Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development

Abstract

Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the Six2-expressing cap mesenchyme represents a multipotent nephron progenitor population. Six2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of Six2-expressing nephron progenitors at the onset of kidney development suggests that the Six2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some Six2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, Six2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking Six2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that Six2 activity cell-autonomously regulates a multipotent nephron progenitor population.

Figure 1. Six2-eGFPCre transgenes are expressed in the cap mesenchyme

Kidneys from Six2-TGC^tg/+ BAC transgenic (A,D,G), and Six2^TGC/+ (B,E,H), and Six2^GCE/+ (C,F,I) knock-in alleles at 11.5 dpc (A,D), 12.5 dpc (B,C,E,F) and 15.5 dpc (G-I). (A-F) Whole-mount kidneys. (A-C) Bright view. (D-F) GFP expression. Backgrounds from non-tissue regions were subtracted from images in D and F. (G-J) Confocal immunofluorescence images of GFP (green), Six2 (red) and cytokeratin (purple). Cytokeratin is expressed in the ureteric tip and collecting duct. Arrow and arrowheads indicate Six2⁺ GFP⁺ and Six2⁺ GFP^- cells, respectively.

Figure 2. The Six2+ population contributes to all cells of the nephron tubule

Fate mapping of the Six2+ population in Six2-TGC^tg/+ ; R26R^lacZ/+ embryos. (A-E) β-gal staining. (A) Whole-mount embryos at 10.5, 11.5, and 12.5 dpc. (B,D) Dissected whole-mount posterior region of embryos at 11.5 and 12.5 dpc, respectively. (C,E) Sections counter-stained with nuclear fast red of B and D, respectively. (F) Epi-immunofluorescence imaging of the kidney with anti-GFP (green), anti-β-gal (red), and anti-cytokeratin (purple) staining. (G-I) Confocal immunofluorescence imaging of the cortical region of kidneys with anti-GFP (green), anti-β-gal (red), and Hoechst (blue) staining. (G) Early pretubular aggregate. (H) Comma-shaped body. (I) S-shaped body. (J-O) Confocal immunofluorescence imaging of kidneys with anti-β-gal (green) and Hoechst (blue) staining. (J-L) The glomerulus stained with Wt1, PECAM-1 and PDGFRb in red, respectively. (M) Cytokeratin (white) staining in the cortical region of the kidney. Endogenous GFP is also visible in the cap mesenchyme localized to the nucleus (white arrowheads). The yellow arrow indicates the collecting duct-nephron junction. (N) Cytokeratin (white) and uromodulin (red) immunofluorescence in the medullary regions of kidneys. (O) Smooth muscle actin (SMA, red) immunofluorescence. White arrows, white arrowheads, black arrows and black arrowheads indicate the developing nephron tubule, cap mesenchyme, ureteric tip and collecting duct, respectively.

Figure 3. Six2+ cells in the cap mesenchyme contribute to the nephron tubule throughout kidney organogenesis

β-gal stained kidneys from Six2^GCE/+ ; R26R^lacZ/+ embryos at 18.5 dpc after injection of oil only (A,C) or 6 mg Tamoxifen (B,D) at 16.5 dpc. (A,B) Whole-mount view. (C,D) Sections counter-stained with eosin. a, adrenal gland; cm, cap mesenchyme; k, kidney; rv, renal vesicle; sb, S-shaped body; ut, ureteric tip.

Figure 4. The nephron progenitor population is maintained by duplication of Six2+ cells

(A,B) Models for maintenance of the nephron progenitor population. (A) A β-gal-labeled (blue) Six2+ cell produces another β-gal-labeled Six2+ cell, which results in retention of β-gal-labeled cells in the cap mesenchyme (cm). (B) A progenitor population that generates Six2+ cells in the cap mesenchyme resides outside of the Six2+ population. Non β-gal-labeled cells from the progenitor population (renal precursor, rp) dilute the β-gal-labeled cells in the cap mesenchyme. pa, pretubular aggregate. (C-Q) β-gal stained kidneys at 19.5 dpc from Six2^GCE/+ ; R26R^lacZ/+ embryos after injection of 2 mg Tamoxifen at 9.5 dpc (C,H,M) and 10.5 dpc (E,J,O), and oil only at 10.5 dpc (D,I,N), and from Wnt4^GCE/+ ; R26R^lacZ/+ embryos after injection of oil only (F,K,P) and 2 mg Tamoxifen (G,L,Q) at 12.5 dpc. (C-G) Whole-mount view. (H-L) Sections counter-stained with eosin. (M-Q) Higher magnification of the cortical region in H-, respectively. a, adrenal gland; cb, comma-shaped body; cd, collecting duct; cm, cap mesenchyme; gl, glomerulus; k, kidney; nt, nephron tubule; pa, pretubular aggregate; rp, renal precursor; sb, S-shaped body; ut, ureteric tip.

Figure 5. A Six2+ cell can duplicate to generate Six2+ cells in the cap mesenchyme and contribute to multiple domains of the nephron tubule

(A,B) Models for developmental potential of Six2+ cells. Different cell types are generated along the glomerular-collecting duct (G-CD) axis of the nephron. (A) A Six2+ cell retains multipotency for nephrogenesis. (B) Developmental potential of a Six2+ cell is restricted to limited cell types of the nephron. (C-M) Kidneys from Six2^GCE/+ ; R26R^lacZ/+ embryos at 19.5 dpc after injection of oil only (C,E,G,L), and 0.1 mg (D,F,H,I-K’,M) Tamoxifen at 12.5 dpc. (C-K’) β-gal staining. (C,D) Whole-mount view. The inset in D is a higher magnification of a clone in the dashed region. (E,F) Sections counter-stained with eosin. Arrows in D and F indicate β-gal+ clusters. (G-K’) Higher magnification of the cortical region. H shows the dashed region in F. White arrows in I indicate β-gal+ cells in the cap mesenchyme. K and K’ are adjacent sections 96 μm apart showing the same nephron. (L,M) Confocal immunofluorescence imaging of the cortical region with anti-β-gal (red), anti-GFP (green) and Hoechst (blue) staining. Arrows and arrowheads in M indicate β-gal+ GFP+ cells in the cap mesenchyme and β-gal+ GFP- cells in the developing nephron tubule, respectively. a, adrenal gland; cd, collecting duct; dt, distal tubule; cm, cap mesenchyme; k, kidney; mc, mesangial cell; nt, nephron tubule; pc, podocyte; pt, proximal tubule; sb, S-shaped body; ut, ureteric tip.

Figure 6. Ectopic nephron tubules are derived from the cap mesenchyme in Six2-null kidneys

Kidneys from Six2^TGC/+ ; R26R^lacZ/+ (A,C,E,G,I) and Six2^TGC/GCE ; R26R^lacZ/+ (B,D,F,H,J) embryos at 13.5 dpc. (A,B) Whole-mount β-gal staining. (C,D) β-gal stained sections counter-stained with eosin. (E,F) Higher magnification of the cortical region in C and D, respectively. (G-J) Confocal immunofluorescence imaging of the cortical region with anti-β-gal (blue), anti-laminin (red), anti-cytokeratin (green) and Hoechst (gray) staining. Laminin is expressed strongly in the epithelium of the ureteric tip, collecting duct and developing nephron tubule, and weakly in the cap mesenchyme, while cytokeratin is expressed only in the ureteric tip and collecting duct. White arrows, yellow arrows, white arrowheads and black arrows indicate the nephron tubule, ectopic nephron tubule, cap mesenchyme and ureteric tip, respectively. a, adrenal gland; cb, comma-shaped body; cm, cap mesenchyme; rv, renal vesicle; ut, ureteric tip.

Figure 7. Six2 function is cell-autonomously required in the cap mesenchyme

(A) Schematic illustration of the strategy for Six2 chimera analysis. To facilitate genotyping of chimeras, two different null alleles of Six2, Six2-GCE and Six2-CE, were crossed to generate Six2^GCE/CE (Six2^-/-) morulae. To genetically label wild-type cells, R26^tg/+ morulae were collected and aggregated with Six2^-/- morulae to generate Six2^-/- ↔ Six2^+/+ ; R26^tg/+ chimeras. For a control, we used Six2^+/- ↔ Six2^+/+ ; R26^tg/+ chimeras. (B-O) Kidneys from Six2^+/- ↔ Six2^+/+ ; R26^tg/+ (B,D,F,H,J,L,N) and Six2^-/- ↔ Six2^+/+ ; R26^tg/+ (C,E,G,I,K,M,O) chimeras at 12.5 dpc. (B,C) Kidneys with β-gal staining counter stained with eosin. (D,E) Higher magnification of B and C, respectively. (F-O) Confocal immunofluorescence imaging of the cortical region with anti-β-gal (red), anti-Pax2 (green), anti-cytokeratin (blue) and Hoechst (gray) staining. Pax2 is expressed in the ureteric tip, cap mesenchyme and developing nephron tubule, while cytokeratin is expressed only in the ureteric tip. White arrows and arrowheads in L and M indicate β-gal⁺ Pax2⁺ and β-gal^- Pax2⁺ cells in the cap mesenchyme, respectively. cm, cap mesenchyme; pa, pretubular aggregate; rv, renal vesicle; sr, stroma; ut, ureteric tip.

Figure 8. Absence of nephron induction in Six2 ; Wnt9b compound mutants

Kidneys from Wnt9b^+/- (A,E,I,M,Q), Wnt9b^-/- (B,F,J,N,R), Six2^-/- (C,G,K,O,S) and Six2^-/- ; Wnt9b^-/- (D,H,L,P,T) embryos at 12.5 dpc (A-H,Q-T) and 11.5 dpc (I-P) with dorsal to top and ventral to bottom. (A-D) H&E staining. (E-H) Confocal immunofluorescence imaging with anti-laminin (red), anti-cytokeratin (green) and Hoechst (blue) staining. (I-P) Whole-mount in situ hybridization. (I-L) Wnt4. (M-P) Pax8. (I-L) Confocal immunofluorescence imaging with anti-Pax2 (red) and anti-cytokeratin (green) staining. White arrows, yellow arrows, white arrowheads and black arrows indicate the nephron precursor, ectopic nephron precursor, cap mesenchyme and ureteric tip, respectively.