A missense mutation in Fgfr1 causes ear and skull defects in hush puppy mice

Abstract

The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP(3) pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting.

Fig. 1

The hush puppy phenotype is caused by a mutation in Fgfr1. a Fgfr1 sequence showing the point mutation T2128A (arrow), located in exon 15 of the gene. Sequences from wild-type animals (top), heterozygotes (middle), and homozygotes (bottom) are shown. b The point mutation causes the missense mutation W691R in a highly conserved tyrosine kinase motif, subdomain IX (complete domain shown). Numbers shown are the residue number for the equivalent amino acid in each sequence. c Development of homozygous embryos is arrested at ~E8.5. Mutant embryos (E8.5) show a characteristic restriction at the embryonic/extra-embryonic boundary (arrow). Heterozygous embryos have no phenotype at this stage. Scale bar = 250 μm. h human, r rat, m mouse, x Xenopus, z zebrafish, n Eastern newt, d Drosophila, ys yolk sac, hf head fold, s somites, A anterior, P posterior

Fig. 2

Reverse transcription PCR of mRNA (E7.5) from each genotype. The results show that the mutation caused a reduction in the amount of transcript present. a PCR products for wild-type (+/+, n = 3 animals), heterozygote (Hspy/+, n = 7 animals), and homozygote (Hspy/Hspy, n = 8 animals) are shown for both Fgfr1 and β-actin, the internal control. Control used water in place of cDNA to check for contamination. bp, base pairs. b Levels of Fgfr1 transcript per genotype, normalised to β-actin transcript. Fgfr1 transcript is significantly reduced compared to wild-type in heterozygotes (P < 0.05) and homozygotes (P < 0.001)

Fig. 3

Fgfr1^Hspy-flag is expressed at the cell membrane. Immunocytochemistry using an anti-flag FITC-conjugated antibody showed that the protein is localised to the cell membrane in cells expressing either Fgfr1^WT-flag or Fgfr1^Hspy-flag (n = 3 experiments). Scale bar = 20 μm

Fig. 4

Changes in [Ca²⁺]_i in response to FGF1. Example traces are shown of changes in [Ca²⁺]_i when FGF1 (100 ng/ml) with heparin (5 mg/ml) is applied to the cells, followed by application of CCh (1 mM, to ensure functional Ca²⁺ stores are present in the cells). a There is no response to FGF1 in nontransfected cells (n = 194 cells). b Cells expressing Fgfr1^WT give a transient increase in [Ca²⁺]_i on application of FGF1 (n = 194 cells). c Addition of the flag tag to the receptor (Fgfr1^WT-flag) causes a small reduction in the amplitude of the FGF1 response (n = 189 cells). d Cells expressing Fgfr1^Hspy-flag gave no increase in [Ca²⁺]_i in response to FGF1 (n = 376 cells)

Fig. 5

The hush puppy mutation prevents phosphorylation of MAP kinase. Cells were stimulated with 100 ng/ml FGF1 and 5 mg/ml heparin and then examined for MAPK expression and phosphorylation via Western blotting (n = 5 lysates). The top blot shows total MAP kinase in the cell lysates. The bottom blot indicates the amount of MAP kinase that is phosphorylated when stimulated. There was no significant difference between cells expressing Fgfr1^Hspy-flag and nontransfected cells. +, stimulated cells; −, unstimulated cells

Fig. 6

Examples of stapes from the backcross mice, all heterozygotes for the Fgfr1 ^Hspy mutation. a The most modified stapes. Both crura appear relatively normal (score 1). b Stapes with thinner than normal posterior crus (score 2). c Posterior crus is quite thin in the middle and may be lacking bone in the centre (score 3). d Posterior crus lacks bone in the middle but tissue is still present forming a very thin link (score 4). e Posterior crus is missing but the stapedial muscle attachment is present at the top right (score 5). f Posterior crus is missing, including the stapedial muscle attachment but anterior crus appears normal (score 6). g Posterior crus completely missing. Anterior crus is affected and appears thin and often more widely splayed than normal (score 7). h Stapes is bony through area between crura. The stapedial artery did not pass through the stapes (score 8). This feature was rarely seen (n = 4 ears), and as it appeared to be qualitatively different to the other stapes defects, it was not included in subsequent analyses. i Diagram to illustrate the backcross used to map the modifier

Fig. 7

Genome scans using the sum of the stapes defect scores from both left and right ears. a Polymorphic microsatellite markers (Table 2) were used on the DNA of animals that had a combined left and right score of 2 or 3. These are therefore the most modified mice from the backcross (n = 39 animals). b Polymorphic microsatellite markers (Table 2) were used on the DNA of animals that had a combined left and right score of 10 or more. These are therefore the most abnormal (least modified) mice from the backcross (n = 36 animals). Mice with a solid bony stapes (Fig. 6H, score 8 on either side; n = 4) were not included in the genome scan

Fig. 8

The distribution of the sum of the stapes scores for mice carrying distal chromosome 7 C57BL/6J DNA (marker D7Mit259) from their father (violet) and for mice carrying distal chromosome 7 C57BL/6J DNA from their mother (maroon). There is a noticeable bias toward the more modified end (scores 2–4) for those mice from a heterozygote father (Shapiro-Wilks test score for mice from a heterozygote father: 6.85 × 10⁻⁷; score for mice from a heterozygote mother: 0.0501)