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First published online 20 August 2003
doi: 10.1242/dev.00682

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Presenilins are required for the formation of comma- and S-shaped bodies during nephrogenesis

Pei Wang1,2, Fred A. Pereira1,2,3, Duane Beasley1 and Hui Zheng1,2,4,*

1 Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
3 Department of Otolaryngology, Baylor College of Medicine, Houston, TX 77030, USA
4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA



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  		Fig. 1. Generation, morphological and biochemical characterization of presenilin rescue mice. (A) Mice carrying a single copy of the human PSEN1 transgene onto endogenous mouse Psen1- and Psen2-double knockout (Psen1–/–Psen2–/–PSEN1, or presenilin rescue) background were produced by three generations of breeding as outlined. (B) Whole-mount photographs of presenilin rescue embryos (mutant) along with their littermates (control) at E13.5 (left) and P0 (right). (C) Dramatically reduced kidney size in presenilin rescue (mutant) compared with that of a littermate control (control). (D) Western blot analysis of PSEN1 expression of P0 kidneys from control (lanes 1 and 2), presenilin rescue mutant (lane 3) and PSEN1-null (lane 4), documenting the absence of PSEN1 expression in the presenilin rescue kidney. Lower panel is hybridization with an anti-ß-actin antibody as a loading control.

 


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  		Fig. 2. Hematoxylin and Eosin analysis of kidney development. Right column, presenilin-null (Mutant); left column: littermate controls (control). At E12.5 (A,B), condensed mesenchyme (asterisks) surrounding ureteric bud could be identified in both PSEN-null mutant (B) and the control (A). At E13.5 (C,D), while pretubular aggregates (arrows) could be seen in both the control (C) and the mutant (D), further advanced structures, such as comma- and S-shaped bodies (arrowheads), could be detected only in the control but not in PSEN-null kidney. At E15.5 (E,F) and P0 (G,H), numerous glomeruli were formed in the control (E,G, arrows). These structures were absent in the mutant (F,H). Scale bar: 100 µM.

 


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  		Fig. 4. Immunohistochemical characterization of renal derivatives. (A) Double staining with anti-NCAM (green) and anti-pan-laminin (red) antibodies at E14.5 (a,b) and E16.5 (c,d) of development. NCAM-positive, laminin-low (or negative) aggregates (asterisks) and NCAM- and laminin-positive renal vesicles (thin arrows) and tubules (thick arrow) were present in both the controls and PSEN-null (mutant). (B) E16.5 sections labeled with an anti-laminin {alpha}1 antibody, which identified polarized renal epithelium in PSEN-null mutant (b, thin arrow). (C) Double staining with anti-cytokeratin 8 (CK8, red, ductal only) and anti-E-cadherin (renal and ductal, green) antibodies. (a,b) E14.5. (c,d) E16.5. (e,f) Magnified view of highlighted structures in c,d, respectively. E-cadherin-positive, cytokeratin 8-negative renal epithelial derivatives could be identified that were either in close proximity (white arrowheads) or appear to have connected with the ureteric bud terminals (thick arrows). Open arrowheads in e,f indicate site of fusion. Scale bars: in A, part a: 20 µm for A,B and C, parts a-d; in C, part e, 8 µm for C, parts e,f.

 


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  		Fig. 3. In situ hybridization analysis of Pax2 (A), Wnt4 (B), Pax8 (C) and Ret (D) expression. In A-C, the top panels are dark field and lower panels are corresponding bright field. (A) At E12.5, the levels of Pax2 were comparable between the control (a,c) and PSEN-null (mutant) (b,d). At E13.5, reduced Pax2 expression was observed in the mutant (f,h) compared with the control (e,g). This was also the case for Wnt4 (B; compare b with a) and Pax8 (C; compare b with a). Arrows indicate aggregate structures that are positive for all the markers. (D) At E13.5, Ret-positive structures were similar in the control (a) and the PSEN-null mutant (b). At E15.5, the number of Ret-positive tips was reduced in the mutant (d) when compared with the control (c). Scale bars: in A part a, 100 µm for A-C and D, parts a,b; in D, part c, 100 µm for D parts c,d.

 


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  		Fig. 5. Analysis of proximal derivatives. (A) Lotus Tetragonolobus lectin (LTL) staining revealed complete absence of mature proximal tubules in PSEN-null (mutant) (b,d). (c,d) Magnified view of highlighted structures in a,b, respectively. Residual positives in d probably result from background staining of blood vessels. (B) Double staining with anti-E-cadherin (green) and anti-WT1 (red) antibodies documented the lack of WT1-expressing podocyte precursors in PSEN-null kidney (b). (C) Whole-mount immunostaining of kidney organ cultures with anti-pan-cytokeratin (green) and anti-WT1 (red) antibodies. (c,d) Magnified view of highlighted structures in a,b, respectively, revealing the connection of weak-cytokeratin positive, truncated renal tubules with strong cytokeratin positive ureteric bud terminal in PSEN-null kidney culture (d). Scale bars: in A, parts a,b, 100 µm; in A, parts c,d and B, 20 µm; in C, parts a,b, 50 µm; in C, parts c,d, 10 µm.

 


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  		Fig. 6. Cell proliferation and apoptosis analysis. Left column, littermate controls; right column, presenilin-null. (A) Anti-phosphorylated histone H3 was used to stain for mitotic cells (a,b). Double staining with anti-phosphorylated histone H3 (red) and anti-NCAM (green) antibodies labeled the renal derivatives undergoing active division (c,d). (B) TUNEL assay revealed that the percentage of apoptotic cells (arrowheads) was increased dramatically in the mutant (b) when compared with the control (a). (c,d) Double staining with anti-cleaved caspase 3 (red) and anti-NCAM antibodies (green) showed increased apoptosis in NCAM-positive renal derivatives (arrow) as well as in cortical mesenchyme (arrowheads). The yellow dots are auto-fluorescence of the blood. Scale bar: 50 µm.

 


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  		Fig. 7. Notch pathway analysis in the control (left) and PSEN-null (right) kidneys. (A) Immunohistochemical staining of presenilin-dependent NICD (green) expression. Ureteric bud was marked by cytokeratin 8 (CK8, red). (B-D) In situ hybridization analysis of Notch downstream target Hesr1 (B), Notch ligands Jag1 (C) and Dll1 (D). All are at E14.5 except C, parts c,d, which are at E16.5. Thin arrows indicate pretubular aggregates/renal vesicles; arrowheads indicate comma- and S-shaped bodies. Scale bars: in A, 20 µm for A; B, 100 µm for B-D.

 

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© The Company of Biologists Ltd 2003