Impaired Collagen X Assembly and Growth Plate Abnormality

<div><p>(A) Cell-free translation demonstrating normal assembly of wt α-chains into α1(X)₃ homotrimers, but impaired assembly of 13del α-chains into trimers. Heterotrimers, which would migrate slightly faster than the α1(X)₃ homotrimers, are absent.</p> <p>(B) Diagram of the 10.4-kb <i>Col10a1-13del</i> transgene. The NC1-encoding domain is indicated by the shaded region.</p> <p>(C) Ten-week-old wt and 13del mice.</p> <p>(D) Left: histology of the proximal tibial growth plates of 10-d-old (10d) and 4-wk-old (4wk) wt and 13del mice. Right: differences in the total height of the growth plate and in the heights of the RZ, PZ, and HZ between wt and 13del mice in each age group (10 d [<i>n</i> = 6] and 4 wk [<i>n</i> = 13]). Bar indicates 100 μm.</p> <p>(E) Retarded longitudinal bone growth in 13del mice. Calcein (10 mg/kg body weight) was injected intraperitoneally into 5-d-old mice that were sacrificed 5 d later. Sections of proximal tibia were examined in an incident-light fluorescence microscope. The distances at different positions between the zone of vascular invasion at the growth plate and the proximal endpoint of the calcein label in the trabecular bone were determined using a micrometric eyepiece. The mean distance represented the rate of longitudinal bone growth over 5 d. Error bars represent ±1 standard deviation (SD). Longitudinal growth was retarded in 13del mice over 5–10 d, being only about half that in wt littermates (0.498 ± 0.013 mm for wt and 0.277 ± 0.025 mm for 13del; <i>n</i> = 3 for each genotype; <i>p</i> = 5.2 × 10⁻⁶).</p> <p>(F) RNase protection assay. The protected fragments of [α-³³P]UTP-labeled probes when hybridized with wt <i>Col10a1</i> transcript are 354 bases; whereas hybridization with 13del transcripts gives two fragments of 201 bases and 140 bases (the unpaired 13 bases are digested by RNase).</p> <p>PS, primary spongiosa.</p>