Figure 1.
dSmad2 RNAi induces vein tissue differentiation.
(A) Control wing showing the location of the longitudinal veins 2–5 (L2–L5); anterior crossvein (ACV); posterior crossvein (PCV). (B) dSmad2 RNAi driven by MS1096-Gal4 leads ectopic vein tissue formation in the vicinity of L5 (arrows). (C) A similar phenotype is observed in flies overexpressing Mad. (E) Overexpression of the dSmad2 homologue human Smad3 results in a partial loss of L4 and L5; an identical phenotype is seen in some Dpp partial loss-of-function mutants [48]. (F) human Smad3 rescues the dSmad2 RNAi phenotype (arrows), demonstrating dSmad2 RNAi specifically depletes dSmad2.
Figure 2.
Knockdown of dSmad2 resembles Dpp overexpression in the wing disc.
(A) Control imaginal wing disc at third instar stage, expressing Scalloped (Sd)-Gal4 throughout larval development of the wing. (B) Overexpression of Dpp causes overgrowth of the wing blade region of the disc. (C) dSmad2 RNAi phenocopies Dpp overexpression, expanding the wing blade region. (D) Conversely, downregulation of the Dpp pathway by Mad RNAi reduces the size of the wing disc.
Figure 3.
dSmad2 depletion leads to transformation of intervein into vein tissue.
(A) Wild-type wing retains normal size and vein patterning when grown at 29°C. A’ and A” insets show higher magnification of L3 and L5 regions to illustrate the size differences between vein and intervein cells, each of which have a single hair (trichome) (B) Driving dSmad2 RNAi with MS1096-Gal4 at 29°C results in small wings due to increase in vein tissue mostly near L3 (B’) and L5 (B”). (C) Dpp-overexpressing flies display a similar wing phenotype to dSmad2 RNAi, although stronger. High levels of Dpp/Mad lead to an overproduction of vein, resulting in wings of reduced size that are composed entirely of vein tissue (C’ and C”). Flies overexpressing Dpp in the wing only survive to adult stages when grown at low temperature (20°C).
Figure 4.
pMad is expressed in ectopic veins induced within dSmad2 RNAi clones.
(A) Pupal wing 25 hrs after puparium formation (APF) stained with pMadcter antibody. Nuclear pMad staining is visible in the crossveins and longitudinal veins, most prominently at the distal tips. (B and B’) dSmad2 RNAi clone marked with GFP induced an ectopic pMadcter expressing vein (arrowhead) posterior to L5 (arrow). (C–D’) Similar result shown in an independent experiment. Note the outgrowth of the wing surrounding the ectopic vein (arrowhead in D). (E) dSmad2 clone marked by yellow (y+) in anterior margin bristles induced the formation of an ectopic vein between the anterior margin and L2. (F) Ectopic vein induced by dSmad2 clone in the posterior compartment (arrow). (G) dSmad2 clone inducing ectopic vein (arrow in G’) and intervein tissue (arrowhead in G’).
Figure 5.
Medea is required for the effects of dSmad2 on vein formation.
(A and B) Medea RNAi causes marked reduction of veins in the distal part of the wing. (C) Simultaneous depletion of Medea and dSmad2 affects vein formation in a similar way as knockdown of Medea alone. Note that the wing blade is smaller. The remaining wing veins are numbered.
Figure 6.
(A and B) MS1096-Gal4 driving dSmad2 RNAi causes an increase of vein tissue in L5. (C) Depletion of Mad by RNAi results in small wings lacking veins. (D) Mad;dSmad2 double RNAi wings show identical phenotypes to Mad depletion alone – complete loss of veins and a small wing blade – showing that Mad is epistatic to dSmad2. (E) Wild type third instar wing imaginal disc. (F) Wing discs become enlarged when dSmad2 is depleted using RNAi driven by sd-Gal4. (G) Mad depletion reduces wing disc size. (H) Mad is required for the enlargement of wing discs caused by dSmad2 depletion, compare panels F and H.
Figure 7.
Hypothetical mechanisms for the opposing effects of dSmad2 on wing vein formation.
(A) The Dpp signal is transduced by the receptors Thickveins and Punt that activate Mad via C-terminal phosphorylation. A heterotrimeric complex consisting of pMad and Medea is formed. This complex is translocated into the nucleus to initiate target gene expression for vein formation and wing growth. The Drosophila Activin (dAct) signal is transduced by the receptors Baboon and Punt; dSmad2 is phosphorylated and associates with Medea forming a heterotrimer. In the present study, we describe an inhibitory effect of dSmad2 signaling on vein formation that is mediated by an as yet unknown molecular mechanism. (B) The competition for Medea model places Medea as the limiting factor between Mad and dSmad2 signaling pathways. The phosphorylated transcription factors compete for association with rate-limiting amounts of Medea. Thus, when the level of dSmad2 is experimentally lowered, the Dpp/Mad branch receives an excess of Medea with which it can form heterotrimeric complexes. This leads to an elevated Mad/Medea response, which is manifested in the formation of extra vein tissue. (C) The mixed pMad/pdSmad2 complexes model is based on the finding by Gesualdi and Haerry [17] that the type I receptor Baboon is able to phosphorylate C-terminal serines of both dSmad2 and Mad. Mixed complexes composed of pMad, pdSmad2 and Medea may be responsible for inhibiting Mad-mediated vein formation. In parallel, the canonical pdSmad2/Medea complex could contribute to wing growth [17] or be involved in other unknown processes.