Col25a1 expression is required for muscle development in mice. (A) Immunofluorescence analysis of myosin positive myofibers on transverse sections of forelimbs from wild-type (WT) and Col25a1−/− E14.5 embryos. Total nuclei were stained with Hoechst. Scale bar: 30 µm. edc: extensor digitorum communis; s: supinator; ecrb: extensor carpi radialis brevis; ecrl: extensor carpi radialis longus. (B) Quantitation of myosin positive myofibers on transverse sections of forelimbs from E14.5 WT and Col25a1−/− embryos. (C) Distribution of myosin positive myofiber cross-sectional areas (n = 3 animals of each genotype). *P < 0.05; **P < 0.01 vs WT. (D) Myosin immunofluorescence on forelimb longitudinal muscles from WT and Col25a1−/− E14.5 embryos (n = 3). Multinucleated myofibers were observed in WT sections. Col25a1−/− myofibers were myosin positive but shorter. Scale bar: 50 µm. Arrows indicate bulges. bbl: biceps brachii long head. (E) E12.5 forelimbs (n = 4) were immunostained with an antibody directed against the embryonic isoform of MyHC (emb-MyHC). Col25a1−/− myoblasts fusion seems to be delayed. High magnification of emb-MyHC staining (right) corresponds to area in white box from the left panel. Scale bar: 50 µm. Arrows indicate breaks between aligned myofibers.