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First published online 28 April 2004
doi: 10.1242/dev.01126

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Hypomorphic expression of Dkk1 in the doubleridge mouse: dose dependence and compensatory interactions with Lrp6

Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA

View larger version (27K): [in a new window]   Fig. 1. Structure of the transgene insertion site in the doubleridge mutant. (A) Sequence of the two junction fragments from overlapping cosmid clones. (B) Position of the mouse genomic sequence in cosmid clones (black) aligned with the assembled genomic sequence of a 400-kb region of mouse chromosome 19. The position of the 60-kb doubleridge deletion and nearby genes is shown. Open box, noncoding exon; filled box, coding exon; arrow, direction of transcription. The promoter of Prkg1 is located at 31.3 Mb. (C) Confirmation of the 60-kb deletion in doubleridge genomic DNA. Primer pairs located between the cosmid junction sequences do not amplify doubleridge genomic DNA.

View larger version (88K): [in a new window]   Fig. 2. Non-complementation between the doubleridge mutation and a null allele of Dkk1. (A) Normal limb development in Dkk1d/+ heterozygote; (B) Postaxial polysyndactyly in Dkk1d/– compound heterozygote; (C) Postaxial polysyndactyly in Dkk1d/d homozygote, (D) Postaxial polysyndactyly in Dkk1–/–. Forelimbs from E18.5 embryos were stained with alcian blue and alizarin red.

View larger version (48K): [in a new window]   Fig. 3. Hypomorphic expression of the Dkk1d (doubleridge) allele detected by primer extension and wholemount in situ hybridization. (A) Quantitative assay of allelic Dkk1 transcripts in heterozygous mice. Primers 1 and 2 are used for RT-PCR amplification of transcripts, and the 25 bp primer 3 for primer extension of the amplified RT-PCR product. The single nucleotide polymorphism in the 3' UTR that distinguishes the C3H and SJL transcripts is shown in bold. The extension products obtained in the presence of 33P-ddGTP are 31 bp in length for the C3H allele and 33 bp in length from the SJL allele, the parental allele for the doubleridge insertion. (B) Primer extension products are separated on a 10% acrylamide gel and visualized using BIOMAX film. Triplicate assays of four RNA samples from pooled hindlimbs at E13.5. Samples 1 and 2, (C3H X SJL)F1-Dkk1d/+ doubleridge heterozygotes; 3 and 4, (C3H X SJL)F1 wild-type heterozygotes. (C-F) Wholemount in situ hybridization of Dkk1d/d embryos and +/+ littermates with a Dkk1 cDNA probe demonstrates reduced expression in multiple domains. (C,D) E9.5 forelimb; (E,F) E9.5 tail bud.

View larger version (99K): [in a new window]   Fig. 4. Effect of varying Dkk1 expression on development of anterior head structures. (A) Normal head development in Dkk1d/d homozygote; (B,C) two viable Dkk1d/– compound heterozygotes with closed eyes and hydrocephaly. (D-G) Skeletal preparations stained with Alcian Blue and Alizarin Red: (D) wild type at P0; (E) severely affected Dkk1d/– compound heterozygote with anophthalmia at P0; (F,G) head development in two Dkk1–/– null homozygotes at E18.5.

View larger version (96K): [in a new window]   Fig. 5. Vertebral defects in Dkk1 mutants. (A-C) Kinked tail in adult mice. (D) Vertebral fusions in the tail of Dkk1d/– compound heterozygote, E18.5. (E-G) Vertebral fusions in sacral and lumbar regions of E18.5 embryos; arrow, hemivertebra. Dkk1 genotypes are indicated.

View larger version (59K): [in a new window]   Fig. 6. (A-C) Genetic interaction between Dkk1 and Lrp6 indicated by rescue of postaxial polysyndactyly in double mutants. All mice are homozygous for the doubleridge allele, Dkk1d. The extra digit in the doubleridge single mutant is marked by the arrow. The frequency of partial and complete rescue is dependent on Lrp genotype (Table 2). E16.5 forelimbs were stained with alcian blue to reveal skeletal elements.

View larger version (71K): [in a new window]   Fig. 7. Rescue of axial truncation in Lrp6 null mice by reduced expression of Dkk1. Genotypes are indicated with Dkk1 above and Lrp6 below. (A-C) E16.5 embryos demonstrating axial truncation; (D) small size of Dkk1d/dLrp6–/– double mutant at P1; (E) fusion of lumbar and sacral vertebrae, compare with wild-type control in Fig. 5E; (F) Correction of posterior digits in E16.5 forelimbs. (G) Cross-section of forelimb at E17.5 stained with hematoxylin and eosin. Arrow, ectopic ventral tendon (v). (H) Normal dorsal tendons in Dkk1d/d homozygote.

View larger version (87K): [in a new window]   Fig. 8. Rescue of anterior head truncation in Dkk1 null mice by reduced expression of Lrp6. (A) Single mutants that are null for Dkk1 lack anterior structures of the head, which results in neonatal lethality. The null phenotype is rescued in double mutants which are heterozygous for the null allele of Lrp6, shown at E16.5 (B) and P30 (C). Lateral deviation of the lumbar vertebrae in the P30 double mutant is evident in the skeletal preparation (D).