Supplementary Figure 1. Allelic Series of Targeted Mutations

Supplementary Figure 1. Allelic series of targeted mutations

(A) Schematic representation of the targeting vector, wild type locus and targeted locus. Three distinct targeting vectors (Fgfr1F, Fgfr1CPGand Fgfr1FCPG) used identical targeting strategies and so they are depicted as one vector. The Fgfr1C allele was generated by homologous recombination events that incorporated the Y463F mutation from the Fgfr1CPG targeting vector without incorporating the Y766F or Y776F mutations.SacI (S) and NheI (N) restriction sites used for 5’ and 3’ Southern blots are shown. Location of 5’ external, 3’ external, internal and neo probes are indicated. Amino acid substitutions are shown above genomic exons. Abbreviations: ex, exons; neo, neomycin resistance cassette; L, loxP site; DTA, Diptheria toxin A cassette. (B) A representative Southern blot of WT and +/F clones showing representative band pattern (WT 8kb, F 10kb) using SacI digest and 5’ probe, asterisk (*) represents non-specific band. (C) Nucleotide substitutions generated for each allele and alterations in restriction sites to identify the mutation. Note some silent mutations were introduced to generate diagnostic restriction enzyme sites.

Supplementary Figure 2. Fgfr1F/F mutant phenotypes vary in expressivity

Skeletal preparations of neonates and schematic representations of control (A-C) and Fgfr1F/Fmutant (D-F) demonstrating lowly expressed defects of the middle ear and limb. (A,D) View of right middle ear in control (A) and Fgfr1F/Fmutant (D). (B,E) Tracing of middle ear bones styloid process (SP), stapes (ST), incus (I), malleus (M), gonial (G) and tympanic ring (TR) in control (B) and Fgfr1F/F mutant (E). Note the styloid process is abnormal while other middle ear bones are smaller but morphologically similar to controls. (C,F) Left forelimb of control (C) and Fgfr1F/F mutant (F) note extra digit consisting of cartilage without the bone element of the most distal phalange (*). The extra digit of the least severely affected mutants consisted of only soft tissue without cartilage or bone elements (not shown).

Supplementary Figure 3.Fgfr1FCPG/FCPG mutants form mesoderm

Whole mount in situ hybridization of mesoderm marker Fgf3 (control n=9, Fgfr1FCPG/FCPG n=7) and somite marker Meox1 (control n=4, Fgfr1FCPG/FCPG n=4) indicate that Fgfr1FCPG/FCPG mutants form mesoderm. For Meox1in situ, Fgfr1FCPG/FCPG embryos were dissected at E10.5 were stage matched with E9.5 control embryos to account for developmental retardation.

Supplementary Figure 4.Frs3 transcriptsare lowly detectable at E7.5 and E8.5

Expression of Frs3 mRNA reported relative to β2microblobulin loading control determined by RT-qPCR reported as mean ± standard deviation at indicated stages. E13.5 central nervous system was used as a positive control. Frs3 transcripts were detected at similar low levels in controls and Fgfr1FCPG/FCPG mutants at E7.5 (control n=5,Fgfr1FCPG/FCPG, n=3) and E8.5 (control n=4, Fgfr1FCPG/FCPGn=3).

Supplementary Figure 5. Loss of Stat3 does not exacerbate Fgfr1FCPG/cKO phenotype

(A) Immunoblot with cytoplasmic (Cyto.) and nuclear (Nuc.) fractionation demonstrated Stat3 nuclear translocation following treatment with 50ng/mL FGF1, 5μg/mL heparin treatment. β-tubulin and lamin-B are used as cytoplasmic and nuclear loading controls respectively. (B) Stat3 and Fgfr1-Flag3x were co-immunoprecipitated in 3T3 cells stably expressing Fgfr1WT-Flag3x or Fgfr1FCPG-Flag3x following treatment with 50ng/mL FGF1, 5μg/mL heparin. (IP) immunoprecipitation, (IB) immunoblot. (C) Ventral view of P0 skulls indicated that loss of Stat3cKO/cKO, Fgfr1FCPG/cKOmutants (n=4) did not exacerbate the penetrance of expressivity of cleft palate in Fgfr1FCPG/cKOmutants (n=2/13).

Supplementary Figure 6. FGF1 activates multiple pathways in primary cells

(A) Mouse embryonic fibroblasts serum starved and treated with 50 ng/mL FGF1 and 5 μg/mL heparin for the indicated times. EGF and 10% calf serum (CS) were used as positive controls for pathway induction. Phospho-blots were stripped and reblotted for either total protein or β-tubulin as loading control. (B-C) FPCs derived from indicated genotypes, serum starved overnight and stimulated with 50 ng/mL FGF1, 5 μg/mL heparin for indicated times. (B) Phospho-blots were stripped and reblotted with total protein for loading controls. Results are representative of at least three biological replicates. (C) Phospho-blots were stripped and reblotted with β-tubulin as a loading control. Four independent experiments with biological replicates are shown.

Supplementary Table 1.Fgfr1C/Cmutants have normal physiology

Physiology panel and complete blood cell count collected from indicated ages and genotypes (n=4) suggest Fgfr1C/C mutants are physiologically normal. P-values represent unpaired, two-tailed t-tests.

Supplementary Table 2.Fgfr1CPG/CPG mutants have normal physiology

Physiology panel and complete blood cell count collected from indicated ages and genotypes (n=5)suggest Fgfr1CPG/CPG mutants are physiologically normal. P-values represent unpaired, two-tailed t-tests.

Supplementary Table 3. Primers table

The name, sequence and utility of each primer are indicated in the table.

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