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Lineage Specification of Parietal Epithelial Cells Requires b-Catenin/Wnt Signaling

† ‡ Stephan Grouls,* Diana Margarita Iglesias, Nicolas Wentzensen,§ | ‡ Marcus Johannes Moeller, Maxime Bouchard,¶ Rolf Kemler,** Paul Goodyer, †† ‡‡ || Felix Niggli, Hermann-Josef Gröne, Wilhelm Kriz,§§ and Robert Koesters

*Department of Nephrology and †Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany; ‡Department of Pediatrics, McGill University-Montreal Children’s Hospital Research Institute, McGill University Health Centre Research Institute and McGill University, Montreal, Quebec, Canada; §Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, Maryland; |Department of Nephrology and Clinical Immunology, University Hospital of Rheinisch-Westfaelische Technische Hochschule Aachen University, Aachen, Germany; ¶Goodman Cancer Centre, Department of Biochemistry, McGill University, Montreal, Quebec, Canada; **Department of Molecular Embryology, Max Planck Institute for Immunobiology, Freiburg, Germany; ††Children’s Hospital, University of Zurich, Zurich, Switzerland; ‡‡Division of Cellular and Molecular Pathology, Deutsches Krebsforschungszentrum, Heidelberg, Germany; §§Ruperto-Carola University of Heidelberg, Anatomy and Developmental Biology, Medical Faculty Mannheim, Mannheim, Germany; and ||INSERM Unit 702, University of Paris 6, Hospital Tenon, Paris, France

ABSTRACT b-Catenin/Wnt signaling is essential during early inductive stages of development, but its role during postinductive stages of development and maturation is not well understood. In this study, we used Pax8Cre mice to target b-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional b-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated replaced parietal epithelial cells in Bowman’s capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional b-catenin knockout mice revealed that these “parietal podo- cytes” derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that b-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

J Am Soc Nephrol 23: 63–72, 2012. doi: 10.1681/ASN.2010121257

Development of the mammalian kidney is initiated Whereas the visceral epithelial cells become podo- by mutual interactions of the outgrowing ureteric cytes, parietal epithelial cells form the parietal layer bud and the metanephric blastema. The latter of Bowman´s capsule. induces branching of the leading to formation of the , whereas the tips of the ureteric bud induce epithelial trans- formation of the metanephric mesenchymal cells. Received December 12, 2010. Accepted August 9, 2011.

The induced cells of the metanephric blastema Published online ahead of print. Publication date available at aggregate to form renal vesicles and upon successive www.jasn.org. invaginations become S-shaped bodies, which give Correspondence: Mr. Stephan Grouls, University of Heidelberg, rise to several distinct cell types of the nephron Department of Nephrology, Im Neuenheimer Feld 162, 69120 including parietal and visceral glomerular epithelial Heidelberg, Baden-Württemberg, Germany. Email: stephan1.grouls@ cells. On invasion by blood vessels, parietal and med.uni-heidelberg.de visceral epithelial cells build up the . Copyright © 2012 by the American Society of Nephrology

J Am Soc Nephrol 23: 63–72, 2012 ISSN : 1046-6673/2301-63 63 BASIC RESEARCH www.jasn.org

Wnt molecules play important roles in the development of mice lacking Wnt7b.16 In contrast, the Pax8Cre-mediated many organs using different signaling pathways (reviewed in knockout of dkk1, an antagonist of Wnt signaling, resulted in Refs. 1, 2). Among these, the canonical pathway has attracted an overgrown papilla.17 This suggests that Wnt7b along with exceptional attention. Canonical Wnt signaling triggers stabi- dkk1 coordinate proper formation of the renal papilla in vivo. lization of b-catenin, its nuclear translocation, and the forma- The signaling mechanisms that control later stages of tion of active transcriptional complexes using members of the nephrogenesis such as differentiation of parietal and visceral T cell factor (TCF)/Lef family of transcription factors as bind- epithelial cells of the have not yet been defined. The ing partners.3 Using genetic mouse models, Wnt signaling has essential requirement for b-catenin during earlier stages of kid- been shown to be functionally involved in different aspects of ney development and the lack of appropriate genetic tools to (reviewed in Ref. 4). During renal vesicle target specifically later stages of nephrogenesis have prevented formation, Wnt9b expression in the ureteric bud is required any such analysis. In this study, we report the phenotype of for induction of the mesenchymal–epithelial transition of conditional b-catenin knockout mice in which Ctnnb1 (mouse metanephric blastemal cells.5 Similarly, metanephric blaste- b-catenin gene) was removed during later stages of nephrogen- mal cells of mice lacking Wnt4 do not undergo epithelial esis through the use of Pax8Cre deleter mice. The conditional transformation.6 Because Wnt4 can induce renal vesicle for- b-catenin knockout mice were found to be viable; however, re- mation in vivo and explant culture even in Wnt9b mutant nal function was impaired. Several kidney abnormalities were metanephric mesenchyme, it was suggested that Wnt4 acts observed including lack of the outermost cortex, presence of in an autocrine fashion downstream of the paracrine Wnt9b glomerular cysts, and underdeveloped glomerular capillary tufts. factor.5,7 The blockage of mesenchymal-to-epithelial transfor- Notably, podocytes were located in an abnormal parietal posi- mation results in rudimentary kidneys in both Wnt9b and tion replacing normal flat parietal epithelial cells. We conclude Wnt4 knockout mice. Secondary to the absence of nephro- that b-catenin/Wnt signaling is required for proper cell fate de- genesis, ureteric branching was also reduced, although to a cision of parietal epithelial cells and is responsible for maintain- larger extent in Wnt9b than in Wnt4 knockout mice.5,6 Both ing the gross architecture of the mouse renal corpuscle in vivo. Wnt9b and Wnt4 signaling activities may primarily involve canonical Wnt signaling. Park et al.8 used Six2Cre mice to remove b-catenin activity specifically from the cells of the RESULTS metanephric blastema. These b-catenin loss-of-function mu- tants lacked early and late markers of an inductive response, We have previously analyzed canonical Wnt signaling activity including Wnt4 expression; they did not form and during nephrogenesis using TCF/lacZ reporter mice and noted showed early cessation of ureteric branching thus mimicking that specific b-galactosidase expression was occasionally the phenotype of Wnt9b knockout mice.8 Furthermore, acti- found in a parietal position of developing glomeruli (Figure vation of a b-catenin gain-of-function mutation initiated a full 1A and Ref. 13). We therefore reasoned that activation of the inductive response in the metanephric mesenchyme even in a Wnt signaling pathway in parietal epithelial cells may be in- Wnt9b mutant background.8 Evidence that linked b-catenin volved in their differentiation. To examine a potential role for function to Wnt4 signaling was provided by the demonstra- b-catenin during late stages of nephrogenesis, mice with a tion of Lhx1 activation in Wnt4 mutants upon activation of floxed b-catenin allele18 were crossed with mice that express b-catenin in the metanephric mesenchyme.8 Lhx1 expression Cre recombinase under control of the Pax8 promoter.19 Using has previously been shown to be dependent on Wnt4.9 Wnt4 this approach, b-catenin deficiency can be targeted to the cells signaling during epithelial-to-mesenchymal transformation of the late S-shaped body stage and ureteric bud cells, whereas may also involve noncanonical calcium/nuclear factor of ac- cells of the metanephric blastema are not affected.19 We con- tivated T cells signaling.10,11 Canonical Wnt/b-catenin signal- firmed the known pattern of activity of Pax8Cre mice by using ing has also been implicated in ureteric branching directly. b-galactosidase staining of embryonic kidneys derived from dou- b-Catenin reporter genes in transgenic mice have shown ble transgenic Pax8Cre/Rosa26 lacZ reporter mice (Figure 1B). strong activity in the tips and stalks of the branching ureteric Both visceral and parietal epithelial cells of maturing glomeruli as bud.12,13 Using Hoxb7Cre mice to remove b-catenin from the well as ureteric bud cells revealed Pax8Cre activity (Figure 1B). developing ureteric bud, it was found that b-catenin is re- Next, we used Pax8Cre mice to remove b-catenin from quired for ureteric branching.14,15 Finally, Wnt7b/b-catenin developing kidneys. b-Catenin protein deletion was wide- signaling has been shown to direct collecting duct growth spread in the adult kidneys of b-catenin–deficient mice (lox/ and extension of the in the medullary zone.16 lox Pax8Cre) (Figure 1, C–H). Collecting duct, proximal and When Wnt7b was removed from the collecting duct system by distal tubular epithelial cells, and parietal epithelial cells were Sox2Cre-mediated recombination, the failed to essentially negative for b-catenin expression. Only individual form. However, in this case, the ligand Wnt7b signals in a para- cells, scattered throughout the kidney in a mosaic fashion, had crine fashion to interstitial cells of the surrounding mesen- escaped the b-catenin knockout (Figure 1, D, F, and H). These chyme. Accordingly, removal of b-catenin from the interstitium findings prove the efficiency of the b-catenin knockout in our but not from the collecting duct mimicked the phenotype of system.

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We did not observe prenatal or perinatal lethality until 2 weeks of age. b-Catenin– deficient mice, however, showed signifi- cantly reduced survival time and kidney function (Supplemental Figure 1, A and B). Their kidneys appeared hypoplastic with the outermost cortex deleted and with the pres- ence of glomerular cysts (Supplemental Figure 1, C and D). The remaining tubular system presented the different segments with intact mor- phology. The proximal tubules consisted of a regular columnar epithelium with clearly detectable brush borders in periodic –Schiff (PAS) staining and electron microscopy (data not shown). The loop of Henle and distal tubules were morpho- logically normal, and connecting tubules and collecting ducts were positive for aqua- porin 2 expression (Figure 1, E–H). In b-catenin–deficient mice, most of the glomeruli had an abnormal morphology (Figure 2, A and B). They frequently showed underdeveloped capillary tufts and cystic dilation of Bowman’scapsule, which was properly connected to the prox- imal (Figure 2B). Notably, juxtame- dullary glomeruli, which represent the earliest developing nephrons, mostly ap- peared normal (Figure 2A). A striking phenotype at the cellular level was the regular presence of podocytes in a parietal position (Figure 2C). At the ultra- structural level, these parietal podocytes were fully differentiated and apparently

and b-catenin–deficient (lox/lox Pax8Cre) mice at 3 months of age. (E) Control kidneys show b-catenin in all tubular epithelial cells and parietal epithelial cells (arrow) at the ba- solateral surface. 2 is detected at Figure 1. TCF and Pax8Cre activity are found in presumptive parietal epithelial cells during the apical surface of collecting duct cells. glomerulogenesis. (A) Enzymatic b-galactosidase staining of embryonic kidneys (embryonic UP, urinary pole. (F) In kidneys of b-catenin– day 15.5 [E15.5]) derived from TCF/lacZ reporter mice. Specific b-galactosidase expression deficient mice, b-catenin is efficiently removed in is seen in parietal epithelial cells of maturing glomeruli. (B) Enzymatic b-galactosidase nearly all epithelial cells. (arrow = parietal epi- staining of cryosections of embryonic kidneys (E15.5) derived from Pax8Cre/Rosa26R mice. thelial cells). A few epithelial cells show persistent b-Galactosidase positivity is seen in all epithelial cells of the kidney including cells of the b-catenin expression (arrowhead). (G) Aquaporin S-shaped body (SS), collecting duct (CD), visceral epithelial cells (VECs), and parietal epi- 2 positive tubules in control kidneys show strong thelial cells (PECs) of a maturing glomerulus and tubules (T). Cells of the metanephric b-catenin expression. (H) In b-catenin–deficient blastema (MB), interstitial cells, and blood vessels are negative. (C and D) Immunostaining mice, expression is similar in distri- for b-catenin in control (lox/lox) and b-catenin–deficient (lox/lox Pax8Cre) mice at 3 months bution and intensity compared with those in of age. (C) Control kidneys show b-catenin expression in all tubular epithelial cells; podo- control mice. A few epithelial cells show persis- cytes are negative. (D) In kidneys of b-catenin–deficient mice, b-catenin is efficiently re- tent b-catenin expression (arrowhead). Original moved in all cells. Only a few single cells show persistent positive staining for b-catenin magnifications: 3500 in A; 3125 in B; 3500 in (arrows). (E–H) Double staining for aquaporin 2 (red) and b-catenin (green) in control (lox/lox) CandD;3750 in E–H.

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Figure 2. b-Catenin–deficient mice show abnormal glomeruli featuring parietal podocytes, parietal capillaries, and underdeveloped capillary tufts. Semithin sections (A and B) and electron microscopy (C–H) of adult b-catenin–deficient (lox/lox Pax8Cre) kidneys. (A) The outermost cortex is missing. Glomeruli show underdeveloped capillary tufts and are cystically widened. Juxtamedullary glomeruli mostly appeared nor- mal. (B) The glomeruli have a normal tubular pole (TP). (C) Electron microscopy reveals that the parietal epithelial cells are parietal podocytes (PP) with foot processes extending and covering Bowman’s capsule. Capillaries (asterisks) are arranged on the outer aspect of Bowman’scapsule adjacent to parietal podocytes. VP, visceral podocytes. (D) Parietal podocytes (PP) and foot processes at higher magnification. Large parietal capillaries (PC) have formed adjacent to the parietal podocytes. (E) Parietal podocytes form a filtration-like barrier consisting of three layers: fenestrated endothelial cells (FE), a parietal (BM), and foot processes (FP). The filtration slits between the foot pro- cesses are bridged by a slit membrane (arrows), and the endothelial fenestrae are bridged by diaphragms (arrowheads). (F) Parietal capillaries (asterisks) associated with parietal podocytes; one capillary bulges into Bowman’s space (BS). (G) Bulging capillary with a well developed capillary neck (boxed area enlarged in I). (H) A schematic draft of the image of (G). Parietal podocytes (blue; PP) are covering the surface of a bulging parietal capillary (red; PC). Parietal basement membrane is dark blue. BS, Bowman´s space. (I) At the neck, the peripheral en- dothelial basement membrane reflects into the parietal basement membrane (white arrows); the bridging portion of the endothelium is underlain only by a thin and fragmented basement membrane (arrowheads). The cells within the neck region have many actin-filled processes (black arrows) that are connected to the basement membrane at the turning points. (J and K) Both visceral and parietal podocytes (arrows) stain positive for WT1. (L and M) Both visceral and parietal podocytes (arrows) stain positive for VEGF. (N and O) Double staining for synaptopodin (red) and WT1 (green). Synaptopodin in control kidneys is exclusively expressed in visceral podocytes. Parietal epithelial cells are negative for synaptopodin. In b-catenin–deficient mice, synaptopodin is expressed in both visceral and parietal podocytes (arrow). Original magnifi- cations: 3125 in A; 3500 in B, J, K, N, and O; 3570 in C; 33400 in D; 340,000 in E; 32000 in F; 36300 in G; 312,500 in I; 3750 in L and M. had attracted parietal capillaries (Figure 2D). A three-layered were stuffed with actin filaments, and they were connected to the capillary-to-urinary space barrier similar to the glomerular turning points of the peripheral endothelial basement mem- filtration barrier was formed. It consisted of a fenestrated cap- brane into the parietal basement membrane (Figure 2I). illary endothelium (the fenestrae were mostly bridged by dia- At the molecular level, parietal podocytes were found to be phragms) followed by a thick basement membrane consisting positive for Wilms’ tumor suppressor gene 1 (WT1) protein of a lamina rara interna, densa, and rara externa like the glomer- (Figure 2, J and K), vascular endothelial growth factor (VEGF) ular basement membrane at the tuft, finally by a layer of inter- (Figure 2, L and M), and synaptopodin (Figure 2, N and O). digitating foot processes with slit membranes indistin- Notably, parietal capillaries were in continuity with peri- guishable from those of visceral podocytes proper (Figure 2E). tubular capillaries (Figure 3A). Furthermore, affixation of the Occasionally, parietal capillaries protruded into Bowman´s space to the outer aspect of Bowman’scapsulewas (Figure 2, F,G, and H). Like glomerular capillaries within the tuft, frequently observed leading to efferent arterioles that ran parietal capillaries formed a capillary neck that was filled with alongside Bowman’scapsuleforhalf(orevenmore)ofits arborized cells reminiscent of mesangial cells. Their processes circumference (Figure 3B). As a consequence, the glomerular

66 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 63–72, 2012 www.jasn.org BASIC RESEARCH

b-catenin expression declined in presump- tive podocytes but remained high in parietal epithelial cells (Figure 4, B and C). Staining of b-catenin–deficient mice showed that b-catenin deficiency started in late S-shaped bodies and was complete in the maturing glomerulus stage (Figure 4, D and E) affect- ing presumptive parietal and tubular epithe- lial cells. We did not detect any change in E-cadherin and zonula occludens-1 protein (ZO-1) expression in b-catenin–deficient mice (Supplemental Figure 2, B and C). In control and b-catenin–deficient mice, vis- ceral and parietal epithelial cells did not ex- b – press E-cadherin in the S-shaped body stage. Figure 3. Parietal capillaries are in continuity with in -catenin fi deficient mice. (A) Superficial parietal capillary in continuity with a peritubular capillary (arrow). To trace podocyte-speci c differentia- T, tubule; G, glomerulus. (B) Glomerular profile with an efferent arteriole running alongside tion, we analyzed WT1 expression. At the the outer glomerular circumference (arrows). (C) Cross section through an efferent arteriole S-shaped body stage, WT1 was expressed in associated with parietal podocytes and Bowman’s capsule. Note the continuous endothelium both presumptive podocytes and cells of and the many profiles of smooth muscle cells (asterisks). G, glomerulus. (D) Glomerular profile Bowman’s capsule (Figure 4F). Whereas with a misshapen vascular pole. The afferent (a) and efferent (e) arterioles are widely sep- WT1 expression remained high in the po- arated; the vascular pole in between appears to be unorganized. (E) Cells of the proximal docyte lineage in control mice during later tubule (left) appear cuboidal with microvilli at the apical side forming the brush border. They stages of maturation, it faded out in the fl are densely packed with mitochondria. Parietal podocytes (right) are at and characterized cells of Bowman’s capsule (Figure 4G). In by foot processes. Between those cell types, there is always a morphologically different b-catenin–deficient mice, however, WT1 epithelial cell (EC), which has none of the morphological characteristics of the neighboring expression remained persistently high in cell types. Original magnifications: 3500 in A, B, and D; 35000 in C; 32500 in E. the parietal lineage throughout glomerulo- genesis (Figure 4G); also, VEGF was ex- vascular pole became misshapen with widely separated affer- pressed by these cells (Figure 4, H and I). Furthermore, electron ent and efferent arterioles. Parietal podocytes were not only microscopy revealed developing podocytes at the parietal cell associated with capillaries but also with arterioles (Figure 3C). layer during S-shaped body maturation from early S-shaped A slim glomerular stalk was lacking; instead, capillaries were body to later stages (Figure 4, J–L). Those presumptive podo- pushed into Bowman’s space at several sites within the ex- cytes attracted capillaries already at these early stages (Figure 4, panded pole (Figure 3D). Notably, at the urinary pole parietal K and L). In maturing glomeruli, developing parietal podocytes podocytes and proximal tubular cells were separated by a dis- formed foot processes and attracted capillaries (Figure 4, tinct cell (Figure 3E) that had none of the morphologic char- M and N). Using TUNEL staining, we did not observe parietal acteristics of the neighboring cells. epithelial cells undergoing apoptosis (data not shown). The origin of parietal podocytes can be explained by at least Together, our findings demonstrate a switch in lineage two different scenarios. Either the precursors of the parietal differentiation of parietal epithelial cells toward a podocyte- epithelial cells have chosen a different (visceral-like) cell fate or like cell fate when precursor cells of the late S-shaped body stage parietalepithelialcellsdiedandtheparietalcompartmenthasbeen become deficient for b-catenin expression. Secondary changes subsequently invaded by visceral epithelial cells. To address this included underdeveloped glomerular tufts and substantial question,westudiedearlyglomerulogenesis.Histologicanalysisof reorganization of Bowman’s capsule by adopting a glomerular- developing metanephros showed normal progression of nephro- like architecture apparently orchestrated by parietal podocytes. genesis at all stages from comma-shaped bodies to capillary loop stages until neonatal day 6. From neonatal day 6 on, however, we observed immature cells at the outer cortex in the kidneys of DISCUSSION b-catenin–deficient mice. The glomerular anlagen of the last generations of developing nephrons were unusually organized As has previously been shown, the mesenchymal–epithelial with cells at the parietal cell layer that were morphologically sim- transition of metanephric blastemal cells, a key step during ilar to presumptive visceral podocytes (Supplemental Figure 2A). nephron development, is strictly dependent on Wnt4 expres- Using immunohistochemistry, we assayed b-catenin ex- sion in cis and Wnt9b expression in trans (from the ureteric pression in embryonic kidneys. In the renal vesical stage, bud). Conditional knockout experiments have demonstrated b-catenin expression was found in all epithelial cells including that b-catenin is the likely intracellular mediator of these ef- ureteric bud (Figure 4A). During maturation of the glomeruli, fects. Because nephrogenesis was completely blocked at very

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Figure 4. Parietal podocytes develop directly from the parietal layer of S-shaped bodies in b-catenin–deficient mice. (A) Immunostaining for b-catenin reveals b-catenin expression in all cells of the comma-shaped body in both control (lox/lox) and b-catenin–deficient (lox/lox Pax8Cre) mice. (B and C) At the S-shaped body stage, b-catenin is expressed in precursor cells of the tubules and the parietal epithelial cells (PECs) (arrowheads) but only weakly in visceral epithelial cells (VECs) (asterisks) (boxed areas enlarged in C). There is no difference between control and b-catenin–deficient mice at this stage. (D and E) In maturing glomeruli, b-catenin staining is only observed in PECs (arrowheads) of control mice. VECs (asterisks) are neither positive in control nor in b-catenin–deficient mice (boxed areas enlarged in E). (F) At the S-shaped body stage, WT1 stains positive in both VECs (asterisks) and PECs (arrowheads). (G) In maturing glomeruli of control mice, WT1 expression is still observed in VECs (asterisks) but not in PECs (arrowheads). In b-catenin–deficient mice, both VECs and PECs remain positive for WT1 expression. (H) At the S-shaped body stage, VECs (asterisks) are strongly positive for VEGF, whereas PECs (arrowheads) are only weakly positive. (I) In maturing glomeruli, positive VEGF staining is observed in VECs (asterisks) but not in PECs (arrowheads) in control mice. In b-catenin–deficient mice, both VECs and PECs remain positive for VEGF. (J through N) Transmission electron micrographs of developing glomeruli. (J–L) S-shaped body stage. (M and N) Capillary loop stage. At the S-shaped body stage, presumptive PECs (asterisks) of control mice are flat (J), whereas the presumptive PECs from b-catenin–deficient mice (K and L) are columnar in shape (as- terisks) and associated with capillaries (arrows). At the maturing glomerulus stage from a b-catenin–deficient mouse (M) (accompanied by a schematic draft in N), PECs have developed foot processes lining up along the parietal basement membrane (blue in the scheme) as have the VECs on the glomerular basement membrane (red in the scheme). Large capillaries (lumina are orange in the scheme) are found at the outer side of the parietal basement membrane. E18.5. Original magnifications: 3750inA–I; 35000 in J; 32000 in K and L; 31500 in M.

68 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 63–72, 2012 www.jasn.org BASIC RESEARCH early stages, however, no functional kidneys were formed in The parietal podocytes observed in our model were highly these previous studies. Our study was designed in such a way differentiated cells and almost indistinguishable from their that b-catenin was removed from developing kidneys at later visceral counterpart. At least three different possible scenarios stages only. This was achieved by a specific Cre-deleter strain, may explain the origin of these parietal podocytes. Failure of Pax8Cre, which is active in late S-shaped bodies and develop- normal parietal epithelial cells to develop and/or premature ing collecting ducts but not within the metanephric blastema death followed by invasion of podocytes from the visceral pole or the branching ureteric bud tips. This approach allowed us into the unoccupied parietal compartment may provide one to study the role of b-catenin during late nephrogenesis and possible explanation. Second, transdifferentiation of mature nephron maturation. parietal epithelial cells into podocytes may have occurred, or, As expected, early steps of nephron development occurred third, a switch in cell fate decision from a parietal to a visceral normally in conditional b-catenin–deficient mice. In later lineage may have happened during early nephrogenesis. Strong steps, characteristic deficiencies were found. The main find- evidence has been compiled from our studies in support of the ings were a virtually normal collecting duct system demon- latter. There was no loss of parietal cells apparent during early strating no absolute requirement for b-catenin in maintaining nephron development, and we could exclude transdifferentia- this compartment, a normal although shortened tubular sys- tion of mature parietal epithelial cells because podocyte-specific tem along with a lacking outermost cortex, and, most prom- differentiation could be traced back to a parietal position at inently, abnormal glomeruli with underdeveloped capillary the earliest stages possible. Therefore, one can conclude that tufts and transformation of parietal epithelial cells into podo- b-catenin is required in prospective parietal epithelial cells cytes. Whereas the lack of a superficial tubular layer and an for proper differentiation and maturation. In the absence overall shortening of the tubular system might reflect an of b-catenin/Wnt signaling, however, prospective parietal intrinsic requirement for b-catenin for tubular cell proliferation, epithelial cells acquire the phenotype of visceral epithelial it could equally well result from the glomerular maldevelopment cells, which seems to represent the default pathway of differ- indicating simply the lack of downstream growth-promoting entiation in these closely related cell types (Figure 5). signals from the glomerulus to the tubule. It is unclear why It is interesting to note that b-catenin–deficient mice the earlier generations of nephrons were not affected in a similar showed a reduction but not a complete absence of renal func- way, but similar observations have previously been made in a tion. We think that, in addition to the intact juxtamedullary conditional knockout model of bone morphogenic protein-7.20 glomeruli, the abnormal midcortical and superficial glomeruli The prominent glomerular phenotype is difficult to un- contributed to the overall filtration rate at a significant rate. derstand in terms of simple b-catenin deficiency. First, endo- It is interesting to discuss the potential functional role of thelial cells are not targeted by Pax8Cre activity, which is parietal podocytes, which constituted a significant proportion specific for epithelial cells. Second, although podocytes will of podocytes in this model. Parietal podocytes apparently had become recombined by Pax8Cre activity, because they lack attracted capillaries and mesangial-like cells toward their b-catenin expression they are unlikely to become phenotypi- outer aspect mimicking formation of glomerular capillaries. cally changed. Accordingly, no phenotype has been observed These findingssuggestthatformationofthefiltration barrier in a podocyte-specific knockout model of b-catenin.21 Hence, and capillary loops may be entirely orchestrated by podocytes. the observed growth retardation of the capillary tuft can only Considering the likely differences in transmural pressure gra- be explained by indirect scenarios such as a distorted VEGF dients between tuft and parietal capillaries (blood pressure gradient caused by ectopic production of VEGF in the periph- in parietal capillaries should be much too low to generate ery of Bowman’s space, by altered glomerular flow caused by an effective filtration pressure) and the different morphology the parietal capillaries, or gross architectural constraints re- of parietal capillaries (endothelial fenestrae having dia- sulting from the inappropriate anchoring of the tuft to the phragms), it is unlikely that parietal capillaries contribute to extraglomerular mesangium. Alternatively, as it has been filtration at all. Whereas fenestrated capillaries without dia- shown previously,22 asignificant proportion of podocytes is phragms constitute just a weak barrier, fenestrated capillaries recruited from the parietal cell compartment. As shown in this with diaphragms will form a much stronger barrier. The reason study, inactivation of b-catenin replaced parietal cells by po- why visceral capillaries develop fenestrae without diaphragms docytes, which can no longer undergo cellular division. There- but parietal capillaries develop fenestrae with diaphragms is fore, it can be speculated that only insufficient numbers of unclear. podocytes could be recruited onto the glomerular tuft, be- The close developmental relationship of parietal and cause the cellular pools on Bowman’s capsule were depleted visceral epithelial cells becomes independently evidenced by prematurely. Insufficient recruitment of cells then resulted in a the complete switch in cell fate decision seen in the b-catenin hypoplastic but intact capillary tuft. It can also be speculated knockout mice presented in this study. that aberrant VEGF secretion by parietal podocytes in the In the current study, we showed that Pax8Cre-directed periphery indirectly disturbed the gradient of VEGF seen by knockout of b-catenin leads to developmental defects affect- glomerular endothelial cells thereby negatively interfering ing the gross architecture of glomeruli. A reduced number of with directed capillary outgrowth. functional nephrons may lead to chronic renal disease and

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Animal Housing We housed mice in groups of up to five. They received regular laboratory chow, and was supplied ad libitum. We maintained a 12-hour day and night cycle. For timed pregnancies, it is assumed that the morning of plug detection cor- responds with embryonic day 0.5. Embryo ages are rounded to the nearest half day.

Genotyping For the identification of the b-catenin alleles (RM41/RM42 primers), the Pax8Cre trans- gene (LC-1 primers), and Rosa26R transgene (Rosa26R primers), DNA was isolated from tail biopsies of adults and embryos. After lysis in buffer containing proteinase K, genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen GmbH) and dissolved in TE buffer; 0.5 mg of genomic DNA was used for PCR. The PCR conditions were set as follows: 1 Figure 5. The cell fate decision of parietal epithelial cells of the glomerulus is dependent cycle of 3 minutes at 94°C; 35 cycles of 30 sec- – on b-catenin/Wnt signaling. b-Catenin/Wnt signaling directs proper differentiation of onds at 94°C, 30 seconds at 52°C 60°C, 30 sec- prospective parietal epithelial cells. In the absence of b-catenin/Wnt signaling, parietal onds at 72°C; 1 cycle of 7 minutes at 72°C. epithelial precursor cells switch to the visceral, podocyte-specific cell fate and become b-Catenin primers were as follows: RM41, 59- parietal podocytes (PPs). DT, distal tubule; PT, ; VECs, visceral epithelial AAGGTAGAGTGATGAAAGTTGTT-39;RM42, cells; PECs, parietal epithelial cells. 59-CACCATGTCCTCTGTCTATTC-39; gener- ating 324-bp and 221-bp products from the eventually to renal failure. These findings prove an important floxed and wild-type alleles. LC-1 primers were as follows: Cre3, role for b-catenin during late stages of nephrogenesis. 59-TCGCTGCATTACCGGTCGATGC-39;Cre4,59-CCATGAGT- The frequent occurrence of parietal podocytes suggests that GAACGAACCTGGTCG-39. Rosa26-R primers were as follows: b-catenin is involved in the cell fate decision of parietal epi- Rosa26R-fwd1, 59-AAAGTCGCTCTGAGTTTGTTAT-39;Rosa26R- thelial cells. rev1, 59-GCGAAGAGTTTGTCCTCAACC-39.

b-Galactosidase Staining CONCISE METHODS To reveal b-galactosidase activity, we prepared dissected tissues. We washed the samples in PBS, fixed them in ice-cold 3% paraformal- Transgenic Mice dehyde for 1 hour, washed them in PBS again, and incubated them Animal experimental procedures were performed according to overnight in 18% sucrose in PBS at 4°C. The next day, we froze the German and European Legislation and approved by the Governing samples on dry ice and stored them at –80°C. We incubated 5-mm Office for Baden-Württemberg (Regierungspräsidium, Karlsruhe, cryosections overnight in the dark in 5-bromo-4-chloro-3-indolyl Germany). b-D-galactopyranoside (X-gal) solution (50 mM Tris HCl pH 7.5, 18 Mice carrying homozygote floxed alleles of the b-catenin gene (in 2.5 mM ferriferrocyanide, 15 mM NaCl, 1 mM MgCl2, fl fl this study referred to as b-catenin ox/ ox) were interbred with Pax8Cre and 0.5 mg/ml X-gal). We counterstained cryosections with eosin mice19 generating mice heterozygous for Pax8Cre and b-cateninflox and mounted them in glycerol–gelatin. fl fl (in this study referred to as Pax8catenin ox/wt). Pax8catenin ox/wt mice flox/flox and b-catenin mice were interbred again creating mice heterozy- Electron Microscopy gous for Pax8Cre and homozygous for b-cateninflox (in this study re- The mice were fixed by retrograde total body perfusion-fixation as ferred to as b-catenin–deficient mice [lox/lox Pax8Cre]). Littermates previously described24 using 3% glutaraldehyde, 0.1 M cacodylate, were used as controls (in this study referred to as lox/lox). Rosa26R is a and 0.1% picric acid as fixative. We processed tissue by standard Cre reporter line in which a gene encoding cytosolic b-galactosidase is procedures and embedded it into EPON. We cut 1-mm sections under control of the ubiquitously active Rosa26 promoter.23 However, with an Ultracut microtome (Leica). Semithin sections were stained expression is conditional on the prior removal of a neomycin expression with methylene blue25 and examined with light microscopy; ultrathin cassette that is flanked by loxP sites. Rosa26R mice were used as Cre sections were stained with uranyl acetate and lead citrate and studied recombinase reporter mice. with transmission electron microscopy.

70 Journal of the American Society of Nephrology J Am Soc Nephrol 23: 63–72, 2012 www.jasn.org BASIC RESEARCH

For immunocytochemistry, hematoxylin and eosin staining, PAS ACKNOWLEDGMENTS staining, we perfused mice anterograde with 3% paraformaldehyde (PFA)inPBS and immersedthe organsinthe same fixative for 2–4 hours We thank Jeanette Wei§, Hiltraud Hosser, Brunhilde Hähnel, Claudia before embedding them in paraffin. We performed hematoxylin and Schmidt, and Charlotte Holler for technical assistance and the Nikon eosin staining, PAS staining, and TUNEL staining according to standard Imaging Center at the University of Heidelberg for their support. protocols. Ellen Krautkrämer contributed valuable critique. Immunocytochemical detection of b-catenin (polyclonal mouse Part of this work was presented in abstract form at the ISN–Nature anti-human b-catenin antibody; 1:100; BD Transduction Laborato- Genetics Forefronts Symposium on Nephrogenetics—from Devel- ries, BD Biosciences), wt1 (polyclonal rabbit anti-human WT-1 an- opment to Physiology, March 8–11, 2007, Danvers, Massachusetts, tibody; 1:100; Santa Cruz Biotechnology, Heidelberg, Germany), and at the 6th International Renal Tumours Biology Meeting, March VEGF (polyclonal goat anti-human VEGF antibody; 1:140; 13–14, 2008, Chamonix, France. R&D Systems), and E-cadherin (monoclonal mouse, anti-human E-cadherin c-terminal recombinant protein antibody; 1:25; BD Transduction Laboratories, BD Biosciences) was performed on DISCLOSURES 4-mmparaffin sections of PFA-perfused tissue according to stan- None. dard protocol. For detection, we applied Vectastain Elite ABC-Kit Peroxidase (VectorLabs) and diaminobenzidine as substrate–chromogen (Vector Labs). Immunofluorescence of b-catenin (monoclonal REFERENCES mouse anti-mouse b-catenin antibody; 1:20; BD Transduction Laboratories, BD Biosciences), aquaporin 2 (polyclonal goat anti- 1. Clevers H: Wnt/beta-catenin signaling in development and disease. human aquaporin 2 antibody;1:800; Santa Cruz Biotechnology), Cell 127: 469–480, 2006 ZO-1 (polyclonal rabbit anti–ZO-1 antibody; 1:100; Invitrogen, 2. Veeman MT, Axelrod JD, Moon RT: A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 5: Karlsruhe, Germany), and synaptopodin (goat anti-synaptopodin 367–377, 2003 P-19 antibody; 1:100; Santa Cruz Biotechnology) was performed 3. 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