BIOLOGICAL OVERVIEW

In Drosophila, two Frizzled proteins, Frizzled and Frizzled-2, have been reported to serve as receptors of Wingless. A third member of the Frizzled family has been identified in Drosophila, Drosophila frizzled-3 (fz3). fz3 is expressed in late embryos and imaginal discs and encodes a Wg receptor whose signal transducer activity is much less efficient than Fz2. In contrast to fz2, fz3 expression is positively regulated by Wg signaling. Fz3 was discovered on the basis of an expression pattern similarity to Wg. Genes whose expression is under the control of Wg signaling may exhibit expression patterns similar to those of wg. Enhancer trap line analysis led to the discovery of a trap line, J29, that exhibits a wg-like reporter gene (lacZ) expression in wing discs. Flies lacking fz3 activity are viable and fertile, with few morphological defects. The absence of fz3 suppresses the effects of hypomorphic wg mutants and restores target gene expression to wild-type levels without change in wg expression. Fz3 may thus serve to attenuate Wg signaling (A. Sato, 1999).

The absence of fz3 produces no apparent phenotype. Binding studies reveal that Wg can interact with Fz3 in cultured cells. In order to reveal a role for fz3 in development, the possiblity of a genetic interaction of fz3 with wingless has been investigated. fz3 may be involved in Wg signaling required for adult appendage formation. For example, fz3 may serve as an attenuator of Wg signaling, at least in a wg hypomorphic mutant background; the absence of fz3 may increase Wg signaling and stimulate wing formation. For analysis of this possiblility, a study was made to find possible interaction between Fz3 and Wg signaling in various wg mutant backgrounds. Wing blades are frequently absent from flies mutant for wg ¹. Thus, the first question to be examined was is the wg ¹ phenotype affected by the absence of fz3? The absence of wing blades is partially rescued through the elimination of fz3 activity. On a wg ¹/wg ^CX4 background, fractions of flies with two wings increased from 46% to 87%, while those flies with one wing and wing-less flies, respectively, reduced from 44% and 10% to 13% and 0.5%. The wing-less phenotype of wg ¹ is enhanced in a heterozygous apterous (ap) mutant background: no wing blade is generated at approx. 90% of the presumptive wing-blade-forming sites in wg ¹ homozygous flies heterozygous for ap. Wing blade formation increases 3-fold in the absence of fz3 activity. Since wg ^CX4 and wg ¹ are null and regulatory mutant alleles, respectively, these effects are not due to possible change in Wg protein conformation. Thus, wild-type fz3 may serve as an attenuator of Wg signaling at least in a wg hypomorphic mutant background; the absence of fz3 may increase Wg signaling and stimulate wing formation (A. Sato, 1999).

To confirm that Fz3 attenuates Wg signaling, an examination was made of the effects of fz3 absence in a different developmental context. Nearly all wg^11en/wg^CX4 flies lack antennal structures. This antenna-less phenotype is significantly rescued by removing fz3 activity; complete antennal structures, as well as incomplete ones, areregenerated at more than 70% of putative antennal sites. Distal antennal segment formation requires the circular expression of Bar homeobox genes. Dachshund (Dac) is required for the formation of proximal leg structures and expressed circularly in leg and antenna discs. Thus, wg ^11en/wg^CX4 fly discs with or without fz3 activity were stained for Wg, BarH1 and Dac. When there is fz3 activity, antennal discs are small and no or little expression of BarH1 and Dac is detected. In the absence of fz3 activity, about 10% of the discs, probably corresponding to the completely rescued type, exhibit circular BarH1 and Dac expression similar to that of wild-type discs. In about 50% of discs, presumably corresponding to the partially rescued type, Dac expression is partially restored without recovery of BarH1 expression. In contrast to BarH1 and Dac, no Wg expression is detected in the rescued mutant discs, indicating that wg expression is not enhanced by the absence of fz3. That wg^CX4 and wg ^11en are regulatory mutant alleles of wg suggests again that the genetic interactions found here would not be due to possible change in Wg protein structure, but simply to reduction in transcription products of wg. Thus it follows that in wg hypomorphic mutants, fz3 reduces Wg signaling activity required for antennal formation without changing wg expression; accordingly, fz3 would appear to function as a negative factor or attenuator of Wg signaling at least on a wg hypomorphic mutant background (A. Sato, 1999).

Wg signaling transduced by high levels of frizzled2 is suppressed by low levels of fz3 in a wg hypomorphic mutant condition, suggesting that Fz2 and Fz3 function in opposite directions during Wg signal transduction, with Fz2 acting to promote Wg signal transduction and Fz3 acting to interfere with Wg signal transducition. Fz2 and Fz3 are downregulated and upregulated, respectively, by Wg signaling and Wg signaling activity in some wg hypomorphic mutant discs is restored to levels similar, if not identical, to those of wild type upon removing fz3 activity. Thus, it may be suggested that, in ventral cells of wg hypomorphic antennal discs, fz2 expression is de-repressed and fz3 expression is repressed while the de-repression of fz2 expression is canceled by the removal of fz3 activity. This possibility was tested directly by staining wild-type and mutant antennal discs for fz2 and fz3 mRNA. As anticipated, fz2 expression is de-repressed in the ventral cells of wg^11en/wg^CX3 antennal discs, and repressed to a marginal level at least in 10% of antennal discs upon removing fz3 activity as in the case of wild type. fz3 expression to reduce to a marginal level in wg hypomorphic mutants. Thus, it may follow that, at least in a wg hypomorphic mutant condition, very low levels of fz3 are capable of effectively suppressing Wg signaling transduced by high levels of fz2. Thus Fz3 may serve as an attenuator of wing-less and antenna-less phenotypes of wg hypomorphic mutants (A. Sato, 1999).

Since Fz3 is a transmembrane protein similar in structure to Fz2 and a putative Wg receptor, and is also capable of binding to Wg in vitro, Fz3 may be a Wg receptor capable of acting as a Wg-signaling attenuator. Antagonistic interactions have been shown to be involved in vertebrate Wnt signaling. sFRP is a CRD-like protein capable of competing with Wnt receptors for Wnt ligands (Wodarz, 1998). WIF is a putative Wnt-binding protein presumed to suppress Wnt signaling (Hsieh, 1999). Thus, fz3- dependent attenuation of Wg signaling might be due to antagonistic interactions at the level of Wg receptors, in which fz3 competes with Fz2 for Wg and possibly Dsh, with consequently abortive Wg signal transduction. However, this simple antagonistic interaction model may not fully account for fz3-dependent suppression of Wg signaling, since (1) in wg hypomorphic mutants, only a trace of Fz3 appears effective enough to suppress Wg signaling transduced with high levels of Fz2, and (2) unlike sFRP and WIF, fz3 overexpression has little apparent effect on Drosophila morphology. Rather, preliminary experiments suggest that Fz3 may possess some positive roles in Wg signaling, since fz3 overexpression causes weak misexpression of Dll, a Wg-signaling-target gene, in the future wing pouch. Thus, possible Wg/fz3 signaling might suppress the redundant inputs of Wg signaling or activate factors that inhibit Wg signaling (A. Sato, 1999).

One of the most intriguing findings of the present study is that, in the absence of Fz3 activity, very low levels of Wg, produced in wg hypomorphic mutant discs and hardly detectable with anti-Wg antibody staining, are still capable of inducing normal levels of target gene expression and repression. The reduced Fz2 expression area in the ventral antennal disc may serve as a measure of the distribution of Wg signals. Reduced Fz2 expression area and hence Wg signal distribution is recovered in a significant fraction of wg hypomorphic mutant discs lacking fz3 activity. Thus, it might be considered that (1) only very low levels of wg activity are essential for normal (antennal) disc development and (2) in wild type, a considerable fraction of Wg signals are either dispensable or neutralized by a negative mechanism, which may include Fz3 (A. Sato, 1999).

Recent experiments have shown that frizzled and fz2 are functionally redundant to one another in embryos (Kennerdell, 1998). However, fz may not be involved in Wg signaling at late-embryonic and larval stages. Interestingly, fz3 is expressed in late embryos and imaginal discs but not early embryos. Thus, in early embryos, Fz, Fz2 and possibly additional Fz members other than Fz3 may form a Wg receptor system, while Wg receptor systems in subsequent developmental stages may be comprised of Fz2, fz3 and additional Fz members other than Fz. fz3 expression is intimately related to Wg expression. fz3 expression is possibly independent of wg activity in two tissues: dorsal ectodermal edge of stage 11-13 embryos and differentiated photoreceptor cells. In the former, DWnt4 but not Wg is expressed, while no Wnt expression has been reported in the latter. In vertebrates, Wnt protein can bind to plural Fz. Fz3 may thus interact not only with Wg but also with DWnt4 or other Drosophila Wnt proteins (A. Sato, 1999 and references).

GENE STRUCTURE

Exons - 2

PROTEIN STRUCTURE

Amino Acids - 581

Structural Domains

fz3 is a TATA-less gene and associated with 5'GTCG, a downstream-promoting- element-like element (Burke, 1997) at about +30 bp. As with other Fz members, Fz3 contains a cysteine-rich domain (CRD) in the amino-terminal region and seven transmembrane domains. Fz3 also contains SXV (X= an arbitrary amino acid), a putative PDZ domain-binding motif, at the carboxy end. Analysis of amino acid sequence homology has shown that Fz3 is much less similar to Fz2 than is Fz (A. Sato, 1999).

date revised: 12 January 2022

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