Fig. 7. Id2 transcription is upregulated in Ovol1-deficient germ cells and is repressed by Ovol1 protein in vitro. (A) Increased levels of Id2 mRNA in P16 Ovol1^-/- testis. (B) In situ hybridization of P16 wild-type (A',C') and Ovol1^-/- (B',D') testis using an Id2 cRNA probe. (C' and D') High magnification images of individual tubules showing the strongest Id2 expression. Arrowheads and arrows indicate the Id2-expressing primary spermatocytes and the non-expressing spermatogonia, respectively. (C) Temporal expression of Id2 during normal prepubertal testis development. (D) Repression of Id2 promoter (Id2-P)-luciferase reporter expression by Ovol1. (E) Activation of Id2-P-luciferase expression by VP16-Ovol1. The triangles indicate increasing concentrations of expression vectors. The VP16 alone control is at a concentration corresponding to the highest one used for VP16-Ovol1. (F) Repression or activation is partially dependent on the CCGTTA sequence in Id2 promoter. Each bar represents the average of triplicate samples in a single experiment, and results are representative of several independent experiments. Luciferase activities are normalized for transfection efficiency by using a ß-actin promoter driving lacZ as an internal control.