1. Pathway Specificity by the Bifunctional Receptor Frizzled Is Determined by Affinity for Wingless 
Eric J. Rulifson, Chi-Hwa Wu, Roel Nusse 
Molecular Cell 
Volume 6, Issue 1, Pages (July 2000)
DOI: /S (05)

2. Figure 1
Measuring Differences in Affinity for Wg between Frizzled Proteins
(A) Scheme for assaying binding affinities between Fz CRD-Alkaline phosphatase (CRD-AP) fusion proteins, present in conditioned medium and active Wg tethered to S2 cell plasma membrane (pm) via fusion with the transmembrane region of Neurotactin (Nrt). DFz2 CRD-AP and Fz CRD-AP both bind Wg-Nrt expressing S2 cells to saturation (B). Scatchard plots and derived Kd values from binding data in (B) for the Fz CRD-Wg interaction (C) and DFz2 CRD-Wg interaction (D).
Molecular Cell 2000 6, DOI: ( /S (05) )

3. Figure 2
Wg Affinity for Fz Proteins Is Correlated with Wg Stabilization In Vivo
Wg protein accumulation on cells expressing various Myc-tagged Fz transgenes under ptc-GAL4 is shown in the wing blade region of wandering third instar wing discs. Discs are labeled with anti-Myc antibody (green) and anti-Wg (red). Wingless protein is stabilized away from its site of synthesis and secretion at the D/V compartment boundary (vertical Wg stripe) by the membrane tethered high Wg affinity DFz2-GPI, seen as overlap of Myc and Wg signals (horizontal yellow stripe in [B]). The stabilization of Wg is also seen on cells expressing DFz2-Fz (comparable to DFz2, data not shown) but less so than DFz2-GPI (D). No detectable Wg accumulation is observed with the low Wg affinity Fz CRD-GPI cells (A) or Fz-DFz2 cells (C), though both proteins are expressed highly at the membrane as evidenced by the cell outline staining of the Myc epitope. Bar = 50 μM.
Molecular Cell 2000 6, DOI: ( /S (05) )

4. Figure 3
Expression of Chimeric Frizzled Proteins in the Wing Reveals that Wg Signaling Activity Is Correlated to Affinity for Wg
Wild-type wing (A). Normal Wg-dependent anterior margin bristles indicated by arrow and normal planar tissue polarity pattern of epidermal hairs shown in the enlarged view (right). Abnormal planar tissue polarity pattern resulting from loss of fz function in fzK21/fzD21 wing (B). Planar polarity defects resembling loss of fz function result from ectopic expression of fz seen in UAS fz-myc;71B-GAL4 (C). Ecoptic wing margin bristles arise (arrows) in positions near the normal wing margin bristles in UAS Dfz2-myc;71B-GAL4 wings, but planar polarity patterning is normal. An exchange of the low-affinity Fz CRD onto the remainder of DFz2 produced the chimera diagrammed at the left (E). Wings of UAS fz-Dfz2;71B-GAL4 are normally formed ([E], 1X). Wings of UAS fz-Dfz2/UAS fz-Dfz2;71B-GAL4 that express two copies of Fz-DFz2 show one or two extra wing margin bristles (arrow in [E], 2X), though planar polarity patterning is normal. An exchange of the high-affinity DFz2 CRD onto the remainder of Fz produced the chimera diagrammed at the right (E). Wings of UAS Dfz2-fz;71B-GAL4 show ectopic wing margin bristles ([F], 1X). The activity is similar to UAS Dfz2-myc;71B-GAL4 wings (compare to [D]). In addition, these wings show whorl patterns of epidermal hairs (open arrows; [F], 1X) similar to those seen in UAS fz-myc;71B-GAL4 wings (compare to [C]). Both the extra bristle and planar polarity defects are increased further in UAS Dfz-fz/UAS Dfz2-fz;71B-GAL4 wings that express two copies of DFz2-Fz ([F], 2X).
Molecular Cell 2000 6, DOI: ( /S (05) )

5. Figure 4
Both Fz Chimeras Are Active Wg Receptors and Can Transduce All Wg Signaling in the Embryo
A wild-type embryonic ventral cuticle pattern (A). The strong denticle lawn phenotype of a ventral cuticle that lacks maternal and zygotic fz and Dfz2 functions resembles wg loss-of-function (B). Weak wg loss-of-function phenotype in embryos with same genotype as (B) combined with da-GAL4 driving expression of Fz-DFz2 (C) and DFz2-Fz (D) (see Experimental Procedures for details of genetics).
Molecular Cell 2000 6, DOI: ( /S (05) )

6. Figure 5 The DFz2-Fz Chimera Requires Wg Function to Activate Ac
Antibody labeling of anterior wing margin Ac (black), the target of Wg signaling from the D/V boundary in the normal wandering third instar wing disc (A). Open arrows indicate the position of Wg expression and the D/V boundary. Ac levels are elevated, and intense labeling is seen in cells at a distance from the region of normal expression in UAS Dfz2-fz;71B-GAL4 discs (arrows, [B]). Ac is lost in wg-ts;UAS Dfz2-fz;71B-GAL4 discs where the temperature-sensitive wg function is severely reduced at the restrictive temperature (C). Ac expression in (C) is similar to expression in the treated wg-ts disc alone (data not shown). Other visible nonmargin Ac is not Wg dependent (Phillips and Whittle 1993). Bar = 100 μM.
Molecular Cell 2000 6, DOI: ( /S (05) )

7. Figure 6
The DFz2-Fz Chimera Does Not Require the Polarity Pathway Downstream of Dsh to Induce Wing Margin Bristles
Proximal-distal planar polarity is roughly normal at the distal wing tip in the dorsal epidermis of dsh1 mutants (A). An area of polarity disruption is present in the same position in UAS fz;71B-GAL4 wings (B). The planar polarity defect in dsh1;UAS fz;71B-GAL4 wings (C) resembles that in dsh1 mutant wings (A). The extra bristle phenotype of dsh1;UAS Dfz2-fz;71B-GAL4 wings (arrows in [D]) is comparable to that of UAS Dfz2-fz;71B-GAL4 wings (2F).
Molecular Cell 2000 6, DOI: ( /S (05) )

8. Figure 7
The DFz2-Fz Chimera Activation of Ac Is Blocked by Negative Regulators of the Canonical Wg Pathway
Small Act5C-GAL4;UAS Dfz2-fz mitotic clones (see Experimental Procedures) are marked by GFP (green), anti-Ac (red), and anti-Wg (blue). Most Act5C-GAL4;UAS Dfz2-fz clones have elevated Ac expression (yellow) near the D/V boundary Wg stripe, though in a few clones cytoplasmic GFP expression is not in the same focal plane as nuclear Ac signal and appears green (A). Elevated Ac expression is lost cell autonomously from Act5C-GAL4;UAS Dfz2-fz mitotic clones (green) when UAS Daxin (B) or dominant-negative pangolin (UAS dTCFDN) (C) are simultaneously expressed. Bar = 100 μM.
Molecular Cell 2000 6, DOI: ( /S (05) )