Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo

Abstract

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. We have analyzed the phenotype of Fgf8(-/-) embryos and discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate their expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak.

Figure 1

Morphology of Fgf8^−/− embryos. In all figures, anterior is to left and posterior to right; intact embryos are viewed laterally, unless otherwise noted. The genotype of each embryo is indicated: Normal embryos were either Fgf8^+/− or Fgf8^+/+. (A) Offspring at E7.5 of a cross between Fgf8^+/− mice. A characteristic bulge of cells in the amniotic cavity (arrow) identifies the two embryos at right as mutant homozygotes. The open arrowhead points to the border between the embryonic (em) and extraembryonic (ex) regions in a normal littermate. (B–D) Scanning electron micrographs of the embryonic region at E7.5. (B) Arrows indicate the direction of epiblast cell movement toward the primitive streak and of mesoderm cell migration away from the streak in a normal embryo. The transverse plane of section was somewhat oblique (higher at right). (C) Fgf8 mutant embryo illustrating the distortion of the primitive streak region, which bulges into the amniotic cavity. Embryos in B and C are shown at the same magnification. (B,C) Red arrowheads indicate the most anterior extent of mesoderm cell migration away from the streak. (D) Higher magnification view of the primitive streak region of the embryo shown in C. Note the accumulation of cells with mesenchymal morphology. (E–L) Sagittal sections of normal and mutant embryos; E and G show embryos sectioned in utero. The times at which they were collected are indicated. The A-P and P-D axes are indicated in E. (E,F) Mid-streak-stage embryos. (*)The nascent exocoelom, which consists of a layer of extraembryonic ectoderm and a layer of mesoderm. (G,H) Late-streak-stage embryos. The extent of the primitive streak and the anterior ectoderm (prospective neuroectoderm) is indicated. (I,J) The normal embryo is at the head-fold stage. In a mutant embryo collected at the same stage, head mesenchyme and a morphologically distinct node are absent. (K,L) The normal embryo contains several somites, a heart, and foregut; the mutant embryo does not contain any such mesoderm- or endoderm-derived tissues. The arrow in L points to a small, isolated patch of mesodermal cells in the mutant embryo. (M,N) Whole-mount in situ hybridization assay for Fog RNA in E8.5 embryos. Signal is detected in blood islands (arrows). (A) Anterior; (ac) amniotic cavity; (al) allantois; (am) amnion; (bl) blood island; (ch) chorion; (Di) distal; (ec) anterior ectoderm (prospective neuroectoderm); (ecc) ectoplacental cone; (em) embryonic region; (en) endoderm; (ex) extraembryonic region; (exo) exocoelom; (fb) forebrain; (fg) foregut; (hm) head mesoderm; (ht) heart; (mes) mesoderm; (nd) node; (ne) neuroectoderm; (P) posterior; (Pr) proximal; (ps) primitive streak; (so) somite; (xe) extraembryonic ectoderm; (xm) extraembryonic mesoderm; (+) normal; (−/−) mutant.

Figure 2

Gene expression in the primitive streak. (A–F) RNA in situ hybridization using a probe for T RNA. (A,B) Embryos at E7.75 in whole mount. (C,D) Sagittal sections of an E7.75 normal embryo stained in whole mount and the mutant embryo shown in B. (E,F) Transverse sections through the normal embryo shown in A and an E7.75 mutant embryo stained in whole mount. The broken white lines in A and B indicate the approximate level of the sections shown in E and F, respectively. Arrows in B and D point to T-expressing cells anterior to the primitive streak. (G,H) Tbx6 expression at E7.75. (I–L) Lim1 expression in an E7.25 normal embryo and an E7.5 mutant embryo. (K,L) Transverse sections through the embryos shown in I and J, respectively. Broken white lines indicate the approximate level of the sections shown. Arrows point to Lim1-expressing cells in the AVE/axial mesoderm. Red arrowheads indicate the most anterior extent of mesoderm cell migration. Abbreviations as in Fig. 1. (axm) Axial mesoderm.

Figure 3

Analysis of markers for the anterior streak and its derivatives. (A,B) Gsc expression in a normal embryo at E7.0 and a mutant embryo at E7.25. Expression was detected in the AVE of the normal but not the mutant embryo. (C,D) Hnf3β expression in a normal embryo at E6.75 and a mutant embryos at E7.5. (E) Parasagittal section of the mutant embryo shown in D. The asterisk (*) indicates the nascent exocoelom. (F,G) Shh expression in a normal (lateral view) and a mutant (posterior view) embryo at E8.0. The arrow points to Shh-expressing cells in the mutant embryo that remain near the distal end of the primitive streak. In most mutant embryos examined there were fewer Shh-expressing cells than are seen here. (H,I) Hnf3α expression in a normal (anterior view) and a mutant (lateral view) embryo at E8.25. Expression is detected in the definitive endoderm of the normal embryo. (J–O) Ndl expression as detected by β-gal staining. (J,K) Normal and mutant embryos at E7.5. (L,M) Transverse sections of the embryos shown in J and K, respectively. The broken white lines indicate the approximate level of the sections. Note that Ndl continues to be detected in the ectoderm on the anterior side of the mutant embryo. (N,O) A normal (posterior view) and a mutant (lateral view) embryo at E8.0. Abbreviations as in Figs. 1 and 2.

Figure 4

Patterning of the neuroectoderm in Fgf8^−/− embryos. (A,B) Hex expression in normal and mutant embryos at E7.5. (Arrows) Hex-expressing AVE cells. (C,D) Hesx1 expression at E8.0. (E,F) Six3 expression in a normal embryo at E7.75 and a mutant embryo at E8.25. (G,H) Otx2 expression at E7.5. (I,J) Gbx2 expression at E7.75.

Figure 5

Expression of FGF family members in Fgf8^−/− embryos. (A–F) Fgf3 expression in normal and mutant embryos at E7.5. Broken lines in A and B indicate the levels of the sections shown in C, E, and D, F, respectively. (G,H) Fgf5 expression at E7.25. (I,J) Fgf4 expression at E7.5. As illustrated in J, Fgf4 RNA is not detected in mutant embryos.