Figure 7.

Over- and under-expression of miR-196 disrupts patterning of the axial skeleton. (A) Normal axial skeleton stained for cartilage (Alcian blue) and bone (Alizarin red) showing 4, 10, 15 and 3 vertebrae in the Weberian apparatus (region W), rib-bearing precaudal vertebra (region P), caudal vertebra (region C) and caudal fin vertebra (region T), respectively. (B, D) Over-expression of mir196 showing deletion of one Weberian vertebra and three ribs with their precaudal vertebrae. (C, D) Morpholino knockdown of miR-196 resulted in extra ribs and extra rib-bearing precaudal vertebrae. (D) Skeletal element counts after miR-196 manipulation showing reciprocal effects of over-expression and knockdown in the rostral axial skeleton. Asterisks indicate statistical significance compared to UIC (uninjected control) (Student T-test, p < 0.001). UIC, n=79; miR-196oe (over-expression), n=56; mir196-Mo (miR-196 knockdown), n=122. (E, F, I, J) Weberian apparatus sketch (after (Grande and Young, 2005)) and skeleton staining, lateral and dorsal views, respectively. (G, H, K, L) Over-expression of miR-196, lateral (G, H) and ventral (K, L) views of the Weberian apparatus from four different individuals. In wild-type fish, the Weberian apparatus has four highly modified vertebrae, each with bone and cartilage morphologies that specify their identity (E, F, I, J). Typically, the first vertebra (v1) has a pair of anterior-laterally projecting bones called the lateral process-1 (lp1) and dorsally, a small bone called intercalarium (in) that contacts with vertebra-2 (v2). V2 normally has a pair of laterally projecting bones (the lateral process-2, lp2) on each side. Lp2 is larger than lp1 (E, I). Vertebra-3 (v3) has a pair of lateral tripus (tr) bones, a triangular shaped bone, and neural arch-3 (na3) dorsally. Vertebra-4 (v4) has a pair of highly modified ribs (r4) protruding ventral-laterally that bears os suspensorium bones ventrally and dorsally, a neural arch-4, on top of which, supranueral-4 (sn4) usually fuses anteriorly with supranueral-3 (sn3) on v3. Precaudal vertebrae are defined by their parapophyses, which support ribs, and their lack of hemal arches and hemal spines, which are found on v15–v28 (Bird and Mabee, 2003). Treated animals are discussed in the text. Abbreviations: #1, #2, #3, vertebrae numbers from anterior without regard to identity; in, intercalarium; lp1, lp2, lateral processes; na3, na4, neural arches; os, os suspensorium; r4, r5, ribs; tr, tripus; v1, v2, v3, v4, v5, v7 vertebrae with identities.