Figure 1. COLXV-B deposition during primary myogenesis. A–I, Immunostaining of embryos at 8–10 somites (A–D) and 18 hpf (E–I) with COLXV-B antibodies (red) and anti-β-catenin (green). Dorsal views of anterior and posterior part of the same embryonic trunk show morphological changes of adaxial cells at 8–10 somite stage: elongated adaxial cells (A), cuboid adaxial cells (D, arrow), and interleafing stage (D, asterisk). A, COLXV-B is deposited by elongated adaxial cells. Arrowheads indicate somite boundaries. B, C, Orthogonal views of areas 1 and 2 as indicated in A. Dorsal views (E, G), cross sections (F, H), and lateral view (I) of anterior and posterior trunk of 18 hpf embryos. E, Arrowheads indicate vertical myosepta. COLX-B is deposited in the motor path and vertical myosepta. J–M, Immunostaining of embryos at 18 hpf (J, K) and 24 hpf (L, M) with COLXV-B antibodies (red) and F59 (green). Cross section at 24 hpf (M) shows the persistence of COLXV-B staining in the motor path when F59-positive slow fibers (green) have migrated to the myotome periphery. M, Arrowhead indicates COLXV-B staining in contact with MPs. N, Production, characterization of polyclonal antibodies against zebrafish COLXV-B. Left, Schematic representation of the domain structure of the zebrafish COLXV-B α1 chain and location of the NC10 (residues 255–270) epitope. Collagenous domains (green) are numbered from the C terminus. Black represents noncollagenous linker domains. NC10 domain contains a TSPN (thrombospondin N-terminal like domain) domain and the NC1 the restin domain. Right, Western blotting with anti-COLXV-B antibodies of conditioned HEK293 media transfected with zebrafish col15a1b full-length cDNA incubated with (+) or without (−) collagenase. Lane 1: no treatment; lanes 2, 3: incubated at 37°C without collagenase (lane 2) and with collagenase (lane 3). The COLXV-B α1 chain is detected as an immunoreactive band migrating >200 kDa in undigested samples incubated (lane 2) or not (lane 1) at 37°C. After collagenase digestion, the band corresponding to COLXV-B α1 chain disappears and a collagenase digestion product is observed at the expected size for NC10 domain, attesting to the specificity of the anti-COLXV-B antibodies. O–Q, COLXV-B coincides with chondroitin sulfate signal in the motor path (P, Q, arrowheads). Single confocal plane of immunofluorescence staining of 24 hpf embryo cross section with antibodies to COLXV-B (O, red) and C-6-S (P, green) and merge image (Q). Hoechst solution was used to reveal nuclei. SC, Spinal cord (F, H, M, dotted line); N, notochord; ant, anterior trunk; post, posterior trunk. For lateral and dorsal views, anterior is left.