Loss of function of Colgalt1 leads to defects in musculoskeletal structure. (A) Colgalt1^fosse/fosse embryos are smaller, rounded and swollen compared to unaffected littermates at E18.5. (B) Skeletal preparations of Colgalt1^fosse/fosse embryos at E18.5 clearly demonstrate a reduction in the size of the bones in the wrist and the rib cage. (C) Hematoxylin and Eosin (H&E)-stained sections through the proximal tibiae indicate that there is no gross disorganization in the growth plates of this skeletal element at E18.5. (D) H&E-stained sagittal sections through the forelimbs show disorganized, ragged and shorter muscle fibers in the bicep and brachioradialis compared to wild type. Right hand panels show magnified views of sections in left hand panels. Scale bars: 200 μm (C, 10×; D, left) and 100 μm (C 20×; D, right).

Collagen IV accumulates intracellularly and exhibits decreased stability and molecular mass in Colgalt1 mutant MEFs. Immunoblot detection of the α2 chain of type IV collagen in lysates (A) and conditioned media (B) from wild-type and Colgalt1^fosse/fosse MEF cultures. Ponceau S staining is shown as a loading control. Arrows indicate the band corresponding to full-length collagen IV. Graphs show densitometric quantitation of COL4A2 signal intensities normalized to Ponceau S. (C) Immunoblot detection of monomer and dimers of the NC1 domain of COL4A2 in collagenase digests of the ECM isolated from wild-type and fosse MEFs. The graph shows densitometric quantitation of the total (monomers and dimers) signal intensity from five different samples in each MEF group. Results are shown as mean (s.d.) and P-values ≤0.05 and ≤0.01 are indicated by one (*) or two (**) asterisks, respectively. (D) Immunofluorescent detection of type IV collagen in muscle tissue from wild-type and fosse mice. Magnification: 40×.

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Molecular mass of collagen VI is reduced upon loss of Colgalt1 function in MEFs. Immunoblot detection of the α1 chain of type VI collagen in lysates (A) and conditioned media (B) from wild-type and fosse MEF cultures. The double arrows indicate the difference in electrophoretic mobilities between the collagen VI molecule derived from wild-type and fosse MEF. Immunoblots were subjected to densitometric analyses with ImageJ using Ponceau S staining for normalization. (C) Immunofluorescent detection of type VI collagen in muscle tissue from wild-type and fosse mice. Magnification: 40×. (D) Phalloidin staining of wild-type and double-homozygous colgalt1 mutant zebrafish at day 9 post-fertilization reveals muscle disorganization.