BIOLOGICAL OVERVIEW

Each sensory unit, or ommatidium, of the compound eye is composed of eight photoreceptors. During the late third instar larval stage, photoreceptor axons grow from the eye across the optic stalk to reach their synaptic target cells in the brain's optic lobe. The lamina is the outermost of three cell layers or ganglia constituting the optic lobe. Six photoreceptors (R1-6) synapse with lamina neurons; this connection constitutes the first relay station for visual information flowing into the brain. The generation of lamina neurons from their precursor cells (lamina precursor cells or LPCs) is coordinated with the arrival of photoreceptor axons; LPC divisions then take place in a stereotyped and highly ordered pattern (Selleck, 1991). Earlier studies have shown that LPC divisions are controlled by an intercellular signal delivered by photoreceptor axons (Selleck, 1991). This signal is required for LPCs to enter their final S phase from the preceding G1 (Selleck, 1992). The signal consists of the morphogen Hedgehog, carried from the eye to the brain along photoreceptor axons (Huang, 1996). The number and location of LPC divisions is dictated by the number and placement of photoreceptor axons arriving in the CNS. division abnormally delayed (dally) was characterized in a screen for genes that affect cell division control of LPCs. Molecular cloning of the dally cDNA shows it encodes a putative integral membrane proteoglycan of the Glypican family (Nakato, 1995).

LPCs are derived from a set of neighboring neuroblasts in the anterior segment of the outer proliferative center (aOPC). The aOPC and LPCs form an epithelial sheet on the surface of the brain. OPC neuroblasts that produce LPCs are at the anteriormost extent of this epithelium, with the lamina marking its posterior limit. LPCs are produced continuously from OPC neuroblasts and complete two cell cycles before differentiating into neurons. LPCs therefore enter the cell cycle as they are produced from aOPC neuroblasts and exit after the second division to differentiate into lamina neurons. As a result of this "assembly line" organization, LPCs in successive phases of the cell cycle are found in sequence along the proliferative epithelium. LPCs in different phases of the cycle are found at discrete positions relative to anatomical landmarks. Most notable of these is a furrow in the aOPC/LPC epithelium, located at the anterior boundary of the developing lamina. This lamina furrow, like the morphogenetic furrow (MF) in the eye disc, sweeps forward as neurons are added to the differentiating lamina. LPC divisions are synchronized across the furrow, with cells in different phases of the cell cycle at specific positions (Selleck, 1992). The distribution of LPCs can be revealed using two markers, antibodies to Cyclin B, which is expressed at highest levels in late G2-early M phase, and propidium iodide, a fluorescent dye that binds to DNA and permits the visualization of mitotic chromosomes. The two LPC division cycles are evident as two regions of peak cyclin B expression and corresponding to two domains of mitotic figures, along the anterior and posterior segments of the lamina furrow respectively. Cells between the two cyclin B-expressing domains reside in G1, (low levels of cyclin B) and the subsequent S phase. Newly arrived photoreceptor axons specifically run along the base of the G1-phase LPCs and trigger their entry into S phase (Nakato, 1995).

Third instar larvae homozygous for several dally alleles were evaluated for the organization and cell cycle progression of LPC divisions. All dally mutants examined showed the same constellation of defects, with varying degrees of abnormalities either as homozygotes, or in combination with dally P2 . dally P2 mutation severely affects the level and size of the Dally mRNA. Larval brains were stained with both anti-cyclin B antibodies and propidium iodide, allowing for the simultaneous visualization of G2- and M-phase cells. In every brain examined the G2- and M-phase cells of the second LPC division were absent. In wild-type larvae, G2- and M-phase cells of the second division are located near the surface of the brain, at the posterior limit of the proliferative epithelium. cyclin B-expressing cells and mitotic figures are not found in this region of dally P2 homozygous larval brains. The complete absence of the second LPC division in dally homozygotes, as evidenced by the loss of the second cyclin B-expressing domain, is particularly clear from lateral views of brain lobes. Abnormalities in the first LPC division cycle are also found. Normally, G2- and M-phase cells of the first division are found exclusively along the anterior segment of the lamina furrow. In dally P2 homozygous larvae, mitotic figures are frequently found in the posterior part of the furrow. The domain of Cyclin B immunoreactivity marking the G2 phase of the first division extends up to these abnormally positioned mitotic cells. The extended Cyclin B domain, and the misplacement of the M phase cells of the first division, suggests that this division cycle is delayed somewhere along the G2-M transition in dally P2 mutants. Given the complete absence of the second LPC division and the dependence of this division on an intercellular signal from photoreceptor axons, whether axons do in fact arrive from the eye disc in dally mutants was examined using an antibody that recognizes axonal membranes. Axons do in fact reach the lamina, and yet the second LPC division fails to take place. The absence of the second LPC division is therefore not a consequence of photoreceptor axons failing to reach the CNS. dally P2 homozygous larvae LPCs do not enter the S phase of the division cycle triggered by photoreceptor axons. Therefore, despite the presence of photoreceptor axons in dally mutants, the second LPC division does not take place as assessed by the absence of the S, G2 and M phases of this division cycle. Cell division defects in dally mutants prove not to be secondary to a gross morphological abnormality (Nakato, 1995).

Integral membrane proteoglycans are cell surface glycoproteins, implicated in regulating growth factor signaling. Proteoglycans bear long unbranched disaccharide polymers (glycosaminoglycans) attached to serine residues of the core protein. These sugar polymers bind a host of extracellular molecules including many growth factors. Cell-associated glycosaminoglycans affect signaling mediated by Fibroblast Growth Factor (FGF), Wingless (Wg) (Reichsman, 1996), TGF-beta, Hepatocyte Growth Factor (HGF) and Heparin Binding-Epidermal Growth Factor (HB-EGF). Betaglycan, a molecule identified on the basis of its affinity for TGF-beta, is a transmembrane proteoglycan that potentiates TGF-beta responses in transfected cells by promoting the interaction of TGF-beta with its signaling receptors. Both syndecans and glypicans, two different types of integral membrane proteoglycans, can affect responses to FGF in tissue culture cells (Jackson, 1997 and references). These studies have made it clear that cell surface proteoglycans can affect growth factor signaling but do not address the role of these molecules in vivo, or during development. For these reasons, the role of Dally was evaluated in the potentiation of Decapentaplegic signaling in Drosophila (Jackson, 1997).

decapentaplegic acts as a genetic enhancer of dally The ability of dpp mutants to affect the severity of the phenotypes found in dally adults was examined. Flies heterozygous for dpp and dally show phenotypes never observed in animals heterozygous for either dpp or dally alone, and the reduction in the eye observed for dally homozygotes is greatly enhanced by reducing dpp function. Heterozygosity for several dpp alleles also increases the penetrance of phenotypes found in dally homozygotes for eye, antenna and genitalia defects. The severity, or expressivity, of the phenotypes is also increased by reducing dpp function. However, the wing phenotypes found in dally mutants (incomplete wing vein V and wing notching) are suppressed by reducing dpp function, suggesting that dally is doing something different in the wing disc than it is in other imaginal tissues. dally mutants also show reduced expression of dpp target genes. The level of expression of two genes that are activated by Dpp signaling, optomotor blind and spalt, is severely reduced in the antenna and eye discs of dally mutants and a similar reduction in spalt expression is observed in the genital discs of dally mutants (Jackson, 1997).

dally mutants are shown to suppress phenotypes resulting from ectopic expression of Dpp in the wing disc. Ectopic Dpp results in overgrowth and wing vein patterning defects limited to the anterior segment of the wing. These phenotypes are rescued in a graded fashion by reducing dally function. A partial rescue is observed in dally heterozygotes and a complete suppression of defects in dally homozygotes. A dally enhancer trap insertion shows that dally is expressed along the wing margin, where reductions in its function could rescue the effects of ectopic Dpp expressed along the anterior segment of the future wing margin. Consistent with the effects in the adult wing, dally mutants reduce the ectopic activation of dpp target genes in the wing (Jackson, 1997).

These findings are entirely consistent with Dally serving as a co-receptor, where Dally binds Dpp and participates in forming a signaling receptor complex. However, other molecular mechanisms are possible: dally could affect a parallel signaling pathway that alters the responses of cells to activation of Dpp signaling. If Dally does affect the Dpp signaling pathway directly, it could influence the distribution or availability of Dpp. It is also possible that Dally and its associated heparan sulfate affect the activity of extracellular enzymes that regulate Dpp. Tolloid, a metalloprotease related to BMP-1, potentiates Dpp activity and could potentially be a target for glycosaminoglycan regulation of its protease activity. Heparin, a short chain glycosaminoglycan synthesized by mast cells and basophils activates the protease inhibitor Antithrombin III, providing a precedent for a glycosaminoglycan controlling extracellular enzyme activity. Whatever the mechanism, cell surface proteoglycans add another dimension to the regulation of growth factor signaling at the cell surface. Further study will be required to determine if Dally signaling somehow suppresses the activity of Dpp at the wing margin via Wg (Jackson, 1997 and references).

GENE STRUCTURE

Bases in 5' UTR - 689 (dally) and 453 (dally-like)

Exons - 7 (dally-like isoform A)

Bases in 3' UTR - 837 (dally) and 998 (dally-like)

PROTEIN STRUCTURE

Amino Acids - 626 (Dally); 765 (Dally-like isoform A)

Structural Domains

Sequencing of the dally cDNA clone reveals a predicted protein sequence homologous to a family of mammalian integral membrane proteoglycans of the glypican type (glypican related integral membrane proteoglycans, or GRIPs). GRIPs are a group of heparan sulfate proteoglycans (HSPGs) that are covalently attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) linkage. Proteoglycans are proteins with covalently attached glycosaminoglycan (GAG) chains GAGs are unbranched polysaccharide chains composed of repeating disaccharide units. Proteoglycans bear long unbranched disaccharide polymers (glycosaminoglycans) attached to serine residues of the core protein. Because sulfate or carboxyl groups are present on most of their sugar residues, GAGs are highly negatively charged. GAGs tend to adopt highly extended conformations that occupy a large volume relative to their mass. The entire predicted Dally protein sequence shows 23.9-25.9% identity and 45.8-48.3% similarity to members of the GRIP family; rat and human Glypican, OCI-5, and Cerebroglycan. Dally shows a similar degree of homology to the different GRIPs as they do to each other. The putative Dally protein also shows several features characteristic of this protein family including (1) a set of 14 conserved cysteine residues found at specific positions in all vertebrate GRIPs, (2) a potential signal peptide at the amino terminus, (3) glycosaminoglycan attachment site consensus sequences and (4) a stretch of hydrophobic amino acid residues at the carboxy terminus required for GPI-anchoring to the cell membrane (Nakato, 1995).

date revised: 20 November 2006

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