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First published online 3 August 2006
doi: 10.1242/dev.02513

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Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.

View larger version (62K): [in a new window]   Fig. 1. Sax antagonizes Gbb function, whereas Tkv is required to mediate both Gbb and Dpp. (A,F) Overexpression of gbb at 25°C (A) or dpp at 18°C (F) in the wing disc produce wings divided into four classes by phenotypic severity. (B-E,G,H) Percentage of wings in each class produced by gbb or dpp overexpression alone (yellow) or in combination with changes in Tkv or Sax levels (red). (B) gbb overexpression wing phenotypes (yellow) are very strongly suppressed by overexpression of wild type sax (red), while (C) increasing Tkv levels in the presence of excess Gbb enhances the gbb overexpression phenotype. (D) Reducing the level of endogenous Sax strongly enhances gbb overexpression. (E) Reducing the level of endogenous Tkv results in a strong suppression. (G) Reducing the level of endogenous Sax mildly suppresses dpp overexpression wing phenotypes. (H) Reducing Tkv levels strongly suppresses dpp overexpression. (I) Ligand overexpression in different mutant backgrounds.

View larger version (79K): [in a new window]   Fig. 2. Loss of sax function expands pMad distribution in wing discs. Intensity plots (A,B,C,D) of the relative fluorescence (y-axis) of (A',B',C',D') pMad (red) across equivalent regions of the wing pouch (broad white line). (A) Wild type, (B) sax4/Df(2R)H23, (C) Df(2R)H23/+ and (D) gbb1/+. (A'',B'',C'',D'') brk expression (green) (brkX47-LacZ). Thin white lines mark the extent of brk repression from the center of wing pouch. All images were captured using the same microscope settings.

View larger version (67K): [in a new window]   Fig. 3. Ectopic veins produced by sax clones depend on gbb function. In a wild-type background (A-C), sax-null clones produce an ectopic vein, while in a gbb4/gbb4 mutant background (D) sax-null clones fail to produce vein duplications. Higher magnification of clones: A`-D', bright field; A"-D", dark field). sax clones are marked with shaIN. Elimination of trichomes on both surfaces of the wing (i.e. a double-sided clone) appears black in dark field. (A,B,D) Anterior border of each double-sided clone (white arrowhead), ectopic vein (black arrowhead). (C) Posterior border of clone (white arrowhead). (A) sax clone with anterior border between L2 and L3 produces an ectopic vein at the anterior border of the clone. (B) sax clone with its anterior border between L1 and L2 produces an ectopic vein anterior to L2. (C) A sax clone in the posterior compartment that generated an ectopic L5 outside the clone boundary. (D) sax clone in a genetic background reduced for gbb function is very similar in position and shape to that shown in B but it lacks an ectopic vein. (E-H) Schematics depicting effect of sax clone (white box) on graded pMad (purple) in the anterior compartment of wing discs. x-axis indicates distance from Bmp source near the AP boundary. y-axis indicates level of pMad or Bmp signaling activity. (E) Relative domains of kni, sal, omb and brk expression and the position of L2 primordia (thick blue line). (F-H) Interpretation of clone in A,B,D, respectively. (F,G) Loss of Sax results in a reduction in Bmp signaling (gray arrows) within the clone and an increase in Gbb (blue), which influences the level of Bmp signaling in surrounding cells (black arrows). (H) Increased availability of mutant Gbb protein (pale blue) is not sufficient to raise pMad levels to the point necessary for ectopic L2 specification.

View larger version (69K): [in a new window]   Fig. 4. Small sax clones reveal both signal promotion and antagonism functions. Wing pouch of third instar wing discs (dorsal is upwards; anterior is leftwards) stained for pMad (red), brk-lacZ (blue), GFP (green). AP boundary is indicated by vertical arrows. White lines indicate transect of fluorescence intensity profiles. (A) Wild type. (B-C''') sax-null clones (absence of GFP, B,C) result in reduced pMad levels (B,C'''), but increased pMad levels (asterisks) in cells adjacent to the clone (B'',C''', clone position indicated by the gray bar). (C,C') Ectopic brk expression occurs in some sax mutant cells. Pattern of ectopic brk expression highlights where contribution of Sax to signal promotion is highest and where Tkv-Tkv is unable to mediate sufficient signaling to repress brk.

View larger version (24K): [in a new window]   Fig. 5. Model of Bmp signaling and the effect of Sax modulation. (A) A model reconciling both the antagonistic and the signaling functions of Sax in wing disc cells. Dpp and Gbb have different binding preferences (indicated by the thickness of the arrows) for receptor complexes with different combinations of type I receptors. Tkv-Sax receptor complexes contribute more significantly to signaling (indicated by the thickness of the black arrow) than Tkv-Tkv, whereas Sax-Sax complexes fail to phosphorylate Mad. (B) tkv-null cells lack p-Mad. (C) sax-null clones with receptor complexes solely of the Tkv-Tkv type are not as efficient at signaling as wild-type cells. (D) Wild-type cells adjacent to sax-null cells exhibit a higher level of pMad than normal. The antagonistic function of Sax is preferentially directed at Gbb and the high affinity of Gbb for Sax ensures that in the absence of Sax, a region of the Gbb pool is available for signaling not only in the mutant cells via Tkv-Tkv but also in wild-type neighboring cells. (E) Gene expression domains for brk (green) and omb (orange) in the wing pouch of wild-type (top) and sax-null wing disc (bottom). Gbb derived from cells along the AP boundary (black vertical line) is required to repress brk expression in the lateral wing pouch of both compartments (Bangi and Wharton, 2006). Sax limits the range of Gbb by acting antagonistically on the ability of Gbb to signal; thus, in the absence of Sax, the range of Gbb signaling expands, repressing brk expression throughout the wing pouch.