The similarity of the phenotypes of dSmad2 loss-of-function and Dpp/Mad gain-of-function in imaginal discs (Figure 2) and in adult wings (Figure 3) suggested that an antagonism exists between the dSmad2 and Mad signaling pathways during Drosophila wing differentiation.
C-terminal phosphorylation of Mad marking Dpp pathway activity becomes visible around 22 hrs after puparium formation (APF) in the crossveins, and soon after in the longitudinal veins [41].
In the Dpp pathway, Mad (Mothers against decapentaplegic) and Yki interact to form a transcription complex to activate their common targets [ 29].
If Medea were a rate-limiting factor shared by the Mad and dSmad2 pathways, the knockdown of dSmad2 would free Medea to form complexes predominantly with pMadcter, transducing a stronger Mad signal that manifests itself by extra vein formation.
An attractive candidate for this gene is moleskin (msk) which encodes Drosophila Importin-7, a protein involved in nuclear translocation that has been shown to regulate the TGF-β pathway by controlling Mad localization.
Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype.
The consensus sequences for a combination of signaling pathways such as E-twenty six (Ets) [NSYGGAWRY] [13] downstream of RTK pathway, Mothers against Dpp (Mad) [GCCGNCGC] [59] an activator of Dpp pathway, Cubitus interruptus (Ci) [TGGGWGGTC] [60] of Hh pathway, and Brinker (Brk) [TGGCGYY] [61], [62] a transcriptional repressor of Dpp and Wg pathways were used.
The expression of interference RNA against Mad, the transducer of the Dpp pathway, in the central region of the wing disc (sal EPv-Gal4 UAS-GFP/+; UAS-Mad-i) results in smaller than normal wings that fail to differentiate the veins L2, L3 and L4 (Fig. 6A,B).
To confirm that oridonin stabilizes RARα through the NF-κB pathway, we used NB4/GFP-MAD cells to perform further experiments.
