Fig. 3.

Overexpression of miR-27b in somite explant cultures and transgenic embryos reduces Pax3 protein levels and reduces the ratio of proliferating (Pax3⁺) to differentiating cells in the limb bud. (A) Newly formed somites in explant culture were transduced with lentivirus expressing GFP only (GFP) or GFP and miR-27b (GFP-miR-27b). After 3 days in culture, immunohistochemistry with Pax3 or Pax7 antibodies (red) indicates down-regulation of Pax3 for somites transduced with GFP-miR-27b, but not GFP only. Pax7, not a target of miR-27b, was not affected. (B) Immunohistochemistry with a Pax3 antibody (red), a Lbx1 antibody (green), and the two merged on serial sections through the complete forelimb bud of tg[miR-27b] embryos at E10.5 indicates a marked reduction in Pax3⁺ cells migrating to the limbs of transgenic, compared with wild-type embryos. (C) An example of Pax3⁺ cells per serial section spanning the limb bud is shown for control and transgenic embryos. (D) Immunohistochemistry with Pax3 (red) and Myogenin (green) antibodies on serial sections through the forelimb buds and interlimb somites of tg[miR-27b] and wild-type embryos at E11.5 shows a decrease in the ratio of Pax3/Myogenin (MyoG)-positive cells when miR-27b is overexpressed. Blue marks DAPI staining of nuclei. (E) Summary of results illustrated in D. Reduction in Pax3⁺ cells corresponds to an increase in myogenic differentiation, as indicated by the ratio of Pax3/MyoG expression.