Establishment of Motor Neuron-V3 Interneuron Progenitor Domain Boundary in Ventral Spinal Cord Requires Groucho-Mediated Transcriptional Corepression
Figure 3
Lack of effect of AES overexpression on ventral spinal cord Pax6+ and Nkx2.2+ progenitor populations.
(A) Schematic comparison of the structure of AES to that of full-length TLE. AES lacks the WDR domain involved in Nkx2.2 binding but retains the amino-terminal Q domain [8]. (B and C) Western blotting analysis of lysates from chick embryo spinal cords electroporated with plasmids encoding GFP together with FLAG epitope-tagged AES using either anti-FLAG (B) or anti-AES (C) antibodies. (B) “n.s.” indicates a non-specific band detected by the anti-FLAG antibody. (C) Exogenous AES was dramatically overexpressed in electroporated spinal cords. (D) Quantification of the number of GFP+ cells expressing Nkx2.2 or Pax6 in chick embryos electroporated with GFP alone (Control) or together with AES (AES). AES had no significant effect on the number of either Nkx2.2+ or Pax6+ progenitor cells. (E) Coimmunoprecipitation experiments performed using lysates from chick embryo spinal cords electroporated with plasmids encoding either Myc-tagged TLE4 or FLAG-tagged AES, as indicated. TLE4 or AES were immunoprecipitated (IP) using anti-Myc or anti-FLAG antibodies, respectively, followed by Western blotting (WB) analysis of input lysate (10%) and immunoprecipitated material using an anti-TLE1 antibody that does not cross-react with TLE4 or a panTLE antibody that recognizes all full-length TLE proteins because it is directed against the WDR domain [19]. Endogenous TLE1 coimmunoprecipitated efficiently with exogenous TLE4. In contrast, only a modest coimmunoprecipitation of AES with endogenous TLE was detected.