Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development

Abstract

We disrupted the fibroblast growth factor (FGF) receptor 2 (FGFR2) gene by introducing a neo cassette into the IIIc ligand binding exon and by deleting a genomic DNA fragment encoding its transmembrane domain and part of its kinase I domain. A recessive embryonic lethal mutation was obtained. Preimplantation development was normal until the blastocyst stage. Homozygous mutant embryos died a few hours after implantation at a random position in the uterine crypt, with collapsed yolk cavity. Mutant blastocysts hatched, adhered, and formed a layer of trophoblast giant cells in vitro, but after prolonged culture, the growth of the inner cell mass stopped, no visceral endoderm formed, and finally the egg cylinder disintegrated. It follows that FGFR2 is required for early postimplantation development between implantation and the formation of the egg cylinder. We suggest that FGFR2 contributes to the outgrowth, differentiation, and maintenance of the inner cell mass and raise the possibility that this activity is mediated by FGF4 signals transmitted by FGFR2. The role of early FGF signaling in pregastrulation development as a possible adaptation to mammalian (amniote) embryogenesis is discussed.

Figure 1

Targeted disruption of FGFR2. (A) Genomic map and its manipulation. Above the line: H, HindIII; C, ClaI. Under the line: C, KV, and BV, exons 7, 8, and 9 (IIIc loop); TM, exon 10 (transmembrane); K1, kinase domain 1; ATP, exon 12 encoding the ATP binding site; neo, neomycin resistance gene. (B) Map of the recombinant, flanking sequences blocked, abbreviations as in A. (C) Wild-type (6.2-kb) and recombinant (9.4-kb) HindIII fragments. (D) Southern analysis of recombinant ES cells. Clones shown in lanes 1 and 2 were used for aggregation. The recombinant allele was analyzed also by a 3′ probe demonstrating the large deletion and the absence of the EcoRV site of exon 9. Specific probes showed the absence of the transmembrane domain.

Figure 2

Histological analysis of 4.5 d.p.c. (FGFR2+/−)F₂ embryos. (A) Control. (B–D) Presumptive mutants. The mesometrium is toward the left side of the figures. pe, Primitive endoderm; mt, mural trophectoderm. (Bar = 25 μm.)

Figure 3

Mutant defects in cultured blastocysts; FGFR2 expression in preimplantation blastocysts. (A–D) Seven-day cultures. (A) Heterozygote. (B–D) Homozygous mutant. (E–H) Four-day cultures, Sophora japonica lectin staining. (E and F) Heterozygote. (G and H) Homozygous mutant. (E and G) Phase contrast. (F and H) Fluorescence. (I) Blastocyst (3.5 d.p.c.) anti-FGFR2 fluorescent staining, confocal microscopy. (Bars: 250 μm.)

Figure 4

PCR analysis of individual cultured blastocysts. (Upper) Ethidium bromide staining. (Lower) Southern blot hybridization with FGFR2 probe. Lanes 1 and 2, homozygous wild type; lanes 4, 5, 6, 8, and 11, heterozygotes; lanes 3, 7, 9, and 10, homozygous mutant; lanes 12 and 13, blank control; lane 14, water. This is an analysis of the experiment visualized in part in Fig. 3 A–D.

Figure 5

Localization of FGFR2 transcripts in the early embryo by in situ hybridization. (A) Blastocyst 3.5 d.p.c. (B and D) Bright field. (C and E) Dark field. (B and C) Embryo 5.5 d.p.c. (D and E) Embryo 6.5 d.p.c. (Bars: A = 20 μm; B–E = 200 μm.)