Figure 2. Effect of TLE1 overexpression in the developing chick ventral spinal cord.

(A) Schematic representation of the TLE domain structure. Notice the Q domain involved in oligomerization and transcriptional repression and the WDR domain important for protein-protein interactions [8]. Nkx2.2 binds to the TLE WDR domain using an Eh-1 motif. (B) Western blotting analysis of lysates from chick embryo spinal cords electroporated with plasmids encoding GFP alone or together with FLAG epitope-tagged TLE1 demonstrating the expression of exogenous TLE1 using anti-FLAG antibody. “n.s.” indicates a non-specific band detected by this antibody. (C) Double-labeling analysis of the expression of GFP and the indicated proteins in embryos electroporated with GFP alone (control) or GFP+TLE1 (TLE1). Nkx2.2+ cells were observed in both the ventricular zone (VZ) and mantle zone (MZ). Arrows in the two right-hand columns point to examples of double-labeled cells coexpressing GFP and either Hb9 or Isl1. (D and E) Quantification of the numbers of electroporated cells (GFP+) expressing Nkx2.2 [in either the VZ (D) or the MZ (E)], Pax6, Nkx6.1, Hb9, or Isl1, as indicated. TLE1 overexpression caused an increase in the number of Nkx2.2+ cells in the VZ, with a concomitant decrease in Pax6+ cells. The number of cells expressing Nkx6.1 was not altered. These changes were associated with an increase in Nkx2.2+ cells in the MZ and a decrease in the number of electroporated cells expressing the MN markers Hb9 and Isl1. Data are expressed as mean ± SEM (*p<0.05). Scale bars = 50 µm.