frizzled 3

Transcriptional Regulation

To determine whether Wg signaling is required for fz3 expression, examination was made of the effects of reduction and misexpression of Wg signals on fz3-lacZ expression. In a wg hypomorphic mutant (wg^CX3) background, the area of fz3-lacZ expression in leg discs decreases with a reduction of Wg expression. All embryonic fz3 expression other than that occurring along the dorsal edge and weak expression in brain disappears in a wg null mutant, (wg^CX3). When UAS-wg^ts is driven by ptc-Gal4, fz3-lacZ misexpression occurs in anterior cells along the anteroposterior compartment border in a cell-non-autonomous fashion. Similar but cell-autonomous misexpression of fz3-lacZhas been noted in flip-out clones expressing DArm, a constitutively active form of Arm. From these findings and expression of fz3-lacZ, it is concluded that fz3 expression is positively regulated by Wg signaling, which gives the opposite effect on fz2 expression (Cadigan, 1998). Consistent with this conclusion, at least in leg and wing discs, fz2 and fz3 show virtually complementary expression. That fz3 expression along the dorsal edge of an embryo where DWnt4 is expressed is insensitive to the absence of wg activity suggests that dorsal-edge fz3 expression may be due to DWnt4 signaling (A. Sato, 1999).

Fz3 is a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Fz2, Fz3 is a target gene of Wingless (Wg) signaling, but in contrast to fz2, it is activated rather than repressed by Wg signaling in imaginal discs. Fz3 is not required for viability but is necessary for optimal Wg signaling at the wing margin. Fz3 was identified by characterizing a P-element line from a large scale Gal4 enhancer trap screen that allows direct visualization of gene expression patterns in living flies. A Gal4 insert found in the cytological position 1C exhibits an adult expression pattern resembling that of wg. The Gal4 expression pattern of this line has been visualized by a UAS-lacZ reporter gene. Depending on the tissue analysed, Fz3-Gal4 is expressed in a broad domain centered over, or in a domain coinciding with, the wg expression domain. Fz3 is expressed throughout the wing pouch but appears to be upregulated by Wg signaling at the presumptive wing margin and in a ring around the pouch. In the notum the expression is similar to the thoracic expression of wg. In the leg disc Fz3 is expressed in a broad ventral wedge centered on the wg domain. Fz3 expression in the eye disc is also coincident with wg expression and can be detected at the dorsal and ventral margins, which give rise to the head capsule (Sivasankaran, 2000).

Since the expression pattern in the wing disc strongly suggests that Fz3 is a Wg target gene, an examination was carried out to see whether its expression is controlled by Wg signaling. When the Wg pathway is ectopically activated in clones of cells expressing a constitutively activated form of Armadillo, Fz3 expression is upregulated in these cells in a cell-autonomous fashion. This supports the view that Fz3 is a target of Wg signaling, and indicates that Wg acts directly and at long range in the wing pouch to control the expression of Fz3 (Sivasankaran, 2000).

DFz3 protein manifests characteristic features of the Frizzled family: it has a cysteine-rich amino terminal domain followed by seven transmembrane domains, and ends with an S/T-X-V motif at the C terminus. The DFz3 protein sequence shows 18% identity to Fz and 28% identity to DFz2. The P-element insertion has been determined to be 201bp upstream of the ATG. The P-element was mobilized and a PCR based screen was carried out to select for imprecise excision events. Three such chromosomes were identified in which the ATG start codon, 220 bp of the coding region, and an undetermined fraction of intron sequences were removed. These molecular null mutants are perfectly viable and show no abnormal phenotype. It is possible that Fz3 acts to transmit the Wg signal but that its function, like that of Fz and Dfz2 is masked by redundancy. Evidence supporting a functional role for Fz3 in Wg signaling comes from a genetic interaction with Serrate (Ser), a gene that encodes a Notch ligand involved in establishing wg expression in the wing margin. Ser^D is a dominant mutation that results in reduced wg expression in wing margin cells. Fz3 mutations enhance the Ser^D phenotype in a dose-dependent manner. With one copy of Fz3 removed the Ser^D wings show stronger notching and a loss of wing margin bristles. Removing the second copy of Fz3 enhances this phenotype further and results in additional posterior margin notching. It is suggested that Fz3 may function in concert with Dfz2 and Fz to transduce or transport the Wg signal in imaginal discs (Sivasankaran, 2000).

Protein Interactions

To determine whether fz3 is capable of binding to Wg and transducing Wg signals, fz2 and fz3 were expressed under the control of the metallothionein promoter in Schneider line 2 (S2) cells. To examine Wg binding, either fz2 or fz3 was transiently expressed in S2 cells. Transfectants were then incubated with Wg-conditioned medium and stained with anti-Wg antibody. Not only fz2- but also fz3-transfectants showed strong surface staining while no surface signals could be detected in pMK33-transfected cells, indicating tight binding of Wg to Fz3. To determine whether fz3 is capable of transducing Wg signals, Arm stabilization in response to added Wg was assessed. Arm is phosphorylated by Zeste-White 3 kinase and the phosphorylation is suppressed by Dsh (activated by Wg signals), thus permitting assay of Wg signaling activity by Western blotting using anti-Arm antibody. S2 cells were transfected by fz2 or fz3 cDNA, whose expressions were controlled by the metallothionein promoter. Arm always accumulates in fz2-expressing cells in a Wg-dependent manner. In fz3-expressing cells, there is little or no Wg-dependent Arm accumulation; Wg-dependent Arm accumulation in S2/Fz3-C1 and C2 is marginal while that in S2/Fz3-C3 and C4, is low but significant. Thus it may be, at least in cultured S2 cells, that Fz3 is capable of serving as a transducer of Wg signals but its activity is much less than that of Fz2 (A. Sato, 1999).

DEVELOPMENTAL BIOLOGY

Using reporter gene expression and in situ hybridization, fz3 expression was examined. DFZ3 mRNA expression is essentially identical to that of fz3-lacZ. Staining for Wg and fz3-lacZ indicates that fz3 expression is similar to wg expression, but it occupies a much wider area than wg expression, suggesting that fz3 is a general target gene of Wg signaling. As with Wg signals, strong fz3-lacZ signals are detected along the dorsoventral compartment border of the late-third-instar wing pouch and in future hinge and notum regions. In the wing pouch, Wg is present at high levels in cells expressing WG RNA, but drops off sharply on moving away from the stripe. Consistent with this, weak broad fz3-lacZ expression is detected in most wing pouch cells expressing Distal-less (Dll), a target gene of Wg signaling, in addition to strong reporter gene expression along the dorsoventral compartment border. Coexpression of fz3 and Bar homeobox genes is detected in the lateral prescutum (A. Sato, 1999). Bar expression in the lateral prescutum has been shown to be positively regulated by Wg produced in scutum cells (M. Sato, 1999).

Although Wg accumulates in a striped pattern in early embryos, neither the ectoderm nor CNS of early embryos is associated with fz3 expression, indicating that fz3 is unrelated to early Wg stripes. Striped fz3 expression can initially be seen in ventral ectoderm at later stages. Similar delayed striped expression has also been seen in DWnt4. As with wg, fz3 is expressed strongly in brain, proventriculus, parasegment (PS) 8 of the visceral mesoderm (VM-PS8), Malpighian tubules and anal plate. DWnt4 maps close to wg and both genes are coexpressed in many cells, although their expression differs in several respects. wg is specifically expressed in Malpighian tubes, while DWnt4, but not wg, is expressed in the nerve cord, VM-PS4 and the dorsal edge of an embryo. fz3 is expressed in the dorsal edge of the embryo but not in the nerve cord and VM-PS4 (A. Sato, 1999).

At late third instar, fz3 expression in leg discs is ventrally restricted, as noted for H15. Late fz3 expression should thus be negatively regulated by a dorsal factor. In late-third-instar eye discs, wg is expressed in undetermined cells along the periphery of the eye disc anterior to the morphogenetic furrow FZ3 RNA is detected in cells within several cell diameters from Wg sources. Although no Wg is expressed in ommatidial cells, fz3-lacZ expression is detected strongly and considerably in R8 and R7 photoreceptors, respectively; fz3-lacZ is also expressed in other photoreceptors at later stages. Ommatidial fz3 expression may not be related to Wg signaling (A. Sato, 1999).

In leg discs, wg is expressed in anteroventral cells along the anteroposterior compartment border and required for fate determination of both dorsal and ventral cells. At early third instar, fz3 expression is evident in both ventral and dorsal cells within about 6 cell diameters from the Wg sources. fz3 expression differs considerably from that of H15, an enhancer trap of a leg-specific gene whose expression is positively and negatively regulated by Wg and Decapentaplegic (Dpp) signaling, respectively (A. Sato, 1999).

Effects of Mutation or Deletion

A fz3 mutant was generated by imprecise P-element excision. DNA was extracted from 108 balanced mutant stocks, digested with restriction enzymes, size fractionated with agarose gel electrophoresis and subjected to Southern blotting. Lines with abnormal mobility of Fz3 fragments were sought. A deletion mutant line, Fz3^G10 was obtained. In Fz3^G10, the RNA start site, the first exon and a portion of the intron of fz3 were deleted and no FZ3 mRNA expression could be detected in both embryos and imaginal discs, indicating that Fz3^G10 is a null mutation of fz3. Surprisingly, flies homozygous for Fz3^G10 were found viable and fertile, with few appreciable morphological defects. Planar polarity was also normal. Double knockout flies of fz and fz3 were also constructed and found viable and fertile, with no morphological defects other than those in planar polarity, which is characteristic of fz homozygotes (A. Sato, 1999).

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Sato, M., Kojima, T., Michiue, T. and Saigo, K. (1999). Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted function of decapentaplegic and wingless. Development 126, 1457-1466.

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date revised: 20 July 2000

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