| |
Fig. 2. gli2 participates in the early induction of the NC. (A–E, H–M) Dorsal views of Xenopus laevis embryos at mid-neurula stage; anterior side is on the left. Arrowheads indicate the injected side, shown in turquoise and by GFP fluorescence in A–E. The expression of gene markers is shown in purple. (A–F) Analysis of gli2MO efficiency in vivo and in vitro. (A–E) in vivo assay. Merged fluorescence and clear field images of each embryo are shown as insets. (A) Normal GFP fluorescence was detected in the embryo coinjected with gli2GFP mRNA (2.6 ng/E) and CoMO (27 ng/E). (B–E) Embryos injected with gli2GFP and increasing concentrations of gli2MO (2.5 to 27 ng/E) evidenced a reduction in GFP fluorescence intensity. (F) in vitro analysis by Western blot shows a marked reduction in the expression of gli2GFP. α-tubulin expression was used as loading control. (G) Schematic representation of the gain- and loss-of-function experiments. (H–J) Analysis of gli2 requirement on NC induction. gli2MO-injected embryos show a reduced expression of the NC induction markers pax3, msx1 and snail1. (K–M) gli2 mRNA-injected embryos show an increase in the expression of the NPB markers pax3, msx1 and snail1. (N–O) Quantification of phenotypes shown in H–J and K–M, respectively. (P–R) The specific inhibition of Gli2 in NC explants incubated from stage 12.5 to 16 with GANT61 diminishes the expression of the NC marker foxd3. |
