Figure 1.

A canonical BMP signaling pathway acts in neurons. (A) Schematic representation of the canonical fly BMP signaling pathway and binding of pMad and Medea to cis-regulatory BMP-AE sequences. (B) Schematic of the BiFC method. Mad and Medea are fused to two split non-fluorescent fragments of Venus YFP. Upon co-expression of both constructs, direct interaction between VN::Mad and VC::Medea allows for reconstitution of fluorescence. (C–E”) Bimolecular Venus YFP fluorescence is specific to nuclei with BMP activity in L3 VNCs, as revealed by overlap with immunoreactivity to pMad in control genotypes (C–D”), and its absence in wit mutants (E–E”). (F–K”) β-galactosidase expression of the dad enhancer trap dad^j1E4 (F–G”) and the dad13-nlsLacZ reporter construct (H–K”), shown with pMad co-immunostaining in controls (F,H-I”) and wit mutants (G,J–K”). (I–I”, K–K”) Close up of immunoreactivity at the dorsal midline taken from the region of the square dotted box in H and J, respectively. The mean±SEM number of nuclei per VNC that express the reporter is indicated at the bottom of each images. Reporter activity of both dad^j1E4and dad13-nlsLacZ was significantly reduced in wit mutants as dad^j1E4: P = 0.0022 and dad13-nlsLacZ: P = 0.0012 (two-sided Wilcoxon rank-sum test). At least 5 VNCs were analyzed for each genotype. Genotypes: (C, D): pUbi-VC-Medea/+; pUbi-VN-Mad, wit^A12/+. (E): pUbi-VC-Medea/+; pUbi-VN-Mad, wit^A12/wit^B11. (F) dad^j1E4, wit^A12/+. (G) dad^j1E4, wit^A12/wit^B11. (H, I) dad13-nlsLacZ/+; wit^B11/+. (J, K) dad13-nlsLacZ/+; wit^A12/wit^B11.