Fgfr1 and the IIIc isoform of Fgfr2 play critical roles in the metanephric mesenchyme mediating early inductive events in kidney development

Authors

  • Sunder Sims-Lucas,

    1. Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
    2. Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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  • Brian Cusack,

    1. Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
    2. Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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  • Jeffrey Baust,

    1. Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
    2. Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
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  • Veraragavan P. Eswarakumar,

    1. Department of Orthopaedics and rehabilitation, Yale University School of Medicine, New Haven, Connecticut
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  • Hagiwara Masatoshi,

    1. Department of Functional Genomics, Tokyo Medical and Dental University, Bunkyo-Ku, Tokyo, Japan
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  • Akihide Takeuchi,

    1. Department of Functional Genomics, Tokyo Medical and Dental University, Bunkyo-Ku, Tokyo, Japan
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  • Carlton M. Bates

    Corresponding author
    1. Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania
    2. Division of Nephrology, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
    • Rangos Research Center, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA
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Abstract

Fibroblast growth factor receptors (Fgfrs) have critical roles in kidney development. FgfrIIIb is thought to act in epithelium, while FgfrIIIc functions in mesenchyme. We aimed to determine roles of Fgfr2IIIc in kidney development. Mice with deletion of Fgfr2IIIc (Fgfr2IIIc−/−) had normal kidneys. Combination of Fgfr2IIIc−/− with conditional deletion of Fgfr1 in metanephric mesenchyme (MM) (Fgfr1Mes−/−Fgfr2IIIc−/−) had small but identifiable MM at embryonic day (E) 10.5, expressing mesenchymal markers including Eya1, Six2, Pax2, and Gdnf (unlike Fgfr1/2Mes−/− mice that have no obvious MM). E11.5 Fgfr1Mes−/−Fgfr2IIIc−/− mice had rudimentary MM expressing only Eya1. Control, Fgfr2IIIc−/−, and Fgfr1Mes−/−Fgfr2IIIc−/− kidney mesenchymal tissues also express Fgfr2IIIb. In ureteric lineages, E10.5 Fgfr1Mes−/−Fgfr2IIIc−/− embryos had ureteric outgrowth (sometimes multiple buds); however, by E11.5 Gdnf absence lead to no ureteric elongation or branching (similar to Fgfr1/2Mes−/− mice). Beyond E12.5, Fgfr1Mes−/−Fgfr2IIIc−/− mice had no renal tissue. In conclusion, Fgfr2IIIc and Fgfr1 in kidney mesenchyme (together) are critical for normal early renal development. Developmental Dynamics, 2011. © 2010 Wiley-Liss, Inc.

INTRODUCTION

Development of the permanent (metanephric) kidney begins when the metanephric mesenchyme stimulates an outgrowth of the Wolffian (nephric) duct (termed the ureteric bud) that then invades and branches within the metanephric mesenchyme. Subsequently, ongoing reciprocal signals between these two structures lead to continued ureteric branching, eventually forming the collecting duct system (Saxen and Sariola, 1987; Kuure et al., 2000; Dressler, 2009; Costantini and Kopan, 2010). Conversely, the metanephric mesenchyme condenses around the ureteric tips and undergoes a series of differentiations to form the functional units of the kidneys the nephrons (Saxen and Sariola, 1987; Kuure et al., 2000).

Fibroblast growth factor receptors (Fgfrs) have been shown to mediate several steps of renal development. Fgfrs are receptor tyrosine kinases with four signaling family members with 22 ligands (Powers et al., 2000; Turner and Grose, 2010). In addition to roles for Fgfr signaling in ureteric branching (Zhao et al., 2004) and nephron development (Perantoni et al., 1995, 2005; Barasch et al., 1997; Grieshammer et al., 2005), recent studies have shown roles for Fgfr2 (with and without Fgfr1) in early mesenchymal and ureteric development (Poladia et al., 2006; Hains et al., 2008). Although deletion of Fgfr1 or Fgfr2 alone in the metanephric mesenchyme is not (usually) associated with severe early inductive abnormalities, removal of Fgfr1/2 together in the metanephric mesenchyme with a transgenic Pax3cre line (Fgfr1/2Mes−/−) leads to severe renal dysgenesis, characterized by no recognizable metanephric mesenchyme on general staining and absence of many mesenchymal markers (Poladia et al., 2006). Fgfr1/2Mes−/− mice also have initial ureteric induction, but no ureteric elongation or branching; interestingly, the mice do occasionally develop multiple ureteric buds per nephric duct. A subsequent study showed that, while metanephric mesenchyme forms normally in mice with conditional deletion of Fgfr2 alone in the mesenchyme, the mice do have ureteric induction abnormalities characterized by duplex ureters and/or anterior/cranial displacement or the ureteric bud that can lead to different congenital anomalies of the kidney and urinary tract (CAKUT; Hains et al., 2008, 2010).

Of interest, Fgfr1–3 can be alternatively spliced at the third external Ig-like domain into IIIb and IIIc isoforms (Powers et al., 2000; Turner and Grose, 2010). These Fgfr isoforms often have different ligand-binding specificities, and it is generally thought that IIIc isoforms largely mediate signaling in the mesenchyme, while IIIb isoforms are active in developing epithelium (Turner and Grose, 2010). The Fgfr2IIIb isoform binds ligands such as Fgf1, Fgf7, and Fgf10 and deletion of this isoform and the latter two ligands lead to ureteric branching defects (Zhang et al., 2006). While the Fgfr2IIIc isoform can bind ligands such as Fgf1, Fgf2, and Fgf8 (Zhang et al., 2006; and conditional deletion of Fgf8 in mesenchyme interrupts nephron development), there are no reports about the role of Fgfr2IIIc in renal development.

The major objective for this study was to determine the role of the Fgfr2IIIc isoform in kidney development. We subsequently examined mice with a nonfunctional Fgfr2IIIc isoform (Fgfr2IIIc−/−); these mice have been previously shown to have significant bone and chondrocyte malformations (Eswarakumar et al., 2002). Exhaustive evaluation of Fgfr2IIIc−/− mice revealed no kidney and urinary tract abnormalities. Due to known redundancy between Fgfr1 and Fgfr2 in the metanephric mesenchyme, we then generated compound mutant mice with conditional deletion of Fgfr1 in the metanephric mesenchyme and global inactivation of Fgfr2IIIc (Fgfr1Mes−/− Fgfr2IIIc−/−). These mice developed early defects characterized by a small, but discernable metanephric mesenchyme at E10.5 that expressed appropriate markers such as Eya1, Six2, Pax2, and Gdnf. At embryonic day (E) 11.5, Fgfr1Mes−/−Fgfr2IIIc−/− mutant metanephric mesenchyme had failed to expand in size; Eya1 expression remained but Six2, Pax2, and Gdnf was down-regulated. While ureteric buds did form in the compound mutants, including occasional multiple buds per nephric duct, the buds failed to elongate or branch. While the Fgfr1Mes−/−Fgfr2IIIc−/−-ureteric defects are similar to those in Fgfr1/2Mes−/− mice, the mesenchymal defects were not as severe as in Fgfr1/2Mes−/− mice.

RESULTS

Fgfr2IIIc−/− Kidneys Are Phenotypically Normal

To determine the role of Fgfr2IIIc in the developing kidney, we examined mice with global deletion of this isoform. Comparison of histological sections revealed no obvious differences between wild-type (WT), Fgfr2IIIc+/−, and Fgfr2IIIc−/− E13.5 and E16.5 kidneys (Fig. 1A,B and data not shown). To determine whether deletion of the IIIc isoform of Fgfr2 had any effect on branching morphogenesis and nephron formation, we cultured E12.5 wild-type, Fgfr2IIIc+/−, and Fgfr2IIIc−/− kidneys for 48 hours and then stained for Calbindin and Wt1; in serum-free media. As in the histological sections of embryonic kidneys in vivo, gross explant ureteric branching and nephron development appeared similar between all genotypes (Fig. 1C,D, and not shown). Quantitatively, there were no differences in ureteric tip number (WT 46 ± 3 [N = 9], Fgfr2IIIc+/− 47 ± 4 [N = 19], Fgfr2IIIc−/− 46 ± 3 [N = 8], P = 0.67) or nephron number (WT 42 ± 4 [N = 9], Fgfr2IIIc+/− 40 ± 5 [N = 19], Fgfr2IIIc−/− 39 ± 3 [N = 8]; P = 0.42). To further characterize these mice, we also examined E18.5 wild-type and Fgfr2IIIc−/− kidneys and found no differences between the long axis (WT 0.35 ± 0.03 cm, Fgfr2IIIc−/− 0.37 ± 0.02 cm, P = 0.25; N = 8) and the surface area of the total kidney (WT 0.075 ± 0.016 cm2, Fgfr2IIIc−/− 0.082 ± 0.008 cm2, P = 0.29; N = 8). Finally, we noted no evidence of ureteric induction abnormalities (duplex ureters and/or anterior/cranial displacement of the ureteric bud) or other congenital anomalies of the kidney and urinary tract such as ureteral obstruction, renal hypoplasia, or renal agenesis (all of which is seen in Pax3cre-mediated deletion of both isoforms of Fgfr2; Poladia et al., 2006; Hains et al., 2008). Thus, Fgfr2IIIc−/− mice have phenotypically normal kidneys.

Figure 1.

Comparison of Fgfr2IIIc−/− and control embryonic kidneys. A,B: Images of hematoxylin and eosin (H&E) stained embryonic day (E) 13.5 kidney sections from control (A) and Fgfr2IIIc−/− (B) kidneys appear similar (arrows = developing nephrons; arrowheads = ureteric epithelium). C,D: Whole-mount immunostaining for ureteric epithelium with Calbindin (green) and for nephrons with Wt1 (red) in explanted kidneys from control (C) and Fgfr2IIIc−/− (D) embryos also appear similar. Scale bars = 75 μm in A,B, 100 μm in C,D.

Fgfr1Mes−/−Fgfr2IIIc−/− Mice Have Early Developmental Kidney Defects

Given potential redundancy between Fgfr1 and Fgfr2IIIc in kidney mesenchyme, we generated compound mutant Fgfr1Mes−/−Fgfr2IIIc−/− mice (global deletion of Fgfr2IIIc and conditional deletion of Fgfr1 in metanephric mesenchyme, including nephron progenitors and renal cortical stroma, and in stromal cells between the nephric duct and metanephric mesenchyme with a transgenic Pax3cre line; Poladia et al., 2006; Hains et al., 2008). As early as E10.5, there were clear renal abnormalities in Fgfr1Mes−/−Fgfr2IIIc−/− mice characterized by a dramatic reduction in the size of the condensed metanephric mesenchyme on hematoxylin and eosin (H&E) sections compared with cre-negative littermate controls (Fig. 2A,B). In fact, all combinations of alleles were phenotypically normal except for Pax3creTg/+Fgfr1Lox/LoxFgfr2IIIc−/− (not shown). To quantify the differences between Fgfr1Mes−/−Fgfr2IIIc−/− mutant and control developing kidneys, we performed three-dimensional (3D) reconstructions of the metanephric mesenchymal tissues from serial H&E-stained sections. As shown (Fig. 2C,D and Supp. Movies S1 and S2, which are available online), the 3D reconstructions revealed that the E10.5 Fgfr1Mes−/− Fgfr2IIIc−/− mice had a 70% reduction in the volume of metanephric mesenchyme from the onset of kidney development (Control [N = 6] 1.49 × 106 ± 6.79 × 105 μm3; mutant [N = 6] 4.52 × 105 ± 2.05 × 105 μm3; P = 0.02). By E11.5, the Fgfr1Mes−/−Fgfr2IIIc−/− kidney mesenchyme was still small and appeared less compacted than controls on H&E staining (Fig. 2E,F). PCNA (proliferating cell nuclear antigen) and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) assays through serial sections at E10.5 and 11.5 did not reveal any obvious differences in mesenchymal proliferation (data not shown) or apoptosis (Supp. Fig. S1) in mutants versus controls. By E12.5, however, there was a clear increase in apoptosis in Fgfr1Mes−/−Fgfr2IIIc−/− mutant kidneys throughout the small remnant mesenchymal tissues compared with controls (Fig. 3). By later stages (E13.5 and E18.5), there were no apparent kidney tissues remaining in the mutants compared with controls (Fig. 2G,H). Thus, Fgfr1Mes−/− Fgfr2IIIc−/− mice have severe defects in early metanephric mesenchyme formation, although they differ from Fgfr1/2Mes−/− mice that have no obvious metanephric mesenchyme by H&E staining at any stage of development (Poladia et al., 2006).

Figure 2.

Comparison of Fgfr1Mes−/−Fgfr2IIIc−/− and control embryonic kidneys. A,B: In embryonic day (E) 10.5 sections, hematoxylin and eosin (H&E) staining reveals that Fgfr1Mes−/−Fgfr2IIIc−/− mesenchyme (B, arrows) appears reduced in size compared with controls (A, arrows) (arrowheads = ureteric bud or nephric duct) C,D: Still images of three-dimensional (3D) reconstructions confirm that Fgfr1Mes−/−Fgfr2IIIc−/− metanephric mesenchyme (D, green) is reduced in size compared with controls (C, green; ND, nephric duct, blue; MM, metanephric mesenchyme, green; UB, ureteric bud, pink). E,F: In E11.5 sections, H&E staining reveals that in controls (E) the metanephric mesenchyme has expanded greatly (arrows) and the ureteric buds have branched (arrowheads), while in Fgfr1Mes−/−Fgfr2IIIc−/− mice (F), the mesenchyme is small and less condensed (arrows) and the ureteric buds remain unbranched (arrowheads) G,H: Whole-mount E18.5 images of urogenital systems reveal that in controls (G) the kidneys (K), bladder (B), and adrenals (A) are clearly evident while in the mutants (H), the kidneys are absent. Scale bars = 50 μm in A,B,E,F, 200 μm in G,H.

Figure 3.

TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) assays in embryonic day (E) 12.5 Fgfr1Mes−/−Fgfr2IIIc−/− and control kidneys. A,B: Sections with fluorescent TUNEL staining overlying adjacent hematoxylin and eosin (H&E) -stained sections reveals minimal apoptosis (green dots) in control kidneys (A), but high levels of apoptosis in Fgfr1Mes−/−Fgfr2IIIc−/− remnant metanephric mesenchyme (B, arrowheads). ×25 magnification.

We next examined Fgfr1Mes−/− Fgfr2IIIc−/− mice for defects in the early ureteric lineage. The mutants did appear to have ureteric bud formation at E10.5 on H&E sections (Fig. 2B, right arrowhead); however, at E11.5, Fgfr1Mes−/−Fgfr2IIIc−/− mice appeared to have unbranched ureteric epithelium, in contrast to controls (Fig. 2E,F). To confirm these findings, we performed whole-mount in situ hybridization in E11.5 mice with Ret, which stains the nephric duct and the ureteric epithelium. This finding confirmed the histological observations as none of the ureteric buds elongated into the metanephric mesenchyme (Fig. 4). Unlike WT control and Fgfr2IIIc−/− mice, that had elongation and branching of the ureteric epithelium, the Fgfr1Mes−/− Fgfr2IIIc−/− mutants had small, unbranched buds (Fig. 4C). Of interest, however, a subset of the mutants had an anteriorly displaced secondary ureteric bud (Fig. 4D). These abnormalities appear similar to the Fgfr1/2Mes−/− mice.

Figure 4.

Ret whole-mount in situ hybridization of embryonic day (E) 11.5 control, Fgfr2IIIc−/−, and Fgfr1Mes−/−Fgfr2IIIc−/− kidneys. A–D: While control (A) and Fgfr2IIIc−/− (B) embryos have phenotypically comparable elongated ureteric bud trunks (arrowheads) and branches (arrows), arising from the nephric duct (concave arrowheads), Fgfr1Mes−/−Fgfr2IIIc−/− ureteric buds (C,D) fail to elongate or branch (arrowheads) although a subset have multiple ureteric buds (D). Scale bars = 150 μm.

Fgfr2IIIb Is Expressed in Kidney Mesenchymal Tissues

Given that the apparent partial rescue of the renal abnormalities in Fgfr1Mes−/−Fgfr2IIIc−/− mice compared with Fgfr1/2Mes−/− mice could be due to actions of the Fgfr2IIIb isoform in mesenchyme, we examined expression of Fgfr2IIIb by in situ hybridization. Initially, we performed whole-mount in situ hybridization and as expected, Fgfr2IIIb was strongly expressed in epithelial tissues such as Wolffian duct in E10.5 controls, Fgfr1Mes−/−Fgfr2IIIc−/−, and Fgfr2IIIc−/− mice and throughout the ureteric tree in E12.5 controls and Fgfr2IIIc−/− mice (not shown). We then paraffin embedded and sectioned the whole-mount stained tissues. While the E10.5 tissues were too fragmented, the E12.5 sections revealed Fgfr2IIIb expression in stromal mesenchyme surrounding the invading ureteric trunk, that appeared stronger in Fgfr2IIIc−/− mice compared with controls (Supp. Fig. S2). There also appeared to be some Fgfr2IIIb expression in the cortical nephrogenic zone surrounding the positive ureteric tips in the postsectioned E12.5 Fgfr2IIIc−/− and control kidneys (Supp. Fig. S2). To clarify expression in metanephric mesenchyme proper, we then performed in situ hybridization on tissue sections. At E10.5, while controls demonstrated minor Fgfr2IIIb signal in metanephric mesenchyme near the Wolffian ducts (Fig. 5A), Fgfr1Mes−/−Fgfr2IIIc−/− mutants had more robust signal in the MM (Fig. 5B). In situ hybridization in E12.5 sections revealed discreet areas of Fgfr2IIIb expression in cap mesenchyme (nephron progenitors descended from MM) surrounding the positive ureteric tips in both controls and Fgfr2IIIc−/− kidneys (Fig. 5C,D). Sense probes for Fgfr2IIIb showed no signal in either whole-mount or section in situ hybridization experiments (not shown). Thus, in addition to epithelial tissues, Fgfr2IIIb appears to be expressed in developing renal mesenchymal tissues, and may be up-regulated in the absence of Fgfr2IIIc.

Figure 5.

Fgfr2IIIb in situ hybridization in mutant and control developing kidney sections. A,B: At embryonic day (E) 10.5, light Fgfr2IIIb staining is apparent in control metanephric mesenchyme adjacent to the Wolffian duct (A, arrow), while in Fgfr1Mes−/−Fgfr2IIIc−/− mutants, expression in the metanephric mesenchyme is more robust (B, arrow). C,D: At E12.5, Fgfr2IIIb signal is apparent in cap mesenchyme surrounding the epithelial ureteric tips in both controls (C, arrowheads) and Fgfr2IIIc−/− mutants (D, arrowheads). WD, Wolffian duct. Original magnification: ×400 in A,B, ×200 in C,D.

Fgfr1 and Fgfr2IIIc Control Expression of Key Regulators of Mesenchymal and Ureteric Development

To understand how Fgfr1 and Fgfr2IIIc act in the metanephric mesenchyme, we examined expression of key molecules that are themselves necessary for mesenchyme formation and related in a molecular hierarchy (i.e., Eya1>Six2> Pax2; Torres et al., 1995; Xu et al., 1999; Nishinakamura et al., 2001; Vainio and Lin, 2002; Brodbeck and Englert, 2004; Sajithlal et al., 2005). At E10.5, the compound Fgfr1Mes−/−Fgfr2IIIc−/− mutants displayed robust expression of all of the markers, Eya1, Six2, and Pax2 (Fig. 6). In contrast, by E11.5, while Eya1 expression was apparent in the remnant mutant metanephric mesenchymal tissues, Six2 and Pax2 mesenchymal signal was absent (Fig. 7). By E12.5 Eya1 expression persisted in mutant remnant mesenchymal tissues. Also, Foxd1, a marker of developing renal cortical stroma that is clearly present in E12.5 controls, was not visible in Fgfr1Mes−/−Fgfr2IIIc−/− kidney mesenchymal tissues (Supp. Fig. S3). In total, these findings appear less severe than we found in Fgfr1/2Mes−/− mice that expressed Eya1 but did not obviously express the other downstream molecules, even at E10.5.

Figure 6.

Comparison of mesenchymal gene expression at embryonic day (E) 10.5 in control and Fgfr1Mes−/−Fgfr2IIIc−/− kidneys. A–C: Control section in situ hybridization at E10.5 reveal normal expression of Eya1 (A) and Six2 (B) in metanephric mesenchyme (arrowheads) and of Pax2 (C) in mesenchyme and ureteric epithelium/nephric duct (dotted line). D–F:Fgfr1Mes−/−Fgfr2IIIc−/− section in situ hybridization at E10.5 reveal similar expression patterns for Eya1 (D), Six2 (E), and Pax2 (F), although the mutant metanephric mesenchyme appears generally smaller. Scale bars = 30 μm.

Figure 7.

Comparison of mesenchymal gene expression at embryonic day (E) 11.5 in control and Fgfr1Mes−/−Fgfr2IIIc−/− kidneys. A–C: Control section in situ hybridization at E11.5 reveals ongoing normal expression of Eya1 (A) and Six2 (B) in the expanded metanephric mesenchyme and Pax2 (C) in both mesenchyme and ureteric bud (arrowheads). D–F:Fgfr1Mes−/−Fgfr2IIIc−/− section in situ hybridization at E11.5 reveals persistent expression of Eya1 (D,D′) in small rudimentary metanephric mesenchyme (arrowheads) around unbranched ureteric buds (dotted lines), but no Six2 (E) and Pax2 (F) expression in mesenchyme. Note that Pax2 is still expressed in mutant ureteric epithelium (F, arrowheads); Six2 expression was also maintained in the limb of the mutants (concave arrowhead). Scale bar = 50 μm.

To determine the reason for the elongation and branching defects in mutant ureteric tissues, we examined temporal expression of Gdnf, known to be critical for ureteric induction (Pichel et al., 1996a, b; Sanchez et al., 1996; Vega et al., 1996; Pepicelli et al., 1997; Sainio et al., 1997; Sariola and Saarma, 1999; Costantini and Shakya, 2006). At E10.5, in situ hybridization in tissue sections displayed robust expression of Gdnf in both Fgfr1Mes−/−Fgfr2IIIc−/− and control kidney mesenchymal tissues (Fig. 8A,B). However, by E11.5 Gdnf was apparently absent from the remnant mutant metanephric mesenchyme (Fig. 8C,D). To confirm the finding at E11.5, we performed whole-mount in situ hybridization and once again noted no expression of Gdnf in Fgfr1Mes−/−Fgfr2IIIc−/− renal tissues (Fig. 8E,F). Thus, Gdnf is present at E10.5 allowing ureteric induction, but absent at E11.5 preventing elongation and branching. Furthermore, mutant Gdnf expression parallels Pax2 expression (and is known to be downstream of Pax2 signaling in the kidney; Brophy et al., 2001.

Figure 8.

Comparison of Gdnf expression in embryonic day (E) 10.5 and E11.5 control and Fgfr1Mes−/−Fgfr2IIIc−/− kidneys. A,B: At E10.5, section in situ hybridization reveals robust expression of Gdnf in control (A) and Fgfr1Mes−/−Fgfr2IIIc−/− (B) metanephric mesenchyme (arrows) adjacent to ureteric buds (arrowheads). C,D: At E11.5, section in situ hybridization reveals ongoing Gdnf expression in control (C) metanephric mesenchyme (arrows) surrounding the ureteric epithelia (arrowheads), but no apparent expression in and Fgfr1Mes−/−Fgfr2IIIc−/− (D) mesenchyme (arrows) adjacent to the ureteric epithelium (arrowheads, dotted lines). E,F: At E11.5, whole-mount in situ hybridization confirms Gdnf expression in control mesenchyme (E, dotted lines) but no visible expression in Fgfr1Mes−/−Fgfr2IIIc−/− mutant mesenchyme (F, dotted line). (Arrowheads = nephric ducts). Scale bars = 50 μm.

DISCUSSION

This study reveals that the Fgfr2IIIc isoform is dispensable for formation of the normal kidney and urinary tract. We hypothesize that this is due to redundant roles of other receptors or receptor isoforms with Fgfr2IIIc. Furthermore, when we generated mice with combined conditional deletion of Fgfr1 from the metanephric mesenchyme and global deletion of the Fgfr2IIIc we observed severe renal dysgenesis. However, we noted that the metanephric mesenchymal abnormalities in the Fgfr1Mes−/−Fgfr2IIIc−/− were not as severe as in conditional deletion of Fgfr1 and both isoforms of Fgfr2 in the mesenchyme; thus, we hypothesize that the presence of other isoforms of Fgfr2 (likely the IIIb isoform) alone may be sufficient to produce a rudimentary metanephric mesenchyme. Finally, it appears that mesenchymal genes critical for metanephric mesenchyme development and ureteric induction are expressed early in the Fgfr1Mes−/−Fgfr2IIIc−/− mice; however, expression of most of these genes is not sustained in the absence of Fgfr1 and Fgfr2IIIc, leading to the failure of the mesenchyme and ureteric lineages to develop further. These findings are discussed below.

As noted above, we found no abnormalities of the kidneys or urinary tracts in Fgfr2IIIc−/− mice. The absence of severe dysgenesis is likely due to some redundant roles of Fgfr1 (as discussed later). The absence of any ureteric induction abnormalities, however, was surprising. First, we previously determined that conditional deletion of all Fgfr2 isoforms with the Pax3cre line (that deletes in the metanephric mesenchyme and in stromal mesenchyme between the nephric duct and MM) leads to aberrant ureteric bud induction abnormalities and associated CAKUT (Poladia et al., 2006; Hains et al., 2008). Second, we recently recapitulated many of the ureteric induction abnormalities including duplex ureters and displaced ureteric induction sites in mice with deletion of Fgfr2 only in the stroma between the nephric duct and metanephric mesenchyme with a Tbx18cre line (C. Bates, unpublished data). Third, given the presumed separate roles of Fgfr IIIc isoforms in mesenchymal tissue and IIIb isoforms in epithelia, we expected to see ureteric induction abnormalities anomalies in the Fgfr2IIIc−/− mice. It is unlikely that the normal renal phenotype in the Fgfr2IIIc−/− is due to persistent low level expression of the IIIc isoform, given that if anything, conditional deletion with a Cre-LoxP system is more likely to be “leaky” than a global deletion (Naik et al., 2006; Means et al., 2008). Thus, the absence of ureteric induction abnormalities in the Fgfr2IIIc−/− mice strongly suggests that the Fgfr2IIIb isoform has a redundant role with the IIIc isoform in mesenchyme (likely the perinephric duct stromal mesenchyme) to regulate the proper ureteric bud induction site. The presence of Fgfr2IIIb in the stromal mesenchyme adjacent to the ureteric trunk at E12.5 by in situ hybridization (that was even stronger in Fgfr2IIIc−/− kidneys than controls) would support the latter possibility.

Unlike global deletion of Fgfr2IIIc alone, combined deletion of Fgfr2IIIc and Fgfr1 in kidney mesenchyme leads to severe renal dysgenesis. While the ureteric defects in Fgfr1Mes−/− Fgfr2IIIc−/− mice (unbranched and sometimes multiple UBs) are similar to mice with combined conditional deletion of Fgfr1 and Fgfr2 in kidney mesenchyme (Poladia et al., 2006), the mesenchymal defects in the Fgfr1Mes−/− Fgfr2IIIc−/− mutants, however, are less severe than in Fgfr1/2Mes−/− mice. First, Fgfr1Mes−/−Fgfr2IIIc−/− do generate a small but obvious metanephric mesenchyme at E10.5 unlike Fgfr1/2Mes−/− mice that form no histologically recognizable MM. Second, while both lines demonstrate expression of a restricted domain of Eya1 at E10.5, only Fgfr1Mes−/−Fgfr2IIIc−/− mice also robustly express other mesenchymal markers at E10.5, including Six2 and Pax2. (Given that both lines express Gdnf at E10.5 allowing ureteric induction, it is likely that there is low and transient expression of Six2 and Pax2 in Fgfr1/2Mes−/− mice below the level of detection). Considering the reasons for the differences in the phenotypes, as noted above, it is unlikely due to more leakiness in the global Fgfr2IIIc deletion than the conditional deletion of Fgfr2. Another explanation could be that background strain differences between Fgfr1Mes−/−Fgfr2IIIc−/− and Fgfr1/2Mes−/− mice could account for the divergence in phenotypes; while that cannot be completely excluded, both lines are predominantly in an FVB/N background. Finally, a plausible explanation is that the Fgfr2IIIb isoform does act redundantly to allow an early, albeit smaller metanephric mesenchyme to form even in the absence of Fgfr1 and Fgfr2IIIc (Fig. 9). Precedence for this hypothesis is supported by recent findings showing that when Fgfr2IIIc is knocked down, Fgfr2IIIb expression in calvarial bone mesenchyme leads to alterations in craniofacial morphogenesis (Veistinen et al., 2009). Further support for this explanation comes from our in situ hybridization data showing Fgfr2IIIb expression in E10.5 MM (that was even stronger in Fgfr1Mes/−Fgfr2IIIc−/− kidneys than controls) and in E12.5 cap mesenchyme adjacent to ureteric tips (in both control and Fgfr2IIIc−/− kidneys). Thus, it appears that Fgfr2IIIb isoforms can at times act in mesenchymal tissues.

Figure 9.

Schematic of mechanisms by which Fgfrs and receptor isoforms mediate early kidney development. Top: Presence of Fgfr1 and 2 in kidney mesenchyme is necessary for sustained expression of hierarchical genes to allow for continued development of metanephric mesenchyme and ureteric outgrowth, elongation, and branching. Bottom: Absence of Fgfr1 and Fgfr2IIIc (presence of Fgfr2IIIb alone) still permits early expression of all hierarchical genes and therefore early mesenchymal and ureteric development; however, at later stages, only Eya1 expression persists, leading to failure of further mesenchymal and ureteric morphogenesis.

Finally, it appears that mesenchymal genes critical for metanephric mesenchyme development and ureteric induction are expressed early in the Fgfr1Mes−/−Fgfr2IIIc−/− mice; however, expression of most of these genes is not sustained in the absence of Fgfr1 and Fgfr2IIIc, leading to the failure of the mesenchyme and ureteric lineages to develop further. As noted earlier, at E10.5, there is robust expression of several genes critical to formation of metanephric mesenchyme including Eya1, Six2, and Pax2; thus, there is an identifiable metanephric mesenchyme in the compound mutant mice. Furthermore, the presence of Pax2 likely contributes to the expression of Gdnf and therefore successful ureteric outgrowth in E10.5 mutants. By E11.5, however, only Eya1 expression remains in mutant metanephric mesenchymal rudiments; while we detected no excessive apoptosis or alterations in proliferation to explain the small rudiments at E11.5, they were clearly less compacted and had not expanded in size compared with controls. Furthermore, the absence of the other molecules downstream of Eya1 at E11.5, including Pax2, likely contributes to the absence of Gdnf and failure of the ureteric epithelium to branch. By E12.5, there was a clear increase in apoptosis in Fgfr1Mes−/−Fgfr2IIIc−/− remnant renal elements compared with controls, leading to the absence of any recognizable mutant kidney tissues thereafter. A summary of how the molecular pathways are affected in Fgfr1Mes−/−Fgfr2IIIc−/− developing kidneys is outlined in Figure 9.

In summary, deletion of the Fgfr2IIIc isoform alone did not affect kidney or urinary tract development, likely from redundant actions of Fgfr1 and/or Fgfr2IIIb in mesenchymal tissues to prevent ureteric induction abnormalities. However, conditional deletion of Fgfr1 in kidney mesenchyme along with global Fgfr2IIIc deletion led to severe renal dysgenesis. A small, but clearly identifiable metanephric mesenchyme did form at E10.5, suggesting some redundant actions of Fgfr2IIIb in renal mesenchyme at early stages of development. Finally, the presence of Eya1, Six2, Pax2, and Gdnf early in the Fgfr1Mes−/−Fgfr2IIIc−/− mutants likely allowed for an early mesenchyme and ureteric bud to form. Absence of all except for Eya1 later, however, likely leads to failure of either lineage to develop further.

Experimental Procedures

Mice

All of the experiments using mice were approved by the University of Pittsburgh Institutional Care and Use Committee.

Fgfr2IIIc−/− mice.

Generation of The Fgfr2IIIc−/− mouse was previously described (Eswarakumar et al., 2002). In brief, a stop codon was introduced, just after the start of exon 9 (the IIIc-specific exon). This mutation produced a global deletion of Fgfr2IIIc isoform but did not affect transcription of the Fgfr2IIIb isoform.

Combined Fgfr1Mes−/− conditional and Fgfr2IIIc global knockout mice.

The Pax3creTg/+ Fgfr1lox/lox mice (previously described in Poladia et al., 2006) were crossed with Fgfr2IIIc−/+ mice. Mice that were triple heterozygous for Pax3creTg/+, Fgfr1Lox/+, and Fgfr2IIIc+/− were crossed with Fgfr1Lox/loxFgfr2IIIc+/− mice to produce the desired Pax3creTg/+Fgfr1lox/lox Fgfr2IIIc−/− compound mutants (Fgfr1Mes−/−Fgfr2IIIc−/−) and littermate controls. The compound mutants thus had conditional deletion of Fgfr1 in the metanephric mesenchyme and global deletion of the IIIc isoform of Fgfr2.

Genotyping

Genotyping was performed by means of polymerase chain reaction (PCR). Tail clippings and or embryonic tissues were collected and digested and genomic DNA was isolated. To determine if mice were carrying the Pax3Cre cassette the forward primer 5′-AATCTTATGGTCAC CTGAGTGTTAAATGTCCAATTTAC-3′ and reverse primer 5′-CATCTTCAGG TTCTGCGGG-3′ were used, with single band at 230 base pairs (bp) indicating presence of cre. To determine the genotype of the Fgfr1 floxed mice, the forward primer 5′-TTGACCG GATCTACACACACC-3′ and reverse primer 5′-GCACACCGGGGTATGGG GAGC-3′ were used, giving a 602-bp wild-type band and a 672-bp mutant band. To determine the genotype of the Fgfr2IIIc allele, the forward primer 5′-GAGTACCATGCTGACTGCATGC-3′ and the reverse primer 5′-GGAGAGG CATCTCTGTTTCAAGACC-3′ were used, giving a 225-bp wild-type band and a 315-bp mutant band.

Organ Culture and Calbindin/Wt1 Immunohistochemistry

Embryos were harvested from wild-type, Fgfr2IIIc+/− and Fgfr2IIIc−/− embryos at E12.5 and cultured in serum-free media for 48 hours on transwell membranes as previously described (Sims-Lucas et al., 2008). At the completion of the culture period, cultures were immersion fixed in ice-cold methanol. Whole-mount immunohistochemistry was then conducted as previously described (Sims-Lucas et al., 2008). In brief, organ cultures from wild-type, Fgfr2IIIc+/− and Fgfr2IIIc−/− kidney rudiments were blocked in 5% fetal bovine serum, and incubated with primary antibodies (monoclonal mouse anti-CalbindinD28K (Sigma-Aldrich, St. Louis, MO) and polyclonal rabbit anti-Wt1 (Invitrogen, Carlsbad, CA) for 2 hr at 37°C. Cultures were then washed and incubated with secondary antibodies, goat anti-mouse Alexa Fluor 488 (Invitrogen) and goat anti-rabbit Alexa Fluor 594 (Invitrogen), for 2 hr at 37°C. The cultures were then washed mounted and visualized. The number of tips and nephrons for each sample were counted three times and averaged.

Histological Evaluation

Embryos from Fgfr2IIIc−/−, Fgfr1Mes−/−Fgfr2IIIc−/− compound mutants and controls were harvested at ages ranging from E10.5 to E16.5 and fixed with 4% paraformaldehyde overnight. Wild-type, Fgfr2IIIc+/− and Fgfr2IIIc−/− E18.5 embryos were harvested and images of the intact and dissected embryos as well as the dissected kidneys were taken. Embryo and kidney images were then measured using ImageJ software (version 1.32j from Wayne Rasband, National Institutes of Health, USA), the parameters measured included: embryo crown–rump length, kidney long axis, kidney short axis, and kidney surface area.

3D Reconstruction of Fgfr1Mes−/−Fgfr2IIIc−/− Developing Kidneys

3D reconstruction was conducted using Stereoinvestigator (Microbrightfield (MBF), Williston, VT) as previously described (Sims-Lucas et al., 2008). Briefly, E10.5 control and Fgfr1Mes−/− Fgfr2IIIc−/− embryos were serially sectioned at 4 μm and stained with hematoxylin and eosin. The ureteric bud, Wolffian duct, and metanephric mesenchyme were then traced through every section. The developing kidney was then 3D reconstructed and quantitated using Neurolucida explorer (MBF).

In Situ Hybridization

Whole-mount and section in situ hybridizations was carried out on E10.5, E11.5, and E12.5 urogenital systems as previously described (Hains et al., 2008; Sims-Lucas et al., 2009). Digoxigenin-labeled antisense and sense riboprobes were used against various genes, including Eya1, Six2, Pax2, Gdnf, Foxd1, and Ret. To detect the Fgfr2IIIb isoform, we used a probe template from a portion of Fgfr2 Exon 7 (NCBI Reference Sequence: NM_201601.2,bases 1966–1806). Select whole-mount stained tissues were embedded in paraffin and sectioned at 8 μm.

Apoptosis (TUNEL) Assays in Fgfr1Mes−/−Fgfr2IIIc−/− Mice

To visualize the ureteric epithelium of the E10.5 and E11.5 control and Fgfr1Mes−/−Fgfr2IIIc−/−, tissues were labeled for Calbindin. In brief, paraffin-embedded sections were dewaxed and blocked in 10% fetal bovine serum (FBS); sections were then incubated with monoclonal mouse Calbindin antibody (Sigma-Aldrich), sections were then washed and subsequently incubated with Alexa Fluor 594 goat anti-mouse (Invitrogen). Following this, TUNEL assays were carried out using the Fluorescent FragEl DNA160 Fragmentation Detection kit (Oncogene, Cambridge, MA) as previously described (Sims-Lucas et al., 2009). E12.5 control and Fgfr1Mes−/− Fgfr2IIIc−/− sections were assayed for apoptosis using FragEl kits after which images were overlaid and merged with images of adjacent H&E-stained sections.

Proliferation Assays in Fgfr1Mes−/−Fgfr2IIIc−/− Mice

A PCNA kit was used (Invitrogen). In brief, E10.5 and E11.5 control and Fgfr1Mes−/−Fgfr2IIIc−/− paraffin embedded sections were dewaxed and endogenous peroxidases were blocked using 3% H2O2; heat induced antigen retrieval was then carried out using citrate buffer. Once the slides had cooled to room temperature they were washed and blocked. Primary antibody was then added, followed by washing and addition of strepavidin peroxidases. Sections were subsequently washed and a diaminobenzidine (DAB) chromogen was used to form a brown precipitate; slides were counterstained with hematoxylin.

Statistics

Statistical analysis was carried out upon all biological replicates with a Students t-test or a one-way analysis of variance followed by Fisher post hoc tests. All values are represented as means ± SD.

Acknowledgements

The authors thank Ms. Kayle Kish for her assistance in maintaining the mouse colony. We thank Dr. Jason Cain and Dr. Norm Rosenblum for the Gdnf probe template, and Dr. Pin-Xian Xu for the Six2 and Eya1 probe templates.

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