Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia

Abstract

Classic tissue recombination and in vitro lineage tracing studies suggest that condensed metanephric mesenchyme (MM) gives rise to nephronic epithelium of the adult kidney. However, these studies do not distinguish between cap mesenchyme and pre-tubular aggregates comprising the condensed MM, nor do they establish whether these cells have self-renewing capacity. To address these questions, we generated Cited1-CreER(T2) BAC transgenic mice, which express tamoxifen-regulated Cre recombinase exclusively in the cap mesenchyme. Fate mapping was performed by crossing these mice with the Rosa26R(LacZ) reporter line and evaluating the location and cellular characteristics of LacZ positive cells at different time points following tamoxifen injection. These studies confirmed expected results from previous in vitro analysis of MM cell fate, and provide in vivo evidence that the cap mesenchyme does not contribute to collecting duct epithelium in the adult. Furthermore, by exploiting the temporally regulated Cre recombinase, these studies show that nephronic epithelium arising at different stages of nephrogenesis has distinct spatial distribution in the adult kidney, and demonstrate for the first time that the cap mesenchyme includes a population of self-renewing epithelial progenitor cells.

Figure 1. Generation and characterization of Cited1-CreER^T2 mice

A, Schematic of the targeting strategy used to create the BAC Cited1-CreER^T2-IRESeGFP transgene. Hatched lines represent regions of homology. Following BAC targeting Tet_R was removed through Flp mediated recombination E1–Exon 1, F–Frt sites, I–IRES; A–Afl II, N–Not I, X–Xho I. B, Dual immunofluorescence staining of E15 mouse kidneys using anti-Cited1 (red) and anti-ECadherin (green) antibodies demonstrates endogenous Cited1 expression in the cap mesenchyme (arrows), and the absence of Cited1 expression in all ECadherin positive epithelial structures including renal vesicles (arrowhead) and UBs (asterisk). C/D, Low (C) and high (D) power images of the nephrogenic zone of E18 β-Gal stained Cited1-CreER^T2/R26R^LacZ kidneys following E15 maternal injection of tamoxifen. Recombination is observed within the cap mesenchyme (arrows) and early nephronic epithelia including renal vesicles (arrowheads). No recombination is observed in UBs (asterisks). E, β-Gal stained kidney from E18 Cited1-CreER^T2/R26R^LacZ embryo whose mother was not injected with tamoxifen. F/G, Anti-Cited1 antibody staining (F) corresponds to eGFP signal (G) within the cap mesenchyme (arrows) in mice carrying the Cited1-CreER^T2-IRESeGFP transgene. No expression observed in UBs (asterisk). H, Merged image of (F) and (G) demonstrating overlapping expression of Cited1 and eGFP.

Figure 2. The cap mesenchyme gives rise to cells in all regions of the adult kidney

Cited1-CreER^T2 mice were crossed to R26R^LacZ mice and pregnant females injected with tamoxifen at E13. Lineage was assessed using β-Gal staining in 6 week old mice. A–C, Low power images of the entire kidney (A), corticomedullary region (B) and inner medulla (papillary) region (C) show cap mesenchyme derived cells in all areas of the kidney. Cx-cortex, Md-medulla, Pa-papilla. D, In the cortex, some of these cell types are recognizable by morphology, including proximal tubules (arrowheads) and glomeruli (arrows). E/F, In addition to tubular epithelium (arrowheads), the medulla (E) and papilla (F) contain cap mesenchyme derived cells which have a more elongated phenotype (arrows, inset shows higher power image of these structures).

Figure 3. Cap mesenchyme gives rise to diverse populations of renal epithelial cells

Cited1-CreER^T2/R26R^LacZ mice were injected with tamoxifen at E13 and lineage examined at 6 weeks of age. A–C, β-gal positive cells coincide with Na/K atpase expression, a broad marker of nephronic epithelium. Inset shows tubule in cross section. D–F, β-gal (D) and podoplanin (E) expression overlap (F) demonstrating cap mesenchyme lineage in the glomerular epithelial compartment (arrows). G/G′, Staining of sequential sections with anti-WT1 (G, nuclear) and anti-β-Gal (G′, cytoplasmic) shows that the cap mesenchyme gives rise to podocytes (arrows). The glomerular outline is indicated with dashed white lines. H/H′, Staining of sequential sections with anti-Aqp1 (H) and anti-β-Gal (H′) demonstrates that the cap mesenchyme gives rise to thin limb epithelium in the papilla. I/J, Immunoperoxidase staining using anti-Aqp1 on sections which have been β-Gal stained shows cap mesenchyme derived cells in thin limb in the papilla (I) and in proximal tubules in the cortex (J).

Figure 4. Neither collecting duct epithelium or renal endothelium arises from the cap mesenchyme

Cited1-CreER^T2/R26R^LacZ mice were injected with tamoxifen at E13 and lineage was examined by staining with anti-β-Galactosidase antibodies at 6 weeks of age. A–B′, Staining of sequential sections with anti-β-Gal (A, A′) and anti-Aqp2 (B, B′) demonstrates that cap mesenchyme derived cells do not populate the collecting duct epithelium. LacZ positive cells (arrows) are closely opposed to, but distinct from, Aqp2 positive collecting ducts (arrowheads). Position of one of the collecting ducts is indicated with dashed white lines, illustrating lack of overlap with β-Gal staining. C/D, Immunoperoxidase staining using anti-Aqp2 on sections which have been β-Gal stained shows that cap mesenchyme derived cells (arrows) do not overlap with either cortical (I, arrowheads) or medullary collecting ducts (J, arrowheads). Gl–glomeruli. E–G, Cap derived cells (E) and PECAM1 positive endothelial cells (F) do not overlap in glomeruli (G). H–J, Elongated cap mesenchyme progeny in the medulla (H, arrows) do not coincide with PECAM1 positive endothelium (I, arrowheads) but instead run alongside their closely associated vasculature (J).

Figure 5. Cap mesenchyme derived nephrons assume temporally-dependent deep and superfical positions in the adult kidney

Cited1-CreER^T2 mice were crossed to R26R^LacZ reporter mice and injected with 1.5 mg of tamoxifen at times indicated. Mice were sacrificed at 6 weeks of age and lineage assessed with β-Gal staining. A, E11 injections result in recombination in a low percentage of nephrons, but cap derived cells are found in the cortex, medulla, and papilla (arrows). B/C, Injection at E13 (B) and E15 (C) results in recombination in a large proportion of nephrons, some of which extend into the papilla (arrows). D, E18 injection results in recombination in an intermediate number of nephrons, however these structures do not extend into the papilla (arrowheads). Cx–cortex, Md–medulla, Pa–papilla.

Figure 6. Kinetics of CreER^T2 sub-cellular localization following tamoxifen injection

A–D, CreER^T2 localization using anti-Cre antibody staining in E16.5 kidneys; A′–D′ corresponding images with DAPI overlay to illustrate nuclei. Kidneys from uninjected animals (A, A′) show cytoplasmic localization of Cre (arrows). 8 hours after tamoxifen injection (B, B′) Cre is seen in cytoplasmic (arrows) and nuclear compartments (arrowheads). 24 hours after injection (C, C′) Cre is found almost exclusively in nuclei (arrowheads). 96 hours post-injection (D, D′) Cre has redistributed to the cytoplasm in the cap mesenchyme (arrows). UB-ureteric bud.

Figure 7. The cap mesenchyme gives rise to successive generations of renal progenitor cells

Pregnant Cited1-CreER^T2/R26R^LacZ mice were injected with tamoxifen at E13 and lineage was examined at E19.5 using β-Gal staining. A, Low power image of whole kidney shows cells of cap mesenchyme origin have begun to move into distal locations (arrows). B, Nephrogenic zone and primitive corticomedullary junction; cap derived cells are seen in the medulla (arrows, zone 3), inner nephrogenic region (zone 2) associated with comma- and S-shaped bodies (black arrowheads), and outer nephrogenic region (zone 1) containing cap mesenchyme (white arrows) and renal vesicles (white arrowheads). C, Outer nephrogenic zone demonstrating cap mesenchyme lineage in primitive nephrons (arrowheads) and retention of labeled cells in the cap mesenchyme (arrows). D. High power image of the branched UB tip. Persistent labeling of cells in the cap mesenchyme at E19.5 indicates that these are progeny of cap cells originally labeled at E13.