Fig. 3. Ex vivo regulation of the MAP1B promoter by Foxa2 and Engrailed. (A) Primary cultures of mouse mes-metencephalic neurons were transfected with the MAP1B promoter fused to a luciferase reporter (pMAP-luc), or with a modified version of this promoter, in which a 43 bp fragment containing both the HF1 and HF2 sites was deleted (pMAPHF1+2-luc). The expression of the deleted promoter is about threefold that of the full length promoter, indicating that the 43 bp fragment including HF1 and HF2 has regulatory functions. The data are the results of pooling three independent experiments. (B) Expression levels of En1, En2 and Foxa2 mRNAs in E13 mes-metencephalic neurons or N2A cells, as determined by real-time RT-PCR. PCR was performed on cDNA derived from 2 ng of RNA (see Materials and Methods). Results are expressed as a percentage of GAPDH mRNA expression level (100% being the level of GAPDH mRNA expression in mes-metencephalic neurons). Significant amounts of En1, En2 and Foxa2 mRNAs were detected in mes-metencephalic cultures. Foxa2 mRNA low abundance is probably due to the fact that Foxa2 is expressed only by cells located in the ventral mes-metencephalon (the engrailed gene being, by contrast, expressed by most cells of the mes-metencephalon). In N2A cells, En2 mRNA level is extremely low and En1 and Foxa2 mRNAs are not detected (nd). (C) N2A cells were co-transfected with various concentrations of Foxa2- or En2-expressing plasmids, alone or in combination, together with the MAP1B-luciferase reporter plasmid (pMAP-luc). Control cells were transfected with empty pCL9m plasmid. Transfection of high concentration of En2-expressing plasmid activates the promoter. Transfection of low concentration of Foxa2-expressing plasmid has no effect by itself but partially antagonizes the En2-dependent activation of the MAP1B promoter. In a similar way, activation of MAP1B promoter by high concentration of Foxa2 plasmid is partially antagonized by a low dose of En2 plasmid. The data are the results of pooling three independent experiments.