Normal limb development in conditional mutants of Fgf4 - PubMed (original) (raw)

Normal limb development in conditional mutants of Fgf4

A M Moon et al. Development. 2000 Mar.

Abstract

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.

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Figures

Fig. 1

Fig. 1

Conditional mutagenesis of the Fgf4 locus. (A) The wild-type (WT) Fgf4 locus is depicted on the first line; exons are represented by blue boxes; untranslated regions are shown as lines (purple, Fgf4 promoter; black, introns and 3′UT). The F4AP targeting vector (second line) was constructed by insertion of a loxP site (red arrowheads) 59 bp upstream of the translation start site. A cassette containing: MC1neor (white box), another loxP site in the same orientation as that in the 5′ untranslated region and, human placental alkaline phosphatase-coding sequences and the SV40 polyadenylation signal (yellow box), was inserted 89 bp downstream of the polyadenylation signal. Thymidine kinase (TK, green) genes flank the Fgf4 homology to permit negative selection against random integrants. Cre-mediated recombination (large red arrow) of the F4AP-targeted allele deletes all Fgf4 sequences between the loxP sites, generating F4APR. (B) The F4neo targeting vector contains MC1neor between _Xma_III (X) restriction sites at nucleotides 286 of exon 1 and 113 of intron 1. (C) Genotyping at the Fgf4 locus. PCR primer sets are shown in A and B and fragment sizes are as shown in C. The F4AP and F4APR alleles contain WT sequences in the region examined by the F4neo primer set and vice versa. Each animal was analyzed with both primer sets to obtain a complete genotype. PCR products obtained with P1 and P2 on the WT and F4AP alleles are 256 base pairs (bp) and 296 bp, respectively. The P1/P3 primer set amplifies a 461 bp region of the recombined allele, F4APR. The P4/P5 set detects a 368 bp WT product; while P4/P6 detects a 231 bp product from the F4neoallele.

Fig. 2

Fig. 2

Activity of the RARCre transgene. (A) β-galactosidase activity in offspring of RARCre X P2Bc reporter mice. E9.5, E10.0 and E11.5 embryos were stained with X-gal; there is no activity in embryos with only the P2Bc reporter allele at any stage. Littermates that also carry the RARCre transgene (P2Bc/+;T) have detectable β-galactosidase activity, resulting from Cre mediated recombination of the reporter gene, in the forelimb by E 9.5 and in the AER by E 10.0. Note absence of significant hindlimb staining. (B) Alkaline phosphatase (AP) activity requires Cre-mediated recombination of the F4AP conditional allele. E10.0, 30-32 somite, forelimb stage 3 littermates, were stained for AP activity. There is no staining in F4AP/AP mice in the absence of RARCre. Cre-mediated recombination of the F4AP allele results in uniform staining of the entire AER in the forelimb (FL, yellow arrowhead) but only partial staining in the hindlimb (HL, red arrowhead) AER. The difference in recombination between fore- and hindlimb is confirmed by examining the AERs in cross-section. The appearance of AP signal in cells outside the AER could be a technical artifact or reflect actual low-level activity of the Fgf4 promoter in these cells.

Fig. 3

Fig. 3

Fgf4 expression in conditional mutants and their littermates. Whole-mount RNA in situ hybridization with an Fgf4 antisense riboprobe containing exon 3 and 3′UTR sequences. (A) E10.5 whole-mount embryos; transcripts are present in hindlimb, but not forelimb AER of conditional mutants (F4AP/Null;T), confirming that Cre-mediated recombination of the conditional allele in the context of a null allele completely disrupts Fgf4 gene function. (B) E10.0 (30 somite) embryos. Close-up view of forelimb AER. Absence of Fgf4 transcripts correlates with presence of RARCre and confirms disruption of Fgf4 gene function at the earliest stage of normal Fgf4 transcription. (C,D) Close-up views of the forelimb AERs of E10.5 and E11.5 embryos, respectively.

Fig. 4

Fig. 4

Skeletal phenotypes of newborns. Alizarin red stains ossified structures and Alcian blue stains cartilage in these preparations. Note that the limbs of Fgf4 conditional mutants are indistinguishable from wild-type controls.

Fig. 5

Fig. 5

Molecular phenotypes as assessed by RNA in situ hybridization; normal expression of Shh in Fgf4 conditional mutants. For each probe, top row are E10.5 embryos; bottom row, E11.5. Littermates were used when possible. (A) Shh expression in the ZPA. (B) Fgf8 expression in the AER. (C) Bmp2 expression in the AER and mesenchyme. Note that each of these genes is expressed normally in Fgf4 conditional mutants.

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