BIOLOGICAL OVERVIEW

Most if not all biological organisms demonstrate an incredible (though imperfect) symmetry of structure and patterning. Symmetrical patterns in the Drosophila compound eye ommatidia, as well as symmetrical patterns in adult cuticle, wings and legs develop and are regulated by frizzled. By symmetry is meant the positional repetition of a pattern on opposite sides of a dividing line or plane, distributed about a center or axis, resulting in geometric regularity. Frizzled not only contributes to symmetry, but also to tissue polarity. For example, all the hairs of the wing surface point in the same direction. How does the action of frizzled illustrate the biological basis of symmetry and polarity?

One aspect of eye symmetry regulated by frizzled is a mirror image pattern of ommatidia in the dorsal and ventral halves. The symmetry arises from the morphogenetic movement of cells posterior to the morphogenetic furrow, specifically, a rotation of the ommatidia that occurs symmetrically in the dorsal and ventral halves of the eye. Frizzled protein is required so that cells make the correct choice of which direction to turn, since a complete 90 degree turn of ommatidia takes place during development. Frizzled is also involved in the positional fate specification of individual photoreceptor cells.

Frizzled appears to be a receptor of morphogens that diffuse from an organizing center near the morphogenetic furrow.

Frizzled also has a gradient of activity from the equator outward. In non-autonomous signaling Frizzled passes a polarizing signal from cell to cell. Frizzled also acts cell autonomously to regulate graded expression of other genes in the eye and wing. Therefore, paradoxically, frizzled acts both cell autonomously and cell non-autonomously (Krasnow, 1994).

Creation of asymmetry depends on the direction of movement of the ectopic furrow. The self-propagating wave of furrow progession is initiated by Hedgehog and sustained by Decapentaplegic. Artificial initiation of HH signaling at unusual sites in the eye disc will bring about sustainable furrow progession in any direction. Ectopic ommatidia are generated behind the furrow and are oriented in a direction dictated by the direction of propagation without reference to the normal anterior-posterior axis of the disc (Chanute, 1995).

At the very beginning of furrow formation the first ommatidia formed create an initial node that develops into an equator. Apparently Frizzled acts so that these earliest formed ommatidia communicate with each other to ensure that they take opposite dorsal-ventral polarities (Chanut, 1995 and Jarman, 1996).

The gene nemo is a downstream of frizzled, while Spiny legs is considered a potential target (Zheng, 1995). It is not yet known how Frizzled functions as a receptor, what are the ligands, and how it sends its signals from cell to cell and into the interior of cells.

As to the role of Frizzled in polarity, how does it happen that cells manufacture hairs all pointing in the same direction? Each hexagonally shaped pupal wing cell assembles a single prehair at its distal vertex. These prehairs point distally from the earliest time they can be detected, arguing that hair polarity is controlled at an early step in prehair morphogenesis. It has been suggested that hair polarity is controlled by tissue polarity genes regulating the subcellular localization for prehair initiation. In fact, at least half a dozen genes appear to act downstream from frizzled, including prickle, dishevelled, inturned, fuzzy and multiple-wing-hair. All mutations that alter prehair polarity also alter the subcellular localization for prehair initiation. The observation that a prehair does not form perpendicularly to the cell surface, but lies flat on its distal neighbor cell early in development argues for a cell-cell contact or ligand-receptor type interaction as a possible mechanism for the distal growth of prehairs. Alternatively, interactions between the F-actin bundle in the prehair and the cortical actin filaments could be providing orientation guidance (Wong, 1993). It has been suggested that frizzled affects polarity by interacting with the cytoskeleton (Park, 1994).

How can one gene exhibit both cell autonomous and non-cell autonomous effects? As a receptor, Frizzled acts cell autonomously. As an initiator of a signaling pathway with downstream targets, subsequent effects of frizzled signaling could involve non-cell autonomous signaling. For example, one action of Frizzled might be the release of Frizzled ligand, to perpetuate frizzled signaling to adjacent cells. In this cause Frizzled would appear to have non-cell autonomous effects.

Drosophila Frizzled-2 (Dfz2) has been identified as a putative fly Wingless (Wg) receptor. Although Dfz2 shows significant homology with Frizzled (Fz), Dfz2's sister protein that operates in the establishment of planar polarity in the fly, any clear evidence for an involvement of Fz in a Wnt signaling pathway has hitherto been absent. The planar polarity phenotypes of loss-of-function and overexpression of Fz in the developing Drosophila eye are almost identical to the loss-of-function or overexpression of Dishevelled (Dsh - a protein operating in Wnt second messenger systems). Overexpression of Shaggy (Sgg - another component of the Wnt pathway) in the eye also causes a phenotype similar to Fz and Dsh. When Wg is misexpressed in the developing eye it has a potent polarizing effect in the retinal epithelium. Since the overexpression of Fz in the developing eye gives a phenotype consistent with activating the Wnt pathway, overexpression of Fz was tested in the developing embryonic ectoderm. Fz phenocopies overexpression of Wg, giving a subset of phenotypes resembling overexpression of Wingless. In a number of embryos, the cells that normally contribute to the anterior-most three to four rows of denticles in the belts are transformed to smooth cell types, producing narrowed denticle belts. The extreme heat shock wingless phenotype is not observed, possible due to a higher level of Wg signaling activity than can be achieved by overexpression of Fz alone. To check that Fz is indeed able to activate a Wnt pathway, Fz was overexpressed in Drosophila tissue culture cells: the characteristic phosphorylation of Dsh that occurs in response to Wnt signaling was observed. Taken together these results significantly strengthen the case for Fz acting in a Wnt signaling pathway in Drosophila (Tomlinson, 1997).

Two members of the Frizzled (Fz) family of seven-pass transmembrane proteins, Drosophila Fz and Fz2, can bind Wg and are candidate Wg receptors. However, null mutations of the fz gene have little effect on Wg signal transduction and the lack of mutations in the fz2 gene has thus far prevented a rigorous examination of its role in vivo. Here, the isolation of an amber mutation of fz2 is described; this mutation truncates the coding sequence just after the amino-terminal extracellular domain and behaves genetically as a loss-of-function allele. Using this mutation, Wg signal transduction is abolished in virtually all cells lacking both Fz and Fz2 activity in embryos, as well as in the wing imaginal disc. Fz and Fz2 are functionally redundant: the presence of either protein is sufficient to confer Wg transducing activity on most or all cells throughout development. These results extend prior evidence of a ligand-receptor relationship between Wnt and Frizzled proteins and suggest that Fz and Fz2 are the primary receptors for Wg in Drosophila (Chen, 1999).

Wg is normally expressed in a thin stripe of cells straddling the dorsoventral compartment boundary of the mature wing imaginal disc, under the control of the extracellular signals Delta and Serrate. Wg protein emanating from these cells directs the formation of wing margin bristles and organizes gene expression, growth and patterning in surrounding cells of the presumptive wing blade. Hence, mutations that block Wg signal transduction cause a loss of wing margin bristles as well as deletions of nearby portions of the wing. Wg also plays a role in restricting its own expression to cells immediately adjacent to the dorsoventral compartment boundary by down-regulating the transcription of wg itself in neighboring cells that are close to, but not next to, the D/V boundary. When Wg signal transduction is blocked in these cells, they ectopically express Wg. As a consequence, nearby wild-type tissue is induced to form ectopic margin bristles. Approximately 100 mutations were obtained in a screen for mutations that cause wing margin defects in clones of mutant cells that are also homozygous for the fz loss-of-function mutation, fz^H51. Of these, only one is associated with the formation of ectopic bristles in neighboring, wild-type wing tissue. This mutation, designated fz2^C1, appears to be a loss-of-function mutation in fz2 according to the following criteria: (1) the mutation maps meiotically to a location approximately 1 centiMorgan distal to radius incompletus (ri), the expected map position, given the cytological localization of the fz2 gene; (2) both the wing notching and ectopic bristle phenotypes associated with fz^H51; fz2^C1 mutant cells are completely rescued when the fz2 coding sequence is expressed in these cells using either alpha Tubulin a1-fz2 transgene (which should be expressed in most or all cells), or a UAS-fz2 transgene driven by a vg-Gal4 transgene. All of the remaining wing notching mutations obtained in the screen fully complement the fz2^C1 mutation in a fz^H51 mutant background, indicating that they are not in the fz2 gene. (3) The fz2^C1 mutation is associated with a single base change in the fz2 gene that changes codon 320 from TGG to TAG. This creates a stop codon located at the junction between the coding sequence of the amino-terminal extracellular domain (which contains the CRD) and the remainder of the protein, which includes all seven transmembrane domains. It is unlikely that the resulting truncated protein, composed of just the extracellular domain, would retain any signal transducing activity (Chen, 1999).

To assay the possible roles of Fz and Fz2 in Wg signal transduction during embryogenesis, embryos were generated homozygous for the fz^H51 and fz2^C1 mutations that derive from female germ cells that are similarly mutant for the two genes. Such embryos lack the maternal and zygotic contributions of both genes, and hence, should be devoid of Fz and Fz2 activity. These embryos are referred to as fz-fz2- mutant embryos. To assay these embryos for Wg signal transducing activity, an examination was performed in six well defined Wg signaling events: two in the ectoderm, one in the visceral mesoderm, one in the endoderm, one in the central nervous system, and one in the somatic mesoderm. These double mutant embryos appear unable to transduce Wg when assayed for each event. (1) Initially examined was the cuticular pattern formed by such double mutant embryos. The epidermis of wild-type embryos secretes a segmented cuticle, decorated on the ventral side by stereotyped bands of patterned hairs separated by broad swaths of naked cuticle. In embryos devoid of Wg activity, or of Dsh or Arm activity, most signs of segmentation are eliminated and the ventral cuticle forms a 'lawn' of hairs spanning most of the anteroposterior body axis. Embryos devoid of Fz and Fz2 activity show the same characteristic 'lawn' phenotype. (2) The early striped expression of En in the ectoderm is labile, unless maintained by Wg signaling from adjacent cells across the parasegment boundary. In wg-, dsh- and arm- mutant embryos, this expression is lost within 2 hours after the onset of gastrulation. A similar loss of ectodermal En expression occurs in fz-fz2- mutant embryos. (3) Wg signaling is essential in the visceral mesoderm for initiating a series of stereotyped constrictions that partition the midgut. As in embryos lacking Wg, Dsh, or Arm activity, these gut constrictions are absent in fz-fz2- mutant embryos. (4) Wg signaling from the visceral mesoderm of parasegment 7 up-regulates the expression of the homeodomain gene labial in the adjacent endoderm. This up-regulation is not observed in wg-, dsh- or arm- embryos, and similarly, it is not apparent in fz-fz2- mutant embryos. (5) During development of the central nervous system, Wg signaling is essential for specifying the neuroblasts that generate the RP2 neurons in each segment. These neurons can be easily visualized because they express Even-skipped (Eve) protein. These Eve-expressing neurons are not present in the absence of Wg signaling (e.g., in wg- embryos). Similarly, they are absent in fz-fz2- mutant embryos. (6) Finally, during development of the somatic musculature, Eve protein is expressed in a subset of myoblasts that will give rise to the heart and the presence of these Eve-expressing cells is strictly dependent on Wg signaling. These Eve-expressing cells are also absent in fz-fz2- mutant embryos. In sum, embryos devoid of both Fz and Fz2 activity appear unable to transduce Wg in any of the several developmental contexts examined. These results indicate an absolute requirement for these Fz proteins for Wg transduction during embryonic development (Chen, 1999).

During normal development of the embryonic ectoderm, Wg protein moves at least a few cell diameters from secreting cells, as assayed by the accum. Therefore, an investigation was carried out to see whether the movement and apparent uptake of secreted Wg protein depends on Fz and Fz2. The distribution of Wg in fz-fz2- mutant embryos was determined. Wild-type and fz-fz2- mutant embryos show indistinguishable distributions of punctate Wg staining during the first two hours following germ band extension, consistent with the view that neither Fz nor Fz2 protein is required for the movement of secreted Wg during this phase of development. However, the fz^H51 mutation is expected to generate a protein that is truncated after the sixth transmembrane domain. Hence, if this protein is stable and reaches the cell membrane, it might be able to bind and regulate the movement of secreted Wg even though it can no longer transduce Wg signal. Wg expression dissipates in fz-fz2- mutant embryos shortly after this early stage, as expected given the loss of En expression in neighboring cells across the AP compartment boundary, preventing the examination of later aspects of Wg movement in these embryos. Fz and Fz2 transduce Wg via the regulation of Armadillo. Most, if not all, Wg signal transducing events involve the modification and up-regulation of Armadillo (Arm) protein. Two experiments were performed to test whether Fz and Fz2 transduce Wg through the regulation of Arm. These experiments establish that Fz and Fz2 act upstream of Arm to transduce Wg. In the first experiment, Arm expression was assayed in fz-fz2- mutant embryos. In wild-type embryos, Wg signaling is associated with stabilization of Arm protein and its consequent accumulation in a distinctive pattern of segmental stripes, each straddling a stripe of Wg-expressing cells. This up-regulation is not observed in wg minus embryos, and similarly, it is absent in fz-fz2- mutant embryos. In the second experiment, it was asked whether expression of a truncated, constitutively active form of Arm could drive the Wg signal transduction pathway in fz-fz2- mutant embryos. In this experiment, constitutively active Arm was expressed with the UAS/Gal4 method using a hairy-Gal4 driver line that is active in alternating segmental primordia. Expression of constitutively active Arm in alternating segmental stripes in fz-fz2- mutant embryos causes them to form corresponding stripes of naked cuticle. This result indicates that the activity of constitutively active Arm bypasses the normal requirements for Fz and Fz2 in activating the Wg transduction pathway (Chen, 1999).

Wg is expressed in the wing pouch of late third instar discs in a thin stripe of cells straddling the interface between the dorsal and ventral compartments. Wg emanating from this stripe acts at short range to induce the formation of bristles that will decorate the wing margin, and at longer range, to activate the expression of a number of genes, including Distalless (Dll) and vestigial (vg), that define the primordium of the wing blade and control aspects of its growth and pattern. Wg signal transduction is abolished in presumptive wing cells lacking both Fz and Fz2 activity. As a consequence, cells that lack both activities cannot proliferate normally and are lost from the epithelium. Wg signaling is required for the control of growth and pattern in portions of the wing disc other than the wing pouch. The wing imaginal discs also give rise to the fuselage of the adult second thoracic segment, the mesonotum, the anterior dorsal surface of which is decorated with a stereotyped pattern of large bristles. Wg is expressed in a longitudinal stripe in the developing half-mesonotum derived from each wing disc and this stripe is positioned just lateral to a line of four large bristles. These are the anterior and posterior dorsocentral bristles and the anterior and posterior scutellar bristles. It appears that zygotic activity of the fz2 gene is not essential for Wg signal transduction, provided that a wild-type allele of fz is present. Either protein can transduce most or all Wg signaling events during embryogenesis. It is concluded that Fz and Fz2 proteins are functionally redundant, with either protein being able to bear the full burden of Wg signal transduction in most, if not all, contexts throughout development (Chen, 1999).

GENE STRUCTURE

cDNA length -4 kb

Bases in 5' UTR - 718

Exons - five

Bases in 3' UTR - 1451

PROTEIN STRUCTURE

Amino Acids - 581

Structural Domains

The seven pass transmembrane protein is characteristic of G-protein-coupled-receptors (Adler, 1990). These proteins act through heterotrimeric G-proteins targeting various cytoplasmic regulatory systems. There is, however, no sequence homology between frizzled family members and G-proteins.

date revised: 10 December 99

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.