spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 17 January 2007
doi: 10.1242/dev.02773

Development 134, 801-811 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow A corrigendum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheng, H.-T.
Right arrow Articles by Kopan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheng, H.-T.
Right arrow Articles by Kopan, R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron

Hui-Teng Cheng1, Mijin Kim1, M. Todd Valerius2, Kameswaran Surendran1, Karin Schuster-Gossler3, Achim Gossler3, Andrew P. McMahon2 and Raphael Kopan1,*

1 Department of Molecular Biology and Pharmacology and Department of Medicine at Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8103, St Louis, MO 63110, USA.
2 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
3 Institute for Molecular Biology OE5250, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1 D-30625 Hannover, Germany.


Figure 1
View larger version (115K):
[in this window]
[in a new window]

  		Fig. 1. Loss of Notch2 causes hypoplastic kidneys that do not develop glomeruli, proximal tubules or S-shaped bodies. (A-D) The urinary system from postnatal day 1 and day 2 wild-type (A,B) or mutant (C,D) animals. Note the size difference of the urinary bladder (arrow in A and C). The mutant animals show spotty hemorrhage on the kidneys before they die on postnatal day 2 (D). (E-H) Histology of day 2 kidneys from wild type (E,F) and mutant (G,H) stained with H&E. The wild-type genotype is Pax3-cretg/+; N2f/+; the mutant genotype is Pax3-cretg/+; N2f/f. Blue arrows flank the nephrogenic zone; green arrow, proximal tubule; green arrowhead, presumptive renal tubule in mutant; red arrow, glomerulus; yellow arrow, S-shaped body; turquoise arrowhead, collecting duct; D, distal tubule; P, proximal tubule. Scale bars: 1 mm in A-D; 0.1 mm in E-H.

 

Figure 2
View larger version (98K):
[in this window]
[in a new window]

  		Fig. 2. Notch2-deficient kidneys (N2) develop distal tubules without formation of podocytes or proximal tubules. (A,B) Wild-type (A) kidney contains cells expressing high levels of Wt1 in glomerular podocytes and S-shaped bodies. The only cells that express low levels of Wt1 in the mutant (B) are mesenchymal cells surrounding cytokeratin-8-expressing ureteric buds (red). (C,D) LTL-stained proximal tubules found in wild type (D) are absent in the mutant (C). (E,F) Mutant kidney (E) develops numerous E-cadherin-positive, cytokeratin-8-negative distal tubules, some of which are connected to cytokeratin-8-positive ducts (dashed line in enlarged view E'). The wild-type proximal tubules, judged by morphology, also express E-cadherin (arrowhead in F). N2, Notch2 mutant; Wt, wild type. Scale bars: 0.1 mm in B for A,B,E'; in C for C,D.

 

Figure 3
View larger version (121K):
[in this window]
[in a new window]

  		Fig. 3. Notch2-deficient mesenchyme undergoes normal epithelialization but the newly formed nephron fails to resolve into the S-shaped body seen in wild type. (A-E) Wild type (A) showing S-shaped bodies (arrowheads). In the Notch2 mutant, one nephron, expressing N-Cam, can be identified at each ureteric bud tip (arrowheads in B). These synthesize laminin {alpha}1 (arrowheads in D), as do the S-shaped bodies (arrowheads in C). (E) Three progressive stages during nephrogenesis in the mutant are marked as 1, 2 and 3. (F-J) Expression of molecular markers in each of the three segments in the S-shaped body (see text). Distal tubule precursors are Pax2High- and E-cadherin-positive (F,H). Jag1 is localized in the middle segment in the position of proximal tubule precursors (G). Wt1 marks the podocyte precursors (I). N1-ICD is detectable in both the proximal and podocyte precursors (J). Scale bars: 0.05 mm in A for A-D and in J for F-J; 0.1 mm in E.

 

Figure 4
View larger version (106K):
[in this window]
[in a new window]

  		Fig. 4. The segmentation process in Notch2-deficient nephron (N2) is impaired. (A,B) Cadherin-6-expressing cells are adjacent to E-cadherin-expressing cells in wild type (A), but no cadherin-6-expressing cells are detectable in mutant (B). (A'-B''') Serial sections stained with the segmentation markers Wt1, Pax2 and E-cadherin. Circles and arrowheads locate the same cells in adjacent sections to indicate similar structure across all sections. (C,D) Lim1 expression pattern in the renal vesicle is similar in wild-type and Notch2 mutant kidneys (white arrows). The cells at the very proximal end of the mutant nephron express high levels of Lim1 (D). This distribution is different from that in a wild-type S-shaped body (C). (E) In wild type, few Jag1-expressing cells are seen in the renal vesicle (arrowheads), but the population expands with the formation of the S-shaped body (circle). (F) A small cluster of Jag1-expressing cells is seen in each of the mutant nephrons. (G,H) Dll-1LacZ and N1-ICD (arrowhead) are detectable in the renal vesicle in wild type. (I) In some cases, Jag1 is not co-localized with N1-ICD (arrowhead). N1-ICD remains detectable in Notch2-deficient nephrons (arrowhead, inset in I). Scale bars: 0.1 mm in B for A-B''' and in F for C-F; 0.05 mm in G-I.

 

Figure 5
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 5. Notch2 mutant, Pax2-expressing early renal epithelia proliferate as wild-type, whereas Jag1-expressing, Notch2-deficient clusters have few cycling cells. (A,B) Representative images of BrdU incorporation (red) within the Jag1-expressing clusters (green) in wild type and mutant. (C,D) Histograms of results from several embryos indicate the percentage of BrdU-labeled cells within the Pax2- or Jag1-expressing domains. Data are presented as means±s.e.m. Scale bar: 0.05 mm.

 

Figure 6
View larger version (165K):
[in this window]
[in a new window]

  		Fig. 6. Notch1-deficient ES cells contribute to various parts of the nephron. Kidneys were harvested from E16.5 embryos (A-F) or adults (G) and subjected to whole mount ß-gal staining. (A,C,E) Wild-type ES cells in E16.5 kidneys are found in early nephrons (arrowheads in A), PCTs (circled in C) and glomerular podocytes (arrowhead in E). (B,D,F,G) Notch1-/-; Rosa26 ES cells contribute to renal vesicles (arrows in B), S-shaped bodies (arrowhead in B) and podocytes in the capillary-loop stage (star in B). (D,D') Notch1-deficient ES cells (blue) contribute to LTL-labeled PCT together with wild-type cells (unstained). One LTL-labeled cross-section composed entirely from Notch1-deficient cells is shown in D (arrowhead). (F) Glomerular podocytes develop in the absence of Notch1 (arrowhead). The circle indicates the capillary lumen with red blood cells in it. (G) The adult chimeric kidney is stained with LTL (brown). Some of the LTL-labeled tubules in the inner cortex are entirely derived from Notch1-/- cells (black arrow). White and black arrowheads indicate a juxtamedullary glomerulus and a cortical glomerulus, respectively.

 

Figure 7
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 7. Notch1-deficient metanephroi are phenotypically wild type. Genotypes are marked above all panels. Where indicated, floxed alleles were recombined with the Pax2-Cretg/+ strain. (A,B,E,F) Whole-mount staining of LTL (green) and Ck8 (red) detects extensive renal tubulogenesis. Loss of Notch1 did not alter proximal tubule formation (B), whereas loss of Rbp-J resulted in loss of LTL-positive epithelial cells (F). Note that loss of Notch1 (see Notch1 staining in inset in C,D) or of Rbp-J in the duct did not prevent duct branching. (C,D,G,H) Glomerular podocytes are labeled with Wt1 (green). In G,H, whole-mount preparation is also stained with Ck8 (red). Wt1High, synaptopodin-positive (red in C,D, inset in G) glomeruli are present in wild type (C), Notch1-deficient metanephroi (D) and Rbp-J heterozygotes (G), but not in Rbp-J deficient metanephroi (inset in H, no Wt1 or synaptopodin). Scale bars: 0.5 mm in B for A,B; in D for C,D; in F for E-F.

 

Figure 8
View larger version (99K):
[in this window]
[in a new window]

  		Fig. 8. Constitutively active Notch1 promotes proximal tubule formation while inhibiting the progenitor from differentiating into podocytes and distal tubules. (A) Notch1 activation in the mesenchyme causes renal hypoplasia at E17.5. (B) The ureteric bud branches only once. Subsequent planes of sectioning are marked (a-c). Ck8 is visualized using Cy3, but the image is pseudocolored to correspond with Ba-Bc. (C,C') E11.5 metanephroi from mutant (C) or wild type (C') were cultured for 4 days, stained with LTL (green) and Ck8 (red). (Ba-Bc) Serial sections of the mutant kidney stained with markers for renal tubules. Arrowheads locate the same cells in adjacent sections. Scale bars: A,B,C,C', 0.5 mm; Ba-Bc, 0.1 mm.

 

Figure 9
View larger version (79K):
[in this window]
[in a new window]

  		Fig. 9. N1-ICD directs PCT fates independent of Notch2. (A) Ectopic overexpression of Notch1 intracellular domain is sufficient to promote proximal tubule formation in the absence of {gamma}-secretase activity. Genotypes are shown to the left of the image. DMSO, vehicle only; DAPT, {gamma}-secretase inhibitor. (B) Schematic of the proximalization pathway. After RV induction (a Wnt-dependent process), Pax2 and Wt1 begin to separate into distinct expression domains and Lim1 and Dll1 define a distal domain within the RV. However, Notch2 signals are required for separation of proximal from distal fates; in their absence (or when blocked by {gamma}-secretase inhibitors), only distal tubules form. See text for details. GSI, {gamma}-secretase inhibitors.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007