Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog

Abstract

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6-9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/(+);Shh(-/-) mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/(+) embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/(+) limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/(+) mutants, we sequenced approximately 20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a approximately 600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5' of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/(+) mutants. However, we did not detect anologous deletions in cases of Laurin-Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.

Fig. 1

Expression of Gli3 and Hand2 in E9.5 (A–D) and E11.5 (E–H) wt (A, C, E, G) and Dbf/⁺ (B, D, F, H) forelimb buds. Gli3 is expressed in the anterior region of both wt and Dbf/⁺ at E9.5, and Hand2 in the posterior region (arrows indicate the limits of the expression domains). At E11.5 Gli3 is expressed in a broader anterior domain in Dbf/⁺ limb buds, and Hand2 expression extends more anteriorly in Dbf/⁺ than in equivalently staged wt limb buds. Scale bars 0.2 mm.

Fig. 2

Morphology of E13.5 (A–H) and E17.5 (I–L) embryos and limbs, genotypes as indicated. (A and B) The preaxial polydactyly characteristic of Dbf/⁺ embryos (B) can be seen clearly in the hindlimb, which has 8 digits. (C) Shh^−/− embryos show holoprosencephaly, oedema and highly truncated limbs. (D) Shh^−/−;Dbf/⁺ embryos show only minor modification of the Shh^−/− head phenotype; the limbs resemble those of Dbf/⁺ embryos except that the autopods show both preaxial and postaxial polydactyly; the whole embryo shows hypervascularization. (E–H) Cartilage preparations of E13.5 forelimbs. (E) wt forelimb, (F) Dbf/⁺ forelimb showing preaxial polydactyly. (G) The forelimb zeugopod and autopod of Shh^−/− fetuses are represented by single elements and there is no elbow joint. (H) In the Shh^−/−;Dbf/⁺ forelimb an elbow joint and radius are visible. (I) Wild type E17.5 hindlimb. (J) Dbf/⁺ hindlimb showing preaxial polydactyly (7 triphalangeal digits) and medial rotation (luxation) of the autopod due to the hypoplastic tibia. (K) Hindlimb of Shh^−/− mutant showing a femur of approximately normal length and a rudimentary zeugopod with the proximal tibia and attached putative fibula, plus a single short digit. (L) Shh^−/−;Dbf/⁺ hindlimb (upside-down compared to I–K) showing a femur of normal length, full-length zeugopod and an autopod exhibiting both preaxial and postaxial polydactyly (9 triphalangeal digits, one of which is duplicated distally). Scale bars 1 mm.

Fig. 3

Expression of Shh and Ihh in wt and mutant embryo forelimb buds at E10.5 and E11.5. All darkfield except D, which shows Shh and Ihh expression on the same specimen. The Shh expression domain in Dbf/⁺ limb buds is the same as wt (A, B, F, G), and is mutually exclusive with that of Ihh (C and D). In the absence of Shh expression, the Ihh domain of compound mutant limb buds extends into the most posterior (ZPA) mesenchyme (E). Down-regulation of Ihh leaves a small anterior domain in Dbf/⁺ limb buds at E11.5 (H) but both anterior and posterior residual domains are detected in compound mutants (I). Scale bar 0.2 mm.

Fig. 4

Regulation of limb development genes in E10.5 wt, Dbf/⁺;Shh^−/− and Shh^−/−;Dbf/⁺ forelimb buds. The anterior margin of limb buds is uppermost. Ptc1 and Gli1 expression domains are extended more anteriorly in Dbf/⁺ limbs than in wt, and are broader in Shh^−/−;Dbf/⁺ limbs, but absent from Shh^−/− limb buds. Gli3 expression is present in the anterior region of wt limbs; the domain is reduced in Dbf/⁺ limbs, and extended throughout Shh^−/− limb buds; expression is at a very low level in Shh^−/−;Dbf/⁺ limb buds (extended colour development time). Expression of Hand2 is unaltered in Dbf/⁺ limbs, but is present more anteriorly in Shh^−/−;Dbf/⁺ limb buds. Fgf8 is expressed throughout the AER of limb buds of each genotype. Bmp4 expression is similar to wt in Dbf/⁺ limbs but up-regulated in Shh^−/− limbs; in Shh^−/−;Dbf/⁺ limbs expression appears to be lower in the mesenchyme but high in the AER (shown in bright field). Expression of Hoxd13 is extended anteriorly in Dbf/⁺ limbs and both anteriorly and posteriorly in Shh^−/−;Dbf/⁺ limbs, but is absent from Shh^−/− limbs. Fgf4 is expressed in the posterior region of the AER in wt (posterior to the arrowhead), throughout the AER in Dbf/⁺, is absent from Shh^−/− and present in the anterior and posterior AER of Shh^−/−;Dbf/⁺ limb buds. Scale bar 0.2 mm.

Fig. 5

Gli3 processing is disrupted by the Dbf/⁺ mutation. Western blot analysis of protein extracts from the limb buds of E10.5 wt and mutant embryos using anti-N-terminal Gli3 antibody. (A) Representation of an E10.5 limb bud showing the anterior and posterior regions dissected for protein extraction. (B) Approximately 3 μg of protein extracted from forelimb buds was used in each lane; protein bands were immunoblotted and incubated with a Gli3-specific antibody binding to a 190 kDa band and two 83–86 kDa bands, corresponding to full-length (Gli3A) and processed repressor forms (Gli3R), respectively. A, anterior; P, posterior. (C) The histogram shows the relative intensity of each band in arbitrary units following quantification by densitometry.

Fig. 6

(A) Genomic map of the region around the Dbf deletion showing the genes flanking the deletion. The box encloses the deleted region shown at a higher scale. All nucleotide positions refer to the mm9 assembly. (B) DNA sequence chromatogram showing breakpoint sequence and alignment of this sequence compared to wt sequences on either side of the breakpoint. The arrows indicate the range of possible positions of the breakpoint within the three nucleotide identity shared by both normal sequences.