Evidence that the cysteine-rich domain of Drosophila Frizzled family receptors is dispensable for transducing Wingless

Abstract

Members of the Frizzled family of serpentine transmembrane receptors are required to transduce Wingless/Int (Wnt) signals and contain in their N-terminal regions a conserved Wnt-binding cysteine-rich domain (CRD). Each CRD has specific affinities for particular Wnts, and it is generally believed that signal transduction depends on the strength of this interaction. Here, we report in vivo evidence that the CRD is dispensable for Frizzled family receptors to transduce Wingless (Wg), the primary Wnt signal in Drosophila. Thus, we infer that signal transduction does not require binding of Wg to the CRD, but instead depends on interactions between Wg and other portions of the receptor, or other proteins of the receptor complex.

Fig. 1.

Rescue of fz^P21 Dfz2^– homozygotes by ΔCRD forms of Fz and Dfz2. (A and B) Full-length and ΔCRD forms of Dfz2, with the positions of the Dfz2^C1,2,3 and ^-4 mutations, as well as the corresponding position of the fz^P21 mutation, indicated (see Materials and Methods). (C and D) Ventral larval cuticles formed by wg^CX4 and fz^P21 Dfz2^– homozygotes: Both form lawns of ventral hairs, the hallmark phenotype corresponding to lack of Wg signal transduction. (E and F) Ventral larval cuticles formed by fz^P21 Dfz2^– homozygotes carrying the Tubα1-fz^ΔCRD (E) or Tubα1-Dfz2^ΔCRD (F) transgene: Both show normal, segmented patterns of ventral hairs.

Fig. 4.

Rescue of fz^P21 Dfz2^C2 cells in Dfz3^G10 wing imaginal discs by ΔCRD forms of Fz and Dfz2. (A and B) Rescue of Wg transduction in fz^P21 Dfz2^C2 clones in Dfz3^G10 larvae by Tubα1-fz^ΔCRD or Tubα1-Dfz2^ΔCRD (analyzed as twin spots, as in Fig. 3 G–J). (A–D) Clones are marked black by the absence of GFP; Dll is stained in red. (C and D) Rescue of Wg transduction in fz^P21 Dfz2^C2 clones in Dfz3^G10 larvae by Tubα1-fz^ΔCRD or Tubα1-Dfz2^ΔCRD by using the Minute technique: Mutant clones have a competitive growth advantage and generally populate most of the developmental compartment in which they reside. (E and F) Wing discs from Dfz3^G10; fz^P21 Dfz2^C2/fz^P21 Dfz2^– larvae rescued to the late third instar by the presence of a single Tubα1-fz^ΔCRD or Tubα1-Dfz2^ΔCRD transgene. Dll (red) is expressed normally in a broad stripe flanking Wg-secreting cells (blue) along the dorsoventral compartment boundary [discs are of normal size but are shown at lower magnification than in A–D (see Materials and Methods)].

Fig. 2.

Lack of cell surface accumulation of Dfz2^C2 in vivo.(A and B) Doubly tagged forms of Dfz2⁺ and Dfz2^C2 carrying three copies of the Flu-epitope at the N terminus and GFP at the C terminus. (C–J) Flu-epitope staining (red) and GFP fluorescence (green) associated with doubly tagged Dfz2⁺ and Dfz2^C2 proteins expressed in the “pouch” of the wing imaginal disk [the MS1096-Gal4 transgene drives stronger expression dorsally (up) than ventrally]. In living discs (data not shown) and fixed but nonpermeabilized discs (C–F), Flu-epitope staining was observed only for doubly tagged Dfz2⁺ and only at the cell surface (apical plane, C). In discs fixed in the presence of Triton X-100, Flu-epitope staining is observed for doubly tagged forms of both Dfz2⁺ and Dfz2^C2 at both the apical and subapical planes of focus (G–J). GFP fluorescence was readily detected in all conditions.

Fig. 3.

Rescue of fz^P21 Dfz2^C2 double mutant clones in the wing imaginal disk by ΔCRD forms of Fz and Dfz2. (A–E) Anterior edge of wings carrying multiple clones of homozygous fz^P21 Dfz2^C2 cells (see Materials and Methods). In the absence of rescuing transgenes, loss of endogenous fz and Dfz2 gene function blocks the formation of wing margin bristles by mutant cells and causes wing notching, indicating a failure to transduce Wg (A). Cells within the clone are marked by the yellow cuticle mutation, not readily visible at this magnification. (B–E) These phenotypes are fully rescued by single transgenes expressing Fz⁺, Dfz2⁺, Fz^ΔCRD, or Dfz2^ΔCRD under the control of the Tubα1 promoter. (F–J) Wing discs containing “twin spots” composed of fz^P21 Dfz2^C2 double mutant clones (marked black by the absence of GFP expression) and their WT sibling clones (marked bright green by the presence of two copies of the GFP marker gene) induced during the first or second instar. In the absence of rescuing transgenes, double mutant clones survive only briefly in the wing blade primordium (marked by the central domain of Dll expression; red) and stop expressing Dll before being lost from the epithelium, indicating a failure to transduce Wg (F, boxed portion of the GFP image is shown at higher magnification in the Dll and merged images). In the presence of any one of the Tubα1-fz⁺, Tubα1-fz^ΔCRD, Tubα1-Dfz2⁺, or Tubα1-Dfz2^ΔCRD transgenes, such double mutant clones survive in the presumptive wing blade and appear to proliferate equally well, relative to their WT sibling clones (G–J). Further, the pattern of Dll expression appears normal in the rescued clones.

Fig. 5.

Distribution of secreted Wg in Dfz3^G10; fz^P21 Dfz2^C2 mutant wing disk cells rescued by expression of Dfz2^ΔCRD. (A and B) Wing disk carrying large fz^P21 Dfz2^C2 clones obtained from a Dfz3^G10 larvae carrying a single Tubα1-Dfz2^ΔCRD transgene (Minute technique; the clones are marked black by the absence of GFP). Wg (white in A and red in B) is secreted by a thin line of cells along the dorsoventral compartment boundary and accumulates in cytosolic puncta in surrounding cells over a range of several cell diameters even in the triple mutant cells rescued by the presence of Dfz2^ΔCRD.