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First published online December 7, 2008
doi: 10.1242/10.1242/dev.022087

Development 136, 161-171 (2009)
Published by The Company of Biologists 2009
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A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney

Jing Yu1,*,{ddagger}, Thomas J. Carroll1,{dagger}, Jay Rajagopal1, Akio Kobayashi1, Qun Ren2 and Andrew P. McMahon1,{ddagger}

1 Department of Molecular and Cellular Biology and Harvard Stem Cell Institute, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
2 Department of Cell Biology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908, USA.


Figure 1
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  		Fig. 1. Wnt7b is essential for the development of the medullary component of the mouse kidney. (A-C) In situ hybridization analysis of Wnt7b expression in the developing mouse kidney. Prior to ureteric bud outgrowth, Wnt7b mRNA was expressed at low levels in the mesonephric/Wolffian duct (WD) epithelium (A). After ureteric bud invasion into the metanephric mesenchyme, Wnt7b expression was restricted to the ureteric trunk epithelium (B) and its derivatives, the collecting duct epithelium and ureter of the kidney (C). Expression was not observed at the ureteric tips (boundary demarcated by dashed lines in B). Scale bars: 100 µm in A,B; 400 µm in C. (D-M) Hematoxylin and Eosin staining of kidney sections from wild-type littermates (D,F,H,J,L) and Wnt7b mutants (E,G,I,K,M) as indicated. The renal medullary compartment (bracketed) was first evident at E15.5 in wild-type embryos, but was absent from mutants at this and all later stages. The renal pelvis appeared normal (F and G, insets). Arrows in I and K point to renal corpuscles adjacent to the pelvic space. Arrows in panels H and J point to renal corpuscles that lie above the renal medulla. The P10 ureteric epithelium-specific Wnt7b mutants (M) exhibited hydroureter and hydronephrosis. Scale bars: 200 µm.

 

Figure 2
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  		Fig. 2. Development of the renal cortex and patterning of the ureteric bud epithelium are normal in Wnt7b mutants. In situ hybridization to E15.5 kidney sections to examine branching morphogenesis (Wnt11), nephrogenesis (Wnt4), cortical stroma progenitors (Foxd1), nephron number and nephron patterning (Gsh1 for podocytes, Slc34a1 for proximal convoluted tubules, and Slc12a3 for distal convoluted tubules) indicates that these processes were unaffected in Wnt7b mutants. The expression of Wnt11 at the branching tips of the ureteric epithelium, Wnt9b in the non-tip ureteric epithelium, Wnt7b in the more-distal ureteric epithelium, renal pelvic epithelium and ureter, Foxa1 in the prospective medullary collecting ducts, renal pelvic epithelium and ureter, and Foxi1 in intercalated cells of the maturing collecting duct demonstrate normal stratification and differentiation of the ureteric epithelium in Wnt7b mutants. The Wnt7b riboprobe was generated from part of the exon 4 sequence and thus detected Wnt7b mRNA signals in both control and Wnt7b mutant kidneys. Scale bar: 200 µm.

 

Figure 3
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  		Fig. 3. Morphogenesis of the distal collecting duct epithelium is disrupted in Wnt7b mutants. (A) The organization of the developing collecting duct network was visualized through ureteric epithelium-specific, histochemical staining for E. coli β-galactosidase activity in wild-type kidneys from Hoxb7Cre;R26R embryos. Data represent thick (150-300 µm) vibratome sections at the stages indicated and are schematized in the panel below. Arrows highlight a swelling at the intersection between the ureter and the collecting duct epithelium; arrowheads indicate the triangular-shaped renal pelvis. (B) An analysis of freshly dissected kidneys showed that the ureter was not dilated in Wnt7b mutants at E15.5. (C) By contrast, the collecting duct epithelium was dilated in the prospective medullary region of Wnt7b mutants at E15.5 when the R26R reporter was activated in the context of the collecting duct network of the Wnt7b mutant kidney. Arrows indicate prospective medullary collecting ducts.

 

Figure 4
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  		Fig. 4. The failure to initiate renal medulla development in Wnt7b mutants reflects alterations in the plane of epithelial cell division. (A) Cell proliferation was analyzed in wild-type and Wnt7b mutant kidneys at E15.5 by pulse-labeling with BrdU. BrdU incorporation was visualized by anti-BrdU antibodies in conjunction with specific markers, as indicated, to identify specific epithelial compartments. E-Cadherin (E-cad) and cadherin 6 (Cdh6) immunostainings together with DBA lectin demarcate the cell boundaries between the nephron and collecting duct epithelia. No significant difference in the proliferation rate is observed between wild-type (n=3) and Wnt7b mutants (n=4). Scale bar: 40 µm. (B) Diagrams illustrating the effects of oriented cell division on tubule morphogenesis and three-dimensional reconstructions of optical sections showing mitotic configurations in the collecting duct epithelium. Anaphase chromosomes were visualized with phospho-histone H3 staining (PH3, green, arrow) and the collecting epithelium with pan-cytokeratin staining (red; A'-D'). Stacks of single optical sections (A',B') were rendered to generate 3D images (C',D') for the measurement of mitotic angles. (C) Histogram of mitotic angles in the prospective medullary collecting ducts of wild-type and Wnt7b mutant kidneys at E15.5. Mitotic angles were determined relative to the longitudinal axis of the perspective collecting duct epithelium delineated with pan-cytokeratin immunostaining as detailed in the Materials and methods. (D) 3D reconstruction of stacks of optical sections of acetylated {alpha}-tubulin staining demonstrate that normally sized primary cilia (arrow) were present in the collecting duct epithelia of Wnt7b mutants. Moreover, the intraflagellar transport protein Polaris localized to the primary cilium as expected. (E) Apoptosis was compared by TUNEL analysis (red) in the collecting duct epithelium (DBA lectin, green) of kidney sections from wild-type and Wnt7b mutant embryos at E17.5. The apoptosis rate increased markedly and significantly in Wnt7b mutants (n=4) relative to in wild-type controls (n=4).

 

Figure 5
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  		Fig. 5. Wnt7b is essential for coordinated growth of the loop of Henle, a medullary localized component of the developing nephron. (A,B) The loop of Henle was visualized in E18.5 kidney vibratome sections by immunostaining for Uromodulin (Umod, red). Sections are counterstained with DBA (green) to visualize the ureteric bud epithelium. The loop of Henle was greatly reduced in mutant kidneys compared with in wild-type littermates. (C,D) Measurement of BrdU incorporation in the loop of Henle anlagen at E15.5. The loop of Henle was identified as a U-shaped tubule in serial sections within the deeper cortex beneath the outer cortex where S-shaped bodies were localized. Loops of Henle of similar lengths were compared in the wild-type and mutant. (E,F) Measurement of BrdU incorporation in the S-shaped body (white arrows) at E15.5. (G) Cell proliferation was greatly reduced in the loop of Henle anlagen of mutants (P<0.0026). By contrast, no difference was observed between mutant and wild-type littermates at the S-shaped body stage (P<0.3172). S-shaped bodies were identified by immunostaining as being positive for E-cadherin (E-cad) and Cadherin 6 (Cdh6), but negative for DBA. The loop of Henle was positive for Cdh6 but negative for DBA. Scale bars: in B, 400 µm for A and B; in F, 40 µm for C-E.

 

Figure 6
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  		Fig. 6. Wnt7b signals to the adjacent interstitium through the canonical Wnt signaling pathway. (A) Hematoxylin and Eosin staining of E18.5 kidney sections following the removal of β-catenin activity from the renal interstitial mesenchyme. Mutant kidneys lack a renal medulla. Scale bar: 400 µm. (B) In situ hybridization (Axin2) and immunostaining (Lef1) on sections of E15.5 kidneys. A marked reduction was observed in Axin2 mRNA and Lef1 protein expression within the interstitial mesenchyme positioned adjacent to the nascent medullary collecting ducts (arrows) following removal of β-catenin activity from this cell population. By contrast, their expression was not altered in other renal tissues. Cyto, cytokeratin. Scale bars: 200 µm in top four panels; 100 µm in all other panels.

 

Figure 7
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  		Fig. 7. p57Kip2 expression in mesenchyme cells of the medullary interstitium is dependent on a Wnt7b canonical signaling pathway. (A-T) The distribution of p57Kip2 was compared in E15.5 kidney sections from wild-type (A-E,K-O), Wnt7b mutants (F-J) and mutants lacking β-catenin within the interstitial mesenchyme (P-T). p57Kip2 protein was present in a subset of medullary interstitium within wild-type kidneys (nuclear staining in C,M). The interstitial expression of p57Kip2 was lost in this population in Wnt7b mutants (H), but was retained in podocytes (asterisk in H). By contrast, Hoxa11 was present throughout the renal interstitium of wild-type (B) and Wnt7b mutant (G) kidneys. When p57Kip2 was examined in interstitial cells of β-catenin interstitium mutants (β-galactosidase-positive from genetic labeling), p57Kip2 was not expressed in interstitial mesenchyme (R), except in rare cases where cells had escaped recombination (β-galactosidase-negative cells in insets in Q-S). Scale bar: 40 µm.

 

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