Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless

Abstract

Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10(-/-) fetuses continued to develop until birth, despite the complete absence of both fore- and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10(-/-) mice exhibited perinatal lethality associated with complete absence of lungs. Although tracheal development was normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, was completely disrupted. The pulmonary phenotype of Fgf-10(-/-) mice is strikingly similar to that of the Drosophila mutant branchless, an Fgf homolog.

Figure 1

Targeted disruption of the murine Fgf-10 gene. (A) Schematic diagram of the Fgf-10 exon 1 (black box) locus in mice. The translation initiation site (ATG) is indicated by a bent arrow. Restriction sites are indicated as follows: (H) HindIII; (RI) EcoRI; (B) BamHI; and (N) NotI. (B) The targeting vector. The 3.5-kb long arm of homology and the 0.78-kb short arm of homology are placed flanking the PGK–neo casette, which is in reverse orientation. The PGK–tk cassette is adjacent to the short arm. This strategy eliminates the translation initiation codon (ATG) and the predicted signal peptide and places multiple stop codons in-frame. (C) The resulting targeted allele after homologous recombination. (D) Genomic Southern blot using EcoRI-digested DNA. The wild-type allele is a 4.3-kb fragment, whereas the targeted allele is a 1.4-kb fragment. The heterozygote mice (+/−) have both alleles. The small open box in A and C represents DNA probe used for Southern blot experiments. (E) Northern blot analysis of RNA from embryos (E11.5) derived from heterozygous matings. Only wild-type and heterozygote embryos show the 4.0-kb transcript. The same blot is probed with β-actin as an endogenous control for RNA loading.

Figure 2

Gross morphology and bone structure of Fgf-10^−/− mice. Gross photographs of E9.5 (A) and E17.5 (B) Fgf-10^−/− conceptuses are shown with Fgf-10^+/− and Fgf-10^+/+ littermates for comparison. (A, dorsal view) A complete absence of forelimb buds (arrow) in the Fgf-10^−/− embryo relative to the presence of a forelimb bud (FB) in Fgf-10^+/+ and Fgf-10^+/− littermates; (B, lateral view) complete absence of both limbs in the Fgf-10^−/− near-term fetus; (C–H) Gross photographs of E18.5 term Fgf-10^−/− (F–H) and wild-type (C–E) fetuses that have undergone skeletal double staining. Both wild-type (C–E) and Fgf-10^−/− (F–H) fetuses have scapulas with a blue-stained cartilaginous cap (S in C,D,F, and G) and pelvic girdles (arrowheads in E and H). However, the Fgf-10^−/− pelvic girdle is rudimentary and entirely cartilaginous (blue stained), whereas the wild-type pelvic girdle contains both bone (arrowhead directed at red-stained area) and cartilage (arrowhead directed at blue-stained area). The head of the humerus (blue-stained area labeled H) is visible only in the wild-type fetus (C,D) and obscures the scapula in the ventral view (D).

Figure 3

In situ hybridization of the nascent forelimb bud in E9.5 mouse embryos. (A–D) Limb bud of wild-type embryo (bar, 500 μm). Diffuse Fgf-10 expression occurs in limb bud mesenchyme (B). Fgfr2b is expressed throughout the epithelium in the developing limb region (C), whereas Fgf-8 is specifically expressed in AER (D). (E–H) Distribution of Fgf-8 mRNA in Fgf-10^+/+ (E,F) and Fgf-10^−/− (G,H) embryos (bar, 100 μm). Note the absence of an Fgf-8 signal near the presumptive site of limb bud initiation (arrow) in the Fgf-10^−/− embryo. Fgf-8 expression in both Fgf-10^+/+ and Fgf-10^−/− embryos is similar at other sites including the neuroectoderm of the rostral telencephalon (open arrowhead), the junction of the mesencephalon and rhombencephalon (arrowhead), and the pharyngeal epithelium (*). (A,E,G) Stained with eosin plus hematoxylin in A.

Figure 4

H&E-stained sections of E17.5 Fgf-10^−/− fetuses (C,F,I) with Fgf-10^+/+ (A,D,G) and Fgf-10^+/− (B,E,H) littermates for comparison. (A–C) Fetal cross sections showing that Fgf-10^−/− fetuses have a normal trachea (Tr) and thymus (Thy). (Insets in A–C) The tracheal epithelium from Fgf-10^−/− fetuses is ciliated pseudostratified columnar (inset arrowheads indicate cilia) with mucus-secreting goblet cells (clear areas) and is morphologically identical to tracheal epithelium in wild-type and heterozygous fetuses. (D–F) Fetal sagittal sections showing that the Fgf-10^−/− fetal trachea terminates blindly just beyond the anterior thymus (arrow in F), whereas the tracheas in wild-type and heterozygous fetuses continue beyond the thymus and branch to form main-stem bronchi (E). (G–I) Fetal cross sections showing that Fgf-10^−/− fetuses (I) fail to develop main-stem bronchi (Br) or lungs (Lu). (E) Esophagus; and (Ht) heart in A–I.