Maintained hypertrophic cartilage formation and inhibition of bone formation during HIF induction. A. Fish exposed to CoCl2 after fin amputation; for 5-6 days, regenerated very fragile fins that fissure easily, leading to their partial or total loss, while they progress in water. B. Alizarin red-staining of fins exposed or not to CoCl2, shows staining of fin rays, including newly regenerated areas (black arrows). Fish exposure to CoCl2 induced loss of Alizarin red staining in regenerated fin areas. Note the inhibition of bone ossification behind the site of amputation in the presence of CoCl2. Original magnifications: 2-x objective for A, 10-x and 40-x objectives for B. C. Double staining of bone (Alizarin red) and cartilage (Alcian-blue) in regenerated fins exposed or not to CoCl2. Note that while exposure to CoCl2, induced loss of Alizarin red staining in the regenerated fin area, Alcian-blue staining pattern was well maintained throughout regenerated fin area. Original magnifications: 10-x and 40-x objective, respectively. D. Fold change in the expression of the hypertrophic marker collagen 10, as measured by real time PCR analysis of total RNA obtained from control and regenerated fins, exposed or not to CoCl2 (1mM). Note the highly induced expression of collagen 10 during regeneration (lanes 1 and 3) and its maintained high expression during hypoxia (lanes 2 and 4). Data shown represents Mean ± S.E (n=6 per group) from three independent experiments. ***P<0.0001.
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