Calcium/NFAT signalling promotes early nephrogenesis

Abstract

A number of Wnt genes are expressed during, and are known to be essential for, early kidney development. It is typically assumed that their products will act through the canonical β-catenin signalling pathway. We have found evidence that suggests canonical Wnt signalling is not active in the early nephrogenic metanephric mesenchyme, but instead provide expressional and functional evidence that implicates the non-canonical Calcium/NFAT Wnt signalling pathway in nephrogenesis. Members of the NFAT (Nuclear Factor Activated in T cells) transcription factor gene family are expressed throughout murine kidney morphogenesis and NFATc3 is localised to the developing nephrons. Treatment of kidney rudiments with Cyclosporin A (CSA), an inhibitor of Calcium/NFAT signalling, decreases nephron formation--a phenotype similar to that in Wnt4(-/-) embryos. Treatment of Wnt4(-/-) kidneys with Ionomycin, an activator of the pathway, partially rescues the phenotype. We propose that the non-canonical Calcium/NFAT Wnt signalling pathway plays an important role in early mammalian renal development and is required for complete MET during nephrogenesis, potentially acting downstream of Wnt4.

Supplementary Fig. 1

Pan β-catenin antibody staining. A) In E12.5 sectioned kidneys anti-pan-β-catenin antibody fails to detect β-catenin (red) in the condensed MM whilst strong localisation was found in the UB (DAPI-stained nuclei are blue). B) By E18.5 anti-pan-β-catenin localises to epithelial structures including the tubules and those derived from the MM: the renal vesicles, and comma- and s-shaped bodies. Protein can also be seen in the cap mesenchyme at this stage. MM — metanephric mesenchyme; RV — renal vesicle; SB — s-shaped body; Tu — tubule; UB — ureteric bud.

Supplementary Fig. 2

NFATc3 and NFATc4 expression. 70 μm vibratome sections of E13.5 kidneys previously stained whole mount for A–B) NFATc3 and C) NFATc4 expression by in situ hybridisation. MM — metanephric mesenchyme; RV — renal vesicle; SB — s-shaped body; UB — ureteric bud.

Fig. 1

β-catenin signalling is not detectable in early nephrogenic tissues, suggesting that Wnt4 acts through a non-canonical pathway during nephrogenesis. A-F) X-gal staining in the BAT-gal reporter mouse confirms β-catenin signalling activity in the UB and absence of detectable activity in the MM and its derivatives, in A–C) ~ E11.75–E12.5 whole kidneys (not shown to scale), D) cultured kidney rudiments (E11.5 + 72 h), and E) sectioned E13.5 kidneys (counterstained with Nuclear Fast Red). F) Optical Projection Tomography (OPT) analysis of BAT-gal staining and immunolocalisation of Wt1 in an E12.5 kidney (condensed cap MM is denoted by white arrows). G–H) Direct fluorescence (green) from the product of the TCF/Lef:H2B-GFP reporter transgene in cultured (E11.5 + 18 h) kidneys with antibodies against G) Calbindin (red), a UB marker, and H) Pax2 (red), a marker of the MM. I) Immunohistochemistry on cultured kidneys (E11.5 + 48 h) for dephosphorylated (active) β-catenin (green). The signal around the edge of the cultured tissue is auto-fluorescence. MM — metanephric mesenchyme; UB — ureteric bud.

Fig. 2

NFAT genes are expressed during kidney development. A) Quantitative real-time RT-PCR analysis, relative to GAPDH, of E11.5–15.5 whole kidneys reveals that all five NFAT genes are expressed. The main graph shows data on the calcium-regulated NFAT genes (NFATc1-c4); the inlay graph also includes data on the non-calcium-regulated NFAT5 gene. B) Wt1-GFP (green) fluorescence in an embryonic kidney marking the MM and its derivatives. C) Expression analysis of NFATc genes in MM (Wt1-GFP positive) and whole kidney. Expression of D) NFATc3 and E) NFATc4 was confirmed by in situ hybridisation in E13.5 whole kidneys. G — gonad; K — kidney.

Fig. 3

NFATc3 is present in the developing nephrons and UB. Immunoperoxidase antibody detection (brown) on sections of A–C) E12.5 and D–F) E14.5 kidneys, and G–I) fluorescent antibody detection (green) on E11.5 + 72 h cultured kidney rudiments. In (A–F) cell nuclei are stained blue with haematoxylin; in (G–I) nuclei are DAPI-stained blue. Higher magnification images are presented from separate kidneys to increase the range of data shown. CB — comma-shaped body; RV — renal vesicle; SB — s-shaped body; Tu — tubule; UB — ureteric bud. Black arrows: condensed MM.

Fig. 4

Inhibition of calcineurin/NFAT signalling by CSA disrupts renal morphogenesis. A) Cultured kidney rudiments were grown on control (0.1% ethanol) or CSA-supplemented (10 μM) media and the numbers of UB tips and nephrons (epithelised bodies within the MM, at all stages of nephrogenesis) counted. CSA treatment for 24 h (C) or 72 h (G) disrupted renal morphogenesis, when compared to ethanol-treated controls (B, F respectively). The number of both UB tips and nephrons was reduced. D, E) These phenotypes could be rescued by removing the CSA-supplemented media at 24 h and replacing it with control media for 48 h. (B–G) show immunohistochemistry on cultured kidneys using antibodies against Laminin (red; marks basement membrane of epithelia) and Calbindin (green; marker of UB epithelium).

Fig. 5

Ionomycin disrupts renal morphogenesis in a genetic background-dependent manner. A–B) Control and Ionomycin-treated CD1 kidneys after A) 5 or B) 3 days in culture. A) Bright field, B) immunofluorescence using antibodies against Calbindin (green) and Laminin (red). C) Numbers of branches, tips and aggregates in control and Ionomycin-treated cultured CD1 kidney rudiments. D) Comparison of branch, tip and aggregate numbers for control and Ionomycin-treated cultured CD1 and C57BL/6 kidney rudiments (data is not available for C57Bl6 aggregate number in non-supplemented media).

Fig. 6

Disruption of calcium signalling reduces expression of UB genes but ectopic activation promotes nephrogenic gene expression. Quantitative real-time RT-PCR on E11.5 CD1 kidneys cultured for 72 h in the presence of CSA, FK506 or Ionomycin normalised to the relevant solvent-only control. CSA: 10 μM in ethanol; FK506: 10 μM in ethanol; Ionomycin: 1 μM in DMSO; ethanol: 0.1%; DMSO: 0.1%.

Fig. 7

Ectopic activation of Calcium/NFAT signalling partially rescues the Wnt4^−/− phenotype. A) Nephrogenesis is disturbed in Wnt4^−/− kidneys, as demonstrated by the absence of Laminin (red) marked aggregates in an E11.5 kidney grown in non-supplemented media for 72 h (green: Calbindin). B) The total number of renal aggregates (aggregates, renal vesicles, comma shaped bodies, s-shaped bodies) in wild type, Wnt4^+/− and Wnt4^−/− E11.5 kidney rudiments after 72 h culture in control or Ionomycin-supplemented medium. C) Number of UB branches and tips in control and Ionomycin-treated cultures. D–G) Immunofluorescense of control (D) or Ionomycin-treated (E–G) Wnt4-deficient kidney rudiments from the same embryo using Laminin (red, epithelialised nephrons) and Calbindin (green, UB) antibodies. H) Enlarged examples of the different stages of nephron formation found in Ionomycin-treated kidneys (not to scale). I) Quantification of different stages of renal aggregates.