For some such syndromes, such as EDS kyphoscoliosis type and S?cklers, the underlying defects are understood, at least for some subsets of cases. For other ...
DELETION OF Mds1 GENE IN MOUSE RESULTS IN DISK DEGENRATION AND KYPHOSIS THROUGH MIS-‐REGULATION OF MATRIX PROTEIN SYNTHESIS BY TENDON/LIGAMENT CELLS
Subhash C. Juneja1, Lianping Xing1, C. Zeiss2, K. Lezon-‐Geyda2, DG. Reynolds1, Z Yao1, S Lin2, G Steele-‐Perkins2, T Ardito2, Jian-‐ping.Zhang2, W Philbrick2, Hani A. Awad1, Brendan F. Boyce1, Archibald S. Perkins1, University of Rochester1, Rochester, NY and Yale University2, New Haven, CT USA INTRODUCTION DATA and RESULTS The spine experiences mechanical stress constantly. Ligaments and tendons, composed of densely packed and
crosslinked type I, Collagen, as well as paraspinal muscles, provide structural stability and prevent misalignment of vertebrae. Inborn errors in the forma>on of these structures can lead to spinal deformi>es, such as kyphosis, lordosis, and scoliosis. For some such syndromes, such as EDS kyphoscoliosis type and S>cklers, the underlying defects are understood, at least for some subsets of cases. For other subtypes and for other congenital spine syndromes, such as Scheuermann’s juvenile kyphosis, the underlying cause of the deformity is unknown. The genes thus far found to be mutated in syndromes with spinal deformi>es primarily encode for collagens (e.g., collagen type Iα in S>cklers syndrome) or enzymes involved in collagen processing, e.g. LOH in EDS VIA, but at least one regulatory protein has been implicated – ZF transcrip>on factor 452 in cases of EDS VIB. The spine is formed from somi>c structures under the regulatory influence of notochord-‐ and neural tube-‐secreted factors. Vertebrae form through condensa>on of sclerotome-‐derived cells the forma>on of which is ini>ated by notochord-‐derived Shh, with Shh-‐induced Pax1 playing a cri>cal role. While the notochord contributes to the forma>on of the nucleus pulposus, the intervertebral AF is thought to be derived from the ventromedial sclerotome, while intervertebral ligaments and tendons are derived from the adjacent syndetome. Paraspinal muscles derive from the myotome. Recent studies have shown that the syndetome forms under the influence of adjacent myotome and sclerotome structures, and can give rise to both bone and ligament/tendon structures, and expresses SCX as a specific marker for cells in this lineage. The combined Mds1-‐Evi1 locus is a large (>500 kb) locus with established roles in myeloid leukemogenesis and hematopoiesis. The locus harbors two dis>nct transcrip>on start sites located ~450 kb apart. Via these and alterna>ve splicing, the locus produces at least four different nuclear proteins: MDS1-‐EVI1 and three EVI1 isoforms (p135, p123, and p103). These proteins all possess C2H2-‐type ZFs that bind DNA in a sequence-‐specific manner. Among the gene products of Mds-‐Evi1, Mds1 is dis>nct in that it has an N-‐terminal PR domain that shares homology with the SET domain, which is known in some proteins to have histone methyltransferase ac>vity and to play a role in the establishment and maintenance of gene expression pajerns during development. In an effort to define the role of MDS1-‐EVI1 in leukemogenesis and blood cell development, we created a targeted muta>on at Mds1 in mouse, by inser>ng a promoterless lacZ gene into the first exon of Mds1. To our surprise, the major phenotype of Mds1-‐/-‐ mice is kyphosis at young age, which is followed by spine deformity, revealing an unexpected role of the Mds1 transcrip>on factor in regula>ng the forma>on and or maintenance of the spine and its support structures. The Mds1-‐/-‐ mouse provides an instance of muta>on in a regulatory protein leading to kyphoscoliosis. As such, the Mds1-‐/-‐ mouse represents a unique gene>c model of congenital kyphosis. Mds1 is expressed in early stages of development of ligament and tendon cells; and remarkably reduced expression levels of SCX and extracellular matrix genes in Mds1-‐/-‐ tendon cells imply a defect in cells of this lineage. These findings predict the existence of muta>ons in the Mds1 gene that are causally involved in human congenital spine deformity syndromes
Figure 3. Severe spine degenera>ve changes associated with kyphoscoliosis in Mds1-‐/-‐ mouse. X-‐ray analysis of WT and Mds1-‐/-‐ mice at 2, 3, and 10 weeks age as indicated. Narrowing of joint spaces between L4 and L5 is indicated by arrow (anterior) and arrowhead (posterior) (A and B). Blue arrows indicate lumbar lordosis, white arrows-‐ thoracic kyphosis, and red arrow-‐ indicate a dorsally-‐ posi>oned tail. Beetle-‐fed cleared skeleton (g-‐r). At 7 weeks (g-‐j), Mds1-‐/-‐ mice display narrowed IVD spaces, rostrally elongated lateral processes, distorted ar>cular processes and short fused dorsal vertebral processes. By 9 months in lumbar (k-‐n), mutant vertebrae are fused with prominent epiphyses, lateral processes, and ar>cular processes and dorsal processes are fused in lordosis. At 9 months in the sacrum (o-‐r), Mds1-‐/-‐ show scoliosis with an unstable and hypertrophic lumbosacral joint. In all panels (g-‐r), caudal is to the right.
EXPERIMENTAL DESIGN
Figure 6. Pajern of β-‐gal staining: A. Whole-‐mount staining of Mds1+/-‐ embryos with X-‐gal at e8.5 to e14.5 days as indicated. At e8.5, staining is primarily is in the anterior heart field; at e9.5, in the anterior heart field, sclerotome and forelimb bud. At e11.5, staining is primarily in the skeletal structures and the mesenchyme of the limb; at e12.5 and e14.5, staining becomes restricted in vertebral bodies, rib and limb areas. Note: Degree of magnificaGon is arbitrary, so as to allow visualizaGon of stained structures. B. Sec>on through a β-‐gal-‐stained e12.5 Mds1+/-‐ embryo showing staining within the sclerotome; right panel provides a schema>c of the somite anatomy for reference. C. β-‐gal-‐stained spine sec>on from e14 Mds1+/-‐ embryo, showing staining within the car>laginous anlage of the vertebral bodies. D. Sec>ons from P1 pup, showing strong staining in ligament cells linking two intervertebral processes (arrowheads) and tendon like cells (arrows). 1=car>lage; 2=spinal cord; 3=bone marrow; 4=muscles.
Figure 8. Examination of tendon phenotypes in Mds1-/- mice. A. Ultrastructural analysis of collagen fibrils in sacral ligament at 3 months; B. in tail tendons at 3 months; and in IVD at one week (C). Representative TEM show reduced diameter but increased number of collagen fibrils in Mds1-/- mouse. Three equal areas were scanned. pme PCR analysis. Values are the mean ±SD of 3-‐4 mice.
Abundance and modifica>on of collagen in tendons appears normal in Mutant mouse. The decreased strength
and abnormal ultrastructure of Mds1-‐/-‐ tendons suggested a primary defect in collagen or their post-‐transla>onal processing.
Figure 1. Len: A knockout construct was generated by a standard molecular cloning, and contains lacZ within exon 1 of the Mds1 gene, and deletes out the splice donor. Downstream of the lacZ is a human growth hormone polyadenyla>on site, followed by a PGKneo-‐hGHpA casseje. Right: A representa>ve adult WT (above) and its lijermate Mds1-‐/-‐ mouse, as indicated, showing lordosis and kyphosis, note also dorsiflexed tail in Mds1-‐/-‐ mouse.
Figure 2. Growth curves for Mds1-‐/-‐ (n=7) and WT mice (n=20) for 0-‐30 days (A) and 0-‐50 weeks (B) show markedly decreased growth and smaller adult size in mutant mice (pnguishable from WT mice.
Thus, protein extracts from tendons were analyzed by SDS-‐PAGE and coomassie blue staining, which revealed no difference in the abundance and sizes of the major collagen proteins (Figure A below). The prominence of β dimers and lack of any mobility shin in collagen α chains indicates there is no obvious post-‐transla>onal or cross-‐linking differences between these two genotypes. In addi>on, collagen α1(I) and α2(I) chains from tendon and bone (and α1(II) from car>lage) were gel purified and submijed for mass spectroscopy to screen for changes in 3-‐hydroxyproline and lysine hydroxyla>on. The analyses revealed no effect on these post-‐transla>onal modifica>ons by the muta>on in Mds1 (data not shown). To further assess the modifica>on of collagen, skin collagen samples were analyzed for evidence of LOH-‐mediated lysine deamina>on, the first step in lysine modifica>on that leads to collagen crosslinking. The product of lysine deamina>on is allysine, which can be quan>tated by further oxida>on to 2-‐aminoadipic acid or reduc>on to norleucine; crosslinked lysine residues will be resistant to these modifica>ons. A defect in lysyl oxidase would result in lower levels of allysine, and hence lower levels of 6-‐OH norleucine and 2-‐aminoadipic acid per mol lysine. As shown in Figure B, the levels of 6-‐OH norleucine and 2-‐aminoadipic acid in skin collagen were no different between wildtype and Mds1-‐/-‐ mice.
Figure 4. Histologic analysis. H&E-‐stained sec>ons from 6-‐week-‐old mice show the range of spinal abnormali>es in mutant mice. Low mag pictures from a mutant mouse with severe spine phenotype show marked lordosis of the lumbar spine (A) and kyphosis of the thoracic spine (B). Exostoses with compression of the spinal cord are noted at both loca>ons (arrows). Bar=500μm; High mag pictures illustrate various severi>es of disc changes in the lumbar spine. Compared to WT (C), mutant animals show a progression of disc abnormali>es that range from rela>vely normal disc morphology (D), narrowed intervertebral space, reduced nucleus pulposus and loss of car>lage (arrow, E), and fusion of vertebrae (arrows, F). Bar = 50 μm.
Figure 5. Caudal vertebrae (C2-‐C3-‐C4) in Mds1-‐/-‐ show par>al fusion with collapsing IVDs at the post-‐sacral-‐tail angle and spine show scoliosis at lumbar-‐sacral region.
Figure 7. μCT analysis of individual vertebrae (Above): Individual vertebral bodies (T10-‐L5) from 3 months old mice were subjected to μCT analysis; N=3; ±SD; Pes progress with aging and by 8 weeks to 10 weeks (shown here), their thoracic column becomes severely kypho>c, which is associated with IVD abnormali>es, vertebral fusion, bone loss and reduced vertebral biomechanical strength. Interes>ngly, long bones of Mds1-‐/-‐ mice were not affected.
Genera>on Strategy and Genera>on of Mds1 mutant mouse
Figure 12. Lack of endochondral bone defect: Ihh and Pthrp expression remained same in WT and KO mouse. Micrographs of darkfield illumina>on of lumbar spine show pajern of expression of Ihh and Pthrp mRNA by ISH. Sec>ons were hybridized with the probe indicated to the right; photographs taken at the magnifica>on as indicated; genotype is indicated at top
Figure 10B. Analysis of skin collagen samples for allysine, by conversion to either 6-‐OH norleucine via reducTon or 2-‐ aminoadipic acid via oxidaTon. Samples with the genotypes indicated were analyzed.
• LacZ staining of >ssues from Mds1+/-‐ mice showed the expression of Mds1 gene product in the sclerotome and developing limbs at e9.5 embryos and within the car>laginous anlagen of developing vertebral bodies and their processes at e14.5 embryos in newborns, LacZ expressing cells were seen in the tendinous layer surrounding vertebral bodies and anchoring the intercostal muscles to the ribs and in ligaments between vertebral processes. Ultrastructural analyses of sacral ligament and tail tendon of Mds1-‐/-‐ mice showed smaller diameters and the IVDs of Mds1-‐/-‐ mice revealed marked disarray of the collagen fibrils In tail tendon cell cultures from Mds1-‐/-‐, there was a reduced expression of tendon specific and ECM-‐specifc genes as compared to their WT counterparts, i.e., Scx, Tnmd, Dcn,Comp, Bgn and Fmod, whereas Timp3 and Lox were not affected by Mds1 null muta>on • In Conclusion, Mds1-‐/-‐ mice develop progressive IVDD, kyphosis associated with dysfunc>onal tendon and ligament cells. Thus this study showed that the transcrip>on factor, Mds1, not only regulates hematopoiesis as shown earlier, but is expressed during early tendon ligament cells and its homozygous deficiency causes impairment of tendon and ligament fucn>on which, in turn, causes reduc>on in their strength, leading to destabiliza>on of the spine.
