Yonsei Med J.  2008 Dec;49(6):965-972. 10.3349/ymj.2008.49.6.965.

Point Mutation of Hoxd12 in Mice

• Cho KW
• Kim JY
• Cho JW
• Cho KH
• Song CW
• Jung HS

• KMID: 1782945
• DOI: http://doi.org/10.3349/ymj.2008.49.6.965

Abstract

PURPOSE
Genes of the HoxD cluster play a major role in vertebrate limb development, and changes that modify the Hoxd12 locus affect other genes also, suggesting that HoxD function is coordinated by a control mechanism involving multiple genes during limb morphogenesis. In this study, mutant phenotypes were produced by treatment of mice with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). We analyzed mutant mice exhibiting the specific microdactyly phenotype and examined the genes affected. MATERIALS AND METHODS: We focused on phenotype characteristics including size, bone formation, and digit morphology of ENU-induced microdactyly mice. The expressions of several molecules were analyzed by genome-wide screening and quantitative real-time PCR to define the affected genes. RESULTS: We report on limb phenotypes of an ENU-induced A-to-C mutation in the Hoxd12 gene, resulting in alanine-to-serine conversion. Microdactyly mice exhibited growth defects in the zeugopod and autopod, shortening of digits, a missing tip of digit I, limb growth affected, and dramatic increases in the expressions of Fgf4 and Lmx1b. However, the expression level of Shh was not changed in Hoxd12 point mutated mice. CONCLUSION: These results suggest that point mutation rather than the entire deletion of Hoxd12, such as in knockout and transgenic mice, causes the abnormal limb phenotype in microdactyly mice. The precise nature of the spectrum of differences requires further investigation.

Keyword

N-ethyl-N-nitrosourea induced mice; microdactyly; Hoxd12; limb and digit abnormality; point mutation

MeSH Terms

Animals
Base Sequence
DNA/genetics
DNA Primers/genetics
Ethylnitrosourea/toxicity
Genes, Homeobox
Homeodomain Proteins/*genetics
Limb Deformities, Congenital/genetics
Male
Mice
Mice, Inbred BALB C
Mutagens/toxicity
*Point Mutation
Transcription Factors/*genetics

Figure

• 

  Fig. 1 Dorsal views of fore (a-l) and hind (m-z) limbs of ENU-induced microdactyly and WT (BALB/cJ) mice. Digits I, II, III, and V were shorter in microdactyly than in WT mice, and were curved, with only digit IV being the same in the fore limb (a-d). Digit I was widely expanded and shorter in microdactyly than in WT mice (a, c, g, and j). Compared with WT mice, the tip of digit I was missing in microdactyly mice (a, g, and j), the radius and ulna were shorter and thinner (e and f), the interosseous space of the fore limb was larger, all first phalanges were shorter and thicker, all joints were thicker (m, o, and s-z), and the metatarsal of digit I was shorter, whereas the digital phalanx (tip) was normal (m, o, and s). In digit II, the metatarsal bone was shorter, thicker, and curved as in digit I (m, o, t, w, y, and z). Digit V was also curved proximally (m-p, v, and x-z). The hind limb exhibited abnormalities in the tibia and fibula (q and r). Arrows indicate abnormal limb formations. Arrowheads indicate the fibular abnormality. *; interosseous space. R, radius; U, ulnar; T, tibia; F, fibula. Scale bars: 10 µm.

• 

  Fig. 2 Haplotype analysis and the genetic linkage map (a, b), and (c) mutation analysis of ENU-induced microdactyly mice. (a) Haplotype analysis of intraspecific backcross between mutant and C57BL/6. The (mutant × B6) F1 was crossed with (F1 × mutant) F2 and produced 21 mutant homozygous mice. Each column represents a chromosomal haplotype identified in the intercross and inherited from the F1 parents. Each genotype was determined by a single sequence length polymorphism. Black boxes, BALB/cJ alleles; white boxes, C57BL/6 alleles; grey boxes, both alleles. Markers are indicated on the left. (b) Genetic linkage map. (c) Sequencing of PCR products of WT and ENU-induced microdactyly mice. Red arrowheads indicate the mutations responsible for the phenotypes. Sequencing of Hoxd12 in ENU-induced microdactyly mice revealed G-to-T transversion at nucleotide 453, resulting in a change from alanine to serine.

• 

  Fig. 3 Comparison of mRNA expression levels between ENU-induced microdactyly and WT (BALB/cJ) adult mice. Bmp2, Bmp4, En1, Gli3, Fgf4, Fgf8, Lmx1b, Ptch1 and Shh were detected in mictodactyly and WT mice. The expressions of Fgf4 and Lmx1b were significantly higher in microdactyly than in WT mice. The gene expression was normalized to that of the housekeeping gene, GAPDH. Data are mean and SD values (n=6). *p < 0.01.

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