DEV02513 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. β-Galactosidase reporter assay for BMP signaling activity in Drosophila S2 cells. As described by Muller et al. (Muller et al., 2003), a reporter construct expressing lacZ is controlled by the Su(H) response element and a brk silencer element. Transcription is activated by co-transfection of plasmids encoding Su(H) and an activated form of N*. Activation of BMP signaling by co-transfection of a plasmid encoding the activated form of Tkv (tkv-QD) leads to repression of reporter activity, owing to the presence of the brk silencer element, indicating that endogenous signaling components are expressed in S2 cells. The degree of repression depends on the quantity of tkv-QD plasmid added (Muller et al., 2003). β-Galactosidase values were measured using the dual luciferase assay system (Dual-Light, Applied Biosystems) and normalized by co-transfection of 5 ng of a plasmid expressing luciferase. Values depicted are the fold activation of β-galactosidase over the basal activity of the reporter construct alone and represent the average of samples measured in triplicate. One-hundred nanograms of plasmids encoding Dpp, Gbb, Tkv or Sax, or 8 ng of tkv-QD were co-transfected with Su(H) and N plasmids. Co-transfection of plasmids encoding Dpp or Gbb result in repression (indicating that endogenous receptors are expressed) that is enhanced when Tkv encoding plasmid is co-transfected. Addition of the plasmid encoding Sax leads to a suppression or blocking of the ligand-induced repression of reporter activity, consistent with in vivo data that Sax is able to block BMP signaling.

• Supplemental Figure 2 -

  Fig. S2. Tkv and Sax have different ligand preferences in vivo. (A) Wild-type wing. Five longitudinal veins (L1-L5), anterior and posterior cross veins (acv and pcv, respectively), and the AP boundary (red line) are labeled. (B) Weak dpp mutant wing phenotype showing a reduction in the L4/L5 intervein (arrow). (C) Weak gbb mutant wing phenotype, with small gap at distal tip of L5 (arrowhead). Mathematical models of gradient formation predict that a binding affinity of the receptor for a particular ligand directly correlates with the ability of the receptor to impede ligand movement across a tissue (Kerszberg and Wolpert, 1998; Lander et al., 2002). Consistent with this prediction, overexpressing Tkv phenocopies a dpp loss-of-function wing phenotype (Lecuit and Cohen, 1998). If Gbb binds Tkv, we reasoned that an increase in Tkv levels should also interfere with the distribution of Gbb and lead to a gbb loss-of-function phenotype (C,I) (Khalsa et al., 1998; Ray and Wharton, 2001b; Bangi and Wharton, 2006a) versus a strictly dpp loss of function phenotype (B) (Spencer et al., 1982). Thus, it theoretically should be possible to reveal differences in the Tkv-ligand binding preference in a dose-dependent manner by overexpressing Tkv at different levels. (D-F) Increased levels of wild-type Tkv during patterning. (D) Tkv overexpression at low levels results in a weak dpp loss-of-function wing phenotype (reduction in L4/L5 intervein). (E,F) Higher levels of Tkv expression phenocopy a stronger dpp loss-of-function phenotype (loss of L4 and reduction in L4/L5 intervein, arrow), as well as a gbb loss-of-function phenotype (loss of L5, arrowhead). As expected, based on its higher affinity, Dpp is more sensitive than Gbb to changes in the level of Tkv expression. As endogenous Sax can block ectopic Gbb signaling, overexpressing Sax in the absence of excess ligand is also likely to interfere with endogenous Gbb signaling in a manner that correlates with its affinity. (G) Overexpression of wild-type Sax during wing patterning results in loss of the distal region of L5 (arrowhead), a gbb loss-of-function phenotype. (H) Strong overexpression of Sax in gbb mutant wing discs enhances the loss of L5 (arrowhead), resulting in a phenotype similar to a stronger gbb allelic combination (I), which can be enhanced by reducing gbb gene dose (gbb/+; data not shown). No level of Sax overexpression revealed a dpp loss-of-function wing phenotype, indicating that endogenous Dpp is not very sensitive to the presence of ectopic Sax, consistent with the lower affinity of Dpp for Sax in biochemical assays (Brummel et al., 1994). All phenotypes are fully penetrant at the temperature indicated (n=30 for A9-Gal4/Y;UAStkv/UAStkv; n=80-100 for other genotypes). Overexpression of Tkv or Sax with 1348Gal4 (deCelis, 1997) during the later stages of wing development using 1348Gal4 fails to produce such vein and intervein abnormalities (E.B. and M. Chen, unpublished), indicating that the vein and intervein loss phenotypes associated with the overexpression of wild-type receptors shown here clearly reflect defects in wing patterning and not the later function of BMP signaling in vein differentiation. It is important to note that these in vivo assays of ligand-receptor interactions, which highlight the relative binding preference of Gbb and Dpp for Tkv and Sax in the context of the developing wing disc, are in complete agreement with biochemical experiments carried out with both Drosophila proteins and their vertebrate orthologs (Brummel et al., 1994; Penton et al., 1994; Yamashita et al., 1995; Nishitoh et al., 1996; Chalaux et al., 1998; Ebisawa et al., 1999; Piek et al., 1999).

• Supplemental Figure 3 -

  Fig. S3. Complete loss of sax enhances gbb loss-of-function phenotype, indicating that Sax plays a role in mediating Gbb signals. (A) Mild gbb loss-of-function wing phenotype is apparent as a narrowing of L4/L5 intervein and a truncation of distal L4 and L5. gbb⁴/gbb⁴. (B) Reducing the dose of sax by one (sax⁴ gbb⁴/ + gbb⁴) fails to enhance the gbb loss-of-function phenotype, but (C) completely eliminating sax function with a sax null clone encompassing the entire posterior compartment (hsFLP1; FRT^G13 sax⁴ sha^IN gbb⁴/FRT^G13 gbb⁴), shows an enhancement of the gbb mutant phenotype. sax clone is visible as the lighter region just posterior of the AP boundary (arrow) owing to the absence of wing hairs associated with cells homozygous for sha. Severe truncation of L5 and L4 are apparent in this clone, null for sax and homozygous for a hypomorphic gbb allele, sax⁴ gbb⁴. (D) In the presence of wild-type gbb, a sax null clone similar to that shown in C (removes sax function from the entire posterior compartment) shows no patterning defects in the posterior compartment.