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First published online September 2, 2003
doi: 10.1242/10.1242/dev.00697

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{gamma}-Secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney

Hui-Teng Cheng1, Jeffrey H. Miner2,*, MeeiHua Lin1, Malú G. Tansey3, Kevin Roth4 and Raphael Kopan1,5,*

1 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, St Louis, MO 63110, USA
2 Department of Medicine, Renal Division, Washington University School of Medicine, Box 8126, 660 South Euclid Avenue, St Louis, MO 63110, USA
3 Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390, USA
4 Department of Pathology, The University of Alabama at Birmingham, 1530 3rd Avenue, Birmingham, AL 35294-0019, USA
5 Department of Medicine, Division of Dermatology, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, St Louis, MO 63110, USA



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  		Fig. 5. Proximal epithelial structures are missing in DAPT-treated metanephroi. (A,E) E-cadherin (green), cytokeratin 8 (red) and Wt1 (blue, overlay of A on B and E on F with Canvas). Distal tubules (green only) formed in DAPT-treated metanephroi throughout the section (E, asterisk). (B,F): Wt1HIGH (glomerular podocytes) and Wt1LOW (induced/condensed mesenchyme and primitive epithelia) are detected in both DMSO- and DAPT-treated metanephroi. Only a few podocytes and well-defined glomeruli are found in the center of DAPT-treated metanephroi, and an increase in Wt1LOW cells is also evident (F). (C,G) Detection of proximal tubules with Lotus tetragonolobus Lectin (LTL, green). Note the overall reduction in the number and the central location of proximal epithelia in DAPT-treated metanephroi. (D,H) Laminin {alpha}1 (red) was detectable in the basal laminae of tubules in both control and treated metanephroi; fewer renal tubules were detected in DAPT-treated cultures. (I,J) Proximal tubules are abundant in untreated metanephroi (cadherin 6, pink, inset in I) but are missing in DAPT-treated metanephroi (J). Distal tubules (E-cadherin, green) are present in both. (K,L) De novo formation of epithelia occurs in DAPT. Distal tubules (circled in K and L) but no podocytes (Wt1HIGH, K) are present in 11.5 day metanephroi cultured for 6 days in DAPT. (Inset in L) Pax2 (red), expressed in induced mesenchyme, persists in newly formed epithelia at the periphery of DAPT treated cultures (stained with E-cadherin; green). A more central epithelial cluster loses staining of Pax2.

 


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  		Fig. 8. (A,B) Proximal epithelia can form in the absence of podocytes after DAPT removal. The section shown in Fig. 7L is shown here (A) next to an adjacent section stained with laminin {alpha}1 (green) and E-cadherin (blue; B). The sections are aligned to the same orientation (ducts are marked with blue asterisks). NICD-positive (pink) cells are surrounded by laminin {alpha}1 and are E-cadherinLOW (yellow asterisks). In addition to these proximal epithelial, laminin {alpha}1 and E-cadherinHIGH distal epithelia are also detected. Ducts did not stain with laminin {alpha}1 in this experiment. (C) To confirm the proximal tubule identity, another section from the same metanephros was stained for cadherin 6. (D) Contralateral metanephros cultured continuously in DAPT contained no cadherin 6-positive cells. Bars indicate days with DAPT (blue) or DMSO (white).

 


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  		Fig. 1. Inhibition of {gamma}-secretase in cultured metanephroi eliminates Notch activation and reduces tissue complexity. (A,E) Activated Notch1, detected with an antibody against the antigen generated by {gamma}-secretase (VLLS) (Schroeter et al., 1998Go), is present in the nucleus of S- and comma-shaped bodies (red circles) in 14.5 dpc embryonic kidney. (E) Control with no primary antibody. (B-D) Metanephroi cultured in DMSO-containing media or (F-H) 1 µM DAPT-containing media. NICD is present in control (B) but is undetectable after 5 days in DAPT (F). After the first day of culture, phase contrast images of control (C) and DAPT-treated metanephroi (G) are indistinguishable. After 5 days, the control metanephros is filled with high contrast structures (D), while the DAPT-treated contralateral metanephros has branches but less tissue complexity (H).

 


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  		Fig. 2. Whole-mount images of cultured 12.5 dpc metanephroi. Top two rows, DBA stain (ureteric bud/collecting ducts). Bottom two rows, Wt1 stain. In both cases representative metanephroi are shown. The treatment and time in culture are indicated in the figure. With DAPT, less extensive branching is evident after 3 days in culture. Ducts appear dilated, and the epithelium at the tip of a few end branches formed bubble-like structures (day 5, asterisk and insets). Wt1 staining reveals fewer Wt1HIGH structures (white arrows) and more Wt1LOW cells (black arrows) in DAPT-treated metanephroi from day 2 onwards. Scale bar: 0.5 mm.

 


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  		Fig. 3. (A) Distribution of bifurcations in 5 day DMSO- and DAPT-treated metanephroi. DBA-stained metanephroi were examined as described in the Materials and methods, and the frequency of branches with the given number of bifurcations was determined. Note the shift to fewer bifurcations in DAPT. This is not due to loss of Ret signaling, because even in the presence of DAPT, Ret was activated in response to 40 ng/ml Gdnf, as judged by Ret tyrosine phosphorylation (at the autocatalytic tyrosine Tyr905 and at Tyr1062) and subsequent activation of the downstream MAPK pathway (B).

 


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  		Fig. 4. Fewer epithelial structures in DAPT-treated metanephroi. Hematoxylin and Eosin-stained sections of metanephroi cultured for 5 days in DMSO (A) or DAPT (E). Fewer renal tubular epithelia and more interstitial cells are seen in the DAPT-treated metanephroi. Note dilated collecting ducts in the DAPT-treated metanephroi (asterisks in E and G). (B-D,F-H) Expression of several epithelial cell markers. (B,F) Ncam (green) and Pax2 (red). Pax2 only: ductal cells (arrowheads). Pax2 + Ncam: condensing mesenchyme (orange). Ncam only: epithelial cells (B, arrows) or uninduced mesenchyme (asterisks). (C,D,G,H) Expression of E-cadherin (brown), an epithelial marker present in ducts and enriched in distal tubules, on Hematoxylin counterstained (purple) slides. Insets in C and G: detection of cytokeratin 8 (red), a ductal marker, in sections adjacent to C and G. These images were overlaid using Canvas (Deneba software) on C and G to create images D and H. E-cadherin was highly expressed in both collecting duct and nephron epithelia. Fewer non-ductal epithelia (i.e. cytokeratin 8 negative) developed in DAPT-treated metanephroi (circled, H).

 


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  		Fig. 7. Reversibility of DAPT treatment. (A-D) Whole-mount double staining of metanephroi with anti-Wt1 and DBA; (E-M) sections through metanephroi cultured under conditions similar to the wholemounts. The antigens detected are indicated on the right in the appropriate colors. In all sections, bars at the bottom reflect days in DMSO (white bars) and/or in DAPT (blue bars). Note that NICD and Jag1(J) reside in the same cells (insets and frames in J). Expression of both is inhibited by DAPT (K) but recovers after DAPT removal (frames in L,M). After a 2-day inhibition, Wt1HIGH cells are again detected throughout the metanephroi (C,G). After 3.5 days, despite recovery of NICD and Jag1 (K,L), no Wt1HIGH cells appear at the periphery, and only Wt1LOW; Pax2-positive (not shown) cells increase in abundance, and branching resumes (D,H,I). We conclude that a crucial period exists between day 2 and 3 in DAPT, after which restoration of Notch signaling fails to rescue podocyte differentiation. This is summarized in I.

 


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  		Fig. 6. (A) Hypothetical responses of metanephroi to DAPT. At 14.5 dpc, the metanephros contains mature structures in the center (D, podocytes) and immature ones at the periphery (A, uninduced mesenchyme). The peripheral tissue progressively differentiates (vertical box), flanked with mesenchymal stem cells at more peripheral positions. If DAPT only blocks the induction but not differentiation of proximal epithelia (model 1), induced epithelia can still progress to podocytes. If DAPT also blocks podocyte differentiation (model 2), the deficiency in podocytes (Wt1HIGH cells) will manifest earlier, at day 3 (D-D-D-B-B-A in model 1; D-D-B-B-B-A in model 2). After 3 days in culture, there is no appreciable difference in formation of podocyte/glomeruli near the periphery between the control and DAPT-treated cultures (green circles in B), compatible with model 1. (C) Twenty-four hours later, peripheral regions (green arrows in C) have produced mature Wt1HIGH cells in DMSO but not in DAPT. We therefore assign the DAPT-sensitive step at the formation of proximal renal epithelia, with a possible secondary effect reducing the number of distal epithelia. The affected cell population is the one containing NICD.

 

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© The Company of Biologists Ltd 2003