ColVI expression in zebrafish at different developmental stages. (a) qRT-PCR for col6a1, col6a2, col6a3, col6a4a and col6a4b transcripts in zebrafish embryos and larvae from 12 hpf to 96 hpf. Data were normalized to arp. The expression levels of the different genes were compared to col6a1 expression at 24 hpf, which was arbitrarily set to 1, and represent the mean of at least three independent experiments. Error bars indicate s.e.m. (**P < 0.01). (b) Whole-mount in situ hybridization for col6a1 in zebrafish embryos and larvae. The top panels show lateral (lat) and dorsal (dors) views of 1- to 3-dpf embryos labeled with the col6a1 probe. The bottom panels show transverse sections at different head and trunk levels (i-iv, as indicated on the lateral view at the top) and higher magnifications of the tail region (v and vi) of 2-dpf embryos labeled with the col6a1 probe. At 1 dpf, labeling for col6a1 mRNA is restricted to two lines of slow muscle fibers and to the pectoral fin buds (orange arrowhead in the magnified area highlighted by the dotted box). At 2 and 3 dpf, strong signals for col6a1 transcript are detected in vertical myosepta, pectoral fins (orange arrowhead in the magnified areas highlighted by the dotted boxes), branchial arches, axial vasculature and gut. All panels were cropped from the background. (c) Western blot for α1(VI) chain in protein extracts from zebrafish embryos and larvae at the indicated stages. β-actin was used as a loading control. Numbers on the left indicate sizes (in kDa) of protein standard markers. The extra-band observed at 3 and 4 pdf may correspond to post-translational modifications. The cropped blots for α1(VI) chain and β-actin derive from different parts of the same gel. (d) Whole-mount immunofluorescence labeling with anti-ColVI antibody in zebrafish larvae at different developmental stages from 2 dpf to 10 dpf, as indicated. The panels show lateral (lat), ventral (ven) and dorsal (dors) views of different head and trunk regions. From 2 dpf onwards, ColVI immunoreactivity (red) is present in the connective tissue surrounding the eye, in the pectoral fins and in myosepta. At 3 dpf, ColVI deposition extends to craniofacial elements and intestine. From 6 dpf onwards, ColVI labeling is also detected in the connective tissue surrounding olfactory pits. Scale bar, 100 µm. ch, ceratohyal; ey, eye; fb, fin buds; m, myosepta; mc, Meckel’s cartilage; myoseptum, mys; no, notochord; op, olfactory pit; pf, pectoral fins; pq, palatoquadrate; sc, spinal cord; yo, yolk.
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