphologies.34–37 From an experimental perspective, the prone- results in cystic kidney disease.8 These studies underscore noncanoni- phros, which contains a single nephron, offers a simplified system cal Wnt PCP roles upstream or in conjunction with Dishevelled dur- that mirrors events of mesonephric and metanephric nephron ing kidney tubulogenesis. formation.34,38 In addition to roles in the formation and orientation of cilia Multiple signals contribute to nephron formation, includ- and oriented cell division, vertebrate PCP signaling components ing those of the fibroblast growth factor, bone morphogenic have common roles in morphogenic directed cell migration, as protein, and Wnt pathways.31,39,40 Numerous Wnt ligands are occurs in gastrulation, convergent extension, and neurulation. expressed in the developing kidney, including Wnt2, Wnt4, Disruption of vertebrate PCP components results in common Wnt5a, Wnt6, Wnt7b, Wnt8, Wnt9a, Wnt9b, Wnt11, and phenotypic outcomes including defects in gastrulation and neural Wnt11b,41–48 and several of these are required for nephrogen- tube closure and morphogenic processes involving cell migration esis. Although the downstream signaling trajectories for sev- dependent upon Rho family small-GTPase activity.54–61 PCP- eral of these Wnt ligands have been identified, many within the mediated Rho activation involves the formin protein Daam1 (Di- context of kidney development remain unclear. Wnt ligands shevelled-associated activator of morphogenesis), which together may activate the canonical trajectory, defined as occurring with its associated Rho-GEF, WGEF, participates in cytoskeletal through ␤-catenin, and/or they may activate noncanonical trajec- regulation (e.g., convergence and extension movements).61,62 tories involving planar cell polarity (PCP), calcium signaling com- Formin proteins modulate actin polymerization by coordinating ponents, or other constituents. Although practical considerations the association of actin monomers, recruited by profilin proteins, generally constrain investigators to address one or two trajectories to the barbed, elongating ends of filamentous actin.63–65 Because within a single study, it is now thought that each Wnt ligand is most other PCP effectors, including JNK and small GTPases, act likely to activate several trajectories in context-dependent man- in additional non-PCP pathways, Daam1 appears to provide a ners and even within a single context to activate multiple net- useful entry point to more selectively evaluate PCP signaling. worked trajectories (exhibiting crosstalk).49 Upon binding Dishevelled, Daam1’s autoinhibition is relieved, Wnt4 and Wnt9b are required for tubulogenesis within the allowing it to promote Rho activation and actin polymerization.66 metanephric mesenchyme in mice and initially act predomi- Additionally, the actin-monomer binding proteins profilin-1 and nantly via canonical/␤-catenin signaling.1,41,45 However, attempts profilin-2 are direct effectors of Daam1, with each exhibiting dis- to rescue Wnt4 and Wnt9b knockouts with ␤-catenin in explants tinct roles in gastrulation.67–69 Thus, Daam1 provides a molecular of mouse kidney resulted in partial success, permitting metaneph- target linking PCP signaling and cytoskeletal events. Daam1 and ric mesenchyme condensation, but failed tubulogenesis.1 Fur- WGEF each provide an important and accessible experimental thermore, the overexpression of ␤-catenin in developing mouse opportunity to assess the Daam1/WGEF/Rho trajectory’s role in or frog kidneys reduces tubule epithelialization.1,2 These studies tubule morphogenesis. suggested that canonical Wnt signals arise downstream of Wnt4 Although there is growing evidence linking kidney develop- and Wnt9b, but must subsequently be attenuated for proper tube ment and disease with noncanonical Wnt PCP signaling, the role formation.1,50 Indeed, recent studies provide tantalizing evidence of PCP components directly regulating the actin cytoskeleton in that noncanonical Wnt signals attenuate canonical Wnt signals to tubulogenesis has not been established. In this study, we show that promote tubulogenesis.9,14,15 Daam1 and WGEF are expressed in the developing pronephros, Several noncanonical Wnt components of the PCP pathway and we use amphibian (Xenopus laevis) and fish (Danio rerio) are implicated in kidney tubulogenesis, and disruption of these model systems to show that Daam1, WGEF, and Rho are required components is linked to morphogenesis defects leading to cystic for generating proper tubule morphologies. kidney diseases.51 Mouse kidneys with hypomorphic Wnt9b expression in collecting duct stalks exhibit cystic phenotypes and deficient tubulogenesis, consistent with reduced Rho and JNK RESULTS activation and the misorientation of cell divisions within developing nephric tubules.9 Vertebrate Inversin, a ciliary protein dis- Daam1 and WGEF Are Expressed in the Developing rupted in nephronophthisis II cystic kidney disease and homolo- Xenopus Pronephros gous with the Drosophila PCP component Diego, has been likened in To assess whether the temporal protein levels of Daam1 are zebrafish to a molecular switch between canonical and noncanonical consistent with its possible involvement in pronephric devel-

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opment, we conducted immunoblot analysis using a charac- Daam1 Depletion in Xenopus Alters the Expression of terized antibody directed against Daam1.68 Daam1 protein is Late but Not Early Pronephric Markers present in whole Xenopus egg and embryo lysates, maternally As a first test of Daam1’s involvement in pronephric develop- through tadpole stages, overlapping with pronephric kidney ment, we examined effects upon nephric marker expression formation from gastrulation (stage 12.5) through tadpole after its depletion. Knockdown of Daam1 was achieved using stages (stage 41) (Figure 1A). To examine Daam1 and WGEF an established translation blocking Daam1 morpholino (MO) spatial expression across embryonic kidney development, re- targeting the transcript’s 5Ј untranslated region.61 Immuno- verse-transcriptase PCR (RT-PCR) was undertaken of surgi- blot analysis confirmed the reduction of Daam1 protein levels cally isolated pronephric regions from Xenopus, demonstrat- in gastrula-stage embryos as compared with uninjected or ing expression of both transcripts between stages 12.5 and 35. standard control (Std MO)-injected embryos (Figure 2A). Transcript levels intensified at roughly stage 25, before differ- Because Daam1 and WGEF are expressed in the developing entiation and morphogenesis of the pronephric epithelium pronephros (Figure 1), we assessed knockdown effects on early (Figure 1B). In situ hybridization studies in whole animals pronephric markers of patterning and determination, Lim1 showed that Daam1 and WGEF transcripts are expressed over and vHNF. By injecting MO into the V2 blastomere on the left a range of embryonic stages (Supplementary Figure S1). When side of eight-cell stage Xenopus embryos, in combination with compared with sense control probes, Daam1 and WGEF anti- a nuclear-mCherry membrane green fluorescent protein sense probes detected Daam1 and WGEF transcripts within the (GFP) lineage tracer,71 spatial targeting of the pronephric an- pronephric tubule and duct at stage 41 (data not shown), a lagen was achieved and confirmed. Embryos were allowed to stage not assessed in prior in situ studies.62,70 Thus, Daam1 and develop until stage 28 to 30 and then analyzed for Lim1 or WGEF transcripts were detected earlier via RT-PCR (Figure Hnf1␤ expression by in situ hybridization (Figure 2B through 1B) than using in situ methods (data not shown).62,70 Collec- 2D). The small fraction of animals with reduced expression of tively, the temporal and spatial expression profiles of Daam1 Lim1 or Hnf1␤ markers on the Daam1-MO injected as com- and WGEF are compatible with potential contributions to- pared with the uninjected side of the embryo was similar to ward kidney development. that seen with Std-MO-injected negative controls (Figure 2B through 2D). Our in situ hybridization data therefore suggest that targeted Daam1 depletion does not disrupt early development of the pronephros. Because Daam1 and WGEF expression was found to intensify at stage 25 after the pronephros is patterned and determined (Fig- ure 1B), pronephric Daam1 depletion may affect differentiation and/or morphogenesis. We therefore assessed the effect of Daam1 knockdown on epithelial markers within the glomus (similar to the metanephric glomerulus), proximal tubules, distal tubules, and collecting ducts using a similar V2 blastomere targeting strategy as noted above (Figure 3A). Embryos were allowed to develop until stage 38 to 40 and were then analyzed via in situ hybridization for the expression of Nephrin (nphs1; glomus), Chloride Channel (clckb; distal tubule and duct), Sodium Glucose Cotrans- porter (slc5a1; proximal tubules), and Sodium Potassium ATPase (atp1a1; tubules and duct). After Daam1 knockdown, the fraction of animals with reduced expression of nphs1 on the injected side as compared with the uninjected side of the embryo was similar to that of the Std-MO-injected controls (Figure 3B). In contrast, the fraction of animals with reduced expression of clckb, slc5a1,or atp1a1 in Daam1-MO-injected as compared with uninjected em- Figure 1. Daam1 protein is detectable during Xenopus proneph- bryo sides was greater than that for Std-MO-injected controls ric development, with Daam1 and WGEF transcripts expressed in (Figure 3B). Although there was no observable loss in nphs1 ex- the developing pronephros. (A) Immunoblot (IB) of lysates from pression (Figure 3, B and C), Daam1-MO-injected animals exhib- stage 2 to 8 oocytes and stage 1 to 41 embryos (approximately ited significant reductions in expression of clckb within the distal 1/2 oocyte or embryo per lane), showing Daam1 and GAPDH tubules and ducts, slc5a1 within the proximal tubules, and atp1a1 (loading control) protein from oocyte through tailbud stages, a timeline encompassing pronephric development (stages 12.5 to in tubules and ducts (Figure 3, B and D through F). The reduc- 41). (B) RT-PCR of pronephric anlagen isolated from stage 12.5 to tions in expression of these markers appears to result primarily 35 embryos showing expression of Daam1 and WGEF, with in- from reduced tubule elaboration rather than reduced intensity of tensification at stage 25 during pronephric differentiation and expression, suggesting that the epithelium has properly differen- morphogenesis. tiated but that morphogenesis is disrupted. In all cases, these data

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system assigns one point for each of the five proximal tubule components of the mature pronephros (stage 38 visualized using the proximal tubule marker 3G8 antibody or lectin); this results in fully developed proximal tubules receiving a score of 5. Unformed tubules receive a score of 0, and scores for partially formed tubules range between 1 and 4. The PNI is expressed as the number of tubule components on the uninjected (right) sides minus that on the injected (left) sides of the embryo. Thus, experimental conditions that significantly reduce tubule components result in high PNI values, whereas conditions of little consequence result in low PNI values.73 Originally, the PNI system was stringently defined such that values Ն2 were consid- ered significant.73 However, within our studies, PNI values Ͻ2 were shown to be significant in certain experimental cases on the basis of repeated observations Figure 2. Daam1 knockdown in Xenopus does not affect expression of early pronephric and statistical analyses, thereby sup- determination and patterning markers. (A) Single-cell embryos were uninjected, injected porting the existence of subtle changes with 40 ng of Std MO, or injected with 40 ng Daam1 MO. IB analysis from gastrula-stage in pronephric development at later embryos (approximately 1 embryo equivalent per lane) shows Daam1 protein levels were stages of epithelialization and morpho- reduced in Daam1-MO-injected embryos as compared with uninjected or Std-MO- injected embryos. (B through D) The left V2 blastomere of eight-cell stage embryos was genesis. injected with 20 ng of Std MO or Daam1 MO. Embryos developed until stage 29 to 30 Relative to Std-MO-injected controls, and were analyzed for Lim1 or Hnf1␤ expression levels on their injected (left) versus Daam1 pronephric depletion reduced uninjected (right) sides. (B) The fraction of Daam1-depleted embryos with reduced Lim1 tubule elaboration and branching in pro- and Hnf1␤ marker expression on the injected side as compared with the uninjected side nephic proximal segments (Figure 4, B was similar to that of Std-MO-injected controls. (C) Injected sides of Daam1-MO and through D). This was reflected in PNI Std-MO-injected embryos do not have reduced Lim1 expression. (D) Injected sides of values being significantly greater after ␤ Daam1-MO- and Std-MO-injected animals do not have reduced Hnf1 expression. (C, D) pronephric Daam1 knockdown (Figure ␮ Bars are 500 m. 4B). Similar results were obtained using a dominant negative N-Daam1 construct together suggest that Daam1’s function is crucial during subse- (data not shown).61 Tubule and duct morphogenesis was fur- quent differentiation and morphogenesis stages to form the prox- ther assessed, with Daam1 depletion leading to reduced proximal and distal tubules and collecting ducts of the pronephros. imal and distal tubules and ducts. To assess a possible secondary effect contributing to the observed alterations, we Daam1 Depletion Alters Morphogenesis of the examined somite integrity because earlier work implicated Pronephric Tubules and Duct in Xenopus somite involvement in pronephrogenesis.74 After examination Because Daam1 depletion reduces marker expression of the ma- of Daam1-MO- versus Std-MO-injected animals (12/101 anti- ture pronephric epithelium and appears to alter pronephric mor- body/myofiber marker), no significant loss in somite integrity phology, we conducted a more thorough analysis of shape was observed (Figure 4, C and F). changes of the tubules and duct. Proximal tubule architecture was To assess the specificity of Daam1 MO effects, we wished to evaluated using the 3G8 antibody (unknown epitope) or lectin test if the above noted pronephric morphogenic defects could (sugar binding protein) staining, whereas that of distal tubules be rescued using co-injected full-length Daam1 (flDaam1; re- and duct used the 4A6 antibody (P.D. Vize, personal communi- sistant to Daam1 MO).61 First, we assessed if flDaam1 overex- cation) (Figure 4A).72 pression itself within the developing pronephros produced a To quantitatively assess the effects of perturbing Daam1 path- phenotype. However, unlike other PCP components produc- way components on proximal tubule morphogenesis, an estab- ing overexpression phenotypes,54,55 Daam1 is autoinhibited lished pronephric index (PNI) scoring system was utilized that (when not bound to Dishevelled),61 likely accounting for rela- compares the development of proximal tubule components on tively subtle phenotypic effects upon its overexpression (Sup- the injected (left) sides versus uninjected (right) sides.73 The PNI plemental Figure S2). The rescuing capacity of flDaam1 ex-

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Figure 3. Daam1 knockdown reduces the expression of late pronephric markers of differentiation and morphogenesis in Xenopus. (A) Diagram of Xenopus pronephric kidney showing positions of proximal tubules, distal tubules, collecting duct, and glomus. (B through F) The left V2 blastomere of eight- cell stage embryos was injected with 20 ng of Std MO or Daam1 MO. Embryos developed until stage 38 to 40 and were analyzed for nphs1, clckb, slc5a1, and atp1a1 expression levels on their injected (left) side as compared with their uninjected (right) side. (B) The fraction with reduced nphs1 marker expression on the injected side as compared with the uninjected side for Daam1-MO-injected animals was similar to that of Std-MO- injected controls, whereas the fraction with reduced clckb, slc5a1, and atp1a1 expression on the injected side as compared with the uninjected side for Daam1-MO-injected animals is greater than that for Std-MO-injected controls. (C) Injected sides of Std-MO- and Daam1-MO-injected animals do not have reduced nphs1 expression. (D) Injected sides of Std-MO-injected animals do not have reduced clckb expression, whereas injected sides of Daam1-MO-injected animals have significantly reduced clckb expression. (E) In- jected sides of Std-MO-injected animals do not have reduced slc5a1 expression, whereas injected sides of Daam1-MO-injected animals have significantly reduced slc5a1 expression. (F) Injected sides of Std-MO-injected animals do not have reduced atp1a1 expression, whereas injected sides of Daam1-MO-injected animals have significantly reduced atp1a1 expression. (C through F) Bars are 500 nm. pression upon Daam1 MO depletion was then evaluated. likelihood of secondary effects upon the pronephros (Figure 4, ␤-Galactosidase (␤-gal) was used as a negative control for res- J and M). These data together demonstrate that our MO-di- cue. As expected, Daam1 MO depletion in the presence of rected pronephric depletion of Daam1 is specific. ␤-gal continued to result in fewer proximal tubule components (no rescue) relative to Std MO ϩ ␤-gal-injected controls Daam1 Depletion Has Little Effect on Proliferation or (Figure 4G). In contrast, flDaam1 exhibited clear rescuing ca- Apoptosis within Pronephric Tubules pacity (Figure 4G). Similarly, as evaluated using a prior noted In addition to effects upon differentiation or morphogenesis, re- antibody or lectin staining, we determined that flDaam1 sig- duced staining of pronephric markers might result from the loss nificantly rescued proximal tubules (Figure 4, H and K) as well of cells through decreased proliferation, increased apoptosis, or as distal tubules and ducts (Figure 4, I and L). The fraction of from changes in cell size. As assessed by in situ staining using the animals with reduced somite staining suggested a negligible atp1a1 marker, we first observed that Daam1 depletion results in

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reduced cell number rather than cell size (data not shown). We stage embryos, followed by staining for markers of proximal then tested whether Daam1 depletion altered proliferation or ap- tubules or of distal tubules and ducts (Figure 5A).78–80DN Rho optosis at two time points after the increase of endogenous injection resulted in PNI values significantly greater than for Daam1 and WGEF expression at stage 25. Cell proliferation, as- ␤-gal-expressing animals, meaning that the number of proxi- sessed using a phospho-histone H3 antibody,75,76 was not effected mal tubule components was reduced (Figure 5G). Markers of by Daam1 depletion within the pronephric region at stage 27 proximal tubules, distal tubules and ducts, and somites further (data not shown). An apparently small decrease in proliferation indicated that DN Rho has an inhibitory effect upon tubule, was observed in stage 32 pronephric proximal tubules after duct, and somite formation (Figure 5, H through K). Daam1 depletion (an averaged difference of Ͻ1 cell per kidney) (Supplemental Figure S3). Because tubule staining (3G8 anti- Daam1 Depletion in Zebrafish Reduces Kidney body), and likely the total cell number within Daam1-depleted Tubulogenesis and Alters Morphogenesis pronephroi, is reduced compared with controls, this small differ- To support that the role of the Daam1 trajectory in nephro- ence is even less likely to be meaningful. As measured using an genesis is not restricted to Xenopus, we examined Daam1 de- active caspase-3 antibody77 after Daam1 depletion, no measurable pletion in zebrafish (D. rerio) using an established MO.81 The apoptosis within the pronephros was detected at stage 27 (data mature zebrafish pronephric kidney has an architecture simi- not shown) or stage 32 (Supplemental Figure S4). As a positive lar to the Xenopus pronephros, containing a glomus, nephros- control, apoptosis was readily detectable in somite regions at both tomes, tubules, and ducts (Supplemental Figure S6A). Ze- stages, and exposure of embryos to doxorubicin, an inhibitor of brafish pronephroi exhibit a characteristic shape, including DNA replication, produced apoptosis in 100% of pronephroi and nephrostomes that turn in toward the midline and pronephric most other tissues. These data suggest that depletion of Daam1 tubules and ducts that run parallel to each other but never has negligible effects on proliferation and apoptosis and that the touch or branch (Supplemental Figure S6A). We injected sin- observed alterations in pronephric tubular structure are likely due gle-cell zebrafish embryos with Daam1 MO versus Std MO, to effects upon differentiation and morphogenesis. allowing then to develop 72 hours postfertilization. Proneph- ric tubule and duct shapes were assessed using the ␣6F antibody, WGEF Depletion in Xenopus Perturbs Pronephric which recognizes the ␣ subunit of the sodium potassium ATPase. Tubule and Duct Morphogenesis Whereas Std-MO-injected animals shared the characteristic pro- To further test the role of the Daam1 trajectory of the PCP nephric shapes of uninjected embryos, Daam1-MO-injected ani- pathway in pronephric tubulogenesis, WGEF, a Rho-GEF mals exhibited the absence of nephrostomes or those that did not that associates with Daam1, was depleted within the pro- turn inward toward the midline (Supplemental Figure S6C). Fur- nephric field. Staining with proximal and distal tubule thermore, evidencing phenotypes rarely seen under normal con- markers (Figure 5A) was assessed following established MO- ditions, tubules and ducts became wavy (convoluted) after based WGEF knockdown using a strategy similar to that used Daam1 depletion (Supplemental Figure S6, B and D), occasion- with Daam1 knockdown.62 The PNI scoring for reduced tu- ally branching and frequently touching (Supplemental Figure bule components was significantly greater in WGEF-depleted S6E). Because MOs are usually injected at the single-cell stage in than Std-MO-injected animals (Figure 5B). Likewise, the frac- zebrafish, multiple morphogenic tissues or events may have been tion of injected animals with reduced marker staining of the influenced, including convergence-extension of the elongating proximal tubule or distal tubules and duct was greater for embryo. Thus, analysis of convergent-extension was performed WGEF-MO- than Std-MO-injected controls (Figure 5, C after Daam1 depletion; as anticipated, nearly all such embryos through E). Indeed, the pronephric phenotype resulting from exhibited convergent-extension defects in contrast to Std-MO- WGEF knockdown was more severe than that after Daam1 injected animals (Supplemental Figure S6F). In summary, disrup- knockdown (Figure 4, B and C, and Figure 5, B and C). Somite tion of the Daam1 PCP trajectory in zebrafish results in morpho- integrity was similar in WGEF-MO- versus Std-MO-injected genic defects of the pronephros, possibly reflecting Daam1’s embryos (Figure 5, C and F). Possibly because Daam1 and conserved role across vertebrate species. WGEF physically interact,62 their co-depletion caused similar reductions in staining of pronephric proximal tubules, distal tubules, and ducts, as seen for single depletions (Supplemental DISCUSSION Figure S5). These findings support a role of the Daam1/WGEF trajectory of the PCP pathway in pronephric tubulogenesis. Tubulogenesis is integral to the formation and function of multiple organ systems, including the kidney, lung, mammary and Dominant Negative Rho Alters Pronephric Tubule and prostate glands, digestive tract, and cardiovascular and auditory Duct Morphogenesis in Xenopus systems. In each case, Wnt signals are crucial for the induction and Because the Daam1/WGEF PCP trajectory leads to the activa- morphogenesis of these tubular assemblies.17–29 Although Wnt tion of Rho GTPase, we examined whether Rho is indeed re- signaling requirements in kidney formation are accepted, 2,41–48,82 quired for pronephros formation. Dominant negative RhoN19 understanding the relative contributions of varying Wnt compo- (DN Rho) was injected into the V2 blastomere of eight-cell nents and trajectories remains a key question. For example,

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Figure 4. Daam1 knockdown alters morphogenesis of the pronephric tubules and duct and can be rescued by flDaam1 as assessed by markers of the mature pronephros in Xenopus. (A) Diagram of Xenopus pronephric kidney showing positions of lectin staining in the proximal tubules (green) and 4A6 antibody staining in the distal tubules and collecting duct (red). (B through F) The left V2 blastomere of eight-cell stage embryos was injected with 20 ng of Std MO or Daam1 MO. Embryos developed until stage 38 to 40 and were analyzed for lectin, 4A6 antibody, and 12/101 antibody staining within the proximal tubules, distal tubules/duct, and somites, respectively, on their injected (left) side as compared with their uninjected (right) side. (B) The PNI is greater in Daam1-MO-injected animals than in Std-MO-injected animals. (C) The fraction with reduced proximal tubule and distal tubule/duct marker staining on the injected side as compared with the uninjected side for Daam1-MO-injected animals is greater than that for Std-MO-injected controls, whereas the fraction with reduced somite marker staining on the injected side as compared with the uninjected side for Daam1-MO- injected animals was similar to that of Std-MO-injected controls. (D) Injected sides of Std-MO-injected animals do not have reduced proximal tubule marker staining, whereas injected sides of Daam1-MO-injected animals have significantly reduced proximal tubule marker staining. Bar is 100 ␮m. (E) Injected sides of Std-MO-injected animals do not have reduced distal tubule and duct marker staining, whereas injected sides of Daam1-MO-injected animals have significantly reduced distal tubule and duct marker staining. Bar is 100 ␮m. (F) Injected sides of Std-MO- or Daam1-MO-injected animals do not have reduced somite marker staining. Bar is 250 ␮m. (G through M) The left V2 blastomere of eight-cell stage embryos was injected with 20 ng Std MO ϩ 1ng␤-gal, 20 ng Daam1 MO ϩ 1ng␤-gal, or 20 ng Daam1 MO ϩ 1 ng flDaam1. Embryos developed until stage 38 to 40 and were analyzed for lectin, 4A6 antibody, and 12/101 antibody staining within the proximal tubules, distal tubules/duct, and somites, respectively, on their injected (left) side as

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␤-catenin-mediated (canonical Wnt) signaling is essential in in- may play additional roles (beyond the Daam1 trajectory) within ducing kidney tubulogenesis,1,2,83–85 whereas studies have only these developmental processes. begun to examine noncanonical Wnt PCP contributions. Several To this point, disruption of PCP components in the de- PCP components or regulators that establish the plane of polar- veloping kidney, including Wnt9b, Fat4, Vangl2 (homolog ization within cells are recognized players, such as Wnt9b, Fat4, of Van Gogh), Inversin (homolog of Diego), and Seahorse, Vangl2 (homolog of Van Gogh), Inversin (homolog of Diego), and leads to morphologic defects reminiscent of cystic kidney Seahorse.8–10,14,15 However, involvement of PCP effectors that disease in humans.8–10,14,15 These components establish the regulate actin dynamics have not yet been studied in kidney for- plane of polarity to coordinate asymmetric signaling by down- mation or in tubulogenesis. Our study demonstrates that Daam1 stream PCPeffectors such as Daam1, leading to alteration of the actin and WGEF, components of the vertebrate PCP pathway involved cytoskeleton.86,87 Unexpectedly, Daam1 knockdown does not show in cytoskeletal modulation, are expressed in the developing kid- an obvious cystic kidney phenotype in Xenopus (Figure 4), although ney and are required for pronephric tubule morphogenesis in Xe- in zebrafish a small fraction of animals exhibited cystic kidneys.81 Dif- nopus and likely also in zebrafish (Figures 1, 4, and 5 and Supple- ferences in kidney phenotypes after perturbation of distinct PCP mental Figure S6). components may in part be a consequence of their relative positions Several studies support the hypothesis that Wnt signaling ini- within the PCP pathway. For example, components modulating a tially acts via canonical Wnt/␤-catenin signals that become atten- greater number of key effectors might be expected to have a more uated and supplanted by noncanonical Wnt pathways. Support- substantial effect on morphogenic processes than Daam1. Alter- ive primary evidence for this idea includes studies in which natively, Daam1 may play a role in cystic kidney disease but in ␤-catenin overexpression prevents kidney tubules from form- combination with other unresolved factors. Hypomorphic Wnt9b ing.1,2 Additionally, within ex vivo mouse kidney explants derived expression in collecting duct stalks of mouse kidney results in a from Wnt4 or Wnt9b knockout mice, exogenous ␤-catenin res- cystic defect thought to be primarily caused by failure of tubules to cues condensation of the metanephric mesenchyme but fails to elongate in the face of misoriented cell divisions.9 These hypo- rescue subsequent tubule formation.1 This is consistent with the morphs also display reduced Rho and JNK activation, although it idea that signals in addition to ␤-catenin and downstream of is unknown if this is instrumental in forming nephric cysts.9 Thus, Wnts are needed in nephrogenesis. Studies in zebrafish suggest although Rho disruption upon Daam1 knockdown does not no- that the PCP component Inversin (homolog of Diego) attenuates tably produce nephric cysts on its own, it may do so in combina- canonical Wnt signaling and simultaneously induces PCP signal- tion with other (PCP, etc.) disruptions. ing through selective degradation of cytoplasmic Dishevelled.15 Studies to substantiate the role of PCP signaling in kid- However, the nature of the noncanonical components that atten- ney tubulogenesis and cystic kidney disease are accumulat- uate canonical signaling remain poorly resolved. The studies pre- ing,9,10,14,15,51 but the mechanisms underlying PCP involve- sented here indicate that the Daam1/WGEF trajectory of the PCP ment in shaping kidney tubules have remained largely pathway is required for the elaboration and morphogenesis unclear. For example, work has examined PCP participation phases of nephric tubulogenesis rather than for mesenchyme in- in establishing the plane of polarity within tissues in relation duction (Figures 2 and 3); that is, after ␤-catenin induction/ to cilia formation and orientation as well as oriented cell condensation of the nephrogenic mesenchyme, we propose that divisions.10,14,15,51 However, the relevance of PCP signaling the Daam1/WGEF trajectory contributes to the epithelialization to cytoskeletal modulation, leading to directional cell and morphogenesis of developing nephrons. Because the pro- movements in morphogenic processes such as gastrulation, nephric WGEF knockdown phenotype is more severe than those convergent extension, and neurulation, suggests that PCP after knockdown of Daam1 or inhibition of Rho activity, WGEF may also be involved in nephric tubulogenesis.88–93 Daam1

compared with their uninjected (right) side. (G) The PNI is greater in Daam1-MO ϩ ␤-gal-injected animals than in Std-MO ϩ ␤-gal embryos or Daam1-MO ϩ flDaam1-injected rescue animals. (H) The fraction with reduced proximal tubule marker staining on the injected side as compared with the uninjected side for Daam1 MO ϩ ␤-gal-injected animals was greater than that of Std-MO ϩ ␤-gal-injected controls. This reduction in proximal tubule staining seen in Daam1 MO ϩ ␤-gal animals was rescued in Daam1-MO ϩ flDaam1-injected animals. (I) The fraction with reduced distal tubule and duct marker staining on the injected side as compared with the uninjected side for Daam1-MO ϩ ␤-gal-injected animals was greater than that of the Std-MO ϩ ␤-gal-injected controls. This reduction in distal tubule and duct staining seen in Daam1 MO ϩ ␤-gal animals was rescued in Daam1 MO ϩ flDaam1-injected animals. (J) The fraction with reduced somite marker staining on the injected side as compared with the uninjected side for was similar for Std-MO ϩ ␤-gal-, Daam1-MO ϩ ␤-gal-, and Daam1-MO ϩ flDaam1-injected animals. (K) Injected sides of Std-MO ϩ ␤-gal-injected animals do not have reduced proximal tubule marker staining, whereas injected sides of Daam1-MO ϩ ␤-gal injected animals have significantly reduced proximal tubule marker staining. This loss of proximal tubule marker staining is rescued in Daam1-MO ϩ flDaam1-injected animals. Bar is 100 ␮m. (L) Injected sides of Std-MO ϩ ␤-gal-injected animals do not have reduced distal tubule and duct marker staining, whereas injected sides of Daam1-MO ϩ ␤-gal-injected animals have significantly reduced distal tubule and duct marker staining. This loss of staining with the distal tubule and duct marker is rescued in Daam1-MO ϩ flDaam1-injected animals. Bar is 100 ␮m. (M) Injected sides of Std-MO ϩ ␤-gal-, Daam1-MO ϩ ␤-gal-, and the Daam1-MO ϩ flDaam1-injected animals do not have reduced somite marker staining. Bar is 250 ␮m.

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Figure 5. WGEF knockdown and DN Rho alter morphogenesis of the pronephric tubules and duct as assessed by markers of the mature pronephros in Xenopus. (A) Diagram of Xenopus pronephric kidney showing positions of lectin staining in the proximal tubules (green) and 4A6 antibody staining in the distal tubules and collecting duct (red). (B through F) The left V2 blastomere of eight-cell stage embryos was injected with 20 ng of Std MO or WGEF MO. Embryos developed until stage 38 to 40 and were analyzed for lectin, 4A6 antibody, and 12/101 antibody staining within the proximal tubules, distal tubules/duct, and somites, respectively, on their injected (left) side as compared with their uninjected (right) side. (B) The PNI is greater in WGEF-MO-injected animals than in Std-MO-injected animals. (C) The fraction with reduced proximal tubule and distal tubule/duct marker staining on the injected side as compared with the uninjected side for WGEF-MO-injected animals is greater than that for Std-MO-injected controls, whereas the fraction with reduced somite marker staining on the injected side as compared with the uninjected side for WGEF-MO-injected animals was similar to that of Std-MO-injected controls. (D) Injected sides of Std-MO-injected animals do not have reduced proximal tubule marker staining, whereas injected sides of WGEF-MO-injected animals have significantly reduced proximal tubule marker staining. Bar is 100 ␮m. (E) Injected sides of Std-MO-injected animals do not have reduced distal tubule and duct marker staining, whereas injected sides of WGEF-MO-injected animals have significantly reduced distal tubule and duct marker staining. Bar is 100 ␮m. (F) Injected sides of Std-MO- or WGEF-MO-injected animals do not have reduced somite marker staining. Bar is 250 ␮m. (G through K) The left V2 blastomere of eight-cell stage embryos was injected with 200 pg of ␤-gal or DN Rho. Embryos developed until stage 38 to 40 and were analyzed for lectin and 4A6 antibody staining within the proximal tubules and distal tubules/duct, respectively, on their injected (left) side as compared with their uninjected (right) side. (G) The PNI is greater in the DN-Rho-injected animals than the ␤-gal-injected animals. (H) The fraction with reduced proximal tubule and distal tubule/duct marker staining on the injected side as compared with

1662 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1654–1667, 2011 www.jasn.org BASIC RESEARCH and WGEF are central players in PCP effects upon cytoskel- horseradish peroxidase secondary (BioRad) and enhanced chemilu- etal architecture, which occur through the modulation of minscence (Pierce Supersignal West Pico). formin and Rho GTPase activities. Our work, in combination with previous studies of Daam1 in other tissues/systems,61,62,66,68,69 appears consistent with its role in PCP-directed RT-PCR Analysis The pronephric anlagen was dissected from X. laevis embryos ranging cytoskeletal rearrangements in nephric morphogenesis. from stages 12.5 to 35 and homogenized in 150 ␮l XT buffer (300 mM Our understanding of the role of Wnt signaling in tubu- sodium chloride, 20 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1% SDS). logenesis is growing because of the application of multiple Total RNA was isolated from pronephric anlagen and was used to animal models and differing organ systems. Commonalities generate first-strand cDNA.108 The relative quantity of input cDNA exist between tubulogenic systems including kidney, lung, was assessed by comparing ODC (ornithine decarboxylase) PCR band heart, and stomach, such as the requirement of canonical intensities. Linearity of PCR amplification was confirmed with consec- Wnt/␤-catenin signaling at early stages followed by its at- utive doubling dilutions of input cDNA.47 Primers used are as follows: tenuation during further development.1–7 Noncanonical Daam1-forward 5Ј-AGTCGATGGAGCCAGCATTG-3Ј, Daam1-reverse Wnt signals are necessary in direct tubule branching of kid- 5Ј-GCTTGGTCCTAAGTGCAGTC-3Ј, WGEF-forward 5Ј-CTCCA- ney, lung, and mammary gland, likely via contributions to TAGCCGTCGATCATT-3Ј, WGEF-reverse 5Ј-TGTTCCTGATACGC- directional cell migration.8–15 PCP is also linked with orien- CTGGTT-3Ј, ODC-forward 5Ј-GGAGCTGCAAGTTGGAGA-3Ј, and tating the primary cilia of several tissues, including the kid- ODC-reverse 5Ј-TCAGTTGCCAGTGTGGTC-3Ј. ney and inner ear, and ciliary signaling in turn has an effect on noncanonical signaling in the developing kidney.8,10,14,15,94–101 Although our evidence supports the role In Situ Hybridization of PCP signaling in nephric tubulogenesis, the involvement Digoxigenin-labeled RNA probes were prepared using the DIG RNA la- of more downstream signaling components will require fu- beling kit (Roche). The constructs were linearized and synthesized using ture work. The role of PCP signaling in processes such as the listed enzyme and polymerase: CDaam1-antisense EcoRI/T7-sense gastrulation, convergent extension, and neurulation, and NotI/Sp6,61 NDaam1-antisense StuI/T7-sense NotI/Sp6,61 WGEF- specifically in tissues including the kidney, will ultimately antisense HindIII/T7-sense NotI/Sp6,62 Lim1-antisense XhoI/T7,109,110 help to resolve its mechanistic involvement in tissue move- HNF-antisense SmaI/T7,111 Nephrin-antisense SmaI/T7,112 ClcK- ments and cell-shape changes contributing to tube genera- antisense EcoRI/T7,113 NaGluc-antisense SmaI/T7,114 and NaKATPase- tion. In all cases, our findings show that there is a conserved antisense SmaI/T7b.115 Embryos were collected, fixed, and processed as requirement for the Daam1 trajectory of the PCP signaling described, eliminating the triethanolamine and acetic anhydride steps.116 pathway in the morphogenesis of tubular structures.

Molecular Cloning CONCISE METHODS A nuclear-mCherry membrane-GFP construct containing H2b- mCherry and glycosylphosphatidylinositol anchor-GFP71 was cloned into the pCS2 vector using the XhoI and XbaI restriction sites. Embryos X. laevis embryos were obtained, reared,102 and visualized (Nikon model SMZ-U and Zeiss Stemi DV4). Zebrafish embryos, D. rerio MOs and RNA Constructs strain DZ, were reared103 and staged.104 Published X. laevis translation-blocking MOs targeting the 5Ј untranslated region of Daam1 5Ј-CCGCAGGTCTGTCAGTTGCTTCTA-3Ј Western Blots and WGEF 5Ј-TCATTGTGTGAGTCCATCAGTCCCG-3Ј were used Oocytes were isolated from adult female X. laevis.105 Embryos were with Std MO 5Ј-CCTCTTACCTCAGTTACAATTTATA-3Ј.61,62 collected at various stages.106 Protein lysates were made107 and boiled For zebrafish Daam1 knockdowns, a splice-blocking MO targeting for 5 minutes at 95°C in 2ϫ Laemmeli (BioRad) with 100 ␮⌴ dithio- 5Ј-TCTTGACTTGCCCACCTGAAAGCGC-3Ј at the exon 6/in- threitol. Ten to 20 ␮l of lysate (0.5 to 1 oocyte or embryo equivalent) tron 6 junction was used with the above listed Std MO.81 cDNA were run on an 8% SDS-PAGE gel and transblotted to nitrocellulose. constructs included flDaam1 and dominant negative NDaam1,61 Blots were exposed to a rabbit Daam1 antibody (1:1000)68 and/or a DN Rho,78–80 and a bicistronic nuclear-mCherry membrane- rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) anti- GFP.71 cDNA constructs were linearized using NotI, transcribed in vitro body (1:5000) (Santa Cruz FL-335) followed by goat anti-rabbit IgG using the SP6 mMessage mMachine kit (Ambion), and purified.107

the uninjected side for DN-Rho-injected animals is greater than that for ␤-gal-injected controls. (I) Injected sides of ␤-gal-injected animals do not have reduced proximal tubule marker staining, whereas injected sides of DN-Rho-injected animals have significantly reduced proximal tubule marker staining. (J) Injected sides of ␤-gal-injected animals do not have reduced distal tubule and duct marker staining, whereas injected sides of DN-Rho-injected animals have significantly reduced distal tubule and duct marker staining. (I, J) Bars are 100 ␮m. (K) Injected sides of DN-Rho-injected animals do not have reduced somite marker staining. Bar is 250 ␮m.

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Microinjections and Drug Treatment Jessica Tyler for assistance with analysis of staining within the cover X. laevis microinjections were performed as described previously.107 image. This work was supported by an National Institutes of Health For targeting of constructs and MOs to the pronephros, microinjec- (NIH) R01 grant (GM052112), the March of Dimes (1-FY-07-461- tions were made into the V2 blastomere at the eight-cell stage,106 01), an NIH training grant (HD07325 to R.K.M.), an NIH postdoc- which contributes primarily to the pronephros, dorsal and ventral fin toral National Research Service Award (DK082145 to R.K.M.), and a epidermis, trunk crest, dorsal spinal chord, dorsal and ventral somite, postdoctoral fellowship provided by the Odyssey Program and the lateral plate fin mesenchyme, central somite, hindgut, and procto- Theodore N. Law Endowment for Scientific Achievement at the Uni- deum.117,118 Targeting of injected constructs was verified by co-injec- versity of Texas M.D. Anderson Cancer Center (UTMDACC) tion with nuclear-mCherry membrane-GFP.71 Zebrafish microinjec- (R.K.M.). The DNA sequencing and other core facilities at UTM- tions were performed at the single-cell stage.119 Uninjected X. laevis DACC were supported by UTMDACC National Cancer Institute core embryos at stages 27 and 32 were treated with a 1:1000 dilution of grant CA-16672. doxorubicin hydrochloride (EMD Chemicals, KS13305, 5 ␮g/␮l) in 0.1ϫ Marc’s modified ringers solution for 4 hours before fixation. DISCLOSURES Immunostaining None. X. laevis and zebrafish embryos were stained as described previously.2 Proximal tubules were detected using fluorescein-coupled Erythrina ␮ cristagalli lectin at 50 g/ml (Vector Labs) (PD Vize, personal com- REFERENCES munication) or primary monoclonal antibody 3G8 (1:30; unknown 72 epitope), whereas distal tubules and ducts and somites were de- 1. Park JS, Valerius MT, McMahon AP: Wnt/beta-catenin signaling reg- tected using monoclonal 4A6 (1:5) antibody (unknown epitope)72 ulates nephron induction during mouse kidney development. Devel- and monoclonal 12/101 (myofiber marker; 1:100), respectively.120,121 opment 134: 2533–2539, 2007 Cell proliferation and apoptosis were respectively assayed using Up- 2. Lyons JP, Miller RK, Zhou X, Weidinger G, Deroo T, Denayer T, Park JI, Ji H, Hong JY, Li A, Moon RT, Jones EA, Vleminckx K, Vize PD, state anti-phospho-Histone H3 (06-570) and BD PharMingen rabbit McCrea PD: Requirement of Wnt/beta-catenin signaling in proneph- 75–77 anti-active Caspase-3 (551150) antibodies. Staining was visual- ric kidney development. Mech Dev 126: 142–159, 2009 ized using goat anti-mouse secondary antibody (1:2000) coupled to 3. Rajagopal J, Carroll TJ, Guseh JS, Bores SA, Blank LJ, Anderson WJ, alkaline phosphatase (AP) (Zymed) or goat anti-mouse IgG Alexa 488 Yu J, Zhou Q, McMahon AP, Melton DA: Wnt7b stimulates embry- or Alexa-555 (Invitrogen). In zebrafish, monoclonal antibody ␣6F onic lung growth by coordinately increasing the replication of epithelium and mesenchyme. Development 135: 1625–1634, 2008 (␣-1 subunit of NaK ATPase) with goat anti-mouse secondary anti- 4. Shu W, Jiang YQ, Lu MM, Morrisey EE: Wnt7b regulates mesenchy- body (1:2000) AP (Zymed) and nitro blue tetrazolium/5-bromo-4- mal proliferation and vascular development in the lung. Develop- chloro-3-indolyl phosphate AP substrate (Roche) were used to detect ment 129: 4831–4842, 2002 tubules and ducts.122,123 Embryos stained with AP reagents were 5. De Langhe SP, Sala FG, Del Moral PM, Fairbanks TJ, Yamada KM, bleached as described previously.124 Zebrafish embryos were dehy- Warburton D, Burns RC, Bellusci S: Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morpho- drated in methanol and cleared in Murray’s clearing medium.102 genesis of the mouse embryonic lung. Dev Biol 277: 316–331, 2005 6. Okubo T, Hogan BL: Hyperactive Wnt signaling changes the devel- Imaging opmental potential of embryonic lung endoderm. J Biol 3: 11, 2004 Images were captured with an Olympus SZX12 stereomicroscope 7. Schneider VA, Mercola M: Wnt antagonism initiates cardiogenesis in with a DF PLFL 1.6ϫ PF objective and an Olympus DP71 12.5 mega- Xenopus laevis. Genes Dev 15: 304–315, 2001 pixel camera and software (version 3.3.1.292). Images were assembled 8. Saburi S, Hester I, Fischer E, Pontoglio M, Eremina V, Gessler M, Quaggin SE, Harrison R, Mount R, McNeill H: Loss of Fat4 disrupts using Adobe CS4 Photoshop and Illustrator. PCP signaling and oriented cell division and leads to cystic kidney disease. Nat Genet 40: 1010–1015, 2008 9. Karner CM, Chirumamilla R, Aoki S, Igarashi P, Wallingford JB, Carroll TJ: Wnt9b signaling regulates planar cell polarity and kidney tubule ACKNOWLEDGMENTS morphogenesis. Nat Genet 41: 793–799, 2009 10. Borovina A, Superina S, Voskas D, Ciruna B: Vangl2 directs the We are grateful Jun-Ichi Kyuno for his experimental contributions to posterior tilting and asymmetric localization of motile primary cilia. the manuscript. Unfortunately, we are unable to include him as third Nat Cell Biol 12: 407–412, 2010 author because of journal copyright restraints. We thank all members 11. Imbert A, Eelkema R, Jordan S, Feiner H, Cowin P: Delta N89 beta-catenin induces precocious development, differentiation, and of the Houston Frog and Fish Club for helpful suggestions, in partic- neoplasia in mammary gland. J Cell Biol 153: 555–568, 2001 ular laboratory members of P.D. McCrea, A.K. Sater, J. Kuang, and 12. Cervantes S, Yamaguchi TP, Hebrok M: Wnt5a is essential for intes- D.S. Wagner. We also thank D. Gu and J.Y. Hong for training of tinal elongation in mice. Dev Biol 326: 285–294, 2009 R.K.M. in the methodology of Xenopus research. We are particularly 13. Li C, Xiao J, Hormi K, Borok Z, Minoo P: Wnt5a participates in distal grateful to I.B. Dawid, M. Kloc, P.D. Vize, R.R. Behringer, V. Huff, lung morphogenesis. Dev Biol 248: 68–81, 2002 14. Kishimoto N, Cao Y, Park A, Sun Z: Cystic kidney gene seahorse M.C. Barton, and F. Costantini for reagents and advice. Additionally, regulates cilia-mediated processes and Wnt pathways. Dev Cell 14: we thank Y. Furuta for the use of his stereomicroscope and J.L. Polan 954–961, 2008 for microscopy training. We are grateful to Thomas Dunham and 15. Simons M, Gloy J, Ganner A, Bullerkotte A, Bashkurov M, Kronig C,

1664 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1654–1667, 2011 www.jasn.org BASIC RESEARCH

Schermer B, Benzing T, Cabello OA, Jenny A, Mlodzik M, Polok B, progenitors and their epithelial progeny. Kidney Int 74: 1004–1008, Driever W, Obara T, Walz G: Inversin, the gene product mutated in 2008 nephronophthisis type II, functions as a molecular switch between Wnt 41. Stark K, Vainio S, Vassileva G, McMahon AP: Epithelial transformation signaling pathways. Nat Genet 37: 537–543, 2005 of metanephric mesenchyme in the developing kidney regulated by 16. Yates LL, Schnatwinkel C, Murdoch JN, Bogani D, Formstone CJ, Wnt-4. Nature 372: 679–683, 1994 Townsend S, Greenfield A, Niswander LA, Dean, CH: The PCP genes 42. Kispert A, Vainio S, McMahon AP: Wnt-4 is a mesenchymal signal for Celsr1 and Vangl2 are required for normal lung branching morpho- epithelial transformation of metanephric mesenchyme in the devel- genesis. Hum Mol Genet 19: 2251–2267, 2010 oping kidney. Development 125: 4225–4234, 1998 17. Merkel C, Karner C, Carroll T: Molecular regulation of kidney devel- 43. Lin Y, Liu A, Zhang S, Ruusunen T, Kreidberg JA, Peltoketo H, Drum- opment: Is the answer blowing in the Wnt? Pediatric Nephrology 22: mond I, Vainio S: Induction of ureter branching as a response to Wnt-2b 1825–1838, 2007 signaling during early kidney organogenesis. Dev Dyn 222: 26–39, 2001 18. Cardoso WV, Lu J: Regulation of early lung morphogenesis: Ques- 44. Itaranta P, Lin Y, Perasaari J, Roel G, Destree O, Vainio S: Wnt-6 is tions, facts and controversies. Development 133: 1611–1624, 2006 expressed in the ureter bud and induces kidney tubule development 19. Robinson GW, Hennighausen L, Johnson PF: Side-branching in the in vitro. Genesis 32: 259–268, 2002 mammary gland: The progesterone-Wnt connection. Genes Dev 14: 45. Carroll TJ, Park JS, Hayashi S, Majumdar A, McMahon AP: Wnt9b 889–894, 2000 plays a central role in the regulation of mesenchymal to epithelial 20. Rubin DC: Intestinal morphogenesis. Curr Opin Gastroenterol 23: transitions underlying organogenesis of the mammalian urogenital 111–114, 2007 system. Dev Cell 9: 283–292, 2005 21. Verzi MP, Shivdasani RA: Wnt signaling in gut organogenesis. Or- 46. Yu J, Carroll TJ, Rajagopal J, Kobayashi A, Ren Q, McMahon AP: A ganogenesis 4: 87–91, 2008 Wnt7b-dependent pathway regulates the orientation of epithelial 22. Brade T, Manner J, Kuhl M: The role of Wnt signalling in cardiac cell division and establishes the cortico-medullary axis of the mam- development and tissue remodelling in the mature heart. Cardiovasc malian kidney. Development 136: 161–171, 2009 Res 72: 198–209, 2006 47. Te´telin S, Jones EA: Xenopus Wnt11b is identified as a potential 23. Cohen ED, Tian Y, Morrisey EE: Wnt signaling: An essential regulator pronephric inducer. Dev Dyn 239: 148–159, 2010 of cardiovascular differentiation, morphogenesis and progenitor self- 48. Majumdar A, Vainio S, Kispert A, McMahon J, McMahon AP: Wnt11 and renewal. Development 135: 789–798, 2008 Ret/Gdnf pathways cooperate in regulating ureteric branching during 24. Riccomagno MM, Takada S, Epstein DJ: Wnt-dependent regulation metanephric kidney development. Development 130: 3175–3185, 2003 of inner ear morphogenesis is balanced by the opposing and sup- 49. Cha SW, Tadjuidje E, Tao Q, Wylie C, Heasman J: Wnt5a and Wnt11 porting roles of Shh. Genes Dev 19: 1612–1623, 2005 interact in a maternal Dkk1-regulated fashion to activate both canon- 25. Ohyama T, Mohamed OA, Taketo MM, Dufort D, Groves AK: Wnt ical and non-canonical signaling in Xenopus axis formation. Devel- signals mediate a fate decision between otic placode and epidermis. opment 135: 3719–3729, 2008 Development 133: 865–875, 2006 50. Lyons JP, Mueller UW, Ji H, Everett C, Fang X, Hsieh JC, Barth AM, McCrea 26. Freter S, Muta Y, Mak SS, Rinkwitz S, Ladher RK: Progressive restric- PD: Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and tion of otic fate: The role of FGF and Wnt in resolving inner ear binds Frizzled-6 CRD: Functional implications of Wnt/beta-catenin activity potential. Development 135: 3415–3424, 2008 in kidney epithelial cells. Exp Cell Res 298: 369–387, 2004 27. Jayasena CS, Ohyama T, Segil N, Groves AK: Notch signaling aug- 51. McNeill H: Planar cell polarity and the kidney. J Am Soc Nephrol 20: ments the canonical Wnt pathway to specify the size of the otic 2104–2111, 2009 placode. Development 135: 2251–2261, 2008 52. Park TJ, Mitchell BJ, Abitua PB, Kintner C, Wallingford JB: Dishevelled 28. Miller RK, McCrea PD: Wnt to build a tube: Contributions of Wnt controls apical docking and planar polarization of basal bodies in cili- signaling to epithelial tubulogenesis. Dev Dyn 239: 77–93, 2010 ated epithelial cells. Nat Genet 40: 871–879, 2008 29. Ueno N, Greene ND: Planar cell polarity genes and neural tube 53. Matakatsu H, Blair SS: Interactions between Fat and Dachsous and closure. Birth Defects Res C Embryo Today 69: 318–324, 2003 the regulation of planar cell polarity in the Drosophila wing. Devel- 30. Saxe´n,L: Organogenesis of the Kidney, New York, Cambridge Uni- opment 131: 3785–3794, 2004 versity Press, 1987 54. Goto T, Keller R: The planar cell polarity gene strabismus regulates 31. Davidson, AJ: Mouse kidney development. In: Stembook [Internet], convergence and extension and neural fold closure in Xenopus. Dev edited by Melton D, Girard L, Cambridge, MA, Harvard Stem Cell Biol 247: 165–181, 2002 Institute, 2008 55. Veeman MT, Slusarski DC, Kaykas A, Louie SH, Moon RT: Zebrafish 32. Bouchard M, Souabni A, Mandler M, Neubuser A, Busslinger M: prickle, a modulator of noncanonical Wnt/Fz signaling, regulates Nephric lineage specification by Pax2 and Pax8. Genes Dev 16: gastrulation movements. Curr Biol 13: 680–685, 2003 2958–2970, 2002 56. Carreira-Barbosa F, Concha ML, Takeuchi M, Ueno N, Wilson SW, 33. Dressler GR: The cellular basis of kidney development. Annu Rev Cell Tada M: Prickle 1 regulates cell movements during gastrulation and Dev Biol 22: 509–529, 2006 neuronal migration in zebrafish. Development 130: 4037–4046, 2003 34. Vize PD, Seufert DW, Carroll TJ, Wallingford JB: Model systems for the 57. Choi SC, Han JK: Xenopus Cdc42 regulates convergent extension study of kidney development: Use of the pronephros in the analysis of movements during gastrulation through Wnt/Ca2ϩ signaling path- organ induction and patterning. Dev Biol 188: 189–204, 1997 way. Dev Biol 244: 342–357, 2002 35. Jones EA: Xenopus: A prince among models for pronephric kidney 58. Heisenberg CP, Tada M, Rauch GJ, Saude L, Concha ML, Geisler R, development. J Am Soc Nephrol 16: 313–321, 2005 Stemple DL, Smith JC, Wilson SW: Silberblick/Wnt11 mediates con- 36. Chan T, Asashima M: Growing kidney in the frog. Nephron Exp vergent extension movements during zebrafish gastrulation. Nature Nephrol 103: e81–e85, 2006 405: 76–81, 2000 37. Brandli AW: Towards a molecular anatomy of the Xenopus proneph- 59. Kim GH, Han JK: JNK and ROKalpha function in the noncanonical ric kidney. Int J Dev Biol 43: 381–395, 1999 Wnt/RhoA signaling pathway to regulate Xenopus convergent exten- 38. Wingert RA, Davidson AJ: The zebrafish pronephros: A model to sion movements. Dev Dyn 232: 958–968, 2005 study nephron segmentation. Kidney Int 73: 1120–1127, 2008 60. Habas R, Dawid IB, He X: Coactivation of Rac and Rho by Wnt/ 39. Costantini F: Renal branching morphogenesis: Concepts, questions, Frizzled signaling is required for vertebrate gastrulation. Genes Dev and recent advances. Differentiation 74: 402–421, 2006 17: 295–309, 2003 40. Schmidt-Ott KM, Barasch J: WNT/beta-catenin signaling in nephron 61. Habas R, Kato Y, He X: Wnt/Frizzled activation of Rho regulates

J Am Soc Nephrol 22: 1654–1667, 2011 Daam1 Signals in Nephrogenesis 1665 BASIC RESEARCH www.jasn.org

vertebrate gastrulation and requires a novel Formin homology pro- 84. Schmidt-Ott KM, Masckauchan TN, Chen X, Hirsh BJ, Sarkar A, Yang J, tein Daam1. Cell 107: 843–854, 2001 Paragas N, Wallace VA, Dufort D, Pavlidis P, Jagla B, Kitajewski J, 62. Tanegashima K, Zhao H, Dawid IB: WGEF activates Rho in the Barasch J: Beta-catenin/TCF/Lef controls a differentiation-associated Wnt-PCP pathway and controls convergent extension in Xenopus transcriptional program in renal epithelial progenitors. Development gastrulation. Embo J 27: 606–617, 2008 134: 3177–3190, 2007 63. Chhabra ES, Higgs HN: The many faces of actin: Matching assembly 85. Schedl A: Renal abnormalities and their developmental origin. Nat factors with cellular structures. Nat Cell Biol 9: 1110–1121, 2007 Rev Genet 8: 791–802, 2007 64. Baarlink C, Brandt D, Grosse R: SnapShot: Formins. Cell 142: 172, 86. Habas R, Dawid IB: Dishevelled and Wnt signaling: Is the nucleus the 172.e1, 2010 final frontier? J Biol 4: 2, 2005 65. Faix J, Grosse R: Staying in shape with formins. Dev Cell 10: 693–706, 87. Wallingford JB, Habas R: The developmental biology of Dishevelled: 2006 An enigmatic protein governing cell fate and cell polarity. Develop- 66. Liu W, Sato A, Khadka D, Bharti R, Diaz H, Runnels LW, Habas R: ment 132: 4421–4436, 2005 Mechanism of activation of the Formin protein Daam1. Proc Natl 88. Roszko I, Sawada A, Solnica-Krezel L: Regulation of convergence and Acad SciUSA105: 210–215, 2008 extension movements during vertebrate gastrulation by the Wnt/PCP 67. Komiya Y, Habas R: Wnt signal transduction pathways. Organogen- pathway. Semin Cell Dev Biol 20: 986–997, 2009 esis 4: 68–75, 2008 89. Tada M, Kai M: Noncanonical Wnt/PCP signaling during vertebrate 68. Sato A, Khadka DK, Liu W, Bharti R, Runnels LW, Dawid IB, Habas R: Profilin gastrulation. Zebrafish 6: 29–40, 2009 is an effector for Daam1 in non-canonical Wnt signaling and is required for 90. Dale RM, Sisson BE, Topczewski J: The emerging role of Wnt/PCP vertebrate gastrulation. Development 133: 4219–4231, 2006 signaling in organ formation. Zebrafish 6: 9–14, 2009 69. Khadka DK, Liu W, Habas R: Non-redundant roles for Profilin2 and 91. Seifert JR, Mlodzik M: Frizzled/PCP signalling: A conserved mecha- Profilin1 during vertebrate gastrulation. Dev Biol 332: 396–406, 2009 nism regulating cell polarity and directed motility. Nat Rev Genet 8: 70. Nakaya MA, Habas R, Biris K, Dunty WC Jr, Kato Y, He X, Yamaguchi 126–138, 2007 TP: Identification and comparative expression analyses of Daam 92. Jenny A, Mlodzik M: Planar cell polarity signaling: A common mech- genes in mouse and Xenopus. Gene Expr Patterns 5: 97–105, 2004 anism for cellular polarization. Mt Sinai J Med 73: 738–750, 2006 71. Stewart MD, Jang CW, Hong NW, Austin AP, Behringer RR: Dual 93. Wallingford JB: Vertebrate gastrulation: Polarity genes control the fluorescent protein reporters for studying cell behaviors in vivo. matrix. Curr Biol 15: R414–R416, 2005 Genesis 47: 708–717, 2009 94. Curtin JA, Quint E, Tsipouri V, Arkell RM, Cattanach B, Copp AJ, 72. Vize PD, Jones EA, Pfister R: Development of the Xenopus proneph- Henderson DJ, Spurr N, Stanier P, Fisher EM, Nolan PM, Steel KP, ric system. Dev Biol 171: 531–540, 1995 Brown SD, Gray IC, Murdoch JN: Mutation of Celsr1 disrupts planar 73. Wallingford JB, Carroll TJ, Vize PD: Precocious expression of the polarity of inner ear hair cells and causes severe neural tube defects Wilms’ tumor gene xWT1 inhibits embryonic kidney development in in the mouse. Curr Biol 13: 1129–1133, 2003 Xenopus laevis. Dev Biol 202: 103–112, 1998 95. Montcouquiol M, Rachel RA, Lanford PJ, Copeland NG, Jenkins NA, 74. Mauch TJ, Yang G, Wright M, Smith D, Schoenwolf GC: Signals from Kelley MW: Identification of Vangl2 and Scrb1 as planar polarity trunk paraxial mesoderm induce pronephros formation in chick in- genes in mammals. Nature 423: 173–177, 2003 termediate mesoderm. Dev Biol 220: 62–75, 2000 96. Montcouquiol M, Sans N, Huss D, Kach J, Dickman JD, Forge A, 75. Agrawal R, Tran U, Wessely O: The miR-30 miRNA family regulates Rachel RA, Copeland NG, Jenkins NA, Bogani D, Murdoch J, War- Xenopus pronephros development and targets the transcription fac- chol ME, Wenthold RJ, Kelley MW: Asymmetric localization of Vangl2 tor Xlim1/Lhx1. Development 136: 3927–3936, 2009 and Fz3 indicate novel mechanisms for planar cell polarity in mam- 76. Saka Y, Smith JC: Spatial and temporal patterns of cell division mals. J Neurosci 26: 5265–5275, 2006 during early Xenopus embryogenesis. Dev Biol 229: 307–318, 2001 97. Wang J, Hamblet NS, Mark S, Dickinson ME, Brinkman BC, Segil N, 77. Schreiber AM, Cai L, Brown DD: Remodeling of the intestine during Fraser SE, Chen P, Wallingford JB, Wynshaw-Boris A: Dishevelled genes metamorphosis of Xenopus laevis. Proc Natl Acad SciUSA102: mediate a conserved mammalian PCP pathway to regulate convergent 3720–3725, 2005 extension during neurulation. Development 133: 1767–1778, 2006 78. Fang X, Ji H, Kim SW, Park JI, Vaught TG, Anastasiadis PZ, Ciesiolka M, 98. Wang J, Mark S, Zhang X, Qian D, Yoo SJ, Radde-Gallwitz K, Zhang Y, McCrea PD: Vertebrate development requires ARVCF and p120 catenins Lin X, Collazo A, Wynshaw-Boris A, Chen P: Regulation of polarized and their interplay with RhoA and Rac. J Cell Biol 165: 87–98, 2004 extension and planar cell polarity in the cochlea by the vertebrate PCP 79. Park JI, Ji H, Jun S, Gu D, Hikasa H, Li L, Sokol SY, McCrea PD: Frodo pathway. Nat Genet 37: 980–985, 2005 links Dishevelled to the p120-catenin/Kaiso pathway: Distinct catenin 99. Wang Y, Guo N, Nathans J: The role of Frizzled3 and Frizzled6 in subfamilies promote Wnt signals. Dev Cell 11: 683–695, 2006 neural tube closure and in the planar polarity of inner-ear sensory hair 80. Gu D, Sater AK, Ji H, Cho K, Clark M, Stratton SA, Barton MC, Lu Q, cells. J Neurosci 26: 2147–2156, 2006 McCrea PD: Xenopus delta-catenin is essential in early embryogen- 100. Qian D, Jones C, Rzadzinska A, Mark S, Zhang X, Steel KP, Dai X, esis and is functionally linked to cadherins and small GTPases. J Cell Chen P: Wnt5a functions in planar cell polarity regulation in mice. Sci 122: 4049–4061, 2009 Dev Biol 306: 121–133, 2007 81. Skouloudaki K, Puetz M, Simons M, Courbard JR, Boehlke C, Hartle- 101. Etheridge SL, Ray S, Li S, Hamblet NS, Lijam N, Tsang M, Greer J, ben B, Engel C, Moeller MJ, Englert C, Bollig F, Schafer T, Ram- Kardos N, Wang J, Sussman DJ, Chen P, Wynshaw-Boris A: Murine achandran H, Mlodzik M, Huber TB, Kuehn EW, Kim E, Kramer- dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 Zucker A, Walz G: Scribble participates in Hippo signaling and is in normal cardiac outflow tract, cochlea, and neural tube development. required for normal zebrafish pronephros development. Proc Natl PLoS Genet 4: e1000259, 2008 Acad SciUSA106: 8579–8584, 2009 102. Sive HL, Grainger RM, Harland RM: Early Development of Xenopus 82. Saulnier DM, Ghanbari H, Brandli AW: Essential function of Wnt-4 for laevis: A Laboratory Manual, Cold Spring Harbor, NY, Cold Spring tubulogenesis in the Xenopus pronephric kidney. Dev Biol 248: 13–28, Harbor Laboratory Press, 2000 2002 103. Brand M, Granato M, Nusslein-Volhard C: Keeping and raising ze- 83. Iglesias DM, Hueber PA, Chu L, Campbell R, Patenaude AM, Dziar- brafish, Oxford, NY, Oxford University Press, 2002 maga AJ, Quinlan J, Mohamed O, Dufort D, Goodyer PR: Canonical 104. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF: Stages of WNT signaling during kidney development. Am J Physiol Renal embryonic development of the zebrafish. Dev Dyn 203: 253–310, 1995 Physiol 293: F494–F500, 2007 105. Kloc M, Etkin LD: Analysis of localized RNA in Xenopus oocytes. In:

1666 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1654–1667, 2011 www.jasn.org BASIC RESEARCH

Advances in Molecular Biology: Comparative Methods Approach to beta2 subunit and in situ localization of alpha1, beta1, and gamma Study of Oocytes and Embryos, edited by Richter JD, New York, expression during pronephric kidney development. Differentiation Oxford University Press, 1999, pp 256–278 68: 115–125, 2001 106. Nieuwkoop PD, Faber J: Normal Table of Xenopus laevis (Daudin): A 116. Harland RM: In situ hybridization: An improved whole-mount method Systematical and Chronological Survey of the Development from the for Xenopus embryos. Methods Cell Biol 36: 685–695, 1991 Fertilized Egg Till the End of Metamorphosis, New York, Garland 117. Moody SA: Fates of the blastomeres of the 16-cell stage Xenopus Publishing, 1994 embryo. Dev Biol 119: 560–578, 1987 107. Kim SW, Fang X, Ji H, Paulson AF, Daniel JM, Ciesiolka M, van Roy 118. Moody SA: Fates of the blastomeres of the 32-cell-stage Xenopus F, McCrea PD: Isolation and characterization of XKaiso, a transcrip- embryo. Dev Biol 122: 300–319, 1987 tional repressor that associates with the catenin Xp120(ctn) in Xeno- 119. Strehlow D, Heinrich G, Gilbert W: The fates of the blastomeres of pus laevis. J Biol Chem 277: 8202–8208, 2002 the 16-cell zebrafish embryo. Development 120: 1791–1798, 1994 108. Barnett MW, Old RW, Jones EA: Neural induction and patterning by 120. Seufert DW, Brennan HC, DeGuire J, Jones EA, Vize PD: Develop- fibroblast growth factor, notochord and somite tissue in Xenopus. mental basis of pronephric defects in Xenopus body plan pheno- Dev Growth Differ 40: 47–57, 1998 types. Dev Biol 215: 233–242, 1999 109. Taira M, Jamrich M, Good PJ, Dawid IB: The LIM domain- 121. Kintner CR, Brockes JP: Monoclonal antibodies identify blastemal containing homeo box gene Xlim-1 is expressed specifically in the cells derived from dedifferentiating limb regeneration. Nature 308: organizer region of Xenopus gastrula embryos. Genes Dev 6: 67–69, 1984 356–366, 1992 122. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, 110. Carroll TJ, Vize PD: Synergism between Pax-8 and lim-1 in embryonic Schier AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever kidney development. Dev Biol 214: 46–59, 1999 W, Fishman MC: Early development of the zebrafish pronephros and 111. von Strandmann EP, Nastos A, Holewa B, Senkel S, Weber H, Ryffel analysis of mutations affecting pronephric function. Development GU: Patterning the expression of a tissue-specific transcription factor 125: 4655–4667, 1998 in embryogenesis: HNF1 alpha gene activation during Xenopus de- 123. Takeyasu K, Tamkun MM, Renaud KJ, Fambrough DM: Ouabain- velopment. Mech Dev 64: 7–17, 1997 sensitive (NaϩϩKϩ)-ATPase activity expressed in mouse L cells by 112. Gerth VE, Zhou X, Vize PD: Nephrin expression and three-dimen- transfection with DNA encoding the alpha-subunit of an avian so- sional morphogenesis of the Xenopus pronephric glomus. Dev Dyn dium pump. J Biol Chem 263: 4347–4354, 1988 233: 1131–1139, 2005 124. Mayor R, Morgan R, Sargent MG: Induction of the prospective neural 113. Vize PD: The chloride conductance channel ClC-K is a specific marker crest of Xenopus. Development 121: 767–777, 1995 for the Xenopus pronephric distal tubule and duct. Gene Expr Pat- terns 3: 347–350, 2003 114. Zhou X, Vize PD: Proximo-distal specialization of epithelial transport See related editorial, “Making a Tubule the Noncanonical Way,” on pages processes within the Xenopus pronephric kidney tubules. Dev Biol 1575–1577. 271: 322–338, 2004 115. Eid SR, Brandli AW: Xenopus Na,K-ATPase: Primary sequence of the Supplemental information for this article is available online at http://www.jasn.org/.

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