Fig. 6

miR-214 ectopic expression downregulates chondrogenic markers and impairs cranial cartilage formation in zebrafish. (A) Expression of marker genes of chondrogenesis. Expression levels of sox9a, sox9b, sox10, col2a1a, col10a1a, runx2a and mgp were evaluated by standard qPCR of 3 dpf zebrafish embryos microinjected with MmiR-214 or NC (18 μM), and normalized using 18 S ribosomal RNA housekeeping gene (similar results were obtained using ef1α housekeeping gene; data not shown). Results are presented as fold change over NC. Asterisks indicate values statistically different from NC (data are the mean ± s.d. of at least 3 independent replicates; one-way Anova, ***p < 0.001; **p < 0.01, *p < 0.05). (B) Histological characterization of miR-214 overexpression on zebrafish cartilage formation. 3 dpf embryos microinjected with NC (a) or MmiR-214 (b) were embedded in paraffin, sectioned and stained with the safranin-O/fast green/Mayer’s haematoxylin. Zebrafish eyes (e) are generally underdeveloped in miR-214-injected embryos when compared to NC. Cartilage associated proteoglycans (evidenced by safranin-O) are either absent or present in low amounts in the Ethmoid plate (Ep) or hyosympletic (Hs), respectively, of miR-214-injected embryos comparing to NC. Sections are in the coronal plane; (‘) and (‘’) indicate magnifications of the same section.