Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity

Abstract

Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.

FIG. 1

Generation of mice with a targeted disruption of the tetranectin gene. (A) The restriction map and genomic structure of the wild-type mouse tetranectin gene are shown at the top. The three exons are numbered and represented by solid boxes. Restriction sites for BglII (B), SacI (Sa), StuI (St), and ApaI (A) are indicated. A diagram of the targeting vector is shown in the middle. The 1.1-kb SacI/StuI and 5.5-kb ApaI fragments were cloned into the pPNT vector. The thymidine kinase (TK) and neomycin-resistance (neo) cassettes of pPNT are represented by hatched boxes, and the plasmid backbone is depicted by an open box. The structure of the targeted allele is shown at the bottom. Homologous recombination between the targeting vector and the wild-type locus leads to deletion of the 1.3-kb StuI/ApaI fragment containing exon 1 and insertion of the neoR cassette. The thick line indicates the 5′ external probe used for Southern blot screening of ES cells and mouse tail biopsy specimens. (B) Southern blot analysis of BglII-cut genomic DNA from normal mice (+/+) and from mice heterozygous (+/−) and homozygous (−/−) for the disrupted tetranectin allele. The blot was sequentially hybridized to the 5′ external probe (a), a fragment of the neoR gene (b), and a genomic probe containing exon 1 (c). In the correctly targeted allele, insertion of the neoR cassette will introduce an extra BglII site, and the 6.7-kb BglII band containing exon 1 will be replaced with bands of 3.2 and 4.5 kb carrying the tetranectin gene 5′-flanking DNA and the neoR gene, respectively.

FIG. 2

Analysis of tetranectin gene expression in mice with a targeted tetranectin allele. (A) RT-PCR analysis of wild-type (+/+) and heterozygous (+/−) mouse lung tissue revealed a 249-bp tetranectin (TET) fragment, while no product was amplified from the lung, spleen, and muscle of homozygous (−/−) mice when RT-PCR was performed using TET-specific primers as described in Materials and Methods. A 292-bp fragment was amplified in all samples using mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) primers. Normal mouse muscle tissue served as a positive control, and an RT reaction in which no reverse transcriptase enzyme was added served as a negative control. (B) Northern blot analysis of total RNA isolated from lung, spleen, and muscle tissue extracted from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. (C) Northern blot of total RNA isolated from primary osteoblasts or muscle cells established from wild-type (+/+) and homozygous (−/−) mice. The 1-kb tetranectin transcript is observed in tissues and cell cultures from wild-type (+/+) and heterozygous (+/−) mice but not in homozygous (−/−) samples. (D) Western blot analysis of duplicate samples revealed the 27-kDa TET monomer in serum from wild-type (+/+) and heterozygous (+/−) mice, but no TET protein was detected in samples derived from homozygous (−/−) mice using a polyclonal antiserum to mouse TET.

FIG. 3

Growth curves of tetranectin knockout mice. The weights of wild-type (+/+) and homozygous (−/−) female and male mice were recorded every week for 17 weeks. Wild-type females are indicated by inverted solid triangles, homozygous females are indicated by solid circles, wild-type males are indicated by open inverted triangles, and homozygous males are indicated by open circles. The slight difference in weight between wild-type and tetranectin-deficient mice is not statistically significant.

FIG. 4

X-ray analysis of the kyphotic spine of the tetranectin knockout mice. Radiographs of a wild-type (+/+) 6-month-old mouse (A), a homozygous (−/−) 6-month-old mouse (B), a wild-type (+/+) 12-month-old mouse (C), and a homozygous (−/−) 12-month-old mouse (D) are shown. Note the pronounced cervical lordosis and the thoracic kyphosis in tetranectin-null mice in panels B and D.

FIG. 5

Histological analysis of the tetranectin knockout mice. Sagittal sections of spines from control (+/+) and tetranectin-deficient (−/−) mice are shown. All sections are in the same orientation and as indicated in panel A. Control mice are demonstrated in panels A and C, and tetranectin-deficient mice are shown in panels B and D to H. Note that the increased thoracic curvature in the tetranectin-deficient mice is associated with a thickening and broadening of the vertebral bodies (asterisk in panel B). In the tetranectin-deficient mice, intervertebral disk material is asymmetrical in shape: expanded and loose in structure at the convex side (arrows in panels B and D) and narrowed and compressed at the concave side. Panels E to H demonstrate various degrees of irregular growth plates associated with protrusions of vertebral disk material into the cavum subarachnoidale (E and F) or into the vertebral bodies (G and H) (arrows). Mice shown in panels A to D are 6 months old, and those in panels E to H are 12 months old. M, medulla spinalis; C, cavum subarachnoidale. Bars, 300 (A and B), 85 (C and D), and 115 (E to H) μm.