BIOLOGICAL OVERVIEW

canoe(cno) has a dual character, interacting as it does with genes in both the Notch and the Ras pathways. In the Notch pathway, canoe interacts with the Notch pathway genes Notch and scabrous. Cno protein and its mammalian homolog AF-6 have also been shown to physically interact with Ras. The cno locus was defined by Jurgens (1984). Extra machrochaetes (sensory bristles) are occasionally seen in the head, notum and scutellum, and some mutants show variable but conspicous wing phenotypes such as a notched blade and the loss of a cross vein. In the eye, clones of mutant photoreceptor cells are devoid of any ommatidia, while small mutant patches have reduced numbers of photoreceptors. Ommatidia of mutant pupae often contain extra primary pigment cells. Some of these ommatidia lack a secondary pigment cell, resulting in the apparent fusion of adjacent facets (Miyamoto, 1995).

Canoe and the Notch pathway

Fused ommatidia have been reported to occur in scabrous mutants; sca mutants are also accompanied by bristle abnormalities. Although the compound eye of sca1 homozygotes contains some fused facets and others with excess or reduced numbers of photoreceptors, the majority of ommatidia have a normal complement of eight photoreceptors. When sca1 homozygotes are also heterozygous for the mutant allele cnomis1, the phenotype is dramatically enhanced: in such cases, many ommatidia are fused to one another. The eyes of double mutants for sca1 and cnomis1 are extremely rough, accompanied by giant ommatidia, formed as a result of fusion. The bristle phenotype of sca1 is also enhanced by cnomis1. Double mutant flies show supernumerary bristles. There is a genetic interaction between the two loci, also revealed by an examination of wing morphology. Double mutants have rumpled wings curved downward, with occasional shortening of the anterior cross vein. cno likewise interacts with the split allele of Notch, for eye, bristle and wing development. An interrupted wing vein in Ax1, one N allele producing an activated form of Notch protein, is dominantly suppressed by cnomis1 (Miyamoto, 1995).

Additonal interactions occur between cno and shaggy, armadillo, and myospheroid. It is intriguing that the genes encoding proteins involved in adhesive cell-cell communications are shown to interact with cno. Proteins coded for by these genes mediate the adhesion of the different cell layers in a variety of developing tissues. It is conceivable that Cno is localized to junctional complexes (Miyamoto, 1995).

Canoe and the Ras pathway

Just when it seemed that Canoe was being understood in terms of its interaction with the Notch pathway, it was also becoming clear that Canoe and its mammalian homolog AF-6 physically interact with Ras (Kuriyama, 1996). Morphological studies of the fly's compound eye suggest that induction of photoreceptors, including the R7 photoreceptor, is unaffected by cno mutations. The linear alignment of the R7 photoreceptor is disrupted in the pupal retina of cno mutants, but without exception a single R7 photoreceptor is formed in each ommatidium in cno mutant retinae (Matsuo, 1997). Since Ras signaling is involved in R7 fate determination (see Sevenless), what exactly is the role of Canoe in eye development?

It has been suggested that R7 photoreceptors occupy unusual positions in pupal discs because the mutant discs fail to form a correct mesh of pigment cells, which shape ommatidia into regular hexagonal structures. A strong mutation in cno eliminates from one to three cone cells from most ommatidia. In contrast, overexpression of cno+ results in the formation of supernumerary cone cells. These results indicate that an increase in cno+ expression promotes cone cell induction and a significant decrease in cno+ expression prevents cone cell precursors from differentiating into cone cells (Matsuo, 1997).

The involvement of Ras in non-neural retinal cell formation has attracted little attention. Freeman (1996) has provided evidence that the EGF receptor is required for the development of all retinal cells, including cone cells and pigment cells. A dominant negative form of EGF receptor prevents cell differentiation, whereas a secreted form of Spitz, the EGF receptor ligand, causes differentiation of supernumerary cells when expressed in the R7 equivalence group cells (cells from which arise a single R7 photoreceptor). Expression of a constitutively active form of Ras1, an important component in the EGF receptor signal transduction, causes a strong rough-eye phenotype. The rough-eye phenotype of active Ras is dramatically enhanced in cno heterozygotes, even though cno mutation is completely recessive and flies heterozygous for cno exhibit no specific phenotype. Overexpression of cno+ in R7 equivalence group cells similarly enhances roughness of constitutively active Ras1, without increasing the number of R7 cells beyond the level found without cno+ overexpression. Since the cno mutations interfere with cone cell formation without affecting photoreceptor induction, it seems plausible that Cno interacts with Ras1 in the developing cone cells (Matsuo, 1997).

These observations raise a question: from what cells do the extra cone cells originate? Two possibilities are considered: (1) they originate from retinal precursors overproduced due to accelerated proliferation and/or (2) they are generated at the expense of pigment cells that differentiate after cone cells, and from cells that are fated to die by apoptosis. Since proliferative activity in third instar larval eye discs is indistinguishable in mutant discs, it is likely that supernumerary cone cells are recruited from a pool of retinal precursors that normally develop into pigment cells and/or from cells that would otherwise be eliminated by apoptosis. It is thought that Cno acts in oppositon to Ras1, but the exact mechanism is not known. Nevertheless, an attractive model is that direct binding of Cno to Ras1 occludes the effector domain of Ras, which otherwise provides the binding site for Raf and consequent transduction of the Ras signal. This hypothesis is based on the results of an in vitro experiment demonstrating that mammalian cRas-1 competes with AF-6 ( a mammalian homolog of Cno) in binding to the H-Ras effector domain (Kuriyama, 1996). The direct binding of Cno to Drosophila Ras1 has been confirmed to occur in yeast, by two-hybrid assay. However, the hypothesis cannot be used to satisfactorily explain why overexpression of cno+ results in enhancement, rather than inhibition, of constitutively active Ras action in cone cell formation (Matsuo, 1997).

An alternative to either of these two models is suggested. It is postulated that there are two counteracting signaling pathways in which Cno participates: one for promoting the induction of cone cells (via Ras) and another for repressing this induction. It is postulated that Cno acts as a negative regulator for both pathways. Cno predominately associates with the repressing pathway when Ras activation is minimal, where absence of Cno causes disinhibition of the repression of the cone cell formation. With maximal Ras activation, extra cone cells form because the extremely strong activation of Ras1 overrides the inhibition by Cno and the action of the repressing pathway. Under such conditions, reduction of the Cno levels results in disinhibition of the promoting pathway, leading to enhancement of extra cone cell formation (Matsuo, 1997).

It is worth noting that reduction in Notch activity results in either an increase or a decrease in the number of cone cells, depending on the timing of Notch inactivation. Thus the Notch action is likewise 'bidirectional' in cone cell formation, as found for Cno action. In this context, it is of interest to note that some components of the EGF receptor pathway display strong genetic interactions with the Notch group of genes in eye, wing and leg morphogenesis. Cno is a reasonable candidate for a molecule that mediates such crosstalk between the Notch pathway and the EGF receptor pathway (Matsuo, 1997).

GENE STRUCTURE

cDNA length - 7296

Transcript lengths - 7.0, 7.5 and 8.2 kb

Bases in 5' UTR - 484

Exons - 15

Bases in 3' UTR - 1133

PROTEIN STRUCTURE

Amino Acids - 1893

Structural Domains

The sequence of Cno cDNA clones isolated from an embryonic cDNA library reveal a long open reading frame that encodes protein with the GLGF/DHR motif, a conserved sequence in Discs large, Dishevelled, and some other proteins associated with cellular junctions (Miyamoto, 1995). The molecular structure of Cno suggests its direct association with Ras. It has significant homology with a mammalian Ras-binding protein AF-6 (Kuriyama, 1996), which as been cloned as a fusion partner of All-1, a protein involved in acute myeloid leukemias in humans and a homolog of Drosophila Trithorax. Cno and AF-6 share two putative Ras-binding domains (RA1 and RAs), located at the N-terminus, as well as a kinesin-like and a myosin-V-like domain, and a Discs large homologous region, (DHR, also known as the GLGF or PDA motif) (Matsuo, 1997 and Kuriyama, 1996). The direct interaction of Raf, phosphatidylinositol-3-OH kinase, Ral GDS and Rin1 with activated Ras has been demonstrated. There is no obvious homology among Ras-interacting interfaces of these proteins with either AF-6 or Canoe, indicating that activated Ras can recognize a variety of target interfaces (Kuriyama, 1996 and references)

date revised: 18  January 98

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