A model for FGFR1 function during A-P patterning and the effects of hypomorphic and Y766F mutations on it. The signals through FGFR1 appear to regulate the expression and activity of Hox gene products. In the hypomorphic situation homeotic transformations are seen predominantly to anterior direction, which are consistent with decreased Hox activity. In the absence of a negative regulatory signal, involving the Y766 autophosphorylation site, homeotic transformations to posterior direction are seen, consistent with increased Hox activity.

 Morphological defects in hypomorphic (n15YF/n15YF) embryos. At E8.5 expansion of the posterior neural folds was seen in n15YF homozygotes (B,C) compared to their wild-type littermates (A). In more severely affected embryos, ectodermal vesicles were often observed (arrows in C). At E10.5, compared to the wild-type embryos (D), the n15YF homozygotes (E,F) showed expansion of both the fore- and hindlimb buds sometimes leading into division of the limb buds into several smaller structures (large arrows). The posteriorly located extra buds appeared to degenerate as seen on the opposite side of the embryo shown in E (F). Expansion of the posterior neural tube was also evident at this stage (small arrow).

 Homeotic transformations in posterior direction in the Y766F mutants. Cervical (A–C) and lumbrosacral (D–F) vertebrae of newborn wild-type (A,D) and Y766Fl homozygous mice (B,C,E,F) are shown. In the Y766F mutants the cervical vertebra C7 often has ribs fusing to ribs on the T1 (B) or to the sternum (C). Other transformations include shift of anterior trabeculi from the C6 to the C5 (arrow in C). At the thoracolumbar level, the T13 often loses its ribs taking a L1 phenotype (E,F, arrow in E points to a remaining distal part of a rib). L6 is frequently fused to the iliac bones (arrow in E), although this phenotype is sometimes seen also in wild-type mice. In Y766F mutants these transformations can occur also over two segments (arrows in F).

 Analysis of Mox-1 and Hox gene expression in the hypomorphic mutants by wholemount mRNA in situ hybridization. Expression of Mox-1 (A–F) in an E9.5 wildtype (A), a IIIcl homozygote (B), a n7 homozygote (C), n15YF homozygotes (D,E), and an E10.5 n15YF homozygote (F). The most anterior somites in n15YF homozygotes were often reduced in size (arrows in D,E). Expression of A-P patterning markers HoxD4 (G), HoxB5 (H,I) and HoxB9 (J,K) in wild type (G,H,J) and n15YF homozygous (G,I,K) embryos. The anterior level of expression of HoxD4 appears shifted posteriorly by one somite level in E8.5 mutant embryo compared to a wild-type littermate (arrows in G). In E9.5 embryos, HoxB5 expression posteriorly was weaker in the n15YF mutants than in the wild-type littermate (arrows in H,I). In a subset of E9.5 embryos hybridized with the HoxB9 probe the anterior level of hybridization appeared posteriorized in the lateral plate mesoderm concomitant to the expansion of the limb fields (arrows in J,K). In all cases the neural expression appeared unchanged (arrowheads in J,K).

 Distal limb defects in the hypomorphic mutants. Skeletal preparations of forelimbs (A–D) and hindlimbs (E–H) of newborn wild-type (A,E), n7 homozygous (B,F), IIIbn homozygous (C,G) and n7/null transheterozygous (D,H) newborn mice. Reduced Fgfr1 function appears to correlate with successive loss of anterior digits. Other defects include syndactyly, delayed ossification of distal phalanges, and occasional appearance of an extra postaxial cartilage condensation in the forelimbs (arrows in B,D). In n7/null transheterozygotes the limb defects are markedly enhanced resulting occasionally in defects in the development of the more proximal limb (H). Analysis of 5′ HoxD gene expression in the limb buds of IIIbn hypomorphic mutants by whole mount mRNA in situ hybridization (I–K). HoxD11 expression at E10.5 (I) in a IIIbn homozygote (IIIbn) and a wild-type littermate (wt), showing no obvious difference. In contrast, HoxD13 expression both at E10.5 (J) and E11 (K) was reduced in the mutants compared to their wild-type littermates.

 Posterior truncations and homeotic transformations in the Fgfr1 hypomorphs. (A) A newborn wild-type (WT) and two n15YF homozygous (n15YF) pups. Marked reduction in the posterior axis occasionally results in fusion of the hindlimbs (mutant on the right). The mutants also show greatly reduced size of the outer ear, distal limb defects, and severe spina bifida (arrows). Skeletal analysis of a wild type (B), n7 homozygote (C), n7/null transheterozygote (D), and n15YF homozygote (E) demonstrates the series of axial skeletal defects seen in the mutants. Compared to the wild-type cervical vertebrae (F), vertebral malformations are seen with a low frequency in the n7 homozygotes (G). Both the penetrance and expressivity of these defects is increased in the n7/null transheterozygotes (H), which commonly show fusion of the C1 to the occipital bones and partial transformation of C2 into C1 phenotype (arrow). At the lumbrosacral level, compared to the wild type (I), the n7 homozygotes frequently had extra ribs on L1 (J, arrows). Again, these homeotic transformations into the anterior direction were more severe in the n7/null transheterozygotes, which also often showed transformation of S1 into L6 phenotype (K, arrows).

 Genomic structure and characterization of the Fgfr1 alleles generated. (A) Schematic structure of the FGFR1 protein is shown at top. The two alternatively spliced receptor isoforms, FGFR1 IIIc and IIIb, differing in their ligand-binding domain, were inactivated separately by introducing stop codons (asterisks) in the beginning of exons 7 and 6, respectively. These stop codons were followed by mutations creating diagnostic restriction enzyme recognition sites BamHI (IIIc) and EcoRV (IIIb). One of the tyrosine autophosphorylation sites of FGFR1, Y766, was mutated into phenylalanine (F) followed by a silent mutation creating a XbaI site. The alleles were produced using three targeting vectors: pPNTLoxP–IIIcSTOP, pPNTLoxP–IIIbSTOP, and pPNTLoxP–Y766F. Depending on the site of homologous recombination, alleles either with (IIIcn, IIIbn, and n15YF) or without (n7 and n15) the above mentioned mutations were generated. The neo cassettes were flanked by loxP sites (black rectangles), allowing their excision in vivo to generate alleles l7, IIIcl, IIIbl, l15, and Y766Fl. (B) BamHI; (E) EcoRI; (H) HindIII; (N) NcoI; (RV) EcoRV; (Xb) XbaI; (Ig) immunoglobulin-like domain; (neo) neomycin phosphotransferase gene driven by PGK-1 promoter and followed by PGK-1 polyadenylation site. (B) Southern blot analysis of the ES cell lines generated. See A for identification of the probes and restriction maps. (C) Northern blot analysis of Fgfr1 mRNA expression in a F₂ litter carrying the n7 allele. Total RNA was isolated from E10.5 embryos and analyzed with a Fgfr1 cDNA probe from the kinase domain region and a Gapdh cDNA probe. Quantification of the hybridization signal revealed a three to five fold reduction in the amount of full-length Fgfr1 transcript in the n7 homozygotes compared to their heterozyous or wild-type littermates.