Excessive Baboon signaling perturbs wing development in a Mad-dependent manner.

<p><i>vestigial-</i>GAL4 (<i>vg</i>) was used to express combinations of Baboon, dSmad2, and RNAi for <i>mad</i> and <i>dSmad2.</i> Normal wing development (A; one copy of <i>vg</i>-GAL4) was disrupted by Babo* expression (B, C; Babo*mod and Babo*strong are UAS insertions with varying activity as characterized in other assays). The Babo* phenotype was abrogated by simultaneous <i>mad</i> RNAi (H) and resembled <i>mad</i> RNAi alone (E). In contrast, the crumpling defect of Babo* wings was enhanced in conjunction with <i>dSmad2</i> RNAi (I) and was more severe than <i>dSmad2</i> RNAi alone (F). Overexpression of FLAG-dSmad2 did not affect wing formation (D), but partially rescued the Babo* overexpression phenotype, producing normal sized flat wings with residual peripheral vein defects (G). For each genotype, a representative wing is shown out of 4–7 wings photographed. Within a genotype there was only slight variation in appearance, except that 3/6 dSmad2 RNAi wings had vein defects and a blister (shown) and 3/6 had vein defects without a blister (not shown).</p

Stimulation of the Baboon receptor leads to phosphorylation of both R-Smads independently of BMP Type I receptors.

<p>S2 cells were transiently transfected with FLAG-tagged Smad expression constructs and analyzed by Western blot for C-terminal phosphorylation (P-dSmad2 and P-Mad). The FLAG-Mad band is shown as a loading control (FLAG-dSmad2 is not a useful loading control because of signaling-induced degradation as described later in the text). For all Western blot figures, a thin horizontal line indicates different infrared channels from the same blot. Co-expression of a constitutively active form of Baboon (Babo*) led to phosphorylation of dSmad2 and Mad (A, left blot). Exposure of cells expressing endogenous Baboon to the Dawdle ligand (Daw) had the same effect (A, right blot). RNAi treatment was used to determine receptor requirement for ligand activity (B). Controls confirmed that Dpp ligand treatment caused Mad phosphorylation independently of Baboon (B, left half). Dpp activity required the Punt Type II receptor and the Tkv and Sax BMP Type I receptors (B, right half). In contrast, Daw signaling to Mad required Baboon and Punt, but not Tkv and Sax.</p

P-Mad elevation in <i>dSmad2</i> null mutant tissues depends on <i>baboon</i>.

<p>The schematic depicts the location of the l(X)G0348 <i>P</i> element insertion in relation to the <i>dSmad2</i> locus (A). The F4 excision product removed the entire coding region of <i>dSmad2</i> and portions of the <i>P</i>-element. The genomic breakpoints are indicated above the <i>dSmad2</i> mRNA; they were determined by sequencing PCR products, indicated by dotted lines. (B–D) P-Mad was detected by IHC of fixed larval tissues from several genotypes. For each image, a merged DAPI (blue) and P-Mad (red) panel is displayed above the isolated P-Mad channel. (B) Single confocal sections of P-Mad staining in the fat body. Under the staining conditions employed, endogenous nuclear P-Mad in a control fat body was barely detected (B1), but was increased in a <i>dSmad2</i> null mutant animal (B2). <i>Baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants had normal P-Mad staining (B3,4). (C, D) P-Mad staining at two representative positions along the digestive tract. Images are Maximal Intensity Projections of 3 micron interval confocal sections through the entire sample. The P-Mad primary antibody was omitted from “No Ab Control” samples to convey any background staining and auto-fluorescence in the red channel. (C) In the gastric caeca near the proventriculus, <i>dSmad2</i> mutants showed elevated P-Mad (C3) compared to wildtype control males (C2). <i>baboon</i> single mutants and <i>dSmad2</i>; <i>baboon</i> double mutants showed wildtype levels (C4 and C5). Distal Malpighian tubule staining (lumpy tubes marked with asterisks) showed the same pattern, with the <i>dSmad2</i> mutant displaying the strongest P-Mad staining (D3 compared to D2, D4 and D5).</p

Baboon phosphorylation of Mad is inhibited by dSmad2.

<p>(A) Phospho-Smad accumulation upon exposure to Daw ligand. S2 cells transfected with Flag-Mad were treated with dsRNA for <i>dSmad2</i> or GFP, and transfected with Flag-dSmad2 as indicated. Phospho-Smad and Flag signals were assayed on samples before Daw exposure and after 1 and 3 hours of incubation. Note that there is a gel artifact affecting the appearance of the Flag bands in several lanes. Quantified P-Mad band intensities are plotted to illustrate that accumulation of P-Mad depends on the level of dSmad2. The experiment was repeated with several batches of reporter cells, and the relative signals for the 3 hour time point were always observed in the same order. The 1 hour time points were near background detection and are less reliable due to noise. (B) Mutated dSmad2 proteins with varying phosphorylation efficiency modulate the rate of P-Mad accumulation. Babo* stimulation revealed the steady-state levels of P-dSmad2 and P-Mad. Daw exposure for 1 or 4 hours showed the difference in response to short-term signaling between the WT and HD forms of dSmad2. In both conditions, P-Mad levels were inversely correlated to P-dSmad2 levels. (C–K) Wing imaginal discs from third instar larvae were stained to detect P-Mad and imaged by confocal microscopy. For each condition at least three discs were imaged, and a representative Maximal Intensity Projection encompassing the wing blade is shown (6 sections @ 3 micron interval for C,D,F–H; all sections @ 3 micron interval for E; 5 sections @ 2 micron interval for I–K). Anterior is to the left and the scale bar shown in panel C applies to C–K. The normal P-Mad staining pattern is shown for <i>vg</i>-GAL4 alone (C; scale bar = 100 microns). Expression of a UAS-<i>dSmad2</i> RNAi construct did not alter the P-Mad pattern or the shape of the wing disc (D). Wing discs from <i>babo^fd4/fd4</i> homozygotes showed nearly normal pMad gradient (E). Expression of Babo* altered P-Mad staining, obliterating the normal gradient in the pouch (F). Note the normal P-Mad staining outside of the pouch where <i>vg</i>-Gal4 is not expressed. Babo* and <i>dSmad2</i> RNAi together generated ectopic P-Mad in the entire wing pouch (G). Providing Babo* with additional Punt also produced ectopic P-Mad (H). <i>tkv</i> RNAi prevented P-Mad accumulation in the middle of the wing pouch (I), and addition of Babo* did not counteract this P-Mad pattern (J). Additional knockdown of <i>dSmad2</i> led to ectopic P-Mad (K), which paralleled the results without <i>tkv</i> RNAi. Data in panels C, D, F, and G were from the same experiment and were stained in parallel. Panel H is from a different experiment, but the pouch signals can be compared to the others because the endogenous P-Mad along the posterior margin has similar staining. Samples in panels I–K were stained and processed in parallel.</p

All isoforms of Baboon and mammalian Activin receptors can initiate cross-talk.

<p>(A) Overexpression of wildtype Babo_a or Babo_b in S2 cells led to phosphorylation of dSmad2 and Mad. In this experiment there was a detectable background level of P-dSmad2 well below the Babo-induced levels. Arrowhead indicates that the displayed image contains two portions of one blot. (B) Constitutively active versions of mammalian Activin receptors Alk4 and Alk7 (Alk4* and Alk7*), like Babo*, induce phosphorylation of <i>Drosophila</i> Mad in S2 cells.</p