Gene name - division abnormally delayed and dally-like
Cytological map position - 66E1--66E2 and 70E5--7
Function - growth factor binding protein
Symbol - dally and dlp
Genetic map position -
Classification - glypican related proteoglycan
Cellular location - membrane bound
|Recent literature||Fernandes, V. M., Pradhan-Sundd, T., Blaquiere, J. A. and Verheyen, E. M. (2015). Ras/MEK/MAPK-mediated regulation of heparin sulphate proteoglycans promotes retinal fate in the Drosophila eye-antennal disc. Dev Biol 402(1):109-18. PubMed ID: 25848695
The Drosophila eye-antennal imaginal disc is a well-characterised system in which to study regional specification; it is first divided into antennal and eye fates and subsequently retinal differentiation occurs within only the eye field. During development, signalling pathways and selector genes compete with and mutually antagonise each other to subdivide the tissue. Wingless (Wg) signalling is the main inhibitor of retinal differentiation; it does so by promoting antennal/head-fate via selector factors and by antagonising Hedgehog (Hh), the principal differentiation-initiating signal. Wg signalling must be suppressed by JAK/STAT at the disc posterior in order to initiate retinal differentiation. Ras/MEK/MAPK signalling has also been implicated in initiating retinal differentiation but its mode of action is not known. This study found that compromising Ras/MEK/MAPK signalling in the early larval disc results in expanded antennal/head cuticle at the expense of the compound eye. These phenotypes correspond both to perturbations in selector factor expression, and to de-repressed wg. Indeed, STAT activity is reduced due to decreased mobility of the ligand Unpaired (Upd) along with a corresponding loss in Dally-like protein (Dlp), a heparan sulphate proteoglycan (HSPG) that aids Upd diffusion. Strikingly, blocking HSPG biogenesis phenocopies compromised Ras/MEK/MAPK, while restoring HSPG expression rescues the adult phenotype significantly. This study identifies a novel mode by which the Ras/MEK/MAPK pathway regulates regional-fate specification via HSPGs during development.
|Ferreira, A. and Milan, M. (2015). Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways. PLoS Biol 13: e1002239. PubMed ID: 26313758
How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The PTEN and TSC-TOR pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. This study used the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. These findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. This study presents evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.
|Norman, M., Vuilleumier, R., Springhorn, A., Gawlik, J. and Pyrowolakis, G. (2016). Pentagone internalises glypicans to fine-tune multiple signalling pathways. Elife [Epub ahead of print]. PubMed ID: 27269283
Tight regulation of signalling activity is crucial for proper tissue patterning and growth. This study investigates the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. Pent was shown to internalise the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and the study proposes that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, a requirement of Pent was found to be required for Wg signalling. These data propose a novel mechanism by which morphogen signalling is regulated.
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).
How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The PTEN and TOR pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. This study used the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic Dpp signaling molecule. The findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. This study presents evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size (Ferreira, 2015).
Evidence is presented that targeted deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways, known to promote tissue overgrowth by increasing the number and/or size of cells, induces a nonautonomous reduction in tissue size of adjacent cell populations. This nonautonomous effect is a consequence of a reduction in both cell size and proliferation rates (cell number), and it is not a consequence of programmed cell death or the withdrawal of nutrients from neighboring tissues, as reducing the levels of proapoptotic genes or subjecting larvae to different amino-acid diets does not have any impact on the size reduction of neighboring cell populations. The glypican Dally, which plays a major role in regulating the spread of Dpp in Drosophila tissues, is up-regulated upon deregulation of these tumor suppressor pathways, and the increase in Dally expression levels contributes to the autonomous effects on tissue size and to the nonautonomous reduction in cell number. Whereas the autonomous effects on tissue size caused by deregulation of these tumor suppressor pathways are most probably due, as least in part, to the capacity of Dally to facilitate Dpp spreading throughout the tissue, it is proposed that the nonautonomous effects on cell number are a consequence of withdrawal of Dpp from neighboring tissues. This proposal is based on a number of observations. First, the width of the Dpp activity gradient as well as the total amount of Dpp activity was reduced in adjacent cell populations upon targeted depletion of tumor suppressor pathways. Second, the nonautonomous effects on tissue size were fully rescued by Dally depletion, which has a rather specific role on the spread of Dpp when overexpressed. Third, the nonautonomous effects on tissue size, growth and proliferation rates, and/or Dpp availability and signaling can be phenocopied by overexpression of Dally or the Dpp receptor Tkv (Ferreira, 2015).
Different strengths of the autonomous and nonautonomous effects were observed upon deregulation of these tumor suppressor pathways or overexpression of Dally in either the A or P compartments. Despite the mild autonomous induction of tissue growth caused by the ci-gal4 driver in A cells, it caused a relatively strong nonautonomous reduction of the neighboring compartment. On the contrary, the en-gal4 driver caused a strong autonomous induction of tissue growth in P cells but a relatively weak nonautonomous reduction of the neighboring compartment. The differential autonomous response might simply reflect different strengths of these Gal4 drivers. By contrast, the strongest nonautonomous effects caused by the ci-gal4 driver (when compared to the en-gal4 driver) might be because Dpp expression is restricted to the A compartment and increased levels of Dally in Dpp expressing cells are more efficient at titrating out the levels of this growth factor from the neighboring compartment. It was noticed that the nonautonomous effects on cell size observed upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways are Dally independent, as overexpression of Dally did not cause a nonautonomous reduction in cell size. Moreover, depletion of Dally did not rescue the nonautonomous reduction in cell size caused by activation of these pathways. These results are consistent with the fact that changes in Dpp signaling do not cause any effect on cell size and indicate that Dally and Dpp are regulating cell number but not cell size. Somatic mutations in tumor suppressor genes such as PTEN or TSC are frequently accumulated in early events of tumor development, and these mutations are thought to contribute to the selection of tumorigenic cells. Competition for available growth factors, by modulating the levels of glypicans, such as Dally, might contribute to the outcompetition of wild-type cells and to the selection of malignant mutation-carrying cells in human cancer (Ferreira, 2015).
The PI3K/PTEN and TSC/TOR signaling pathways play a role not only in disease but also during normal development. These two pathways modulate the final size of the developing organism according to nutrient availability. The current results also identify, in this context, Dally as a molecular bridge between nutrient sensing and wing scaling in Drosophila. In a condition of high nutrient availability, which leads to the activation of the nutrient-sensing PI3K/PTEN and TSC/TOR pathways, increased levels of Dally facilitate the spread of Dpp throughout the growing tissue and contribute to the generation of larger but well-proportioned and scaled adult structures. Depletion of Dally expression levels rescues the tissue growth caused by high levels of nutrients or activation of the nutrient-sensing pathways and gives rise to smaller and, again, well-proportioned and scaled adult structures. Of remarkable interest is the capacity of Dally to induce tissue overgrowth when overexpressed or to mediate tissue growth upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways. Interestingly, deregulation of these pathways, and the resulting tissue overgrowth, leads to the expansion of the Dpp gradient without affecting the total levels of Dpp signaling (Ferreira, 2015).
These results imply that Dpp activity levels do not play an instructive role in promoting tissue growth but rather that it is the range of the Dpp gradient that regulates final tissue size. Consistent with this proposal, depletion of Dally levels in one compartment (which might lead to increased levels of available Dpp in the neighboring cell population) does not cause any visible nonautonomous effect in tissue size. These results are reminiscent of the capacity of Dpp to restrict its own spreading through the repression of Pentagone, a diffusible protein that interacts with Dally and contributes to the expansion of the Dpp gradient. The graded distribution of Dpp leads, via the interaction with its receptor complex, to the graded activation of Mad/Medea, which in turn represses the transcription of brinker (brk). This creates a gradient of Brk expression that is reciprocal to the Dpp gradient. Brk is a transcriptional repressor that acts negatively to establish, in a dose-dependent manner, the expression domain of Dpp target genes like spalt. Thus, Dpp regulates the expression of target genes by repressing brinker. Remarkably, the reduced size of the wing primordium observed in hypomorphic alleles of dpp is restored when combined with brk mutants. This experimental evidence indicates that Dpp controls wing growth entirely via repression of brk. The Dally-mediated increase in the width of the Dpp gradient observed upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways might contribute to restrict the expression domain of brk to the lateral sides of the wing primordium. Similarly, the nonautonomous decrease in the width of the Dpp gradient might cause an expansion of the brkdomain, which is known to repress growth. Interestingly, Dally-mediated spreading of other secreted growth factors might also contribute to the autonomous effects on tissue growth caused by deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways. This is revealed by the fact that Dally depletion rescues both the autonomous and the nonautonomous effects, whereas deregulation of these pathways are still able to induce some growth upon knocking down Dpp (Ferreira, 2015).
Compartments have been proposed to be units of growth control. In other words, the size of each compartment is controlled independently. The results on the lack of nonautonomous effects on tissue growth upon depletion of Dally or Sfl, the enzyme needed for the modification of HS chains within glypicans, indicate that this is the case. Targeted depletion of glypican expression or activity in the developing compartments gave rise to an autonomous reduction in tissue size without affecting the neighboring compartment. However, independent lines of evidence support the view that adjacent compartments buffer local variations in tissue growth caused by different means, including a nonautonomous reduction in tissue size upon depletion of the protein biosynthetic machinery or reduced epidermal growth factor receptor (EGFR) activity. The current results on the capacity of overgrowing compartments to withdraw Dpp from neighboring tissues upon targeted deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways and to cause a nonautonomous reduction in growth and proliferation rates reinforce the view that compartments are susceptible to modulate their growth rates upon different types of stress, including depletion of tumor suppressor genes. Interestingly, the halteres and wings of Drosophila are homologous thoracic appendages, and the activity of the Ultrabithorax (Ubx) Hox gene in the haltere discs contributes to defining its reduced size. Remarkably, it does so by reducing the expression levels of Dally, thus reinforcing the role of Dally in modulating tissue growth in epithelial organs (Ferreira, 2015).
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)
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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