FIG. 5.

Helios is expressed by a specific neuronal precursor downstream of Gsx2 and Dlx1/2 during LGE development. Immunohistochemistry and in situ hybridization analysis of Helios expression was performed in several knockout animals for transcription factors involved in striatal projection neurons development. Gsx2 knock-out mice (Gsx2^EGFP/EGFP) show a complete lack of Helios expression (A). In contrast, normal Helios protein expression was detected in Ascl1^–/– mice (B). In situ hybridization analysis for Helios mRNA in wt and Dlx-1/2^–/– mice show that the expression of this transcription factor is lost in the LGE but conserved in cortical areas at E18.5 (C). Double immunohistochemistry for Helios and eGFP performed in recombined Dlx-5/6-Cre-IRES-GFP/gtROSA (Dlx5/6-GFP) mice shows that Helios does not colocalize with eGFP positive cells at E18.5 (D). In addition, Helios immunohistochemistry performed in wt and Ebf-1^–/– mice at E18.5, shows that its expression is detected in Ebf-1^–/– mice although its distribution changes due to the LGE alterations described in these mice (E). Similarly, Helios protein expression is also preserved in Ikaros^–/– mice (IK^–/–) at E18.5 (F). Over-expression of Gsx2 (G) or Dlx2 (H) in neurosphere cultures does not affect Helios expression levels 3 days after transduction or transfection, suggesting that this transcription factor is not directly regulated by Gsx2 or Dlx2. Results are expressed as the mean of 4 to 5 neurosphere cultures and error bars represent the s.e.m. Statistical analysis was calculated by Student's t-test; ***p<0.001 relative to neurospheres transduced or transfected with the respective control vectors. Scale bars: (A, B) 500 μm; (D) 150 μm; (E) and (F) 500 μm.