Deletion of Frs2α from the ureteric epithelium causes renal hypoplasia

Abstract

Fibroblast growth factor receptor 2 (Fgfr2) signaling is critical in maintaining ureteric branching architecture and mesenchymal stromal morphogenesis in the kidney. Fibroblast growth factor receptor substrate 2α (Frs2α) is a major docking protein for Fgfr2 with downstream targets including Ets variant (Etv) 4 and Etv5 in other systems. Furthermore, global deletion of Frs2α causes early embryonic lethality. The purpose of the study was to determine the role of Frs2α in mediating Fgfr2 signaling in the ureteric epithelium. To that end, we generated mice with conditional deletion of Frs2α in the ureteric epithelium (Frs2α^UB−/−) and mice with point mutations in the Frs2α binding site of Fgfr2 (Fgfr2^LR/LR). Frs2α^UB−/− mice developed mild renal hypoplasia characterized by decreased ureteric branching morphogenesis but maintained normal overall branching architecture and had normal mesenchymal stromal development. Reduced nephron endowment in postnatal mutant mice was observed, corresponding with the reduction in branching morphogenesis. Furthermore, there were no apparent renal abnormalities in Fgfr2^LR/LR mice. Interestingly, Etv4 and Etv5 expression was unaltered in Frs2α^UB−/− mice, as was Sprouty1, an antagonist of Frs2α signaling. However, Ret and Wnt11 (molecules critical for ureteric branching morphogenesis) mRNA levels were lower in mutants vs. controls. Taken together, these findings suggest that Fgfr2 signals through adapter molecules other than Frs2α in the ureteric epithelium. Furthermore, Frs2α may transmit signals through other receptor kinases present in ureteric epithelium. Finally, the renal hypoplasia observed in Frs2α^UB−/− mice is likely secondary to decreased Ret and Wnt11 expression.

metanephric kidney development occurs via reciprocal signaling between two tissues derived from intermediate mesoderm, the ureteric bud and the metanephric mesenchyme. The metanephric mesenchyme, paired densities of tissue in the region of the hindlimb, sends signals causing the ureteric bud to evaginate from the Wolffian (nephric) duct. After the ureteric bud invades the metanephric mesenchyme, the mesenchyme stimulates the ureteric epithelium to branch and elongate, forming the collecting duct system of the kidney (6, 7, 31, 39). Simultaneously, ureteric tips send signals causing the mesenchyme to condense and then convert into epithelial nephrons. In addition to the nephrogenic mesenchyme, a separate mesenchymal lineage, called the stromal mesenchyme, appears early in kidney development, ultimately forming interstitial tissues and macrophages.

While there are many molecular pathways important to kidney development, signaling through fibroblast growth factor receptors (Fgfrs) is critical for many different steps of kidney development. Fibroblast growth factor receptors are receptor tyrosine kinases, with 4 known signaling family members and 23 ligands in mammals. Many studies, including genetic manipulation of Fgf and/or Fgfr expression, have shown the importance of this signaling family in ureteric bud induction and branching, nephrogenesis, and stromal development (2, 3, 15, 25, 29, 30, 32, 33, 36, 37, 47). Due to early embryonic lethality of global deletion in some ligands and receptors, conditional knockout approaches are often necessary to interrogate the roles of these genes in kidney development. Our laboratory used this approach in revealing the importance of Fgfr2 signaling in the ureteric bud. We found that Fgfr2 deletion in the ureteric epithelium (Fgfr2^UB−/−) leads to profound defects in ureteric branching morphogenesis, including long, thin trunks and a dramatic reduction in ureteric bud tips (leading to a decrease in nephron number). In addition, Fgfr2^UB−/− mice had stromal mispatterning, including thickened cortical stroma (as evidenced by expanded FoxD1 expression) and an absence of interdigitating fingers between developing nephron units. Ultimately, these defects resulted in a dramatic reduction in kidney size and abnormally shaped kidneys (47).

Fgfrs transmit intracellular signals by phosphorylating and activating docking proteins, including fibroblast growth factor receptor substrate 2α (Frs2α). Frs2α activation leads to a wide array of downstream signaling cascades including phosphatidylinositol-3 (PI-3), mitogen activated protein (MAP) kinase Erk, and alternative forms of protein kinase C (PKC) (11, 12, 14, 16, 26, 27). Two farther downstream targets of Frs2α via Erk activation include two members of the Ets transcription factor family, Ets variant (Etv) 4 and Etv5 (42).

Although Frs2α was identified through its interactions with Fgfrs, Frs2α also acts as a docking protein for several other tyrosine kinases, including Ret, multiple neurotrophin receptors (Trks), and anaplastic lymphoma kinase (Alk) (20, 21, 24). Not surprisingly, given its actions as an adapter molecule for so many receptors and its links to many downstream signaling cascades, global deletion of Frs2α in mice is lethal early in embryogenesis, before kidney development (16). To circumvent the early lethality of the global knockout, we generated a floxed Frs2α mouse line that in the presence of cre recombinase deletes the largest of the coding exons (exon 5) (22). The floxed line produced effective deletion of Frs2α and its downstream targets in both the heart (22) and prostate (46).

Based on the critical role of Frs2α in Fgfr signaling (which is essential in the ureteric bud lineage), we sought to clarify the role of this molecule in the ureteric bud of the developing kidney. To that end, we generated conditional knockouts of Frs2α in the ureteric epithelium using our Hoxb7cre line (47). To complement this approach, we examined potential renal defects in mice with point mutations in the Frs2α binding site of Fgfr2.

METHODS

Mouse Strains

All experiments involving mice were approved by the local Institutional Animal Care and Use Committee.

Frs2α^UB−/−conditional knockout mice.

The mice used to generate the conditional knockout line have been described previously. Briefly, transgenic mice with a portion of the Hoxb7 promoter driving cre recombinase and enhanced green fluorescent protein expression (Hoxb7creEgfp^Tg/+) (47) were crossed with Frs2α^Lox/Lox mice (22). Progeny hemizygous for cre and with 2 lox-p sites had functional deletion of Frs2α (Hoxb7creEgfp^Tg/+/Frs2α^Lox/Lox) from the ureteric epithelium (Frs2α^UB−/−). Timed overnight matings were set up between the various genotypes; when a vaginal plug was identified, noon of that day would be deemed embryonic day 0.5 (E0.5).

Fgfr2^LR/LR mice.

The insertion of two point mutations into the Frs2α binding site of Fgfr2 has been previously described (9). In brief, amino acid Leu-424 and amino acid Arg-426 were replaced with Ala residues. These point mutations were shown to effectively block the ability of Frs2α to bind to Fgfr2 (9).

Genotyping

Genotyping was performed via PCR. Briefly, Tail clippings and or embryonic tissues were collected and digested and genomic DNA was isolated. PCR amplification was used to identify the various mutant and control alleles. To determine whether mice were carrying the HoxCreEgfp cassette, the forward primer 5′-AGCGCGATCACATGGTCCTG-3′ and reverse primer 5′-ACGATCCTGAGACTTCCACACT-3′ were utilized, and a positive result would give a single band at 230 bp. To determine the genotype of the Frs2a floxed mice, the forward primer 5′-GAGTGTGCTGTGATTGGAAGGCAG-3′ and reverse primer 5′-GGCACGAGTGTCTGCAGACACATG-3′ were utilized, with a wild-type band that was evident at 224 and a mutant band at 319 bp. To determine the genotype of the Fgfr2^LR/LR mice, the forward primer 5′-GAGTACCATGCTGACTGCATGC-3′ and the reverse primer 5′-GGAGAGGCATCTCTGTTTCAAGACC-3′ were used, with a wild-type band that was evident at 225 and a mutant band at 315 bp.

Organ Culture

At E11.5, pregnant mice that would generate Frs2α^UB−/− and Fgfr2^LR/LR progeny (and littermate controls) were killed and embryos were quickly removed. Kidneys were dissected from embryos and placed onto nucleopore filters floating on Dulbecco's modified Eagle medium supplemented with 10% FBS, penicillin/streptomycin, and l-glutamine at 37°C. Following a 3-day culture period, kidneys were submerged in ice-cold methanol for a minimum of 15 min before whole-mount immunohistochemistry for calbindin D_28K as previously described (40). Briefly, kidneys were washed in PBS, permeabilized with 0.3% Triton X-100 in PBS, and then washed again before being blocked in 5% FBS. Kidneys were then incubated with 1:100 monoclonal mouse anti-calbindin-D_28k antisera in 5% FBS for 3 h at 37⁰C before being extensively washed in PBS. Kidneys were treated with 1:500 goat anti-mouse Alexa Fluor 488 for 2 h at 37°C. Kidneys were washed in PBS and mounted. The ureteric bud branch pattern and numbers of ureteric tips were then observed under a fluorescent microscope (Leica, Bannockburn, IL).

Frs2α Immunohistochemistry

Frs2α immunostaining was performed as previously described (45). Briefly, 7-μm paraffin sections in Frs2α^UB−/− and control littermates were cut, and antigen retrieval was performed using citrate buffer. Sections were incubated with anti-Frs2α antisera (Santa Cruz, CA), the primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA), and the nuclei were visualized using TP3.

In Situ Hybridization

Whole-mount nonradioactive in situ hybridization (ISH) was performed on E11.5 on E13.5 Frs2α^UB−/− and control embryos and/or dissected kidneys as described previously (17). Digoxigenin UTP-labeled antisense and sense RNA probes were generated against multiple targets, including Frs2α· [GenBank accession no. BC055334, Etv4 (NM008815), Etv5 (NM023794), FoxD1 (L38607), Gdnf (NM010275), Ret (BC059012), Sprouty1 (NM011896), and Wnt11 (NM009519)]. Select whole-mount in situ hybridization-stained tissues were reembedded in paraffin and sectioned at 10 μm. In addition, radioactive section ISH was performed on E13.5 Frs2α^UB−/− and control embryos with probes against Frs2α as described (34, 47).

Real-Time PCR

Four mutant and four control E13.5 embryos were collected, and the kidneys were harvested. RNA was then extracted using a kit (Qiagen, Valencia, CA). Probes for Etv4, Etv5, Gdnf, Ret, Sprouty1, and Wnt11 were utilized, and Gapdh was used as an endogenous control (Invitrogen, Carlsbad, CA). Quantitative real-time PCR was performed using standard protocols and procedures (43) on an Applied Biosystems ABI 7900 HT (Foster City, CA).

Apoptosis Assay

We conducted terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays on E13.5 wild-type and Frs2α ^UB−/− kidney sections, using a Fluorescent FragEl DNA Fragmentation Detection kit (Oncogene, Cambridge, MA) as previously described (34).

Morphometric Measurements

E17.5 or postnatal day 30 (P30) Frs2α^UB−/− and control mice were killed and weighed. Kidneys were removed, weighed, and photographed. The surface area and kidney long axis were measured using Image J analysis software (version 1.32j from Wayne Rasband, National Institutes of Health). In addition, P30 Frs2α^UB−/− and littermate control kidneys (both n = 6) as well as Fgfr2^LR/LR and littermate wild-type controls (both n = 6) were harvested for cortical-medullary ratios. Briefly the kidneys were cut in the transverse midplane, processed through to paraffin, and embedded on the cut surface. Kidneys were sectioned at three levels 200 μm apart and stained with hematoxylin and eosin. The arcuate vessels were used to separate the medulla from the cortex; points were then tabulated for both the medulla and cortex, and a ratio of the cortex to the medulla was calculated for each sample.

Nephron Number Counts

Nephron number counts were performed on P30 kidneys as previously described (40). In brief, six control and six mutant kidneys were immersion fixed and processed through to glycolmethacrylate. Kidneys were then exhaustively sectioned at 20 μm. Kidney volume, glomerular number, and various glomerular volumes were then collected and tabulated using the physical dissector/fractionator method.

Statistical Analysis

Statistical analysis were carried out on all biological replicates with a Student's t-test or a one-way ANOVA followed by Fisher's post hoc tests. All values are represented as means ± SD.

RESULTS

Frs2α Expression in Control and Frs2α^UB−/− Developing Kidneys

To determine the role of Frs2α in the ureteric bud, we first characterized expression in controls and then wanted to confirm its deletion from the ureteric epithelium in Frs2α^UB−/− mice. At E11.5, Frs2α is expressed in the nephric duct and the ureteric bud trunk with a low-level signal at the ureteric tips; expression is also seen in the metanephric mesenchyme (Fig. 1, A–D). At E13.5, Frs2α appears strongly expressed throughout control kidneys by whole-mount staining, compared with a much lighter signal in Frs2α^UB−/− kidneys (Fig. 1E). Sections of the E13.5 tissues reveal strong expression in control ureteric tips and trunk (Fig. 1F) and the nephrogenic zone (not shown). In contrast, there was no signal in the ureteric epithelium above background in Frs2α^UB−/− E13.5 sections while expression was maintained in the nephrogenic zone (Fig. 1G). Similar expression patterns were observed when the protein was visualized by immunostaining in E13.5 mutants and controls (Fig. 2). While controls had expression in both early nephron structures (condensing mesenchyme) and ureteric tips (Fig. 2, A–C), Frs2α^UB−/− kidneys showed Frs2α labeling in the nephrogenic zone only and no expression in the ureteric epithelium (Fig. 2, D–F). Thus at early stages of renal development, Frs2α is expressed in both metanephric mesenchyme and ureteric lineages. Furthermore, Frs2α is deleted in ureteric epithelium in Frs2α^UB−/− kidneys.

Fig. 1.

Fibroblast growth factor receptor substrate 2α (Frs2α) expression by in situ hybridization (ISH) in the developing kidney. A and B: at embryonic day 11.5 (E11.5), whole-mount ISH with sense (A) and antisense (B) probes reveal Frs2α staining in the nephric duct (arrowhead) and the ureteric bud (arrow). Scale bar = 100 μm. C and D: following whole-mount staining in E11.5 tissue sections, the antisense probe shows Frs2α expression in the nephric duct (C, arrowhead) and ureteric trunk (C, arrow) and a lower level signal in ureteric tips (D, arrowheads); labeling is also seen in mesenchyme surrounding the tips (D, arrow). Scale bar = 100 μm. E: at E13.5, whole-mount ISH in control (left) and Frs2α^UB−/− (right) kidneys reveals reduced level of expression in the mutant. Scale bar = 200 μm. F and G: following whole-mount staining, E13.5 tissue sections from control (F) and Frs2α^UB−/− kidneys (G) demonstrate strong Frs2α expression in control ureteric tips (F, arrowheads) and trunk (arrow), but background levels of signal in Frs2α^UB−/− ureteric tips (G, arrowheads) and trunk (arrow). Some mesenchymal Frs2α expression is present in both controls and mutants (concave arrowheads). Scale bars = 25 μm.

Fig. 2.

Frs2α is deleted from the ureteric epithelium by dual-labeling. immunofluorescence. A–C: in E13.5 control kidney sections, anti-TP3 (showing cell nuclei; A) and anti-Frs2α (B) reveal Frs2α staining in ureteric tips on the merged image (C, arrowheads). D–F: in E13.5 Frs2α^UB−/− renal sections, anti-TP3 (D) and anti-Frs2α (E) demonstrate a lack of Frs2α expression in ureteric tips on the merged image (F, arrowheads). In both controls and mutants, Frs2α is expressed in surrounding mesenchymal tissues including condensing mesenchyme (CM). Scale bars = 50 μm.

Frs2α^UB−/− Kidneys are Hypoplastic

We next characterized whether deletion of Frs2α from the ureteric bud led to any renal abnormalities. While cre-negative, heterozygous, and homozygous embryos appear similar at E17.5 (not shown), Frs2α^UB−/− kidneys are smaller than cre-negative and Frs2α^UB+/− kidneys (Fig. 3, A and B, and not shown). Image J measurements confirmed that E17.5 Frs2α^UB−/− kidneys were significantly smaller than cre-negative and Frs2α^UB+/− kidneys by weight, long axis, and surface area (Fig. 3, C–E). In contrast, there were no significant differences between embryo body weight or crown rump length (Table 1). Histological analysis of the mutant kidneys also revealed that the general architecture of the kidneys was unchanged across all three genotypes (Fig. 4 and not shown). Thus Frs2α^UB−/− mice appear to have hypoplastic kidneys compared with both cre-negative and heterozygous mice. Given that the Frs2α^UB+/− and cre-negative kidneys were not different, we used them together as controls for the remaining studies.

Fig. 3.

E17.5 Frs2α^UB−/− kidneys are smaller than controls. A and B: images of cre-negative (A) and Frs2α^UB−/− (B) kidneys reveal that the mutants appear smaller. Scale bars = 500 μm. C–E: bar graphs showing that Frs2α^UB−/− kidneys have reduced weight (C), volume (D), and surface area (E) compared with littermate cre-negative and Frs2α^UB+/− kidneys. *P < 0.01. **P < 0.001. †P < 0.0001.

Table 1.

Measurements of Frs2α^UB−/− embryos and kidneys at E17.5

Genotype	Body Wt, g	Crown Rump Length, cm	Kidney Wt, mg	Long Axis, cm	Surface Area, mm2
Cre negative (n = 46)	1.11±0.10	2.17±0.08	5.10±0.67	0.281±0.019	0.042±0.005
Frs2αUB+/− (n = 12)	1.09±0.05	2.18±0.04	4.97±0.63	0.283±0.017	0.043±0.005
Frs2αUB−/− (n = 14)	1.09±0.08	2.16±0.09	3.77±0.40*†	0.261±0.013*†	0.038±0.004‡§

Values are means ± SD; n = no. of animals. Frs2α, fibroblast growth factor receptor substrate 2α; E17.5, embryonic day 17.5.

^*P < 0.01 vs. cre negative.

^†P < 0.01 vs. Frs2α^UB+/−.

^‡P < 0.001 vs. cre negative.

^§P < 0.001 vs. Frs2α^UB+/−.

Fig. 4.

E17.5 Frs2α^UB−/− histological sections have a normal overall appearance. A and B: low-power hematoxylin and eosin-stained sections reveal that compared with cre-negative (A) Frs2α^UB−/− E17.5 kidneys (B) have a normal overall appearance of the cortex and medulla. Scale bars = 500 μm. C and D: higher-power sections show normal developing ureteric tips (arrows) and developing nephrons including mature glomeruli (arrowheads) in cre-negative (C) and mutant E17.5 kidneys (D). Scale bars = 50 μm.

Similar to the findings at E17.5, gender-matched P30 mutants and controls have comparable body weights, while Frs2α^UB−/− kidneys were smaller as measured by weight, long axis, and surface area than gender-matched controls (Table 2 and not shown). Also, gross histological evaluation of these mutant kidneys revealed no phenotypic abnormalities (not shown), and there were no differences between mutant and control cortex-to-medulla ratios (Table 2 and not shown). Nephron number, as determined by the physical dissector fractionator method in a separate set of P30 mice, was reduced by 22% in mutants compared with controls (Table 3); however, the total and individual glomerular and glomerular capillary (corpuscle) volumes were not different between the groups. Thus, as in the embryos, Frs2α^UB−/− P30 kidneys appear hypoplastic. Interestingly, the reductions in kidney size of the Frs2α^UB−/− mutants was less dramatic than in Fgfr2^UB−/− mice (47).

Table 2.

Measurements of Frs2α^UB−/− embryos and kidneys at P30

Genotype	Sex	Body Wt, g	Kidney Wt, g	Long Axis, cm	Surface Area, cm2	Cortex-to-Medulla Ratio
Control (n = 10)	Female	16.2±1.6	0.11±0.01	0.83±0.03	0.37±0.03	1.85±0.24:1
Control (n = 6)	Male	19.5±1.3	0.14±0.02	0.91±0.05	0.44±0.04	1.93±0.19:1
Frs2αUB−/− (n = 7)	Female	15.3±1.1	0.09±0.01	0.78±0.03	0.32±0.02	1.77±0.35:1
Frs2αUB−/− (n = 9)	Male	16.1±1.5	0.10±0.01	0.82±0.05	0.34±0.04	1.81±0.22:1

Body Weight	Kidney Weight
Male control vs. male Frs2αUB−/−, P < 0.01	Male control vs. male Frs2αUB−/−, P < 0.001
Female control vs. female Frs2αUB−/−, NS	Female control vs. female Frs2αUB−/−, P < 0.01
Female control vs. male control, P < 0.01	Female control vs. male control, P < 0.001
Female Frs2UB−/− vs. male Frs2αUB−/−, NS	Female Frs2αUB−/− vs. male Frs2αUB−/−, NS

Long Axis	Surface Area
Male control vs. male Frs2αUB−/−, P < 0.001	Male control vs. male Frs2αUB−/−, P < 0.001
Female control vs. female Frs2αUB−/−, P < 0.01	Female control vs. female Frs2αUB−/−, P < 0.001
Female control vs. male control, P < 0.001	Female control vs. male control, P < 0.001
Female Frs2αUB−/− vs. male Frs2αUB−/−, NS	Female Frs2αUB−/− vs. male Frs2αUB−/−, NS

Cortex-to-Medulla Ratio
All NS

Values are means ± SD for body wt, kidney wt, long axis, and surface area; n = no. of animals. P30, postnatal day 30; NS, not significant.

Table 3.

Characterization of glomerular number and volume in P30 kidneys

	Control Males (n = 6)	Frs2UB−/− Males (n = 6)
Body weight, g	18.53±2.00	15.13±0.92
Kidney weight, g	0.133±0.018	0.102±0.013
Kidney volume, mm3	109.0±8.0	87.7±15.4*
Total glomerular number	1,0823±1,094	8,387±1,265†
Mean glomerular volume, ×10−4 mm3	2.39±0.33	2.50±0.27
Total glomerular volume, mm3	2.58±0.38	2.10±0.38
Mean renal corpuscle volume, ×10−4 mm3	2.91±0.34	3.08±0.25
Total corpuscle volume, mm3	3.14±0.33	2.59±0.46*

Values are means ± SD; n , no. of mice.

^*P < 0.05.

^†P < 0.01.

To further determine whether the effects of deleting Frs2α from the ureteric epithelium were different than in Fgfr2^UB−/− kidneys, we analyzed ureteric branching morphogenesis and stromal patterning in Frs2α^UB−/− and controls. To determine whether there were any differences between mutants and controls at the early stages of ureteric development, we performed whole-mount in situ hybridization for Ret (to visualize the Wolffian duct and ureteric epithelium) at E11.5. As shown (Fig. 5, A and B), initial ureteric invasion and branching appear similar. To examine subsequent ureteric development, we cultured E11.5 mutants and controls for 3 days and performed whole-mount immunostaining against calbindin. As shown (Fig. 5, C–E) the mutant explants were smaller and had significantly fewer tips than controls. Interestingly, however, the overall pattern of ureteric branching in Frs2α^UB−/− kidneys was very similar to controls. This is in contrast to Fgfr2^UB−/− mice that had alterations in branch patterning, including long, think trunks and a more severe reduction in ureteric tip number (47).

Fig. 5.

Ureteric branching morphogenesis is inhibited in E11.5 Frs2α^UB−/− kidney explants. A and B: images of E11.5 whole-mount ISH for Ret show similar initial ureteric elongation and branching controls (A) and mutants (B). Scale bar = 100 μm; n = 6 in each group. C and D: images of whole-mount immunohistochemistry for calbindin-D_28k show that controls (C) have more branching than mutants (D) after 3 days of growth, although the overall branch pattern in the mutants appear normal. Scale bar = 100 μm. E: bar graph revealing that mutants have a significant reduction in ureteric tips compared with controls (Frs2α^UB−/− 47.6 ± 2.0, control 30.5 ± 2.8). Values are means ± SE. P < 0.0001; n = 17 (control) and n = 12 (mutant).

We also assessed stromal mesenchymal patterning first by expression of Foxd1 in Frs2α^UB−/− mutants and controls. As shown, at E13.5, Foxd1 has a comparable expression pattern in cortical stroma of control (Fig. 6, A and A′) and Frs2α^UB−/− (Fig. 6, B and B′) kidneys, including interdigitating fingers (arrowheads). Also, TUNEL staining was similar in control and Frs2α^UB−/− kidneys with few apoptotic cells in the developing medulla (Fig. 6, C and D). These findings are in stark contrast to Fgfr2^UB−/− mice that develop aberrantly thickened cortical stroma with no interdigitating fingers and that demonstrate excessive cortical stromal apoptosis (47). Thus Frs2α^UB−/− mice appear to have much milder renal abnormalities than Fgfr2^UB−/− mice.

Fig. 6.

Foxd1 ISH and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in E13.5 Frs2α^UB−/− kidneys. A and A′: lightfield (A) and darkfield (A′) images of control tissue section stained for Foxd1 and counterstained with hematoxylin show cortical stroma including interdigitating fingers (arrowheads). B and B′: lightfield (B) and darkfield (B′) images of Frs2α^UB−/− section stained as above also reveals normal stromal pattern in the mutants including the interdigitating fingers (arrowheads). C and D: merged lightfield and darkfield images from control (C) and Frs2α^UB−/− (D) tissue sections with TUNEL and hematoxylin staining reveal similar low levels of apoptosis in mutants and controls (green dots). Scale bars = 100 μm.

Mice with Point Mutations in the Frs2α Binding Site of Fgfr2 have Phenotypically Normal Kidneys

To further clarify the relationship between Fgfr2 and Frs2α in the ureteric bud (i.e., whether Frs2α mediates signals from Fgfr2), we generated mice with point mutations in the Frs2α binding site of Fgfr2 (that effectively abrogate interactions between the 2 molecules) (9). Using E11.5 organ culture, we observed no differences in ureteric branching morphogenesis between Fgfr2^LR/LR mice (named for the amino acids changed to alanine), heterozygous (Fgfr2^LR/+), and wild-type littermates (not shown). Ureteric tip counts were not statistically different among any of the genotypes (wild-type 45.15 ± 8.09, Fgfr2^LR/+ 47.00 ± 8.07, Fgfr2^LR/LR 45.88 ± 6.06). At P30, we observed no overt differences between mutants and controls. Measurements in male mice confirmed no differences in body weights (wild-type 19.77 ± 3.32 g, Fgfr2^LR/LR 19.55 ± 5.67 g); kidney weights (wild-type 0.144 ± 0.033 g, Fgfr2^LR/LR 0.152 ± 0.053 g); kidney long axes (wild-type 0.872 ± 0.080 cm, Fgfr2^LR/LR 0.884 ± 0.120 cm); or kidney surface areas (wild-type 0.410 ± 0.070, Fgfr2^LR/LR 0.422 ± 0.115 cm²). Similarly, there were no differences between male Fgfr2^LR/LR and wild-type renal cortex-to-medulla ratios (data not shown). Thus the renal abnormalities in the Frs2α^UB−/− mice are not recapitulated in Fgfr2^LR/LR mice.

Downstream Targets Etv4 and Etv5 are Unaltered in Frs2α^UB−/− Kidneys

To further investigate the reasons for the abnormalities in Frs2α^UB−/− kidneys, we examined the expression of two known downstream targets of Frs2α in other systems, namely, Etv4 and Etv5 in E13.5 kidneys. Surprisingly, we observed no differences in expression of Etv4 and Etv5 in Frs2α^UB−/− kidneys compared with controls (Fig. 7). A robust Etv4 and Etv5 signal was present in ureteric tips in both mutants and controls (arrowheads). Etv4 and, to a lesser extent, Etv5 were also present in mesenchymal-derived tissues in both mutants and controls (arrows). Thus the renal defects seen in Frs2α^UB−/− mice are not secondary to obvious changes in Etv4 or Etv5 expression.

Fig. 7.

Etv4 and Etv5 expression in E13.5 Frs2α^UB−/− kidneys. A and A′: lightfield (A) and dark field (A′) images of control tissues stained for Etv5 and counterstained with hematoxylin showing Etv5 expression in the ureteric tips (arrowheads) and the surrounding mesenchyme (arrows). B and B′: lightfield (B) and darkfield (B′) images of Frs2α^UB−/− section stained for Etv5 showing normal tip and mesenchymal expression of Etv5. C and C′: lightfield (A) and darkfield (A′) images of control tissues stained for Etv4 and counterstained with hematoxylin showing expression of Etv4 in the ureteric tips (arrowheads) and the surrounding mesenchyme (arrows). D and D′: lightfield (D) and darkfield (D′) images of Frs2α^UB−/− section stained for Etv4 showing normal tip and mesenchymal expression of Etv4. Scale bars = 100 μm.

Expression of Sprouty1, Gdnf, Ret, and Wnt11 in Frs2α^UB−/− Kidneys

Given that Sprouty1 is a known antagonist of Frs2α (and Fgfr2) signaling, we assayed for whether its overexpression could explain the hypoplastic phenotype seen in Frs2α^UB−/− kidneys. As shown, however, expression of Sprouty1 appears unaltered in mutants at E11.5 or E12.5 by in situ hybridization compared with controls (Fig. 8). Furthermore, real-time PCR measurements in whole kidneys at E12.5 confirmed no significant differences between mutants and controls (89% Sprouty1 mutant expression vs. controls, P = 0.294). Thus renal hypoplasia in Frs2α^UB−/− mice cannot be explained by an overexpression of Sprouty1.

Fig. 8.

Sprouty1 expression is unaltered in Frs2α^UB−/− kidneys. A and A′: representative images of E11.5 whole-mount ISH for Sprouty1, showing similar levels of expression in the controls (A) and mutants (A′) in the mesenchyme directly adjacent to the ureteric tips. B and B′: whole-mount ISH at E12.5 for Sprouty1 showing less branching but a similar expression pattern in the mesenchyme directly adjacent to the ureteric epithelium in controls (B) and mutants (B′); n = 6 for all groups.

Given the critical importance of Gdnf, Ret, and Wnt11 in ureteric branching morphogenesis, we next examined their expression in Frs2α^UB−/− and control mice at E12.5. As shown (Fig. 9), Gdnf expression appeared similar between mutants and controls by in situ hybridization; however, the Ret and Wnt11 signal appeared less robust in Frs2α^UB−/− compared with controls. Real-time PCR measurements confirmed that Ret and Wnt11 expression was decreased in the mutants (Ret, 47% compared with controls, P = 0.032; Wnt11, 63% compared with controls, P = 0.035). Real-time PCR measurements of Gdnf revealed a trend toward decreased expression in the mutants, although it did not reach statistical significance (63% compared with controls, P = 0.063). Thus Frs2α^UB−/− mice had decreased expression of Ret and Wnt11, key molecules that regulate ureteric branching morphogenesis.

Fig. 9.

Gdnf, Ret, and Wnt11 expression in Frs2α^UB−/− kidneys. A and A′: images of E12.5 whole-mount ISH for Gdnf appear to show a similar level of expression in the mesenchyme adjacent to the ureteric tips in the mutants (A′) and controls (A). B and B′: images of E12.5 whole-mount ISH for Ret appear to show a reduction in expression at the tips of the ureteric epithelium in mutants (B′) compared with controls (B). C and C′: whole-mount ISH for Wnt11 at E12.5 also appears to show a reduced expression pattern at the tips of the ureteric epithelium in mutants (C′) compared with controls (C). Scale bars = 100 μm; n = 6 for all groups.

DISCUSSION

In this report, we observed that Frs2α is expressed in ureteric epithelium starting at E11.5 and that it is deleted from the ureteric lineage in Frs2α^UB−/− mice. The consequence of the deletion is renal hypoplasia secondary to ureteric branching defects. Interestingly, the renal defects do not appear as severe as in Fgfr2^UB−/− kidneys. Furthermore, mice with point mutations in the Frs2α binding site on Fgfr2 have no apparent renal defects. Expression of Etv4 and Etv5, downstream targets of Frs2α signaling in other systems, was unaltered in Frs2α^UB−/− kidneys, as was Sprouty1, a known Frs2α antagonist. Conversely, expression of Ret and Wnt11 was clearly diminished in mutant kidneys and Gdnf trended toward decreased expression. These findings are discussed below.

Frs2α was known to be expressed in mesenchymal-derived tissues in the kidney at later stages of embryonic development (13). We now show that the docking protein is also expressed as early as E11.5 in ureteric lineages, strongly in the ureteric trunk, and to a lesser extent in ureteric tips. As renal development proceeds, ureteric expression becomes much more pronounced in the tips. Thus Frs2α has overlapping expression with two receptor tyrosine kinases that it associates with, namely, Fgfr2, which is present throughout the ureteric tree, and Ret, which is confined to ureteric tips after E12.5 (17, 28, 47). Finally, in the Frs2α^UB−/− mice, Frs2α mRNA and protein appears absent in the ureteric lineage.

Given that Frs2α is a major docking protein for both Fgfr2 and Ret, we strongly suspected that deletion in the ureteric tree would lead to renal abnormalities. Indeed, Frs2α^UB−/− mice do develop renal hypoplasia, including an underendowment of nephrons secondary to a reduction in ureteric branching morphogenesis. Interestingly, however, deletion of this common receptor adapter molecule is milder than conditional ureteric deletion of Fgfr2 or global deletion of Ret, which leads to severe ureteric branching and stromal mispatterning and renal agenesis, respectively (38, 47). One possibility is that the Frs2α^UB−/− mice are actually hypomorphic, thereby leading to less severe abnormalities. This is not likely, however, given that ureteric expression of Frs2α (both mRNA and protein) appears absent in the mutants. Also, the Hoxb7creEgfp line used to generate the Frs2α^UB−/− mice has been shown to delete other floxed lines well, including the Fgfr2^Lox/Lox mice (5, 47). Another possibility is that there may be redundant signaling through other receptor tryosine kinases in the ureteric bud, such as Fgfr1, that may damped the ureteric defects in Frs2α^UB−/− mice. This also appears unlikely given that conditional deletion of Fgfr1 from the ureteric bud resulted in no renal abnormalities and that combined Fgfr1/Fgfr2 conditional targeting in the ureteric bud was no more severe than deleting Fgfr2 alone. The most likely explanation is that signaling through other docking proteins, such as phospholipase Cγ (PLCγ) or Crk 2 in Fgfr2 or SHC or DOK family proteins in Ret, is critical in regulating receptor functions in the ureteric bud (19, 35). To further clarify the role of Frs2α signaling downstream of Fgfr2, we complemented the Frs2α conditional knockout by generating a mouse with point mutations in the Frs2α binding site on Fgfr2. A previous publication has shown that when amino acids Leu-424 and Arg-426 in Fgfr2 (LR) were replaced by Ala residues, Frs2α was unable to dock with Fgfr2, whereas signaling through other adapters such as PLCγ was unaffected (9). Interestingly, the inability of Fgfr2 to engage Frs2α in the Fgfr2^LR/LR mice resulted in no renal abnormalities. In contrast, mice with a point mutation in the Frs2α binding site of Ret (Y1062), develop a remarkably similar phenotype to the Frs2α^UB−/− mice, namely, mild renal hypoplasia (19); thus Frs2α may be acting through Ret in the ureteric bud. Frs2α is also a major docking adapter molecule for a number of other receptor tyrosine kinsases such as neurotropin receptors and anaplastic lymphoma kinase. While several reports have documented expression of Trks in mammalian kidneys (1, 8, 44), only TrkB mutations have resulted in kidney abnormalities (10). Furthermore, TrkB^−/− mice had mild abnormalities in the juxtaglomerular apparatus and no global renal hypoplasia. Finally, Alk knockout mice did not display an abnormal kidney phenotype (4).

To further interrogate the reasons for the renal abnormalities in the Frs2α^UB−/− mice, we examined expression of two known downstream targets in other systems, namely, Etv4 and Etv5. Surprisingly, we detected no changes in ureteric (or mesenchymal) Etv4 or Etv5 signal in Frs2α^UB−/− mutants vs. controls. Excluding the possibility that the mutant mice represent hypomorphs (which is unlikely for reasons discussed earlier), it is possible that there are small changes in Etv4 and Etv5 expression below the sensitivity of in situ hybridization. It is more likely, however, that signaling through other docking proteins compensates for the absence of Frs2α. For example, SHC signaling following Ret activation has been shown to phosphorylate Erk1/2, which is known to stimulate Etv4 and Etv5 expression (41). Thus Etv4 and Etv5 levels appear unaltered in Frs2α^UB−/− mice.

We next examined whether there were perturbations in other developmental genes that could explain the renal hypoplasia in the Frs2α^UB−/− mice. Despite the fact that Sprouty1 is a known Frs2α antagonist, we detected no increase in expression either by in situ hybridization or real-time PCR. In contrast, we did detect a decrease in both Ret and Wnt11 expression by both in situ hybridization and real-time PCR. Moreover, perturbations in Ret (as noted earlier) or Wnt11 signaling can lead to renal hypoplasia (19, 23). Furthermore, there appear to be synergistic actions of the two molecules, in that compound heterozygous mice (Ret^+/−Wnt11^+/−) mice develop renal hypoplasia (23). Wnt11^−/− and Wnt11/Ret compound heterozygous mice also had a reduction in Gdnf expression (23); thus the reduced expression of Wnt11 and Ret in the Frs2α^UB−/− mice may explain the trend toward decreased Gdnf expression. Finally, a reduction in Ret and Wnt11 expression in Frs2α^UB−/− mice likely explains the ureteric bud branching abnormalities and subsequent hypoplasia.

GRANTS

This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK070030 (C. M. Bates) and National Institutes of Health program Grant P50 AR054086 (V. P. Eswarakumar).