A novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation

Signalling by the TGF-beta superfamily member and BMP orthologue Decapentaplegic (Dpp) is crucial for multiple developmental programmes and has to be tightly regulated. Here, we demonstrate that the Drosophila Dpp pathway is negatively regulated by eukaryotic translation initiation factor 4A (eIF4A), which mediates activation-dependent degradation of the Dpp signalling components Mad and Medea. eIF4A mutants exhibit increased Dpp signalling and accumulation of Mad and phospho-Mad. Overexpression of eIF4A decreases Dpp signalling and causes loss of Mad and phospho-Mad. Furthermore, eIF4A physically associates with Mad and Medea, and promotes their degradation following activation of Dpp signalling in a translation-independent manner. Finally, we show that eIF4A acts synergistically with, but independently of, the ubiquitin ligase DSmurf, indicating that a dual system controls SMAD degradation. Thus, in addition to being an obligatory component of the cap-dependent translation initiation complex, eIF4A has a novel function as a specific inhibitor of Dpp signalling that mediates the degradation of SMAD homologues.     

Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription

Brinker is a nuclear protein that antagonizes Dpp signalling in Drosophila. Its expression is negatively regulated by Dpp. Here, we show that Brinker represses Ultrabithorax (Ubx) in the embryonic midgut, a HOX gene that activates, and responds to, the localized expression of Dpp during endoderm induction. We find that the functional target for Brinker repression coincides with the Dpp response sequence in the Ubx midgut enhancer, namely a tandem of binding sites for the Dpp effector Mad. We show that Brinker efficiently competes with Mad in vitro, preventing the latter from binding to these sites. Brinker also competes with activated Mad in vivo, blocking the stimulation of the Ubx enhancer in response to simultaneous Dpp signalling. These results indicate how Brinker acts as a dominant repressor of Dpp target genes, and explain why Brinker is a potent antagonist of Dpp.     

Repression of dpp targets by binding of brinker to mad sites

Signaling by decapentaplegic (Dpp), a Drosophila member of the transforming growth factor (TGF) beta superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negative regulation of brinker (brk). Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity was localized to an amino-terminal region containing a putative homeodomain. Brk bound to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for the Smad protein, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vg quadrant enhancer to repression by Brk. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.