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First published online 30 November 2005
doi: 10.1242/dev.02174

Development 133, 151-161 (2006)
Published by The Company of Biologists 2006
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Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Kenji Osafune1,2,3, Minoru Takasato2,4, Andreas Kispert5, Makoto Asashima2,3 and Ryuichi Nishinakamura1,4,6,*

1 Division of Stem Cell Regulation, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
2 Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Tokyo 153-8902, Japan.
3 ICORP, JST, Saitama 332-0012, Japan.
4 Division of Integrative Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
5 Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
6 PRESTO, JST, Saitama 332-0012, Japan.



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  		Fig. 1. In vitro colony formation from E11.5 metanephric mesenchyme. (A) Sheet-like colonies were formed on 3T3Wnt4, but not on 3T3lacZ. Arrows: fibroblast-like cells. (B) Colonies were not formed on 3T3Wnt4 with the addition of Fz-Fc chimeric protein. (C) RT-PCR analyses of genes expressed in metanephros and fully differentiated epithelia in glomeruli (podocyte), proximal and distal tubules, and the loop of Henle. Lane 1: E11.5 metanephric mesenchyme; 2: 3T3Wnt4 alone; 3: mesenchyme-derived cells cultured on 3T3Wnt4 at day 3; 4: at day 10; 5: at day 20; 6: 3T3lacZ alone; 7: mesenchyme-derived cells cultured on 3T3lacZ at day 3, 8: at day 10, 9: at day 20, 10: mesenchyme-derived cells at day 10 separated from 3T3Wnt4 feeder cells; 11: organ culture of E11.5 mesenchyme rudiments at day 10; 12: embryonic kidney (E17.5); 13: no RT reaction on mesenchyme-derived cells cultured on 3T3Wnt4 at day 10. (D-M) Immunocytochemistry of colonies for Pax2 (D-F), E-cadherin (G-I), Sall1 (J,K) and Aqp1 (L,M). (D-I) The expression of Pax2 and E-cadherin (red) was not detected at day 3 (D,G, respectively) but was observed at day 10 (E,H). (J,K) Sall1 expression (red) at day 10. (L,M) Aqp1 (red, proximal tubule marker) was expressed in some cells of the colony. Feeder cells have larger nuclei (DAPI, blue; arrows) than cells consisting of colonies. Control staining with rabbit (F,K,M) and mouse (I) IgGs. Mesenchyme of Sall1-GFP knock-in mice was used for J and K to visualize Sall1 expression using anti-GFP immunostaining, while EGFP transgenic mesenchyme was used for D-I,L,M. Scale bars: 50 µm.

 


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  		Fig. 2. Colonies are derived from a single multipotent renal progenitor. (A) One colony derived from a single cell of EGFP transgenic mesenchyme at culture day 20. (B) RT-PCR of three independent wells containing a single cell-derived colony after 20-day culture. Lanes 1-3: colonies; 4: organ culture of E11.5 mesenchyme rudiments. (C) Lectin staining of 8-week-old adult kidney (left panel) and a single cell-derived colony (right) with PNA (red, podocyte marker) and with LTL (green, proximal tubule marker). (D) Staining of adult kidney (left panel) and a single cell-derived colony (right) with E-cadherin (red, arrow, distal tubule marker) and with LTL (green). Scale bars: 50 µm.

 


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  		Fig. 3. Colony-forming progenitors exist in the Sall1-GFPhigh subpopulation of the metanephros. (A,B) Cryosections of metanephros of Sall1-GFP knock-in mouse (A: E11.5; B: E17.5). Blue: DAPI. (C) Metanephros contains three subpopulations (Sall1-GFPhigh, Sall1-GFPlow and Sall1-GFPnegative). The percentages of the subpopulations at each fetal stage are shown. Figures are the average of five independent experiments. (D) RT-PCR analysis of three subpopulations included in E11.5 mesenchyme. (E) Numbers of colonies in each subpopulation derived from E11.5 mesenchyme, E14.5 and E17.5 metanephros. The numbers of colony were counted after 20-day culture. The graph shows the average of five independent experiments. ub, ureteric bud; mes, mesenchyme; c, C-shaped body; rt, renal tubule. Scale bars: 50 µm.

 


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  		Fig. 4. Sall1-GFPhigh mesenchyme differentiates into renal epithelia in organ culture. (A) Three subpopulations in E11.5 mesenchyme (Sall1-GFPhigh, Sall1-GFPlow and Sall1-GFPnegative) were cultured on 3T3Wnt4 feeder cells in an organ culture setting. Only Sall1-GFPhigh cells (upper panels) differentiated into kidney structure, while Sall1-GFPlow cells (lower) disappeared. (B) Hematoxylin-eosin staining of sections of Sall1-GFPhigh aggregates at day 10. Tubule- and glomerulus-like structures are seen. (C) Double staining with Wt1 (red, podocyte marker) and LTL (green, proximal tubule marker) of Sall1-GFPhigh aggregates. g, glomerulus-like structure; t, tubule-like structure. Scale bars: 500 µm in A; 25 µm in B,C.

 


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  		Fig. 5. Colony formation from Sall1-mutant metanephric mesenchyme obtained from intercrosses of Sall1-GFP mice. (A-F) Colonies derived from Sall1+/+ (A,B), Sall1+/- (C,D) and Sall1-/- (E,F) mesenchyme were stained both with anti-GFP (A,C,E, green) and with anti-E-cadherin antibodies (B,D,F, red). Sall1-deficient colonies (E,F) were significantly smaller than those from wild-type (A,B) and heterozygous (C,D) mesenchyme. Scale bars: 50 µm.

 


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  		Fig. 6. PCP pathways regulate colony size and the differentiation of colony-forming cells. (A) Immunostaining of activated JNK1 and 2 in the colony. (B,C) The addition of two kinds of JNK inhibitors (JNKI1 and JNKI2, 10 µmol/l) gave rise to smaller colonies than did the control without reagents or HIV-TAT peptide (10 µmol/l). (D) The transduction of CA-Rac1 resulted in an increase in colony size, whereas that of DN-Rac1 resulted in a decrease. (E,F) The addition of Y27,632 (E, 10 µmol/l), or the transduction of DN-RhoA, increased colony size, while activation with CA-RhoA decreased it (F). (G,H) Activation of the ß-catenin pathway by adding LiCl (G, 10 µmol/l) or transducing the active form of ß-catenin (H) gave rise to smaller colonies. (I) RT-PCR analysis of colonies treated with JNKI-1, HIV-TAT peptide, JNKI-2, Y27,632 and LiCl. (J) E-cadherin expression was lost with JNKI-1 and -2 but not with LiCl. EGFP transgenic mesenchyme was used for B,C,E,G and J, while wild type was used for D,F and H, to visualize colonies infected by retrovirus vectors. c, colony. Scale bars: 50 µm.

 


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  		Fig. 7. Effects of reagents on tubulogenesis in organ culture. Whole metanephroi (A-E) and mesenchyme rudiments (F-J) cultured for 7 days. (A,F) Control explants without reagents, (B,G) 10 µmol/l JNK inhibitor 1 (JNKI1), (C,H) 10 µmol/l JNKI2, (D,I) 10 µmol/l Y27,632, (E,J) 20 mmol/l (E) and 10 mmol/l (J) LiCl. (K,L) Section staining using DBA (green), an anti-sFRP2 antibody (red) and DAPI (blue) on whole kidneys treated with 15 µmol/l HIV-TAT peptide (K) and JNKI-1 (L). (M,N) Double labeling with sFRP2 and DAPI on mesenchyme rudiments treated with HIV-TAT peptide (M) and JNKI-1 (N). ub, ureteric bud. Scale bars: 500 µm in A-J; 200 µm in K-N.

 

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