FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development

Abstract

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

Fig. 1.

FGF/EGF signaling maintains an early nephron progenitor phenotype in vitro. (A) Proliferation assay of nephrogenic zone cells (NZCs) treated with recombinant proteins or retinoic acid. Shown is the number of pHH3-positive mitotic events per 50,000 cells. Inset shows NZC morphology after 24 hours in culture medium with or without FGF2. (B) Subcompartments in the nephrogenic zone. Arrows indicate direction of increasing differentiation. (C) Summary of the ability of growth factors to promote CITED1 expression in NZCs (24 hours). (D) Immunofluorescence showing that the RTK ligands FGF2, TGFα and EGF promote the greatest increase in CITED1 (red, 10× image) of all growth factors tested. FGF2 also promotes increased expression of SIX2 (red, 20× image). (E) FGF2 treatment dramatically increases Cited1 transcription after 24 hours. Error bars indicate s.d. (F) Increased CITED1 expression by FGF2 occurs within the PAX2⁺ nephron progenitor population. PAX2⁺ progenitors (green) from Cited1-CreER^T2 mice that drive a GFP reporter co-express GFP (red) when treated with FGF2, but not when cultured in medium alone. PAX2/GFP double-positive nuclei are yellow in the image overlays. Insets show representative positive and negative cells. (G) FGF2 treatment maintains Cited1 transcription up to 48 hours after harvest.

Fig. 2.

FGF candidates are redundantly expressed in distinct subcompartments of the nephrogenic zone. Tissue expression mined from GUDMAP was used to generate the heatmap shown (reprinted with permission from the GUDMAP consortium). Baseline (black) is derived from the median value for each gene across all tissues within each platform-specific dataset. Red denotes expression above baseline, and blue expression below baseline. UB, ureteric bud; UT, ureteric tip; CI, cortical interstitium; RV, renal vesicle; e, embryonic day; p, postnatal day.

Fig. 3.

Select FGF candidates maintain early nephron progenitor cells. (A) Immunofluorescence shows that, in addition to FGF2, the cap mesenchyme-expressed ligands FGF1, 9 and 20 promote maintenance of CITED1 (red) in NZCs cultured for 24 hours, whereas FGFs expressed in other compartments of the nephrogenic zone do not. (B) Similar to FGF2, cap mesenchyme-expressed FGFs (200 ng/ml each, 24-hour treatment) increase transcription of higher-order progenitor markers in cultured NZCs. (C) qPCR analysis of Fgfr1 and Fgfr2 isoforms in freshly isolated NZCs demonstrates over-representation of the c isoform compared with the b isoform for each receptor. Data were corrected for differences in primer efficiency as shown in supplementary material Fig. S3B. (D) Similar to Cited1, transcription of Fgf1, 9 and 20 decreases in cultured NZCs at 24 hours in the absence of FGF2. Error bars indicate s.d.

Fig. 4.

FGF/EGF signaling maintains early nephron progenitors through RAS-mediated signaling. (A) EGF and TGFα promote a transcriptional profile of higher-order nephron progenitor markers, similar to that of FGF2 (see Fig. 1E), including increased expression of Fgf1 and Fgf9 (insets). Error bars indicate s.d. (B) The FGF receptor inhibitor PD173074 blocks CITED1 maintenance promoted by FGF2, but not that promoted by EGF or TGFα. The RAS (FPT III) and PI3K (LY294002) inhibitors block CITED1 protein expression promoted by all three ligands, demonstrating a common RAS/PI3K dependency.

Fig. 5.

Strategy to overexpress the RTK inhibitor Spry1 in the CITED1 progeni tor compartment. (A) FGF2-treated NZCs (24 hours) derived from tamoxifen-inducible Spry1-Tg mice overexpress Spry1 and block CITED1 protein expression when infected with Cre-expressing adenovirus, as compared with GFP-expressing adenovirus controls. Representative images are shown. Error bars indicate s.d. (B) Strategy to overexpress Spry1 in the CITED1⁺ compartment (red cells in diagram) in vivo. (C) X-gal staining of E17.5 Cited1-CreER^T2;R26R^lacZ kidneys demonstrates recombination in the cap mesenchyme 24 hours after a single tamoxifen injection of the pregnant dam. An X-gal staining negative control is shown in supplementary material Fig. S5. IM, induced mesenchyme; RV, renal vesicle.

Fig. 6.

RTK-mediated RAS signaling is required to maintain the expression of primitive nephron progenitor markers in vivo. (A) Overexpression of mouse Spry1 in the Cited1 progenitor compartment reduces CITED1 protein expression, but not PAX2 or WT1 expression, in the E17.5 embryonic kidney 24 hours after tamoxifen injection. (B) Representative images of the nephrogenic zone demonstrate downregulation of CITED1 and SIX2, but not PAX2 or WT1, in Spry1-expressing cap mesenchyme. (C) Quantitation of CITED1 and SIX2 protein expression from kidney sections derived from Spry1-Tg mice with or without the Cited1-CreER^T2 driver. Error bars indicate s.e. (D) Transcription of early progenitor markers is decreased in NZCs derived from tamoxifen-injected Cited1-CreER^T2;Spry1-Tg transgenic mice as compared with Cited1-CreER^T2 negative controls. Shown are average fold changes ± s.e.m. derived from two independent experiments of NZCs extracted from a total of five Cited1-CreER^T2-positive and 11 Cited1-CreER^T2-negative embryonic kidney pairs, respectively.

Fig. 7.

The fate of nephron progenitors is disrupted in Spry1-overexpressing mice. (A) Representative X-gal staining from Cited1-CreER^T2;R26R^lacZ;Spry1-Tg E17.5 frozen kidney sections 24 hours after tamoxifen treatment demonstrates a loss of β-gal⁺ progenitors and a reduced contribution to more differentiated nephron structures compared with Cited1-CreER^T2;R26R^lacZ controls. (B) Representative immunofluorescent images from E17.5 kidney sections of Cited1-CreER^T2;Spry1-Tg mice demonstrate a loss of the induced mesenchymal and renal vesicle marker LEF1 as compared with control Spry1-Tg mice. (C) Quantitation of LEF1 expression from the full panel of immunofluorescence images shown in supplementary material Fig. S10. Error bars represent the s.e.m. from four mice analyzed in each group.

Fig. 8.

Transgenic Spry1 expression increases apoptosis and proliferation in the cap mesenchyme. (A) Representative images of TUNEL-stained E17.5 Cited1-CreER^T2;Spry1-Tg kidneys show increased apoptosis in the nephrogenic zone, as compared with Spry1-Tg controls. Kidneys were harvested 24 hours after tamoxifen injection. (B) Quantification of TUNEL shows a ∼5-fold increase in apoptosis in the cap mesenchyme per bud tip (n=448) compared with the control (n=361). Four kidney sections were counted and averaged from each of three mice per group. (C,D) Cited1-CreER^T2;Spry1-Tg mice display increased levels of (C) the early apoptosis marker cleaved caspase 3 (CASP3) and (D) the mitosis marker pHH3 in the cap mesenchyme 12 hours after tamoxifen injection. One hundred and eighty-five cap mesenchymes were counted and averaged from each of four mice per group. (E) Representative images co-stained for pHH3 (yellow) and SIX2 (green). Arrows indicate the pHH3⁺ cells counted in the mesenchyme, but not the ureteric bud. (F) Frozen tissue section showing cap mesenchyme cells stained for R26R expression by X-gal staining (blue) and for pHH3 by DAB staining (brown) in Cited1-CreER^T2;R26R^lacZ;Spry1-Tg mice. Double-negative cells are red, whereas R26R/pHH3 double-positive cells show intense dark-blue staining. (G) Quantitation of kidney sections (as shown in F) demonstrates a much higher percentage of wild-type progenitors undergoing mitosis compared with β-gal⁺ progenitors that have undergone recombination. Error bars indicate s.d. per kidney section.

Fig. 9.

Antagonism of RTK signaling in the CITED1⁺ compartment alters FGF ligand and Wnt target gene expression in the nephrogenic zone. (A) NZCs derived from Cited1-CreER^T2;Spry1-Tg mice show decreased expression of Fgf1 and Fgf9 compared with control mice, as assessed by qPCR. (B) BIO does not increase the expression of CITED1 in NZC cultures. (C) NZC cultures treated with FGF2 display increased transcription of several genes (including Uncx4.1 and Pla2g7) that have recently been shown to be direct targets of Wnt signaling in the E11.5 kidney; this is not reversed by the Wnt inhibitor IWR1 (10 μM). (D) Freshly isolated NZCs derived from Cited1-CreER^T2;Spry1-Tg mice show decreased expression of Uncx4.1 and Pla2g7. The numbers of mice and statistical analyses performed are as described in Fig. 6D. Error bars in A and D indicate s.e. as shown in Fig. 6D.