Fgfr1 regulates development through the combinatorial use of signaling proteins

Abstract

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo.

Keywords: MAPK; craniofacial development; gastrulation; preimplantation; signaling.

Figure 1.

An allelic series of Fgfr1 knock-in point mutations. (A) Schematic representation of the allelic series generated for this study. Protein complexes and critical residues are shown at the left and right of the wild-type (WT) receptor, respectively. Amino acid substitutions included in each allele are provided at the right of the mutant alleles. (B) Coimmunoprecipitations of signaling proteins with the indicated allele of Fgfr1^-Flag3x in 3T3 cells stably expressing the receptor after treatment with 50 ng/mL FGF1 and 5 μg/mL heparin. Fgfr1^-Flag3x loading was performed on the same membrane as each coimmunoprecipitation. Plcγ and CrkL immunoblots were run on the same membrane and therefore have the same Fgfr1^-Flag3x loading; the Frs2 immunoblot was run on a different membrane. (IP) Immunoprecipitation; (IB) immunoblot.

Figure 2.

Fgfr1 contributes to primitive endoderm formation. (A) Inheritance frequencies of the indicated genotypes at E3.5–E7.5, demonstrating that Fgfr1^−/− mutants fail to be recovered at Mendelian ratios by E6.5. Embryonic fragments recovered at E6.5 and E7.5 had no defined axis or structure (not shown). χ² test P-values were used to evaluate whether inheritance frequencies differ from the predicted Mendelian genotypic ratio of 1:2:1. (n.s.) Not significant. (B) Embryos stained for Gata4, Nanog, and Cdx2 demonstrate that Fgfr1^−/− mutants form fewer primitive endoderm (Gata4⁺) and more epiblast (Nanog⁺) cells in cultured E3.5 blastocysts and uncultured E4.5 embryos. (C) Quantification representing the total number of cells per embryo and the percentages of primitive endoderm or epiblast lineages; data are presented for individual embryos as well as mean ± standard deviation. (*) P < 0.05; (**) P < 0.005; (***) P < 0.0005, P-values represent unpaired, two-tailed t-test.

Figure 3.

Fgfr1 signaling through CrkL, Plcγ, and Grb14 is not required for embryonic development. (A) Inheritance frequency of the indicated genotypes at postnatal days 5–10 (P5–P10); Fgfr1^C/C and Fgfr1^CPG/CPG mutants are inherited at Mendelian ratios. χ² test P-values were used to evaluate whether inheritance frequencies differ from the predicted Mendelian genotypic ratio of 1:2:1. (n.s.) Not significant. (B) Growth curves indicating that postnatal growth is normal in Fgfr1^C/C and Fgfr1^CPG/CPG mutants. Data are indicated as mean ± standard deviation. n = 5. (C) Ventral view of P0 skeletal preparations demonstrating defects of the most posterior thoracic vertebrae and ribs found at a low penetrance in Fgfr1^CPG/CPG mutants (Fgfr1^CPG/CPG, n = 2/15; control, n = 15). (Panels ii,iii) Both affected Fgfr1^CPG/CPG mutants are shown. (Panel i) Note that 13 thoracic vertebrae (numbered) are present and the size of the 13th rib (black arrows) in control embryos. (Panel ii) Thoracic vertebrae are numbered to illustrate that one thoracic vertebra is missing in this Fgfr1^CPG/CPG mutant. The ossification center of the fourth thoracic vertebra is duplicated (white arrow), and the attached rib is bifurcated (black arrowhead). Also note the rib rudiment present on one side of the 12th thoracic vertebrae (black arrow) and the separated distal element of the rib (red arrow). (Panel iii) Thoracic vertebrae are numbered; note the small rib rudiments present on the 13th thoracic vertebra (black arrows) in the Fgfr1^CPG/CPG mutant.

Figure 4.

Fgfr1–Frs2 signaling is essential for embryonic development. (A) Inheritance frequencies of genotypes at postnatal days indicates that Fgfr1^F/F mutants die perinatally. χ² test P-values were used to evaluate whether inheritance frequencies differ from the predicted Mendelian genotypic ratio of 1:2:1. (n.s.) Not significant. (B) Penetrance of the skeletal defects shown in C–J. (C–J) Skeletal preparations of neonatal controls (C–F) and Fgfr1^F/F mutants (G–J). Fgfr1^F/F mutants exhibit multiple skeletal defects, including cleft palate (G), hypoplastic middle ear bones (H), postaxial polydactyly (I), and one additional thoracic vertebra (J). (C,G) Ventral view of the skull; arrowheads indicate the most medial aspect of the palate in control (C) and Fgfr1^F/F mutants (G). (D,H) Side view of middle ears (D) shows that the tympanic ring (TR), malleus (M), incus (I), stapes (S), and styloid process (SP) are hypoplastic in Fgfr1^F/F (H) mutants. (E,I) Dorsal view of the forelimb of control (E) and Fgfr1^F/F mutants (I). The asterisk indicates postaxial polydactyly. (F,J) Ventral view of thoracic (T) and lumbar (L) vertebrae of control (F) and Fgfr1^F/F (J) mutants. (J) Arrows indicate small ribs growing from the 14th thoracic vertebra (T14) in Fgfr1^F/F mutants.

Figure 5.

Fgfr1 signals additively using multiple pathways in vivo. (A) Inheritance frequencies of the indicated genotypes and embryonic days, suggesting that Fgfr1^FCPG/FCPG mutants die between E10.5 and E11.5. χ² test P-values were used to evaluate whether inheritance frequencies differ from the predicted Mendelian genotypic ratio of 1:2:1. (n.s.) Not significant. (B) Fgfr1^FCPG/FCPG embryos exhibit multiple defects in embryonic development, including highly penetrant developmental retardation, posterior truncations, and epidermal blebbing (arrowheads). Bars, 250 µm.

Figure 6.

Fgfr1 signaling requirements are context-specific. (A) Lateral view of E9.5 2nd PAs (arrows) imaged using nuclear fluorescence imaging (Sandell et al. 2012). Fgfr1^F/F mutant 2nd PAs were hypoplastic (n = 3), while Fgfr1^FCPG/FCPG mutants (n = 5) failed to form 2nd PAs. Fgfr1^FCPG/FCPG embryos were dissected at E10.5 and stage-matched with E9.5 control embryos to account for developmental retardation. (B) Ventral views of P0 skulls demonstrating that the palatine (P) and palatal process of the maxilla (Pmx) are clefted (arrowheads) in both Fgfr1^F/cKO (n = 1/7) and Fgfr1^FCPG/cKO (n = 2/13) mutants, while all Fgfr1^cKO/cKO mutants exhibit facial clefting (n = 16/16). (cKO) Conditional knockout achieved with the Wnt1-Cre driver; (Tg) transgene.

Figure 7.

Fgfr1 uses multiple proteins to activate Erk1/2 and PLCγ in vitro. (A) FPCs derived from the indicated genotypes were serum-starved overnight and stimulated with 50 ng/mL FGF1 and 5 μg/mL heparin for the indicated times. Phospho-blots were stripped and reblotted with total protein or β-tubulin for loading controls. (B) Quantification of pathway activation normalized to the loading control, reported as mean ± standard deviation with a minimum of three independent biological replicates. (*) P ≤ 0.05; (**) P ≤ 0.005, unpaired, two-tailed t-test.