InteractiveFly: GeneBrief

division abnormally delayed and dally-like: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - division abnormally delayed and dally-like

Synonyms -

Cytological map position - 66E1--66E2 and 70E5--7

Function - growth factor binding protein

Keywords - wing, optic lobe, Decapentaplegic signaling

Symbol - dally and dlp

FlyBase ID: FBgn0263930 and FBgn0041604

Genetic map position -

Classification - glypican related proteoglycan

Cellular location - membrane bound



NCBI links for Dally: Precomputed BLAST | Entree Gene

NCBI links for Dally-like: Precomputed BLAST | Entree Gene

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
Summary:
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
Summary:
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.

Dear, M. L., Shilts, J. and Broadie, K. (2017). Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis. Sci Signal 10(504). PubMed ID: 29114039
Summary:
Matrix metalloproteinase (MMP) functions modulate synapse formation and activity-dependent plasticity. Aberrant MMP activity is implicated in fragile X syndrome (FXS), a disease caused by the loss of the RNA-binding protein FMRP and characterized by neurological dysfunction and intellectual disability. Gene expression studies in Drosophila suggest that Mmps cooperate with the heparan sulfate proteoglycan (HSPG) glypican co-receptor Dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling and that synaptogenic defects in the fly model of FXS are alleviated by either inhibition of Mmp or genetic reduction of Dlp. This study used the Drosophila neuromuscular junction (NMJ) glutamatergic synapse to test activity-dependent Dlp and Mmp intersections in the context of FXS. Rapid, activity-dependent synaptic bouton formation depended on secreted Mmp1. Acute neuronal stimulation reduced the abundance of Mmp2 but increased that of both Mmp1 and Dlp, as well as enhanced the colocalization of Dlp and Mmp1 at the synapse. Dlp function promoted Mmp1 abundance, localization, and proteolytic activity around synapses. Dlp glycosaminoglycan (GAG) chains mediated this functional interaction with Mmp1. In the FXS fly model, activity-dependent increases in Mmp1 abundance and activity were lost but were restored by reducing the amount of synaptic Dlp. The data suggest that neuronal activity-induced, HSPG-dependent Mmp regulation drives activity-dependent synaptogenesis and that this is impaired in FXS. Thus, exploring this mechanism further may reveal therapeutic targets that have the potential to restore synaptogenesis in FXS patients.
Kanai, M. I., Kim, M. J., Akiyama, T., Takemura, M., Wharton, K., O'Connor, M. B. and Nakato, H. (2018). Regulation of neuroblast proliferation by surface glia in the Drosophila larval brain. Sci Rep 8(1): 3730. PubMed ID: 29487331
Summary:
Despite the importance of precisely regulating stem cell division, the molecular basis for this control is still elusive. This study shows that surface glia in the developing Drosophila brain play essential roles in regulating the proliferation of neural stem cells, neuroblasts (NBs). Two classes of extracellular factors, Dally-like (Dlp), a heparan sulfate proteoglycan, and Glass bottom boat (Gbb), a BMP homologue, are required for proper NB proliferation. Interestingly, Dlp expressed in perineural glia (PG), the most outer layer of the surface glia, is responsible for NB proliferation. Consistent with this finding, functional ablation of PG using a dominant-negative form of dynamin showed that PG has an instructive role in regulating NB proliferation. Gbb acts not only as an autocrine proliferation factor in NBs but also as a paracrine survival signal in the PG. It is proposed that bidirectional communication between NBs and glia through TGF-β signaling influences mutual development of these two cell types. The possibility is discussed that PG and NBs communicate via direct membrane contact or transcytotic transport of membrane components. Thus, this study shows that the surface glia acts not only as a simple structural insulator but also a dynamic regulator of brain development.
Su, T. Y., Nakato, E., Choi, P. Y. and Nakato, H. (2018). Drosophila glypicans regulate follicle stem cell maintenance and niche competition. Genetics. PubMed ID: 29632032
Summary:
Adult stem cells reside in specialized microenvironments, called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. This study shows that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and FSC competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hyper-competitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNAi knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition.
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).

Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways

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).

Pentagone internalises glypicans to fine-tune multiple signalling pathways

Tight regulation of signalling activity is crucial for proper tissue patterning and growth. This study investigates the function of Pentagone (Pent/Magu), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. It was shown that Pent internalises 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, Pent was found to be required for Wg signalling. These data propose a novel mechanism by which morphogen signalling is regulated (Norman, 2016).

Bone morphogenetic protein (BMP) signalling is required in a wide variety of processes across higher organisms, from the establishment of the dorso-ventral (DV) axis in insects to the maintenance of the mammalian gut. Many of the biological functions of BMP signalling require a high degree of spatial regulation; accordingly, multiple mechanisms have evolved that control the movement, stability and activity of BMP ligands (Norman, 2016).

One of the most intensely studied examples of extracellular regulation of BMP comes from Drosophila wing development, a tissue where Dpp (Drosophila BMP2/4) acts as a morphogen to control both patterning and growth. During larval wing development, Dpp is produced in a stripe of cells at the anterior-posterior (AP) boundary and disperses into both compartments, by mechanisms that are still not fully understood, to organize a BMP signalling activity gradient along the AP axis with highest levels in medial and lowest in lateral regions. Dpp, together with a second, uniformly expressed ligand, Glass bottom boat (Gbb), activates membrane bound receptors and induces the phosphorylation of the transcription factor Mad. Phosphorylated Mad (pMad) accumulates in the nucleus with the cofactor Medea, where the activated Smad complex directly regulates BMP-target gene transcription (Norman, 2016).

In addition to the localized production of Dpp, many other determinants impact on proper establishment and maintenance of the activity gradient. Prominent amongst them are membrane-bound BMP-binding proteins, such as Thickveins (Tkv) and Dally, which have dual functions in the establishment of the pMad gradient. Tkv is cell-autonomously required for signalling as it is the main type I BMP receptor in Drosophila. At the same time, Tkv critically affects Dpp tissue distribution through ligand trapping and internalisation, and thus globally shapes the BMP activity gradient. Similarly, Dally, a GPI-anchored heparan sulfate proteoglycan (HSPG), binds and concentrates Dpp at cell surfaces and, together with a second glypican, Dally-like protein (Dlp), is required for both local signal activation and long-range distribution of the ligand. Absence of glypicans- for example in a clone of cells- can result in both a reduction of BMP signalling within the clone and an interruption of Dpp spreading within and beyond the clone. In addition, glypicans can also, by virtue of their ligand-binding capacity, hinder the movement of ligands in several contexts. For example, the diffusion of BMP4 (a vertebrate homolog to Dpp) during early Xenopus embryogenesis has been shown to be restricted through its interactions with HSPGs. Similarly, in the wing disc, increasing the levels of Dally at the source of Dpp causes a local increase of signalling activity and a drastic compaction of the gradient due to ligand trapping. Reflecting the importance of the activity of Tkv and Dally for proper gradient establishment, the levels of both proteins are tightly regulated along the AP axis of the developing disc. Through complex transcriptional regulation, which involves repression by BMP signalling itself, both Tkv and Dally are down-regulated near the ligand source to maintain the proper balance between Dpp signalling and Dpp dispersion (Norman, 2016).

Pentagone (Pent; also known as Magu), is an additional determinant in the establishment, maintenance and scaling of the BMP signalling gradient in the developing wing. The transcription of pent is directly repressed by BMP signalling, hence its production is restricted to the lateral-most cells of the disc. Pent protein is, however, secreted and distributes in a gradient that is inverse to the pMad gradient. Pent mutants have a restricted pMad gradient with abnormally high levels in the centre of the disc and very low levels in lateral regions; consequently, adult wings have growth and patterning defects in lateral regions. The pMad gradient of pent mutants thus resembles the abnormal gradients caused by medial over-expression of Tkv or Dally, suggesting an interaction of the protein with the BMP-reception system. Indeed, past work established that Pent physically associates with Dally on cell membranes, but the consequences of this interaction have remained unclear (Norman, 2016).

This study presents data showing that Pent binds and induces the internalisation of both Drosophila glypicans, resulting in reduction of Dally and Dlp protein levels. Endocytosis of glypicans is dependent on dynamin and Rab5, but does not require clathrin or Dpp signalling. Additionally, it was shown that Pent influences Wg signalling, which also depends on glypicans. It is concluded from these data that Pent modulates glypican levels in order to modify multiple signalling pathways during wing morphogenesis. The data suggest an additional, protein-level feedback mechanism to tightly control levels of signalling, which cooperates with transcriptional regulatory feedback loops to ensure proper morphogen gradient formation and organ development (Norman, 2016). It has been proposed that the function of Pent in Dpp gradient formation could be to either enhance the ability of Dally to displace Dpp, or to reduce the co-receptor function of Dally. From the data presented in this study, it is proposed that Pent reduces the co-receptor function of glypicans by binding them and inducing their internalisation. It is suggested that Pent may promote spreading of Dpp by reducing Dpp co-receptors and therefore Dpp trapping and signal transduction. Internalisation of glypicans is independent of signalling and Dpp itself, which fits the model that removal of glypicans by Pent enhances spreading. Furthermore, this study has presented data showing that by regulating glypican levels, Pent is also able to modulate Wingless signalling (Norman, 2016).

The work proposes that Pent modulates Dpp signalling via the co-receptors Dally and Dlp. The relative contribution of Dally and Dlp to Dpp signalling is unknown, although both must be removed in order for a reduction in pMad to occur. Data showing that Pent influences the co-receptor but not the receptor itself distinguishes Pent from other BMP signalling modifiers in D. melanogaster, such as Crossveinless-2, Short gastrulation and Twisted gastrulation, which bind either the BMP ligand, the receptor, or both. This could reflect the different roles that BMP signalling must fulfill in D. melanogaster, where it forms a long-range gradient in the larval wing disc but short-range gradients in the embryo and pupal wing (Norman, 2016).

The data show that Pent binds and internalises glypicans. Prior to endocytosis, it is probable that glypicans are clustered at the cell surface by Pent, and this might also inhibit their function without necessarily inducing their internalisation. Glypicans share physical properties, notably a GPI anchor and heparan sulphate side chains, upon which Pent binds. This study has shown that internalisation of glypicans by Pent requires dynamin and Rab5 but not clathrin. Cell culture experiments have shown that GPI proteins are commonly endocytosed via clathrin independent mechanisms, but this has not been demonstrated before in Drosophila. Many clathrin independent mechanisms have been described in cultured cells, but in vivo evidence for many of them is lacking. Lipid-rich microdomains, in some cases marked by flotillin, can be involved, but this study found no requirement for flotillin in the endocytosis of glypicans by Pent (Norman, 2016).

One of the key problems cells must overcome to internalise GPI anchored proteins is that they have no cytoplasmic region to mediate recruitment into endocytic pits. A similar process to that described in this study, the Hh mediated internalisation of GPC3, is thought to utilise LRP1 in order to communicate with the endocytic machinery. This does not seem to be the case with Pent and Dally, as knockdown of the Drosophila homologue of LRP1 does not affect internalisation. It is possible that protein clustering, and the membrane deformations this has been predicted to cause, may be involved in the internalisation of glypicans by Pent. The precise mechanism by which Pent internalises glypicans will be an interesting avenue of future research (Norman, 2016).

While the data implicate glypicans as the direct target of Pent's activity, it cannot be ruled out that the effect on Dpp gradient formation involves the regulation of Tkv levels and/or activity. While Pent does not bind to Tkv directly and Pent over-expression seems not to affect the levels of membrane bound Tkv, a substantial amount of the receptor is found in Pent- and Dally positive endocytic vesicles. This raises the possibility that Pent might target a specific subpopulation of Tkv for degradation, and that this interaction requires glypicans as adaptors (Norman, 2016).

The data show that Pent binds and internalises Dally and Dlp. Glypicans, in particular Dally, have been shown to regulate the spreading of Dpp, in addition to being essential for Dpp signal transduction itself. The molecular basis for these activities is the binding of Dpp to the heparan sulphate side-chains of glypicans, probably a first step that serves to concentrate Dpp at the surface of the disc epithelium. Glypican-bound Dpp molecules can follow multiple routes, as they can be passed to receptors (promoting signalling), to glypicans of neighbouring cells (promoting ligand dispersion), or can persist on glypicans of the same cell resulting in local ligand enrichment. It is probable that the specific outcome at any position along the morphogen field will depend on the relative levels and activities of the involved factors, i.e. the ligand, receptors and glypicans. A similar balance between glypicans, receptors and ligands has been proposed to explain the biphasic activity of Dlp in Wg signalling in the wing imaginal disc. In the case of Dpp, levels of glypicans need to be tightly regulated to allow for the optimal balance between ligand release, trapping and receptor binding. The data suggest that Pent contributes to this balance by fine-tuning the levels of glypicans. It is proposed that in the absence of Pent, glypican levels are too high and this results in excessive ligand trapping and enhanced local signalling. Such local effects would be accompanied with a non-autonomous reduction in ligand spreading and shrinkage of the pMad gradient. An approximation mimicking this situation is artificially elevating levels of Dally in medial regions, which has been shown to locally increase pMad. This study has shown that this increase in signalling by Dally is at the expense of Dpp spreading to the rest of the disc and the formation of the long range pMad gradient. This clearly shows that excess Dally can block spreading of Dpp. Notably, pent mutants display a similarly compacted activity gradient with high medial and low lateral pMad levels. Importantly, ligand-binding properties of HSPGs have been described to impede ligand spreading in multiple physiological contexts, including BMP4 in Xenopus early dorso-ventral patterning and FGF10 in its role in branching morphogenesis (Norman, 2016).

Multiple transcriptional feedback loops are required for the maintenance of the Dpp signalling gradient in the wing. Primary amongst these is the repression of Tkv and Dally transcription by Dpp signalling. This ensures that receptor and co-receptor levels are low near the Dpp producing cells, allowing Dpp to spread out from the centre of the disc. These feedback loops are important for proper establishment of the Dpp signalling gradient. However, such direct feedbacks targeting the production of molecules with ligand-binding properties may have limitations. In response to a reduction in spreading of Dpp, Tkv and Dally levels would increase to locally compensate the reduction in Dpp signalling activity. Such an increase would, however, further enhance trapping and internalisation of the ligand and, at the level of the whole wing disc, would further block Dpp spreading. From the data it is suggested that Pent, a secreted negative regulator of Dpp signalling, fine-tunes the signalling gradient at a different level, by directly adjusting glypican levels and reducing the inbuilt increase in co-receptor and ligand-trapping upon a reduction in the extent of the pMad gradient. This might happen at a critical region of the wing disc, the mediolateral cells, where declining levels of the spreading ligand face increasing levels of the receptor and co-receptor (Tkv and Dally, respectively). Pent, secreted by lateral cells next to this region, could reduce the glypican pool to allow Dpp to overcome excessive ligand trapping and thus promote further spreading. Consistent with such a 'remote' activity, Pent can be detected throughout the wing disc. As Pent is transcriptionally repressed by Dpp signalling and, unlike Tkv and Dally, does not bind Dpp, Pent might be a good candidate for how the system overcomes the inherent limitations of feedback loops involving membrane tethered, Dpp-binding proteins (Norman, 2016).

The key extracellular signalling molecules of the wing disc, Dpp, Wg and Hh, all bind to glypicans. The regulatory proteins Pent, Notum and Shifted also bind glypicans, putting glypicans at the centre of signalling regulation in the wing disc. Consequently, any factor that affects glypican function, such as Pent, is likely to modify multiple signalling pathways. This study has shown that Pent is also able to influence Wg signalling, thus providing a possible link between the Wg and Dpp pathways (Norman, 2016).

The role of glypicans in Wg signalling is well described and complex. Dlp can stimulate Wg signalling, Wg accumulates on cells over-expressing Dlp and fails to accumulate on cells mutant for Dlp. Similarly, the data show that excess Pent internalises glypicans and reduces extracellular Wg. Precise in vitro assays have shown that low levels of Dlp enhance Wg signalling, but too much Dlp reduces signalling. Furthermore, recent evidence shows that deacylation of Wg by Notum, which reduces Wg signalling activity, requires glypicans. It is clear, then, that the level of glypicans must be very finely balanced for Wg signalling to be at the correct level. It is proposed that the elevated glypican levels observed in the absence of Pent push this fine balance towards inhibition of signalling, due to the increased levels of glypicans sequestering Wg away from the receptor and also increasing the platform upon which Notum can deacylate Wg. Consistent with this conclusion, the effects of Notum and Dlp over-expression can be suppressed by increasing the level of Pent protein (Norman, 2016).

Interestingly, inactivation of the BMP-response elements in the regulatory region of the pent gene locus results in prominent expression of pent at the DV boundary, hinting at an input into pent transcription from DV signals. Future studies, including quantitative studies and modelling, should give further insight into pathway interaction and coordination during tissue development by molecules such as Pent (Norman, 2016).

A model is proposed that Pent internalises glypicans to modify multiple signalling pathways. Future work should address the influence of Pent on glypican organisation at the nanoscale, and also the type of membranes at which Tkv and Dally localise, questions that are challenging to answer using current methods. In order to fully understand the role of Pent in establishment of the long range Dpp gradient, first it is important to gain a better understand how glypicans function in Dpp signalling and how Dpp is spread throughout the tissue (Norman, 2016).


REGULATION

Transcriptional Regulation

Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. These and other downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern (Liang, 1998).

The expression pattern of dally, monitored by dally::lacZ enhancer-trap expression, in the developing wing along the AP axis shows a peak of expression at the A/P border cells, and dally levels are lowest in cells adjacent to this region. Furthermore, dally levels gradually increase toward the anterior and posterior distal cells. This pattern correlates with the expression patterns of several genes involved in pattern formation along the AP axis, such as tkv and master of thick veins (mtv; also known as scribbler/breakless); this suggests that dally also participates in this process. Expression of the tkv gene is controlled by two distinct pathways. (1) Hh represses tkv expression at the A/P border cells, and En regulates a high basal level of tkv in the P compartment. The activities of both hh and en genes are mediated by a putative transcription factor, Mtv. (2) tkv levels are downregulated by Dpp signaling. By this mechanism, tkv expression is maintained at low levels in the center of the disc and at higher levels toward the anterior and posterior edges. The correlation between expression patterns of dally and tkv prompted an analysis of the dally function in Dpp signaling in this tissue. Regulatory pathways controlling dally expression were analyzed and compared with those controlling tkv expression (Fujise, 2003).

Hh signaling induces dally expression at the A/P border cells. dally expression is absent in smoothened (smo) mutant clones generated in the anterior compartment, where the Hh signaling is blocked, indicating that Hh signaling is required for activation of dally at the A/P border cells. To further determine whether Hh signaling is sufficient for the induction of dally, clones were examined that ectopically express hhCD2, which encodes a membrane-tethered form of Hh, using the FLP-OUT system. In the A compartment, dally expression levels are increased in hhCD2-expressing cells and in cells immediately adjacent to them. This result shows that Hh expression is sufficient to induce dally expression in the A compartment. To determine if dally expression is controlled by en, which upregulates tkv expression, clones of en-mutant cells were induced using the FLP-FRT mosaic analysis system. Within en-mutant clones in the P compartment, dally levels are dramatically increased; this indicates that dally expression is negatively regulated by en (Fujise, 2003).

To determine whether modulation of Dpp signaling affects dally expression, the dally::lacZ expression was compared between wild-type and tkv heterozygous cells. Clones mutant for tkv were generated in a heterozygous background (tkva12/+) using the FLP-FRT system, which should, as a consequence, produce both mutant (tkva12/tkva12) and wild-type sister clones (+/+). However, tkv cells do not survive in the wing pouch since Tkv activity is indispensable for growth and, thus, only wild-type sister clones survive. In resultant mosaic discs with wild-type and tkv-heterozygous cells, dally expression is decreased cell autonomously in wild-type (+/+) clones at the AP border and peripheral to the border. To further confirm this result, the effects were examined of tkv-hypomorphic clones on dally expression. In such clones, where tkv activity is partially compromised, the levels of dally expression are elevated. In the notum region of the wing disc, tkv-null clones can be generated in which a substantial increase of dally expression is observed. Finally, the effect of increased Dpp signaling on dally expression was tested by using the FLP-OUT method to induce clones of cells that express tkvQ253D, a constitutively active form of tkv, in the wing pouch. The level of dally::lacZ expression was found to be autonomously reduced in the tkvQ253D-expressing clones. All of these results consistently indicate that dally expression in the wing disc is negatively regulated by Dpp signaling, as has been shown for tkv. Thus, dally and tkv are regulated by the same set of molecular pathways: Hh, En and Dpp signaling (Fujise, 2003).

Although dally expression is regulated by the same set of signaling pathways that control expression of tkv the effects of Hh and En on dally are the opposite of those on tkv. In addition, dally expression is negatively controlled by Dpp signaling. Through this mechanism, relative levels of dally expression are higher at the anterior and posterior distal edges. Therefore, dally and tkv show similar patterns of expression with one exception: the level of dally expression is high in A/P border cells, where Dpp is synthesized and secreted, but by contrast, tkv expression levels are low in this region. The high levels of dally in the peripheral regions could sensitize cells to low levels of Dpp, as has been shown for tkv. These regulatory pathways appear to form negative feedback loops, which may stabilize the shape of the Dpp morphogen gradient. Thus, the regulated expression and function of Dally are crucial factors in the generation and maintenance of the Dpp morphogen gradient (Fujise, 2003).

Increased avidity for Dpp/BMP2 maintains the proliferation of progenitors-like cells in the Drosophila eye

During organ development, the progenitor state is transient, and depends on specific combinations of transcription factors and extracellular signals. Not surprisingly, abnormal maintenance of progenitor transcription factors may lead to tissue overgrowth, and the concurrence of signals from the local environment is often critical to trigger this overgrowth. Therefore, identifying specific combinations of transcription factors/signals promoting (or opposing) proliferation in progenitors is essential to understand normal development and disease. This study used the Drosophila eye as a model where the transcription factors hth and tsh are transiently expressed in eye progenitors causing the expansion of the progenitor pool. However, if their co-expression is maintained experimentally, cell proliferation continues and differentiation is halted. Hth+Tsh-induced tissue overgrowth was shown to require the BMP2 Dpp and the abnormal hyperactivation of its pathway. Rather than using autocrine Dpp expression, Hth+Tsh cells increase their avidity for Dpp, produced locally, by upregulating extracellular matrix components. During normal development, Dpp represses hth and tsh ensuring that the progenitor state is transient. However, cells in which Hth+Tsh expression is forcibly maintained use Dpp to enhance their proliferation (Neto, 2016).

Abnormal maintenance of transcription factors that promote an undifferentiated, proliferative state is often an initiating event in tumors. However, abnormal growth is dependent on specific non-autonomous signals provided by the microenvironment. This study used an experimental system that results in continuous growth to identify these signals and the mechanism of action. In this system, the GAL4-driven maintenance during eye development of hth and tsh, two transcription factors normally transiently co-expressed in eye progenitors, cause cells to increase their avidity for Dpp. This, in turn, leads to a hyper-activation of the pathway, which is necessary to maintain the proliferative/undifferentiated phenotype. The increased avidity for Dpp was shown to be mediated, at least partly, through increased expression of the proteoglycans components encoded by dally and dlp, functionally modified by slf (Neto, 2016).

Progenitor cells, forced to maintain Hth and Tsh (hth+tsh progenitor-like cells) trap Dpp produced at local sources, which then causes an increased in intracellular signaling. The mechanism responsible of this trapping seems to be the increase of extracellular matrix (ECM) components. First, a cell-autonomous increase was found in dally transcription and Dlp membrane levels, the two glypican moieties of heparane sulphate proteoglycans. Second, the RNAi-mediated attenuation of sfl function, a gene encoding an enzyme required for the biosynthesis of these proteoglycans, is required for the overgrowth/eye-suppression phenotype induced by hth+tsh maintenance. A third line of support comes from examination of the effects of hth+tsh or hth+tsh+slf RNAi on the pMad profiles. Considering that the Dpp production remains unaltered, hth+tsh tissue shows an increase in both pMad signal amplitude and range, which is consistent with the increase in proteoglycans simultaneously augmenting Dpp diffusion and stability. On the contrary, reducing proteoglycan biosynthesis in hth+tsh+slf RNAi cells results in the retraction of the pMad signaling range back towards control values, which again is expected if Dpp's diffusion depends on proteoglycans (Neto, 2016).

By forcing the expression of hth and tsh in eye precursors, these cells are exposed to signaling levels higher than they would normally encounter. This is because during normal eye development Dpp, produced at the furrow, represses first hth and then, closer to the furrow, also tsh, so that the cells approaching the furrow and receiving the highest Dpp levels no longer co-express hth and tsh. The loss of hth marks the transition between proliferation/undifferentiation and cell quiescence/commitment. This transition coincides with a transient proliferative wave (the so-called 'first mitotic wave') that precedes entry into G1. This transition zone corresponds to a region where low, but not null, levels of Hth and pMad signals overlap. If the interaction between hth+tsh and the Dpp pathway described in this study were to hold also in the zone of hth/Dpp signal overlap during normal eye development (remember that hth-positive cells co-express normally tsh too), one prediction would be that the mitotic wave would be lost if either hth or dpp-signaling were removed. Indeed this has been shown to be the case: RNAi-mediated attenuation of hth or abrogation of Dpp signaling result in the loss of the first mitotic wave. However, it is not thought that the mechanisms driving Dpp-mediated proliferation of optix> hth+tsh cells are necessarily the same as those operating normally in hth+tsh-expressing progenitors during eye development, because of the following experiment. Discs were generated expressing in their dorsal domain an RNAi targeting Hth's partner, the Pbx gene extradenticle (exd). In the absence of Exd, Hth is degraded. Therefore, a depletion of Exd causes an effective loss of Hth. Knowing that in optix>hth+tsh the stability and diffusion of Dpp were increased, the prediction would be that the loss of hth (in exd-depleted cells) should cause a decrease in both the stability and diffusion of Dpp. However, when the dorsal ('exd-') with the ventral ('exd+') pMad profiles of D>exdRNAi discs was quantified, it was found that both the stability and diffusion of Dpp increased by the loss of hth. This result suggests that during normal eye development hth (perhaps together with tsh) influences Dpp signaling, but the mechanisms described in this study as triggered by forced hth+tsh expression are likely different (Neto, 2016).

The upregulation of dally and dlp by hth+tsh is likely the consequence of the transcriptional activity of Hth+Tsh in partnership with the YAP/TAZ homologue, Yki, as previous work showed that loss of the protocadherin genes fat (ft) and dachsous (ds) , which causes the activation of Yki, results in an upregulation of dally and dlp in the wing primordium. In fact, previous studies have found, in imaginal tissues, binding of Yki and Hth to nearby sites on the dlp locus, suggesting that some of this regulation might be direct. All these data make Yki a necessary component of the molecular machinery responsible for the increased avidity of hth+tsh cells for Dpp. However, in the eye primordium, the overexpression of Yki induces a different phenotype than hth+tsh. More importantly, in the eye primordium, yki+ clones do not cause the autonomous upregulation of pMad signal that hth+tsh clones do. Therefore, a specific stoichiometry among Hth, Tsh and Yki is likely necessary to induce the Dpp signaling-dependent properties of hth+tsh cells, at least in the developing eye. Alternatively, Hth and Tsh may activate Yki-independent targets that would be required for the full expression of the phenotype. Recently, another study has found that Yki and the Dpp pathway synergize in stimulating tissue overgrowth, both in eye and wing primordia, through the physical association between Yki and Mad. The current results suggest that hth+tsh progenitor-like cells establish a positive feedback, in which the growth promoting activity of the Hth:Tsh:Yki complex would be enhanced by increasing levels of pMad activated by Dpp. This feedback would be region-specific, as it depends on sources of Dpp that are localized within the eye primordium. Further work is needed to investigate the molecular mechanisms behind this feedback. Finally, it has been shown recently that tissue growth promoted by the PI3K/PTEN and TSC/TOR nutrient-sensing pathways also requires Dally, which, in turn, increases the avidity of the growing tissue for Dpp. Therefore, increasing the avidity for Dpp by augmenting proteoglycan levels may be a common strategy of tissues to sustain their growth (Neto, 2016).

Protein Interactions: Potential role for Dally or other glycosaminoglycans in Wingless signaling

The Drosophila gene sugarless encodes a homolog of vertebrate UDP-glucose dehydrogenase. This enzyme is essential for the biosynthesis of various proteoglycans. In the absence of both maternal and zygotic activities of this gene, mutant embryos develop with segment polarity phenotypes reminiscent of the loss of either Wingless or Hedgehog signaling. To analyze the function of Sugarless in cell-cell interaction processes, attention has been focused on the requirement of Sugarless for Wingless signaling in different tissues. sugarless mutations impair signaling by Wingless, suggesting that proteoglycans contribute to the reception of Wingless. Overexpression of Wingless can bypass the requirement for sugarless, suggesting that proteoglycans modulate signaling by Wingless, possibly by limiting its diffusion and thereby facilitating the binding of Wingless to its receptor. Tissue-specific regulation of proteoglycans may be involved in regulating both Wingless short- or long-range effects. For example, dally mutations show wing notching with loss of wing margin structures, an effect seen in wingless and dishevelled mutants, suggesting the potential involvement of Dally in Wg signaling. A Drosophila homolog of vertebrate Syndecans (see Syndecan) has also been characterized. Syndecan is a transmembrane HSPG and represents the major source of HSPGs in epithelial cells. Syndecan has been demonstrated to function as a coreceptor for FGF signaling. Further genetic and biochemical studies should reveal whether Dally and/or Syndecan play a direct role in Wg signaling (Hacker, 1997).

When expressed in S2 cells, the majority (approximately 83%) of secreted Wingless protein (WG) is bound to the cell surface and extracellular matrix through specific, noncovalent interactions. The tethered WG can be released by addition of exogenous heparin sulfate and chondroitin sulfate glycosaminoglycans. WG also binds directly to heparin agarose beads with high affinity. These data suggest that WG can bind to the cell surface via naturally occurring sulfated proteoglycans. Two lines of evidence indicate that extracellular glycosaminoglycans on the receiving cells also play a functional role in WG signaling: (1) treatment of WG-responsive cells with glycosaminoglycan lyases reduces WG activity by 50%; (2) when WG-responsive cells are preincubated with 1 mM chlorate, which blocks sulfation, WG activity is inhibited to near-basal levels. Addition of exogenous heparin to the chlorate-treated cells restores WG activity. Based on these results, it is proposed that WG belongs to the group of growth factor ligands whose actions are mediated by extracellular proteoglycan molecules (Reichsman,1996).

The Drosophila UDP-glucose dehydrogenase gene is involved in Wingless signaling. UDP-glucose dehydrogenase is an enzyme responsible for the production of UDP-glucuronate. UDP-glucuronate, a uronic acid, is a precursor in glycosaminoglycan (GAG) biosynthesis. GAG is a complex carbohydrate which is linked to a distinct class of glycoproteins called proteoglycans. A mutation in UDP-glucose dehydrogenase, called kiwi, generates a phenotype identical to that of wingless. By rescuing the kiwi phenotype with both UDP-glucuronate and the glycosaminoglycan heparan sulfate, it has been shown that kiwi function in the embryo is crucial for the production of heparan sulfate in the extracellular matrix. Injection of heparin degrading enzyme, heparinase (and not chondroitin, dermatan or hyaluronic acid degrading enzyme) into wild-type embryos leads to the degradation of heparin-like glycosaminoglycans and a 'wingless-like' cuticular phenotype. Heparin-like GAGs are complex acidic polysaccharides that are important soluble components of the extracellular matrix. They provide the necessary hydration to the ECM scaffold and hence solubilize and modulate the function of such proteins as fibroblast growth factor. This study provides the first genetic evidence for the involvement of heparin-like glycosaminoglycans in signal transduction (Binari, 1997).

In vitro experiments suggest that glycosaminoglycans (GAGs) and the proteins to which they are attached (proteoglycans) are important for modulating growth factor signaling. However, in vivo evidence to support this view has been lacking, in part because mutations that disrupt the production of GAG polymers and the core proteins have not been available. The identification and characterization of Drosophila mutants in the suppenkasper (ska) gene is described. The ska gene encodes UDP-glucose dehydrogenase, which produces glucuronic acid, an essential component for the synthesis of heparan and chondroitin sulfate. ska mutants fail to put heparan side chains on proteoglycans such as Syndecan. Surprisingly, mutant embryos produced by germ-line clones of this general metabolic gene exhibit embryonic cuticle phenotypes strikingly similar to those that result from loss-of-function mutations in genes of the Wingless (Wg) signaling pathway. Zygotic loss of ska leads to reduced growth of imaginal discs and pattern defects similar to wg mutants. In addition, genetic interactions of ska with wg and dishevelled mutants have been observed. These data demonstrate the importance of proteoglycans and GAGs in Wg signaling in vivo and suggest that Wnt-like growth factors may be particularly sensitive to perturbations of GAG biosynthesis (Haerry, 1997).

In Drosophila, most Wnt-mediated patterning is performed by a single family member, Wingless (Wg), acting through its receptors Frizzled (Fz) and Frizzled2 (Fz2). In the ventral embryonic epidermis, Wg signaling generates two different cell-fate decisions: the production of diverse denticle types and the specification of naked cuticle separating the denticle belts. Mutant alleles of wg disrupt these cellular decisions separately, suggesting that some aspect of ligand-receptor affinity influences cell-fate decisions, or that different receptor complexes mediate the distinct cellular responses. Overexpression of Fz2, but not Fz, rescues the mutant phenotype of wgPE2, an allele that produces denticle diversity but no naked cuticle. Fz is able to substitute for Dfz2 only under conditions where the Wg ligand is present in excess. The wgPE2 mutant phenotype is also sensitive to the dosage of glycosaminoglycans, suggesting that the mutant ligand is excluded from the receptor complex when proteoglycans are present. It is concluded that wild-type Wg signaling requires efficient interaction between ligand and the Fz2-proteoglycan receptor complex to promote the naked cuticle cell fate (Moline, 2000).

When ubiquitously expressed in a wild-type embryo, wgPE2 subtly changes the denticle pattern and shows a slight dominant-negative effect on naked cuticle formation. This contrasts with ubiquitous expression of wild-type wg, which produces uniform naked cuticle. Ubiquitous expression of wgPE2 in a wg null mutant embryo rescues denticle diversity, but does not significantly rescue naked-cuticle formation. However, coexpression of Fz2 and wgPE2 in wg null mutants produces uniform naked cuticle, as does ubiquitous expression of wild-type wg alone. Thus, the ability of wgPE2 to generate the naked-cuticle cell fate depends on overexpression of Fz2. A slight interaction was also detected with Fz under conditions of high-level coexpression, suggesting that amounts of Wg in excess of physiological concentrations permit interaction with Fz receptor (Moline, 2000).

Indeed, this observation offers an explanation for the apparent genetic redundancy of Fz and Fz2 in embryonic Wg signaling. In the absence of zygotic Fz2 receptor, Wg protein may accumulate to a level sufficient to activate Fz receptor, which then promotes normal epidermal patterning. An increased accumulation of Wg protein was detected in embryos zygotically deficient for Fz2, compared either with wild-type embryos or embryos maternally and zygotically deficient for fz. This suggests that Wg ligand is not internalized and degraded as efficiently when Fz2 is absent from the cell surface, thereby permitting interactions with Fz that are not relevant under wild-type conditions. Abnormal accumulation of Wg protein has also been observed in wgPE2 mutant embryos, which similarly show a broader and less punctate pattern of Wg antibody staining. This staining pattern is restored to a more wild-type appearance by overexpressing Fz2 in wgPE2 mutant embryos, further supporting the idea that the wgPE2 lesion compromises interaction with the Fz2 receptor (Moline, 2000).

In wgPE2 mutant embryos that are zygotically mutant for either dally or sugarless (which encodes an enzyme involved in polysaccharide synthesis), a substantial expanse of naked cuticle is produced. Both mutations are hypomorphic, semi-lethal P-element insertions that do not affect embryonic patterning in the context of wild-type Wg. Therefore, mild reductions in sugar modification suffice to restore functionality to the wgPE2 mutant ligand. Moreover, ectopic expression of dally, using a hs-dally transgene, worsens the wgPE2 mutant phenotype. These effects are specific for the wgPE2 phenotype: the hypomorphic Df(2)DE phenotype is not affected by zygotic loss of sugarless or dally and is partially suppressed, rather than enhanced, by providing ectopic dally. Thus, excess Dally improves signaling efficiency for low levels of wild-type Wg, as has been demonstrated for other hypomorphic wg phenotypes, but has the opposite effect on the partial signaling activity of wgPE2 (Moline, 2000).

Finally, overexpression of dally reverses the rescuing effect of overexpressing Fz2 in wgPE2 mutants. This suggests that ectopic Fz2 expression allows interaction with the mutant ligand because it shifts the ratio of Fz2 to Dally molecules at the cell surface, presumably increasing the number of Fz2 receptor complexes that lack Dally co-receptor and that are therefore, as free receptors, able to bind the mutant Wg ligand. As the wgPE2 genetic lesion changes an uncharged valine to a negatively charged glutamic acid, it is conceivable that introduction of a negative charge in the carboxyl terminus prevents proper binding between Wg ligand and negatively charged sulfated sugar groups (Moline, 2000).

In conclusion, it is proposed that interactions between Wg and proteoglycans are required for promoting naked-cuticle specification, but not denticle diversification, and that wgPE2 cannot promote this high-level response because of abnormal interactions with proteoglycans. It is further concluded that the Fz receptor is able to substitute for Fz2 under conditions of excess Wg ligand, but under normal circumstances, does not appear to have a major role in transducing the naked-cuticle cell fate (Moline, 2000).

A novel Drosophila Wingless (Wg) target gene, wingful (wf: termed Notum by FlyBase), encodes a potent extracellular feedback inhibitor of Wg. In contrast to the cytoplasmic protein Naked cuticle (Nkd), the only known Wg feedback antagonist, Wf functions during larval stages, when Nkd function is dispensable. It is proposed that Wf may provide feedback control for the long-range morphogen activities of Wg (Gerlitz, 2002).

A library of 2000 Gal4 enhancer trap P-element insertions was established, each of which reports a gene expression pattern in the wing imaginal disc. This collection was screened with a UAS-GFP reporter for lines that show a wg-like expression pattern. There were 11 insertions identified that reported wg-like gene expression in the embryonic epidermis and all imaginal discs. Four of these lines (S180, ND382, S476, S554) contained an insertion in the wg gene itself; the other seven lines (S141, S145, S163, S330, ND337, ND339, ND634) all carried a P-element insertion at cytological position 72D, only a few base pairs upstream of gene CG13076, referred to as wingful. These enhancer trap insertions indeed report the expression of wf, as revealed by RNA in situ hybridization. wf is ectopically expressed upon wg misexpression, indicating that wf is a Wg target throughout larval development (Gerlitz, 2002).

wf codes for a presumptive protein of 671 amino acids, with an N-terminally situated signal sequence. The wf product is readily secreted from transfected Drosophila cells and has a noticeable propensity to adhere to the surfaces of intact cells. The analysis of the Wf protein sequence reveals a significant structural homology to a subfamily of poorly characterized hydrolases related to plant pectin acetylesterase. Together, these results suggest that the product of the wf gene may catalyze the hydrolytic cleavage of an extracellular substrate (Gerlitz, 2002).

To test the hypothesis that the pan-Wg-target wf encodes an inhibitor of Wg activity, attempts were made to abolish wf function by genetic means. From a collection of six EMS-induced lethal mutations, located between the distal breakpoint of Df(3L)st-f13 and the proximal breakpoint of Df(3L)brm11, a putative null allele of wf was identified with a stop codon at amino acid position 141, encoding a severely truncated protein. Animals homozygous for wf141 or animals of the genotype wf141/Df3(3L)st-f13 die during pupal stages, and show various phenotypes. The most prominent of these are patterning defects in the wing imaginal disc. Wg signaling plays at least two distinct roles during wing development. Early reduction of wg activity results in a wing-to-notum transformation, indicating a requirement for Wg in defining the wing blade primordium, but later reductions cause the loss of wing margin and adjacent tissue, indicating its subsequent role in specifying the wing margin and organizing wing blade development. wf mutants show phenotypes opposite to both classes. Wing discs mutant for wf are enlarged with an extended wing blade region (hence the name wingful). Often these discs contain two wing pouches at the expense of notal structures. Although no apparent expansion of Distalless-lacZ (Dll-lacZ) expression was detected along the dorsoventral axis, there was a significant increase in the number of cells expressing neuralized, a high-threshold Wg target expressed in neural wing margin cells. Consistent with this observation, rare adult escapers mutant for wf show a dramatically increased number of mechanosensory bristles in the wing. Wg signaling also distinguishes between sternite and ventral pleura development in the adult abdomen. wf adult escapers show extra sternite bristles, an effect that was also observed with ectopic expression of wg. Finally, wf adults show extra dorsocentral bristles, sensory organs on the notum whose specification has been shown to depend on wg activity. Taken together, these results show that the absence of wf function causes a gain of Wg activity in developing adult tissues. Therefore, the function of the wild-type wf product is to limit Wg signaling activity (Gerlitz, 2002).

A further prediction of the assumption that Wf functions as a Wg feedback inhibitor is that wf overexpression should lead to wg loss-of-function phenotypes. Three lines of evidence are presented to show that this is, indeed, the case. (1) One of the wf enhancer trap P-element insertions was replaced with an EP element positioning 10 UAS sites upstream of the wf gene, rendering it transcriptionally responsive to Gal4 expression. Alterations of wf expression have unusually potent effects, since all commonly used Gal4 drivers caused lethality in combination with UAS-wf. The only exceptions were S168-Gal4 and scalloped-Gal4, which are expressed in the wing pouch and represent Wg targets, providing a self-regulating circuit in combination with UAS-wf. Adult animals carrying the S168-Gal4 and UAS-wf transgenes have severely reduced wings that lack all wing margin structures. (2) The expression of two target genes were analyzed in this context. S168-Gal4 expression was virtually abolished by UAS-wf expression, whereas the expression domain of wg itself is expanded. Wg is known to narrow its own domain of expression, because a reduction in Wg signal transduction causes ectopic wg transcription. (3) Finally, and perhaps most strikingly, driving expression of wf with scalloped-Gal4 results in a wing-to-notum transformation, the founding loss-of-function phenotype of the wg gene (Gerlitz, 2002).

Based on its structural features as a secreted protein with homologies to pectin acetylesterases, Wf could exert its function by modifying polysaccharide-based properties of cell surface proteins and thereby impeding the intercellular movement of the Wg protein. Alternatively, Wf could counteract Wg signaling by modifying the transducing properties of Wg or one of its receptors. To distinguish between these two possibilities, tests were performed to see whether Wf also antagonizes a derivative of Wg, Nrt-Wg, that is tethered to the cell surface and does not move through tissue. Expression of Wg or Nrt-Wg driven by dpp-Gal4 results in a robust activation of ectopic Dll-lacZ expression. Surprisingly, wf expression extinguishes Dll-lacZ expression induced by tethered Wg, as well as that induced by free Wg. From this experiment it can be ruled out that the primary function of Wf is to impede the extracellular transport of Wg. Therefore, Wf must interfere with the signaling activities of either Wg or its receptor components. Because no physical interaction between Wf and Wg, Frizzled-2 (Dfz2), and its LRP-like partner Arrow could be detected in tissue culture systems, the view that Wf inhibits the activity of a coreceptor component, such as Dally or Dally-like (Dly), proteoglycans that appear to participate in Wg reception, is favored. Wf may inhibit such receptor components via its presumptive esterase activity, for example, by modifying Dally or Dly glycosaminoglycan chains. A definitive proof for this mode of action could be achieved by the genetic demonstration that in larval dally;dly double-mutant situations, loss of wf function has no antagonistic effect (Gerlitz, 2002).

The discovery of Wingful as an essential Wg feedback antagonist may provide an explanation of why Nkd has no apparent role in imaginal tissues of Drosophila . The function of Nkd may be superseded by that of Wf, which functions in a powerful negative-feedback loop in adult development. Conversely, when both maternal and zygotic components of wf are removed, no obvious requirement was observed for Wf in embryonic development, possibly because the nkd system is operative at this stage of development. Both Naked and Wf can, however, inhibit Wg signaling throughout development if they are overexpressed, but each of them is operating more effectively at only one of the two stages. It may not be coincidence that Nkd, as the intracellular feedback antagonist, is used during embryonic patterning, (where Wg functions at short range), whereas Wf, as a secreted extracellular antagonist, primarily regulates patterning processes that depend on long-range Wg signaling. Wf functions nonautonomously and, like Argos (a secreted feedback antagonist of the Drosophila EGF system), may have a different range of action compared with the primary signal, providing an intricate means to shape the range and slope of the cellular responses to a morphogen gradient (Gerlitz, 2002).

Opposing activities of Dally-like Glypican at high and low levels of Wingless morphogen activity

The glypican family of heparan sulfate proteoglycans has been implicated in formation of morphogen gradients. The role of the glypican Dally-like protein (Dlp) in shaping the Wingless gradient was studied in the Drosophila wing disc. Surprisingly, Dlp has opposite effects at high and low levels of Wingless. Dlp promotes low-level Wingless activity but reduces high-level Wingless activity. Evidence is presented that the Wg antagonist Notum acts to induce cleavage of the Dlp glypican at the level of its GPI anchor, which leads to shedding of Dlp. Thus, spatially regulated modification of Dlp by Notum employs the ligand binding activity of Dlp to promote or inhibit signaling in a context-dependent manner. Notum-induced shedding of Dlp could convert Dlp from a membrane-tethered coreceptor to a secreted antagonist (Kreuger, 2004).

Notum encodes a secreted member of the alpha-hydrolase superfamily, which includes serine proteases, lipases, and other enzymes in which a Ser-Asp-His catalytic triad comprises the active site. Mutation of Ser237 to Ala was shown to remove Notum activity in vivo and in vitro (Giraldez, 2002). On the basis of its similarity to pectin acetylesterase enzymes from plants and plant pathogens and on the basis of its ability to compete with the N-deacetylase-N-sulfotransferase enzyme for use of Dlp as a substrate, it has been proposed that Notum might function as a GAG deacetylase. If this were the case, it would be expected that Notum would be unable to modify a form of Dlp lacking GAG side chains (Kreuger, 2004).

Dally and Dlp each contain five putative GAG addition sites and have been shown to carry predominantly heparan sulfate (HS, as shown for vertebrate glypicans). GAG biosynthesis is initiated by addition of a xylose residue to the hydroxyl group of a serine in a serine-glycine (SG) motif in the stem region of the glypican (Lindahl, 1998). Mutant forms of Dally and Dlp proteins were prepared in which all the putative GAG-addition sites were mutated. For Dally, the serine residues in the five SG motifs were mutated to alanine. For Dlp, four SG motifs were deleted by removing a block of 25 residues, and the fifth was mutated to AG (Kreuger, 2004).

The glycosylation states of epitope-tagged Dally and Dlp and the GAG addition site mutants were compared by anion exchange chromatography. Glypicans isolated from mammalian cells in culture are highly negatively charged and are typically retained on a strong anion exchange matrix up to 1 M NaCl. When expressed in Schneider S2 cells, a considerable fraction of Dally bound to Q-Sepharose in 0.3 M salt and some was able to bind in up to 1 M salt. Dally migrated as a broad band in SDS-PAGE. The more negatively charged forms that were bound to Q-Sepharose at higher salt exhibited a higher apparent molecular weight. This may reflect the presence of more and longer HS side chains as well as differences in charge due to sulfation. Dally behaves like a conventional glypican (Tsuda, 1999). In contrast, most of the Dlp protein bound to Q-Sepharose in 0.15 M salt, but none bound at higher salt concentrations. Dlp migrated as a more tightly resolved band in SDS-PAGE. These observations suggest that Dally and Dlp differ in the extent or quality of GAG modification. Removal of the GAG addition sites considerably reduced retention of both mutant proteins on Q-Sepharose. The proportion of the mutant form of Dally that bound was reduced at all salt levels and none was bound above 0.3 M salt. Very little of the mutant form of Dlp was able to bind to Q-Sepharose, even in physiological salt (Kreuger, 2004).

To further evaluate their GAG content, lysates of S2 cells transfected to express wild-type or mutant Dally or Dlp were digested with heparitinase, and the remaining glycan stub was detected with 3G10 antibody. Dally-expressing cells showed elevated GAG labeling in a broad band that comigrated with the major form of endogenous glypican in untransfected cells. Although the level of expression of the mutant form of Dally was comparable, GAG labeling was not detectable above background. The level of GAG modification on overexpressed Dlp was lower than that seen on the endogenous glypicans. This indicates that Dlp has a lower GAG content than Dally, consistent with its poor retention on Q sepharose. The mutant form of Dlp showed much reduced GAG labeling, barely distinguishable from background levels (Kreuger, 2004).

Dally resembles a conventional glypican, being extensively modified by GAG side chains. Dlp appears to have less heterogeneous and less extensive GAG modification than Dally. The mutant form of Dlp has low levels of residual GAG and would be expected to be a poor substrate for Notum, if Notum acts directly on the GAG side chains. Coexpression of Notum and HA-tagged Dlp in S2 cells caused a large shift in the electrophoretic mobility of Dlp in SDS-PAGE to a faster migrating species, and the Notum S237A mutant had no effect. S2 cells contain endogenous Notum, so there is always a background level of Dlp processing. Interestingly, Notum caused a shift in the mobility of the low-GAG form Dlp comparable in magnitude to the shift in the wild-type protein, though the processing efficiency was lower. This would be difficult to explain if the shift were due to alteration in the amount or in the negative charge of the remaining GAG due to deacetylation. Further, no increase in deacetylase activity was detected in lysates of S2 cells overexpressing Notum, nor was there any detectable decrease in the degree of HS acetylation in larvae overexpressing Notum. These observations suggest that Notum does not act as a GAG-modifying enzyme (Kreuger, 2004).

The mobility shift of Dlp caused by Notum is ~15 kDa. Proteolytic cleavage at the N or C termini of Dlp could cause the apparent size reduction, although other modifications such as removal or attachment of lipids, sugars, or phosphate groups also could cause anomalous migration in SDS-PAGE. To test the possibility of proteolytic processing, forms of Dlp were prepared with epitope tags close to the two ends. For Dlp-HA-C, an HA epitope tag was inserted at serine 732, just before the putative cleavage site for addition of the GPI anchor (S733). To produce GFP-Dlp-HA-C, the HA tag was placed at the corresponding position in GFP-Dlp, which contains a GFP moiety inserted in place of residue G68, close to the N terminus of the protein following removal of the signal peptide at residue 41. S2 cells were transfected with Dlp-HA, Dlp-HA-C, or GFP-Dlp-HA-C together with normal or mutant forms of Notum. Notum induced comparable mobility shifts in all three forms of Dlp. The C-terminally HA-tagged forms of Dlp were processed more efficiently than Dlp-HA, so that more of the faster migrating from was observed without addition of wild-type Notum (Notum is expressed in S2 cells). These observations are difficult to reconcile with Notum acting as a protease (Kreuger, 2004).

The possibility was considered that the shift in Dlp mobility could be due to cleavage of the GPI anchor. Lysates were prepared from S2 cells transfected with Dlp-HA with or without Notum, and half of each was treated with phospholipase-C (PI-PLC) to cleave the GPI anchor of Dlp. Interestingly, PI-PLC cleavage caused a mobility shift in Dlp comparable to that caused by Notum. Notum had no additional effect on PI-PLC-treated Dlp. Although the magnitude of this shift in apparent molecular weight is larger than would be expected from the mass of the GPI anchor alone, anomalous migration of proteins in SDS-PAGE has been observed following removal of GPI anchors (Kreuger, 2004).

To further evaluate the possibility that the mobility shift caused by Notum might be due to cleavage of the GPI anchor, a Triton X-114 phase separation analysis was performed. Following phase separation, integral membrane proteins and GPI-anchored proteins are recovered mainly in the detergent phase, while other proteins are mainly in the aqueous phase. In cells cotransfected to express Dlp and Notum, processing of Dlp was efficient and the faster migrating form of Dlp was mainly recovered in the aqueous phase. In cells transfected to express Dlp, the slower migrating form was mainly recovered in the detergent phase, consistent with it having a GPI anchor. The faster migrating form partitioned between aqueous and detergent phases. These observations are consistent with the suggestion that the faster migrating form of Dlp produced by Notum lacks a GPI anchor (Kreuger, 2004).

It was next asked if Notum could act on forms of Dlp that lack a GPI anchor. The predicted GPI anchor site of Dlp consists of the sequence SDA (at S733) followed by a characteristic hydrophobic motif. Mutation of any of the three residues of the GPI anchor site to proline has been shown to reduce GPI addition in other glypicans. The SDA motif was mutated to PDA in GFP-Dlp-S988P (residue numbers are for GFP-Dlp). A truncated version of GFP-Dlp was also prepared in which a stop codon was inserted after S987 to mimic the effects of cleavage of the protein chain at this position without addition of GPI. Coexpression with Notum had little or no effect on these forms of GFP-Dlp. GFP-Dlp-S988P migrated predominantly at an intermediate position, faster than the GPI-anchored form but slower than the Notum-processed form. This presumably reflects the full-length protein that has not been cleaved for GPI addition (this form appears as a minor band in most preparations). The major band of GFP-Dlp-S988P is unaffected by Notum, though a small amount of the protein does appear to have had GPI added (perhaps at S987) and was subject to Notum activity. The truncated GFP-Dlp-S987Z protein did not produce any of the slower migrating GPI anchored form and was not affected by Notum. GFP-Dlp-S987Z migrated slightly faster than the Notum-modified form of GFP-Dlp. In Triton X-114 phase separation analysis, GFP-Dlp-S987Z partitioned into the aqueous phase, also consistent with the lack of a GPI anchor. The form of Dlp cleaved by PI-PLC comigrated more closely with the Notum-processed form, consistent with the possibility that Notum cleavage was in the GPI moiety (Kreuger, 2004).

Next a version of Dlp was prepared that was not GPI anchored, in order to ask if it would be a substrate for Notum. GFP-Dlp was fused at residue S975 to residue R23 of the transmembrane protein CD2 and expressed in S2 cells. This protein contains the entire ectodomain (CRD and stem) of Dlp, but lacks the GPI addition signals (the fusion is at the equivalent position in Dlp to the S987Z construct; numbering differs because the HA tag was removed). GFP-Dlp-CD2 was not cleaved by PI-PLC, verifying that it does not contain a GPI anchor. GFP-Dlp-CD2 was also not a substrate for Notum. If Notum acted as a protease to cleave in the Dlp protein, it would be expected that GFP-Dlp-CD2 fusion would be a substrate for Notum. Likewise, if Notum acted on the GAG side chains or on another posttranslational modification, it would be expected that GFP-Dlp-CD2 would be affected. Taken together, these observations indicate that Notum can induce modification of Dlp in a manner that resembles cleavage of the GPI anchor. Given that the alpha-hydrolases includes lipases, it is possible that Notum might act directly to cleave within the GPI anchor. Alternatively, Notum could induce the activity of an endogenous phospholipase (Kreuger, 2004).

Can cleavage at the GPI anchor cause Dlp to be shed from the cell surface? The faster and slower migrating forms of Dlp-HA were efficiently recovered from the cell lysates. In addition, the faster migrating cleaved form of Dlp-HA was recovered from the culture medium of cells overexpressing Notum. The slower migrating GPI-anchored form of Dlp-HA was not recovered from the medium. Very little Dlp-HA was recovered from medium conditioned by cells transfected with Dlp alone or with the Notum mutant. This indicates that Notum can release Dlp from the cell surface, causing it to be shed into the medium. Notum does not induce detectable shedding of HA-tagged Dally (Kreuger, 2004).

Dlp-HA or Dally-HA were expressed under en-Gal4 control in the wing disc to compare the effects of the endogenous Notum protein on Dlp and Dally. Notum is expressed at the dorsoventral boundary under control of Wg. En-Gal4 produced a uniform level of Dally-HA across the wing pouch. In contrast, Dlp-HA protein levels were lower at the dorsoventral boundary. Notum caused loss of Dlp, but had little effect on Dally. This presumably reflects shedding of Dlp protein from cells in the disc in the region where Wg levels are highest (Kreuger, 2004).

The finding that Notum can cleave and release Dlp from cells raised the possibility that Dlp might be released together with bound Wg. This could explain how Dlp acts to reduce Wg activity where Wg and Notum levels are high. Therefore the possibility was tested of synergistic action of Notum and Dlp on Wg function. Overexpressed Dlp binds and sequesters Wg at the cell surface, leading to reduced Wg activity. Coexpression of Notum with Dlp reduced the ability of Dlp to bind and retain Wg (Giraldez, 2002). To examine the effects of Notum on Dlp activity, transgenes expressing Notum or Dlp were selected at levels that were on the threshold for altering wing morphology. When expressed under en-Gal4 control, each transgene caused loss of some of the posterior wing margin bristles, indicating mild reduction of Wg activity. Expression of the two transgenes together caused scalloping of the wing, a typical defect caused by more severe reduction of Wg activity. This observation suggests that expression of Notum enhanced the ability of Dlp to reduce Wg activity. In view of the finding that Notum can cause Dlp to be released from the cell surface, it is suggested that coexpression of Notum causes Dlp to be released together with bound Wg (Notum reduced the level of Wg bound in the disc; Giraldez, 2002). Release of Wg bound to Dlp could reduce the level of Wg available for signaling and cause the observed wing scalloping phenotype. Notum causes release of Dlp from cells at the DV boundary, where Wg levels are highest (Kreuger, 2004).

Therefore, Notum can induce cleavage of Dlp in a manner that resembles PI-PLC cleavage of the GPI anchor. Notum and PI-PLC produce comparable shifts in the electrophoretic mobility of Dlp. Although the magnitude of this shift is larger than would be expected from the mass of the GPI anchor, other studies have shown that the effects of removing GPI anchors are not predictable. Examples of increased or decreased mobility have been reported, and the magnitude of the shifts can be large. Notum-induced processing also renders Dlp soluble in the aqueous phase following detergent phase separation of soluble and membrane-associated proteins, consistent with removal of the GPI anchor. Also, a Dlp-CD2 fusion protein that is not GPI anchored, and is therefore not a substrate for PI-PLC, is insensitive to Notum. This would not be expected if Notum acted on the GAG side chains or if Notum was a protease cutting within the Dlp core protein. These observations are consistent with two modes of Notum action. Notum might act directly to cleave the GPI anchor or it might modify Dlp in some way that makes Dlp a substrate for an endogenous phospholipase. The exact nature of the Notum-induced cleavage remains to be determined; however, one intriguing possibility is that Notum might cleave within the glycan linker of the GPI anchor (Kreuger, 2004).

Can the effects of Notum on Dlp provide an explanation for the apparently opposing activities of Dlp at high and low levels of Wg? How could Notum-induced cleavage of Dlp lead to reduced Wg activity, whereas removal of Dlp by RNAi increases Wg activity? If Wg remains bound to Dlp when Dlp is cleaved and shed from the cell, bound Wg would also be shed and so become unavailable for signaling. Shedding of Dlp as a consequence of Notum-induced cleavage could reduce peak levels of available Wg. Notum is expressed at the source of Wg and so would be expected to shed Dlp and reduce Wg activity where Wg levels are highest. In this way, removal of Dlp protein as a consequence of Notum-induced cleavage could have a different effect than failure to express Dlp. In the absence of Dlp, Wg would not bind Dlp and be shed with it, so that more Wg might be available to interact with Dally and/or the Wg receptor complex. This could increase the effective concentration of Wg locally near the site of Wg production (Kreuger, 2004).

This model is consistent with the observed synergy between low-level expression of Dlp and Notum. Overexpression of Dlp is thought to increase the number of Wg binding sites and shift the equilibrium toward more Wg bound to Dlp. At high levels of Dlp, this can reduce the amount of Wg available for signaling and produce a Wg loss-of-function phenotype. A level of Dlp overexpression was chosen that produces a mild defect due to reduced availability of Wg. Coexpression of Notum would lead to shedding of the Dlp bound Wg and thus remove this fraction of Wg from the pool on the cell surface so that it would no longer be able to contribute to the pool of Wg in equilibrium with the receptor. This would be expected to further reduce Wg activity and increase the severity of the defect, as observed (Kreuger, 2004).

Localized cleavage of Dlp induced by regulated expression of Notum may provide a unifying explanation for the opposing effects of the Dlp glypican in different regions of the tissue. A mathematical model of Wg gradient formation has invoked a need for an elevated level of Wg turnover in cells close to the source of Wg. The mechanism described here would be sufficient to provide the reduction of Wg activity in cells closest to the source of the secreted ligand that is needed for formation of a robust morphogen gradient (Kreuger, 2004).

Drosophila VAMP7 regulates Wingless intracellular trafficking

Drosophila Wingless (Wg) is a morphogen that determines cell fate during development. Previous studies have shown that endocytic pathways regulate Wg trafficking and signaling. This study showed that loss of vamp7, a gene required for vesicle fusion, dramatically increased Wg levels and decreased Wg signaling. Interestingly, this study found that levels of Dally-like (Dlp), a glypican that can interact with Wg to suppress Wg signaling at the dorsoventral boundary of the Drosophila wing, were also increased in vamp7 mutant cells. Moreover, Wg puncta in Rab4-dependent recycling endosomes were Dlp positive. It is hypothesized that VAMP7 is required for Wg intracellular trafficking and the accumulation of Wg in Rab4-dependent recycling endosomes might affect Wg signaling (Gao, 2017).

There are two models describing how the apically secreted Wg encounters basolateral receptors at receiving cells. One suggests that Wg and receptors can be internalized separately, and then, endosome fusion results in Wg and receptor interaction in the receiving cells. Another model proposes that apically secreted Wg undergoes endocytosis and will be transported to the basolateral surface in the producing cells, then spread to the receiving cells for the interaction with receptors. Therefore, Wg is actively endocytosed in both receiving cells and producing cells (Gao, 2017).

This study found that Wg distribution was affected in both receiving and producing cells in vamp7-/- mutant background. Further investigation indicated that Wg double labeled puncta significantly increased, so did the percentage of Rab4 and Wg double staining puncta. Thus, it is suggested that VAMP7 is required for Wg endocytosis in the both receiving cells and producing cells in Drosophila wing disc, and its mutation leads to Wg accumulating in endocytic organelles but not degradation. Rab4 dependent recycling endosomes can recruit proteins from the early endocytic organelles, which may finally lead to increased level of Wg in Rab4 dependent recycling endosomes (Gao, 2017).

Although endocytosis has been demonstrated for Wg transport, there is still debate about whether endocytosis plays a direct role in the Wg signaling. Classically, the early step of endocytosis is thought to contribute positively to signaling, as early endosomes can recruit signaling components, while subsequent vesicle transport may downregulate signaling by sequestrating signaling components in endosomes or degradating them in lysosomes. This study found that the expression of the Wg target gene sens was reduced in vamp7 mutant cells. One possibility is that Rab4 recycling endosomes may recruit Wg from early endosomes. As a previous report found that the expression of activated forms of Rab4 suppressed the ability of Rab5 to enhance activation of Wg pathway, Wg accumulation in Rab4 recycling endosomes may affect Wg signaling. Another possible reason is that vamp7 mutation enhances the level of Wg signaling inhibitors (Gao, 2017).

Dlp is a membrane-associated glypican that can interact with Wg by its core protein on the cell surface, and suppresses Wg target gene sens. However, the functional significance of interaction between Wg and Dlp inside the cell has not been well elucidated. This study showed that Wg might encounter endogenous Dlp in Rab4 dependent recycling endosomes, and vamp7 mutation could improve the levels of Dlp and Wg in Rab4 dependent recycling endosomes. Previous studies proposed that Dlp competes with Wg receptors to interact with Wg, and the signaling activity may be determined by the relative levels of receptor and Dlp. It is suggested that competition between Dlp and receptors might not only occur on the cell surface but may have started from intracellular vesicles. The increased levels of Dlp and Wg in Rab4 dependent recycling endosomes may lead to Sens reduction (Gao, 2017).

In conclusion, this study showed that an endocytic pathway involving VAMP7 regulates Wg and Dlp trafficking. This route adds another layer of spatial regulation in the Wg signaling pathway. Additional work will be needed to determine the functional significance of this route in other Drosophila tissues and whether vamp7 is required for vertebrate Wnt trafficking (Gao, 2017).

Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein

Wingless is a morphogen required for the patterning of many Drosophila tissues. Several lines of evidence implicate heparan sulfate-modified proteoglycans (HSPGs) such as Dally-like protein (Dlp) in the control of Wg distribution and signaling. dlp is required to limit Wg levels in the matrix, contrary to the expectation from overexpression studies. dlp mutants show ectopic activation of Wg signaling at the presumptive wing margin and a local increase in extracellular Wg levels. dlp somatic cell clones disrupt the gradient of extracellular Wg, producing ectopic activation of high threshold Wg targets but reducing the expression of lower threshold Wg targets where Wg is limiting. Notum encodes a secreted protein that also limits Wg distribution, and genetic interaction studies show that dlp and Notum cooperate to restrict Wg signaling. These findings suggest that modification of an HSPG by a secreted hydrolase can control morphogen levels in the matrix (Kirkpatrick, 2004).

By a number of cellular and molecular markers, compromising dlp function produced ectopic activation of high-threshold Wg target genes in the wing imaginal disc. dlp adult escapers have ectopic mechanosensory bristles and dlp third instar larvae show expanded expression of Ac, a marker for sensory organ precursor cells. Wg signaling was abnormally elevated along the DV boundary of dlp mutant discs by a number of other measures, including expansion of the zone of nonproliferating cells (ZNC) and ectopic expression of Cyclin A. These findings are consistent with the expansion of sensory organ precursor cells produced by RNAi inhibition of dlp function. These phenotypes show that Dlp serves to limit activation of high-threshold target genes near the DV boundary (Kirkpatrick, 2004).

The expansion of Wg signaling found in dlp mutants is accompanied by locally elevated levels of extracellular Wg. The increase in Wg in the matrix of dlp mutant wing discs is not associated with increases in Wg production or altered expression of D-fz2, a known regulator of Wg levels. These findings argue against the model that dlp controls Wg levels indirectly via control of Wg expression or other regulatory genes. It is well established that Wg binds heparan sulfate, and therefore it is likely that Dlp controls Wg levels through a direct interaction (Kirkpatrick, 2004).

The expansion of Wg-directed patterning in the wing disc of dlp mutants was a surprising finding on a number of counts: (1) mutations in genes encoding heparan sulfate biosynthetic enzymes compromise rather than increase the levels of Wg in the matrix; (2) ectopic expression of Dlp results in higher levels of Wg in the matrix. These findings suggest that loss of Dlp would destabilize Wg on cell surfaces and in the matrix, thereby reducing Wg signaling. However, exactly the opposite result was obtained in dlp mutants: loss of Dlp increases extracellular Wg and signaling. These findings emphasize that while mutations compromising synthesis of all HSPGs might destabilize Wg in the matrix, loss of any one proteoglycan can have very different effects (Kirkpatrick, 2004).

The phenotypes of dlp mutants suggest that Dlp serves to limit Wg distribution and/or alter Wg stability, thus affecting the Wg gradient in the wing disc. Genetic mosaic analysis of dlp clearly demonstrates that the ectopic activation of Wg signaling, as measured by Arm, Cyclin A, and Ac expression, occurs non-cell autonomously, providing support for this model. Recent mathematical modeling of morphogen gradients demonstrates that binding of morphogens to receptors or coreceptors on cell surfaces or in the matrix can have profound effects on diffusion and subsequently limit morphogen activity. Consistent with such modeling, it has been demonstrated experimentally that a nonheparin binding isoform of the secreted growth factor VEGF adopts a broader, shallower distribution than heparin binding forms and the differential localization of VEGF-A isoforms in the matrix controls the vascular branching pattern. It is also well established that heparan sulfate proteoglycans mediate endocytosis of extracellular ligands, and the findings suggest the possibility that Dlp might mediate endocytic control of Wg levels in the matrix. dlp mutants display locally elevated levels of extracellular Wg without an increase in production, and therefore Wg turnover must be affected in some way (Kirkpatrick, 2004).

In contrast to the higher levels of extracellular Wg found near the DV boundary in dlp mutant wing discs, there is no apparent change in the low level of Wg at the edges of the gradient. However, genetic mosaic analysis of dlp in wing development shows that loss of dlp can modestly reduce the activation of a low-threshold Wg target gene in those regions where Wg levels are low. Since this effect on Wg signaling is not a consequence of reduced levels of Wg in the matrix, it must result from an alteration of cell responsiveness or the bioactivity of the Wg that is present. The findings are consistent with studies showing that RNAi of dlp in the wing disc also inhibits transcriptional activation of Wg target genes in regions where Wg is low. It is interesting to note that dlp expression, like that of the Wg receptor D-fz2, is highest outside the presumptive wing margin. Perhaps the high level of dlp expression in this region serves, in part, to enhance cellular responses to Wg. The ability of Dlp to enhance Wg signaling as well as limit Wg distribution is not unprecedented: the other glypican in Drosophila, Dally, enhances Dpp signaling in regions of the wing disc where Dpp levels are modest in addition to regulating the distribution of Dpp protein (Kirkpatrick, 2004).

Mutations in Notum produce ectopic activation of Wg signaling and alter the Wg gradient, similar to what was observed in dlp mutants. Notum encodes a protein with homology to plant hydrolases and has been proposed to alter the structure of Dlp, thus affecting its affinity for Wg. Coexpression of Notum with Dlp decreases Dlp-mediated stabilization of Wg protein in the wing pouch. dlp and Notum mutants fail to complement: this is strong genetic evidence that Dlp and Notum cooperate to control Wg-mediated patterning. Biochemical studies show that Notum can cleave Dlp from the cell surface. The finding that heterozygosity for dlp ameliorates patterning abnormalities produced by ectopic Notum suggests that Dlp is a principal substrate for Notum activity. The results suggest that Notum-mediated release of Dlp from the cell surface is required for Dlp to limit Wg signaling (Kirkpatrick, 2004).

Notum and dlp are expressed in complementary patterns in the wing disc, with Notum levels highest near the DV boundary and dlp levels highest elsewhere. Thus, a gradient of Dlp cleavage may be established across the disc, with some Dlp remaining on cell surfaces in regions distant from the DV boundary, enhancing cellular responses to Wg. Cleavage of Dlp would remove this coreceptor activity, but shed Dlp-Wg complexes also could limit Wg signaling by promoting Wg clearance or could serve as dominant-negative inhibitors of signaling. Coexpression of Notum and Dlp in the wing disc produced more intracellular vesicles compared to expression of Dlp alone: this is consistent with a role for shed Dlp in promoting endocytosis and clearance of Wg from the matrix (Giraldez, 2002). Such a role is also consistent with the non-cell-autonomous effects of dlp somatic cell clones on extracellular Wg distribution. Loss of dlp in clones might locally reduce Wg turnover as well as reducing cell surface binding sites for Wg, permitting increased diffusion of Wg and accumulation on wild-type cells at clone boundaries. Spatial regulation of Dlp activity by Notum can explain how Dlp primarily limits Wg signaling near the DV boundary and enhances signaling away from the boundary, but other Wg-dependent factors may also influence the ability of Dlp and Notum to downregulate Wg levels. Notably, recent mathematical modeling suggests that elevated degradation of Wg close to its source is necessary to enhance the robustness of the morphogen gradient. Together, Dlp and Notum may provide this locally increased turnover and hence stabilize the Wg gradient. Enzymatic modification of a proteoglycan to influence its cell surface localization may enable it to play both positive and negative roles in signaling and provides another potential mechanism for regulating morphogen distribution in tissues (Kirkpatrick, 2004).

In humans, mutations in glypican-3 (GPC3) were identified as the genetic basis of a human overgrowth and tumor susceptibility syndrome, Simpson Golabi Behmel Dysmorphia (SGB) (Pilia, 1996). SGB patients display both prenatal and postnatal overgrowth, a number of morphological abnormalities including renal dysplasia and skeletal defects, as well as a high incidence of tumors (Neri, 1998). Subsequently, loss of GPC3 expression has been found associated with a number of cancers, including breast, mesothelioma, and ovarian neoplasias (Lin, 1999; Murthy, 2000; Xiang, 2001). Given the established role of proteoglycans as molecules facilitating growth factor signaling at the cell surface, the effects of loss of GPC3 on growth and tumor development are a bit puzzling. The current findings provide a molecular mechanism for understanding the capacity of glypicans to serve as tumor suppressors. Dlp restricts the cellular domain of Wg signaling during wing development, and loss of dlp results in ectopic growth factor signaling. Clearly, ectopic signaling produced by loss of GPC3 could readily contribute to tumor development and growth in humans. Recently, analysis of a gene-trap mutant in mouse Ext1 has provided further evidence for the capacity of heparan sulfate proteoglycans to limit the range of morphogen activity during chondrocyte differentiation (Koziel, 2004). Mice partially defective for heparan sulfate biosynthesis as a consequence of hypomorphic mutations in Ext1 show ectopic Indian Hedgehog signaling and altered Hedgehog distribution at the growth plate (Kirkpatrick, 2004).

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors

The respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient has been examined. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, it was found that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. These studies reveal that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, this analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient (Baeg, 2004).

Sfl encodes a homolog of the Golgi enzyme HS N-deacetylase/N-sulfotransferase that is required for the modification of HS. It was of interest to determine whether the retention of Wg at the cell surface involves HSPGs in receiving cells, since it has been proposed that HSPGs are unlikely to be required in Wg-receiving cells. In the wing blade, Wg, originating from the D/V border, is detected in an irregular pattern of puncta in receiving cells; these puncta correspond to the internalized Wg protein. The intensity and number of puncta decreases from the source of Wg. Furthermore, using an extracellular labeling method, a gradient of Wg protein that appears broader, shallower, and with less puncta is observed. Both in sfl mutant wing discs and in large sfl mutant clones, a striking decrease in the number of Wg puncta was observed. This decrease is not due to a change in wg transcription because it is not affected in sfl mutant cells. Further, lack of Sfl activity does not appear to disrupt the overall amount of Wg produced by wg-expressing cells but is associated with a dramatic decrease in extracellular Wg. These results suggest that HSPGs are required for sequestering extracellular Wg in receiving cells (Baeg, 2004).

To gain further insights into the role of HSPGs in receiving cells to shape the Wg gradient, an examination has been made of the distribution of Wg in patches of WT cells located within a large sfl mutant territory. In such cases, bright spots of Wg were be detected within the patch of WT cells, indicating that sfl-expressing cells are able to sequester extracellular Wg, unlike neighboring cells that lack sfl. This result is consistent through an analysis of more than 10 clones. Further, clones of cells that overexpress Notum-GT (Golgi-tethered), which acts cell autonomously in receiving cells, were generated. Notum, which encodes a member of the α/β-hydrolase superfamily, antagonizes Wg signaling and it has been proposed that Notum acts by altering the ability of the cell surface glypican molecules Dally and Dlp to stabilize extracellular Wg (Giraldez, 2002). Consistent with the conclusion that HSPGs are required in receiving cells to capture extracellular Wg, a decrease in the formation of Wg puncta was observed in cells overexpressing Notum-GT. These results are consistent with at least two nonexclusive models: (1) HSPGs could be required for Wg stability and/or trapping of Wg at the cell surface such that it does not diffuse away; (2) HSPGs could be involved in promoting Wg movement throughout tissues. The role of HSPGs in sequestering and/or stabilizing the ligand is supported by previous observations that overexpression of either Dlp or Dally results in the accumulation of extracellular Wg (Baeg, 2004).

Because HSPGs have been implicated in endocytosis of ligands such as FGF, possibly HSPGs also play a role in Wg internalization. A role for HSPG in Wg endocytosis would be consistent with the absence of puncta in sfl clones, and also the observed accumulation of extracellular Wg following overexpression of Dlp-HA (Giraldez, 2002). However, much of Wg proteins appeared in intracellular vesicles, instead of outlining the cell surface, in discs overexpressing both Dlp-HA and Notum. If HSPGs were directly involved in Wg internalization, fewer intracellular vesicles would be detected in discs overexpressing Dlp-HA and Notum since Notum acts to decease the affinity of Dlp for Wg. Furthermore, if the primary function of HSPGs were to internalize Wg, then extracellular Wg accumulation in cells lacking HSPGs activity would be expected, which is not the case. However, the data does not rule out the possibility that HSPGs play a direct role in Wg endocytosis, and, thus, further analysis will be required to clarify this issue. Taken together, these results suggest that the primary role of HSPGs is to trap and/or stabilize extracellular Wg in receiving cells where it is then able to interact with its signaling receptor as well as other factors that are responsible for its internalization, and thus contributes to shaping the Wg gradient (Baeg, 2004).

Previous ectopic expression studies have shown that Dlp can trap extracellular Wg and prevent activation of the Wg signaling pathway. Because Dlp appears to be a major HSPG required to regulate Wg signaling, its endogenous distribution was examined in the wing imaginal disc using a polyclonal antibody against Dlp and a staining method that primarily detects extracellular proteins. The specificity of the Dlp antibody was confirmed by misexpressing dlp using the ap-Gal4 driver. In the third instar wing imaginal disc, Dlp was detected throughout the disc; however, a significant decrease in the level of Dlp was detectable at the D/V border. This domain of low Dlp expression correlates with the region where high level of Wg signaling is required to induce the expression of short-range target genes. It is noted that since dlp mRNA expression is uniform throughout the disc, the down-regulation of Dlp at the D/V border must occur post-translationally. Interestingly, an optical cross section of the disc has revealed that endogenous Dlp localizes mostly on the basolateral surface of the cell where extracellular Wg is detected. The subcellular localization of Dlp protein was also examined using a GFP-dlp expressed under the control of a Gal4 driver. Consistent with the Dlp antibody result, it was found that GFP-Dlp localizes predominantly to the basolateral membrane. Altogether, these observations suggest that Dlp can bind to extracellular Wg and that Dlp levels need to be reduced for high-level Wg activity in cells near the D/V boundary (Baeg, 2004).

In a genome-wide RNAi screen in S2R+ cells to identify genes that either up- or down-regulate Wg signaling, Dlp was identified as both a negative and positive regulator of Wg signaling under stimulated and nonstimulated conditions, respectively. The cell-based assay devised consists of the activation of a Tcf/Arm-dependent Wg-reporter gene upon induction of S2R+ cells by expressing a wg cDNA by transient transfection. The activity of the Wg pathway and the effect of the addition of various dsRNAs on the pathway were assayed by monitoring Luciferase-reporter activity using a luminescence-based plate reader. Using this assay, the addition of dsRNAs of positive transducers of Wg signaling, such as arm, decrease the Top12X-HS-Luciferase reporter activity, while dsRNAs to negative Wg regulators, such as Daxin, increase its activity. Interestingly, under condition of Wg induction, dlp was found to act as a negative regulator of Wg signaling, since dlp dsRNA led to a twofold increase in luciferase activity. This increase is significant since it is comparable to that of daxin dsRNA. In the absence of Wg induction, dlp was found to positively regulate Wg signaling, since dlp dsRNA leads to a fivefold decrease in luciferase activity, a decrease that is similar to that observed by the addition of arm dsRNA. These results suggest Dlp acts as a positive regulator of the Wg pathway when Wg level is low and negatively influences signaling when Wg is abundant. These results are consistent with in vivo results that demonstrate that Dlp has both a positive and negative role in Wg signaling (Kirkpatrick, 2004). The observations in S2R+ are consistent with the hypothesis that low Dlp levels at the D/V boundary supports high-level Wg signaling, while further away from the D/V boundary where the Wg concentration is lower, Dlp positively influences Wg signaling. Overall, the result from the S2R+ RNAi experiments indicates that (1) Dlp is not an essential component of the Wg signal transduction pathway; and (2) Dlp can either have a negative or positive impact on Wg signaling depending on the level of Wg available. The negative effect of Dlp is consistent with previous in vivo studies that have shown ectopic Dlp expression can trap extracellular Wg and prevent activation of the Wg signaling pathway. Therefore, a reduction of Dlp levels at the D/V border would be expected to contribute to high-level Wg signaling. The positive effect of Dlp in Wg signaling needs to be understood in the context of the previous findings that loss of HSPG activity results in wg loss-of-function phenotypes, as shown by a decrease in dll expression in clones mutant for enzymes involved in GAG biosynthesis. One attractive model is that Dlp would act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with the signal transducing Fz receptors in cells located at a distance from the D/V boundary where low level of Wg is available. Finally, these observations are consistent with a recent study (Kirkpatrick, 2004) that showed that (1) ectopic activation of Wg signaling at the wing margin occurs in dlp mutant tissues, and (2) a cell autonomous reduction in Wg signaling in dlp clones located distal to the Wg-producing cells (Baeg, 2004).

The distribution of Dlp protein is reminiscent of the down-regulation of Dfz2 transcription near the D/V border. Wg-mediated repression of DFz2 expression has been shown to affect the shape of the Wg gradient, resulting in a gradual decrease in Wg concentration. Because these results indicate that HSPGs affect Wg distribution, the functions of the two seven transmembrane Wg receptors, Fz and DFz2, were examined to evaluate how the signal transducing receptors cooperate with HSPGs in shaping the Wg gradient. To determine the role of Fz and DFz2 in Wg movement, the distribution of Wg in fz DFz2 double-mutant clones was examined. In these clones, an expansion of wg expression was observed, which is consistent with the previously described Wg “self-refinement” process, by which Wg signaling represses wg expression in cells adjacent to wg-expressing cells. Unexpectedly, within these clones, Wg puncta are still present, indicating that Fz/DFz2 receptor activities are not required for Wg spreading (Baeg, 2004).

To determine whether Wg is present in endosomes in the absence of Fz/DFz2 activities, wing discs were labeled with the endosomal marker Texas-red dextran. More than 50% of Wg puncta co-localize with red dextran, indicating that Wg is internalized in the absence of Fz/DFz2 activities. These observations are consistent with results in the embryo, and altogether suggest that internalization of Wg can be accomplished by proteins other than Fz/DFz2. Interestingly, this observation contrasts with the role of HSPGs in Wg distribution, since wing discs lacking GAGs show alteration in Wg puncta in receiving cells. The extracellular distribution of Wg was examined in fz DFz2 mutant clones. Interestingly, accumulation of extracellular Wg was detected throughout these clones, thus revealing that Wg can bind to the cell surface and that Fz/DFz2 receptors are required somehow for Wg degradation. To exclude the possibility that accumulation of extracellular Wg results from increased wg expression or secretion in fz DFz2 clones that cross D/V boundary, small clones that do not include the D/V boundary were generated. Accumulation of extracellular Wg was detected in these clones, which is reminiscent of the finding that overexpression of a dominant-negative form of DFz2 (ΔDFz2-GPI) driven by en-Gal4 in embryonic tissue prevents Wg decay within the en domain. It has been proposed that endocytosis of a Wg/receptor complex is responsible for down-regulating Wg levels. Further, because Wg is still organized in a graded manner in these clones, as shown by the distribution of the Wg puncta, it indicates that Wg movement can occur in the absence of Fz/DFz2 (Baeg, 2004).

There is a third member of the Frizzled family encoded by DFz3 that could influence the distribution of Wg in tissues. DFz3 expression is similar to that of wg, and a constitutively activated form of Arm up-regulates its expression in the wing disc, suggesting that DFz3 is transcriptionally regulated by Wg signaling. Based on these observations, little or no DFz3 protein would be expected to be present in cells that lack Fz/DFz2 activity, suggesting that internalization of Wg in Fz/DFz2 mutant cells is unlikely to be mediated by DFz3. Another candidate that could affect Wg distribution is Arrow, which is a Drosophila homolog of a low-density lipoprotein (LDL)-receptor-related protein (LRP) and has been shown to be essential in cells receiving the Wg signal. However, because a soluble form of the Arrow fails to bind Wg and Fz receptors in vitro, and because Arrow functions after DFz2 engages Wg, it is unlikely that Wg internalization in Fz/DFz2 mutant cells is mediated by Arrow. Finally, as is case for FGF endocytosis, HSPGs themselves possibly play a role in Wg internalization (Baeg, 2004).

In summary, there is a Fz/DFz2 receptor-independent mechanism that organizes Wg distribution, and Fz/DFz2 proteins play a role in Wg gradient formation by decreasing the level of extracellular Wg. Regulation of extracellular Wg levels by Fz/DFz2 may occur through receptor-mediated endocytosis, or by some other mechanisms. If Wg degradation occurs by receptor-mediated endocytosis, it indicates that there may exist more than one way to generate Wg puncta since these are still present in the absence of Fz/DFz2 receptor activity. These findings also emphasize that the amount of Fz/DFz2 receptors at the cell surface must be precisely regulated to achieve the proper spreading of Wg, an observation that is underscored by the transcriptional down-regulation of DFz2 expression near the source of Wg (Baeg, 2004).

To further examine the role of Fz/DFz2 receptors in Wg gradient formation, DFz2 was overexpressed at the D/V boundary using the C96-Gal4 driver, and the effect on Wg distribution and wing patterning was examined. Analysis was focused on DFz2 since DFz2 has been shown to bind Wg with high affinity and to stabilize it. Further, DFz2 expression is down-regulated by Wg signaling, and this regulation has been shown to play a critical role in the overall shape of the Wg gradient. Interestingly, ectopic expression of DFz2 results in wing notching and ectopic bristles at the wing margin of adult wing. Previous studies have shown that wing nick phenotypes result from an inhibition in Wg signaling activity while the presence of ectopic bristles on the wing blade corresponds to an increase in Wg signaling. Thus, based on the wing phenotypes, it appears that overexpression of DFz2 paradoxically both increases and decreases Wg signaling (Baeg, 2004).

Overexpression of the DFz2 could interfere with Wg signaling and its distribution in a number of ways. For example, an increase in DFz2 could increase the efficiency of Wg signaling, if the amount of receptor is limiting. Further, since wg expression in the wing disc is restricted to the D/V margin, and Wg diffuses from it, trapping of Wg near these cells most likely will have an effect on Wg short- and long-range activity since the shape of the Wg gradient will be disrupted. To distinguish between these possibilities, Wg distribution was examined in discs with clones of cells that overexpress DFz2 at the D/V boundary. These clones were associated with two effects on Wg distribution. (1) The level of Wg was increased in the clones of cells where DFz2 was overexpressed, indicating that an increase in the level of DFz2 in receiving cells leads to an increase in trapping extracellular Wg. This observation is consistent with the occurrence of extra bristles on the wing blade since they reflect high levels of Wg signaling activity. (2) A dramatic reduction in Wg puncta was detected in WT cells located adjacent to the cells overexpressing DFz2, suggesting that Wg movement from the D/V margin into the wing blade is impaired as a result of the excess trapping of Wg by cells that overexpress DFz2. To demonstrate that Wg accumulation correlates with an increase in Wg signaling and that the absence of Wg puncta correlate with an absence of Wg signaling, the effect of DFz2 overexpression on the expression of senseless was examined. sen expression is expanded in cells overexpressing DFz2, yet sen is not expressed in WT cells near a clone of cells overexpressing DFz2. This is consistent with the observation that more Wg can be detected in cells overexpressing DFz2 and that less Wg puncta are present in WT cells near a clone of cells overexpressing DFz2 (Baeg, 2004).

It has been proposed that Fz proteins contribute to Wg turnover. Thus, it is intriguing to note that overexpression of DFz2 leads to an accumulation of extracellular Wg. This may reflect saturation of the endocytotic pathway when DFz2 is overexpressed or an inability of the regulatory pathways that normally control Dfz2 endocytosis in the wing disc to appropriately respond under this overexpressed condition. Another possibility is HSPGs themselves might play an important role. Wg endocytosis and the stoichiometry of Fz to HSPGs is essential to promote proper Wg internalization. Detailed biochemical and cell biological studies are now required to clarify the role(s) of these receptors in Wg movement (Baeg, 2004).

Finally, whether overexpression of DFz2 at the D/V boundary could affect long-range activity of Wg was examined, using wing disc overexpressing DFz2 driven by C96-Gal4 driver. Interestingly, Dll expression is dramatically shortened in wing disc overexpressing DFz2 at the D/V boundary when compared to that of WT disc. It is concluded that DFz2 has multiple roles in Wg signaling: First, it transduces Wg signaling and its level is limiting in amount; and second, it affects Wg short- and long-range activity by modulating the availability of extracellular ligand (Baeg, 2004).

In this study, the respective roles of HSPGs and Fz/DFz2 receptors in Wg distribution and gradient formation were examined. Interestingly, it was found that loss of Dlp activity significantly increases the level of Wg activity in S2R+ cells upon Wg induction, indicating that Dlp acts as a negative regulator in Wg signaling and that it is not required for transducing the Wg signal. Interestingly, the in vivo results show that Dlp protein levels are low near the D/V boundary. Thus, low levels of Dlp near the source of Wg production may allow for activation of high threshold Wg target gene. It is of interest to note that Notum is highly expressed along the D/V boundary (Giralez, 2002), which would be predicted to further diminish HSPGs activity. In addition, it was found that Dlp positively influences Wg signaling in S2R+ cells when Wg is not induced, suggesting that it is required for Wg signaling in cells where Wg level is low. A possible explanation for this result is that Dlp may act as a co-receptor that traps/stabilizes extracellular Wg and facilitates its association with signal transducing Fz receptors. In the wing imaginal disc, given that Dlp is required for Wg signaling in cells where Wg levels are low, HSPG activity is possibly required for Wg signaling by somehow facilitating Wg movement. The binding of extracellular Wg to the low-affinity HSPG receptors in receiving cells may result in the association of Wg to cell membranes. Ligand movement could then occur by a mechanism that directly involves HPSGs where subsequent cycles of Wg dissociation/reassociation with HSPGs might promote the movement or require other yet to be identified extracellular molecules. To distinguish these possibilities, the role of HSPGs in Wg movement will require further detailed analysis. Regardless, these results clearly indicate that the primary role of HSPGs is to sequester and/or stabilize extracellular Wg in receiving cells. The imposition of the HSPG-mediated Wg accumulation and the Fz-dependent degradation mechanism would thus contribute to the Wg morphogen gradient. It is important to note that the expression levels of some of the critical components of each systems (i.e., dally, DFz2) are also regulated by the Wg pathway itself, indicating that the slope of the Wg gradient is established by the delicate balance between these two systems (Baeg, 2004).

Hox control of morphogen mobility and organ development through regulation of glypican expression

Animal bodies are composed of structures that vary in size and shape within and between species. Selector genes generate these differences by altering the expression of effector genes whose identities are largely unknown. Prime candidates for such effector genes are components of morphogen signaling pathways, which control growth and patterning during development. This study shows that in Drosophila the Hox selector gene Ultrabithorax (Ubx) modulates morphogen signaling in the haltere through transcriptional regulation of the glypican dally. Ubx, in combination with the posterior selector gene engrailed (en), represses dally expression in the posterior (P) compartment of the haltere. Compared with the serially homologous wing, where Ubx is not expressed, low levels of posterior dally in the haltere contribute to a reduced P compartment size and an overall smaller appendage size. One molecular consequence of dally repression in the posterior haltere is to reduce Dpp diffusion into and through the P compartment. These results suggest that Dpp mobility is biased towards cells with higher levels of Dally and that selector genes modulate organ development by regulating glypican levels (Crickmore, 2007).

Upon comparing Dpp signaling readouts in the wing and haltere, it was noticed that, in addition to a general narrowing of Dpp pathway activity, Dpp signaling was also asymmetric relative to its source (the AP organizer) in the haltere. Specifically, the P-Mad signal was stronger anterior to the AP organizer (roughly demarcated by the domain of peak P-Mad staining) than it was posterior to the organizer. To test if this asymmetry is due to asymmetric ligand distribution or differences in signal transduction, an extracellular staining protocol was used to examine the distribution of a Dpp::GFP fusion protein following its expression in AP organizer cells. In wing cells, Dpp::GFP was detected in a broad gradient on both sides of the AP organizer. In the haltere, the distribution of Dpp::GFP is limited in both directions owing to high tkv expression levels, but this restriction is stronger in the P direction. Dpp::GFP spread was abruptly halted a few cell diameters posterior to the haltere AP compartment boundary, contrasting with a tapering signal seen in the anterior direction. By contrast, the Gal4 driver used to express Dpp::GFP (ptc-Gal4) drove nearly symmetrical expression of a UAS-GFP transgene, demonstrating that the distribution of Dpp::GFP in the haltere is not due to asymmetric activity of the ptc-Gal4 driver. In both the wing and haltere, the pattern of extracellular Dpp::GFP was very similar to the P-Mad pattern, suggesting that Ubx does not affect Dpp signal transduction downstream of ligand binding, at least as detected with the anti-P-Mad antibody. Furthermore, in both the wing and haltere, a similar coincidence of extracellular Dpp::GFP and P-Mad patterns was observed when Dpp::GFP was expressed in clones. The correlation between the P-Mad and extracellular Dpp::GFP patterns in both the wing and haltere allows inference of extracelluar Dpp ligand distribution by visualizing P-Mad in the proceeding experiments (Crickmore, 2007).

In the wing, Dpp::GFP distribution and P-Mad staining were also asymmetric, owing to slightly higher levels of Tkv in the P compartment, which impedes diffusion. By contrast, because Tkv levels are similar on both sides of the AP boundary of the haltere, Tkv levels are unlikely to account for the Dpp signaling asymmetry in this appendage. This idea directly by providing uniform levels of UAS-tkv to both the haltere and wing. Under these conditions, P-Mad staining became symmetric in the wing, but remained asymmetric in the haltere. These results suggest that the more-restricted P-Mad staining in the P compartment of the wild-type haltere is due to a tkv-independent and haltere-specific anterior bias in the diffusion of Dpp (Crickmore, 2007).

In previous work, it was showed how the upregulation of the Dpp receptor, thickveins, in the haltere causes an overall decrease in Dpp mobility as compared with the wing, and consequently contributes to the small size of the haltere. This study shows that the HSPG dally is repressed in the P compartment of the haltere and that this regulation decreases the P:A ratio and overall size of the haltere. Posterior dally repression causes Dpp diffusion to be biased away from P cells, generating an AP asymmetry in Dpp signaling. The findings reported here therefore provide another instance wherein Ubx controls the extracellular signaling environment of the developing haltere and thereby distinguishes it from the wing (Crickmore, 2007).

The movement of most or all signaling molecules through tissues is regulated by HSPGs, including glypicans such as dally. In contrast to receptors, HSPGs control the distribution of multiple signaling molecules. Regulation of HSPG expression and activity by selector genes is therefore a potentially very powerful mechanism for shaping signaling pathway activation profiles and molding organ shapes and sizes. However, the promiscuity of HSPGs also makes it difficult to assign the morphological consequences of their expression patterns to the alteration of individual signaling pathways. Indeed, it is likely that the altered dally expression pattern in the haltere has implications for Hh, Wg and Dpp signaling, all of which control growth and patterning. This study has focused on the relationship between dally expression and Dpp signaling (Crickmore, 2007).

Dpp signaling is increased in dally+ clones and decreased in dally- clones. These and other findings have suggested that Dally participates in the control of Dpp mobility. The current results add to these earlier observations by suggesting that variations in the levels of Dally between the cells of a tissue influence the direction and extent of Dpp diffusion. Specifically, it is proposed that in addition to simply being promoted by Dally, Dpp mobility is biased towards cells with higher Dally levels. This idea derives mainly from the observation that Dally can influence Dpp movement in a cell-non-autonomous manner. For example, when Dally levels are increased in the haltere P compartment, there is a shift in Dpp signaling from the A to the P compartments, as visualized by the levels of P-Mad. Similarly, knocking down Dally levels in the P compartment of the wing influences the extent and levels of P-Mad in the A compartment. If discontinuities in Dally levels can non-autonomously influence Dpp signaling across compartment borders, it follows that differences in Dally levels between cells within a compartment can also shape the Dpp signaling landscape. This might be important for wild-type wing development, where graded Dpp signaling represses dally, resulting in an inverse dally gradient that increases towards the lateral edge of the disc. It is suggested that this inverse dally gradient helps to attract Dpp to more lateral regions of the disc. Accordingly, in a dally-mutant wing disc, the Dpp gradient is less broad than in a wild-type wing disc. It is possible that other HSPGs control the mobility of signaling molecules in a similar manner (Crickmore, 2007).

Altering dally levels in either the A or P compartment changes relative compartment size, but only P compartment dally levels are relevant for total organ size. Two possible explanations are considered that link the P-specific dally repression seen in the haltere to a reduction in final organ size. Both of these scenarios (which are not mutually exclusive) focus on the role of P cells in producing Hh, which diffuses into A cells to instruct Dpp production and, consequently, controls final organ size. Importantly for both models, it was found that there is in fact less Hh detected in the P compartment of the wild-type haltere as compared with the wing. In the first model, the repression of dally reduces overall Hh production simply by reducing the size of the P compartment, which is a consequence of reduced Dpp signaling. In this scenario, fewer Hh-producing P cells result in less total Hh production from the P compartment, and therefore less Dpp produced in the A compartment. The logic of this potential mode of size regulation is interesting: a selector gene (Ubx) restricts growth factors (Wg and Dpp) from the pool of cells (the P compartment) that produces another growth factor (Hh). In the second scenario, dally repression may directly reduce the amount of Hh in the P compartment that can be transported into the A compartment. In support of this idea, Hh staining was found to be reduced in clones of cells where Dally levels are reduced through UAS-dallyRNAi (Crickmore, 2007).

Together, dally and dlp influence the mobility of all known morphogens in Drosophila. In addition to the compartmental regulation of dally, it is also noted that Dlp levels are generally lower throughout the haltere as compared with the wing. The haltere also lacks the domain of dlp repression seen at the DV boundary of the wing. Finally, it was also noticed that the expression of Notum-lacZ, an enhancer trap into a gene that encodes an HSPG-modifying enzyme, is different between the wing and haltere. The combined alteration of dally, dlp and Notum levels in the haltere is likely to have consequences for any signaling molecule that uses HSPGs for transport. When these observations are combined with those of earlier work showing that the levels of both Dpp and its receptor are regulated differently in the haltere and wing, and the observation that wg is repressed in the posterior haltere, a picture emerges in which selector genes alter the expression of multiple components of multiple signaling pathways to change morphogen signaling landscapes between tissues and thereby modify organ shapes and sizes. It is hypothesized that the summation of all signaling pathway changes may be sufficient to understand the size and shape differences between fundamentally similar epithelia such as the wing and haltere imaginal discs (Crickmore, 2007).

Protein Interactions: Potential role for glycosaminoglycans in Hedgehog signaling

Hedgehog (Hh) proteins act through both short-range and long-range signaling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. A new Drosophila gene, named tout-velu and meaning ‘all hair’ has been identified; it is required for diffusion of Hedgehog. ttv was identified in a screen for maternal-effect mutations associated with segment polarity. ttv clones prove to have a non-cell-autonomous effect. Hh signal is shown to be unable to reach wild-type cells located anterior to ttv mutant cells. It is proposed that ttv functions in the receiving cells for the movement of Hh from sending to receiving cells. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Analysis of the Ttv sequence shows that there is a hydrophobic stretch at the amino terminus of the Ttv protein, indicating that Ttv might be a transmembrane protein. Ttv appears to be a type II integral protein, with the C-terminal region displayed extracellularly. These results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, have led to a proposal that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog. EXT-1 has been found in the extracellular reticulum, where it helps to regulate the synthesis and display of cell-surface heparin sulphate glycosaminoglycans (GAGs). Because GAGs have been implicated in receiving signaling molecules, it is plausible to speculate that Ttv is involved in the synthesis of a GAG that specifically interacts with Hh at the cell surface. Such an interaction might result in the endocytosis of Hh or, alternatively, it may facilitate the movement of Hh from one cell to the next by translocating Hh around the surface of the cell. (Bellaiche, 1998).

The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. The signalling properties of these molecules in the embryonic epidermis have been tested. RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, the Hedgehog and Wingless signalling pathways were uncoupled; Dally-like and Dally, separately or together, were found to be unnecessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor (Desbordes, 2003).

Although heparan sulphate modifications have been implicated in several signalling pathways, it remains unclear which proteins are modified by these enzymes, and how the modifications affect a given signalling event. Since most heparan sulphate chains at the cell surface are thought to be carried by proteoglycans of the syndecan or glypican families, this study has examined the function of the two Drosophila homologs of glypicans, dally and dally-like (dlp), in the embryonic epidermis. Unexpectedly, this study has found a restricted and specific role for the fly glypicans. RNAi silencing shows that Dlp is a segment polarity gene that is absolutely required for Hh signalling. This requirement is specific to the Hh pathway; RNAi silencing of dlp does not affect Wg signalling in embryos. In contrast, RNAi silencing of dally, the other homolog of glypicans in Drosophila, does not produce a segment polarity phenotype, suggesting that Dally is dispensable for Wg or Hh signalling in embryos. Furthermore, RNAi silencing of both dally and dlp does not affect Wg signalling, suggesting that they do not function redundantly in this pathway (Desbordes, 2003).

dlp is a bona fide segment polarity gene since dlp RNAi generates embryos that fail to maintain en and wg expression at mid-embryogenesis, and exhibit a full segment polarity phenotype in the cuticle at the end of embryogenesis. The late disappearance of en expression and the single stripe of rho expression in dlp embryos suggest a loss of Hh activity. This is confirmed by the fact that when hh expression is under heterologous control, ectopic wg transcription is lost in dlp RNAi embryos, whether Hh is provided autonomously (armGal4 experiments) or non-autonomously (simGal4 experiments). These experiments demonstrate unambiguously that dlp is required for Hh signalling and rule out a requirement for hh transcription (Desbordes, 2003).

Dlp is a GPI-anchored protein and is likely to be localised at the cell surface. This leaves two plausible roles for Dlp: either it is required for the release of active Hh from the secreting cells, or it is required for the interpretation of the Hh signal on the receiving cells. Several possibilities have been eliminated. First, Dlp is required for the activity of Hh-N, an engineered form of Hh which is pre-processed and unmodified by cholesterol. This suggests that Dlp is necessary downstream of Hh processing and cholesterol modification. Downstream of these events, Hh undergoes another lipid modification, the addition of a palmitoyl moiety. The segment polarity gene rasp codes for an acyltransferase which is thought to be needed for Hh palmitoylation. Thus, Dlp could be required for the function of rasp in the signalling cells. However, whereas palmitolylation is essential for Hh-N activity, a recent report shows that it is not strictly required for the activity of wild-type Hh in Drosophila embryos. This suggests that the cholesterol and palmitoylate modifications might be partially redundant for the activity of wild-type Hh, at least in embryos. Thus, although Dlp could still act at the level of rasp on another function, loss of palmitoylation alone cannot account for the complete loss of Hh signalling seen in dlp RNAi embryos. It seems therefore more likely that dlp functions in the responding cells (Desbordes, 2003).

ptc is epistatic to dlp, indicating that Dlp acts upstream or at the level of the Ptc receptor. One possibility is that Dlp binds Hh and facilitates its interaction with Ptc. Increasing the concentration of Hh in receiving cells in either armGal4/UAShh or armGal4/UAShh-N experiments, does not abolish the requirement for Dlp. This argues against a role of Dlp in merely increasing the concentration of Hh ligand at the cell surface, and suggests a more specific role. Recent evidence supports a model in which, upon Hh binding, Ptc is endocytosed and inactivated by degradation, and this in turn indirectly activates Smoothened and the Hh intracellular pathway. Dlp may localize Hh and Ptc in membrane microdomains required for Ptc endocytosis and subsequent degradation (Desbordes, 2003).

Protein Interactions: Role for Dally in Dpp signaling

Dpp functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. Evidence is shown that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thickveins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. It is proposed that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient (Fujise, 2003).

To examine the effect of dally mutations on the distribution of Dpp morphogen, Dpp-GFP was expressed in the region where it is endogenously expressed using dpp-GAL4. In wild-type discs, Dpp-GFP is detectable as intracellular punctate spots and on the surface of the receiving cells. Dpp-GFP migrates throughout the wing pouch region, forming a shallow but evident gradient. However, in dally-mutant discs, no evident gradient of Dpp distribution could be detected in the receiving cells. In general, mutant discs showed a lower level of cell surface signals, suggesting reduced stability of Dpp (Fujise, 2003).

To determine whether dally overexpression at the A/P border cells, which causes abnormal patterns of pMad, also affects Dpp ligand gradient formation, Dpp-GFP distribution was observed in discs where dally is co-expressed with Dpp-GFP using dpp-GAL4. Consistent with the pMad patterns, Dpp is restricted to the dally-overexpressing region and fails to migrate properly. This suggests that Dally binds to Dpp protein and limits its distribution. Intensity profiles of these discs show that both reduction of dally and overexpression of dally at the A/P border cells result in a shallower gradient and lower levels of Dpp in the receiving cells. Taken together, Dally regulates formation of both Dpp ligand and activity gradients. In addition, the results strongly suggest that Dally plays at least two roles in the formation of the Dpp signaling gradient: (1) it regulates the sensitivity of cells to Dpp in a cell autonomous fashion; and (2) it affects Dpp protein distribution, which is a non-autonomous effect (Fujise, 2003).

This study demonstrates that dally controls shape of both the ligand and the activity gradients of Dpp in the developing wing. How does dally contribute to the Dpp gradient formation? In vitro analyses using mammalian tissue culture cells have established that HSPGs can increase FGF signaling by stabilizing FGF/FGF receptor complexes Several lines of evidence indicate that the dosage of HSPGs is an important factor for FGF signaling. For example, sodium chlorate treatment, which inhibits the sulfation of heparan sulfate, reduces the biological response of cells to FGF; the response can be restored by an exogenous supplement of heparin. However, restoration is seen only at an optimal concentration of heparin; excess heparin competes for FGF with signaling complex, resulting in a reduction of signaling. In the Drosophila wing, ectopic expression of Dally-like, another glypican related to Dally, leads to a massive accumulation of extracellular Wg protein and compromises Wg signal transduction, suggesting that the glypicans can affect ligand stability and distribution (Fujise, 2003 and references therein).

On the basis of these studies as well as the current data, Dally would appear to have both positive and negative roles on Dpp signaling. In its positive role, Dally serves as a co-receptor for Dpp, stabilizing Dpp protein and enhancing signaling. Conversely, given that Dpp is a heparin-binding protein, Dally may bind Dpp through its heparan sulfate chains and reduce the amount of free Dpp ligands. Thus, Dally affects the Dpp gradient at two distinct steps: signal transduction (autonomous effect) and ligand distribution (non-autonomous effect). A model is proposed in which alterations in the shapes of the Dpp ligand and the activity gradients caused by dally mutations and dally overexpression are interpreted as the sum of these plus and minus effects of Dally function. In this model, Dally normally sequesters Dpp protein to some extent in A/P border cells, where dally levels are very high. Therefore, reduced levels of Dally in mutant discs may result in the release of Dpp ligand and, consequently, higher levels of signaling activity in the central region. Therefore, dally mutations may severely reduce the stability of Dpp protein as well as its signaling activity in the receiving cells. When dally is overexpressed in A/P border cells, Dpp is trapped by binding to excess Dally and fails to distribute properly (Fujise, 2003).

Although it is thought that Dally regulates the diffusion of Dpp, the results do not rule out the possibility that Dally plays a more active role in facilitating Dpp diffusion or 'carries' Dpp protein. For example, it is possible that Dally is required for the Dpp movement through the transcytosis pathway or other transport systems, such as cytonemes (Fujise, 2003).

Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans

The Drosophila transforming growth factor ß homolog Dpp acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing. Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, it was found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. A blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). These results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly (Belenkaya, 2004).

One new observation in this work is that the extracellular Dpp is broadly distributed in the wing disc. Consistent with these findings, previous biochemical analysis demonstrated that the majority of mature Dpp signaling molecules are extracellular. Importantly, the overall shape of the extracellular Dpp gradient coincides well with its activity gradient, suggesting that the extracellular Dpp gradient contributes to Dpp activity gradient in the wing disc. The observation of broadly distributed extracellular Dpp led to a re-examination of the role of Dynamin-mediated endocytosis in Dpp movement and signaling. These analyses argue that Dynamin-mediated endocytosis is not essential for Dpp movement: (1) both Dpp signaling activity and extracellular GFP-Dpp levels are not reduced in the wild-type cells behind the shits1 clones that are defective in endocytosis; (2) the extracellular GFP-Dpp is also broadly distributed in endocytosis-defective wing discs homozygous for shits1 at nonpermissive temperature. These data demonstrate that Dpp molecules are able to move across Dynamin-defective cells. Finally, it was found that extracellular Dpp accumulates on the cell surface of shits1 mutant clones, suggesting that Dpp is able to move into shits1 mutant cells and that Dynamin-mediated endocytosis is normally involved in downregulating levels of the extracellular Dpp. No accumulation of extracellular Dpp on wild-type cells was observed in front of shits1 mutant clones; this would be expected if endocytosis were required for Dpp movement (Belenkaya, 2004).

While Dynamin-mediated endocytosis does not appear to be critical for Dpp movement, Dpp signaling activity is reduced cell autonomously in shits1 mutant cells. This result argues that Dynamin-mediated endocytosis is an essential process for Dpp signaling. Studies in mammalian cell culture system have demonstrated the critical role of Dynamin-mediated internalization of activated TGF-β receptors in TGF-β signaling. SARA (Smad anchor for receptor activation), a FYVE finger protein enriched in early endosomes is involved in this process. Although the exact mechanism of endocytosis-mediated TGF-β signaling is still unclear, current data suggest a role of early endosomes as a signaling center for TGF-β. Consistent with this view, it has been shown that ectopic expression of the dominant-negative form of Rab5 (DRab5S43N) using engrailed-Gal4 leads to a reduction of Dpp signaling, while overexpression of Rab5 broadens the Dpp signaling. Rab5 localizes in early endosomes and is required for endosome fusion. Taken together, it is proposed that dynamin-mediated endocytosis is not directly involved in Dpp movement but is essential for Dpp signaling. Furthermore, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient (Belenkaya, 2004).

To investigate the role of HSPGs in Dpp morphogen gradient formation, Dpp signaling and its extracellular distribution was examined in sulfateless (sfl) and dally-dly mutant clones. dally and dly are shown to be required and partially redundant in Dpp signaling and movement in the wing disc. Two lines of evidence support the role of Dally and Dly in Dpp movement across cells: (1) Dpp signaling activity is reduced in cells behind sfl or dally-dly mutant clones; (2) extracellular Dpp levels are diminished in cells behind sfl or dally-dly mutant clones. Importantly, it was found that sfl or dally-dly mutant clones only a few cells wide can effectively block GFP-Dpp movement, suggesting that Dpp movement does not occur through 'free diffusion', by which extracellular Dpp would be expected to move across sfl or dally-dly mutant cells. Based on these observations, it is proposed that Dpp moves from cell to cell along the epithelium sheet through restricted diffusion involving Dally and Dly (Belenkaya, 2004).

If the HSPGs Dally and Dly are indeed involved in Dpp movement, observation of extracellular GFP-Dpp accumulation in front of sfl, dally-dly mutant clones would be expected. Indeed, extracellular GFP-Dpp accumulation is visible in front of sfl or dally-dly mutant clones. Consistent with this observation, Hh has been observed to accumulate abnormally in clones mutant for tout-velu (ttv) and brother of tout-velu (botv), two Drosophila EXT members involved in HS GAG chain biosynthesis. Both Wg and Dpp accumulation in front of ttv-botv clones are also observed, albeit less pronounced, compared with the case of Hh. Similarly, extracellular GFP-Dpp accumulation is relatively weak, compared with Hh accumulation. One possibility is that extracellular Dpp molecules bound by Dally and Dly in wild-type cells can still be internalized by adjacent sfl or dally-dly mutant cells through cell-cell contact, leading to a reduction of extracellular Dpp accumulation in front of sfl or dally-dly mutant cells. Consistent with this view, it was noticed that, within sfl or dally-dly mutant clones, the first row of the mutant cells immediately adjacent to wild-type cells and facing Dpp-expressing cells is still capable of transducing Dpp signaling (Belenkaya, 2004).

In addition to being required for Dpp movement, Dally and Dly are also essential for Dpp signaling in its receiving cells. Dpp signaling is reduced in sfl or dally-dly mutant cells. Reduced levels of extracellular Dpp were observed in sfl or dally-dly mutant clones. Consistent with the results in this work, clones mutant for ttv or botv as well as sister of tout-velu (sotv), members of Drosophila EXT, led to reductions in Dpp signaling and its ligand distribution when analyzed by a conventional staining protocol that revealed both extracellular and intracellular Dpp. Collectively, these data suggest that the main function of Dally and Dly in Dpp signaling is to maintain and/or concentrate the extracellular Dpp available for Dpp receptors (Belenkaya, 2004).

This study has shown that Dynamin-mediated endocytosis is not essential for Dpp movement. Dpp movement is through a cell-to-cell mechanism involving the HSPGs Dally and Dly. On the basis of these findings, it is proposed that secreted Dpp molecules in the A-P border are immediately captured by the GAG chains of Dally and Dly on the cell surface located in either the A or P compartments. The differential concentration of Dpp on the cell surface of producing cells and receiving cells drives the displacement of Dpp from one GAG chain to another toward more distant receiving cells. Alternatively, Dpp molecules bound by Dally or Dly could also move along the cell surface through a GPI linkage that is inserted in the outlet leaflet of the plasma membrane and is enriched in raft domains. In the receiving cells, Dally and Dly may present Dpp to its receptor, Tkv, that transduces Dpp signal through the Dynamin-mediated internalization process, which further downregulates extracellular Dpp levels and cell surface Tkv. Based on this model, extracellular Dpp and its receptor, Tkv, would be accumulated on the surface of Dynamin-deficient cells, and extracellular Dpp would be able to move across Dynamin-deficient cells to reach more distal cells. In sfl or dally-dly mutant clones, extracellular Dpp molecules can not be attached on the cell surface and therefore can not be transferred further to more distal cells. In this model, endocytosis is not directly involved in Dpp movement; however, through receptor-mediated internalization, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient. It remains to be determined how Dpp is transferred from one cell to another by the GAG chains of Dally and Dly and whether Dally and Dly play a role in preventing extracellular Dpp from degradation. Further studies are needed to determine whether other mechanisms are also involved in Dpp movement (Belenkaya, 2004).

Dally and Dally-like shape the extracellular Wingless morphogen gradient in the wing disc

During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly result from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).

One important finding in this work is that Dally and Dlp are required for Wg gradient formation. Several recent studies have shown that extracellular Wg distribution is compromised in clones mutant for HS biosynthesis enzymes, including sfl, slalom and members of the Drosophila EXT gene. However, it is unclear which HSPG cores are involved in this process. This study shows that Wg morphogen distribution is defective in either dally or dlp mutant clones. These new findings clearly establish the requirement of Dally and Dlp in Wg morphogen gradient formation. Thus, as in the case of Hh and Dpp, the glypican members Dally and Dlp, rather than Drosophila syndecan or perlecan, are the main HSPGs involved in Wg gradient formation (Han, 2005).

Interestingly, Dally and Dlp differentially regulate the Wg extracellular gradient in distinct regions of the wing disc. Both Dally and Dlp are glypican members of HSPG family. One would expect that differences in the structure of Dally and Dlp, and their attached HS GAG chains may determine their abilities to interact with Wg, thereby leading to their specificities. This is probably one of the factors, since overexpression of Dally and Dlp has very different effects on extracellular Wg gradient. Consistent with the data in this work, previous studies have shown that Dlp is much more potent in accumulating Wg protein than Dally when overexpressed. However, the regional effects of Dally and Dlp on extracellular Wg gradient correspond well to their expression patterns. The regions with higher expression levels of Dally or Dlp have stronger extracellular Wg defects when Dally or Dlp is removed, respectively. Based on these data, it is suggested that the differential roles of Dally and Dlp in extracellular Wg distribution are at least partially determined by their restricted expression (Han, 2005).

What exact roles do Dally and Dlp play in shaping the extracellular Wg gradient? Loss-of-function results suggest that removal of Dally or Dlp leads to reduced extracellular Wg levels on the cell membrane. Furthermore, extracellular Wg levels are reduced in wild-type cells behind sfl or dally-dlp clones. These data suggest that the primary function of Dally and Dlp in Wg gradient formation is to maintain extracellular Wg proteins so that locally concentrated Wg proteins can further move to more distal cells through diffusion (Han, 2005).

Despite a positive role of Dlp in extracellular Wg distribution, surprisingly, Dlp negatively regulates Wg signaling at the DV boundary. However, ectopic Wg signaling at the DV boundary of the dlp mutant is not as great as expected. This relatively weak effect is most probably due to the low level expression of Dlp, which is downregulated by Wg signaling. The results are consistent with a previous observation that overexpression of Dlp in the wing disc leads to a blockage of Wg signaling. Dlp may compete with Fz proteins for available Wg protein at the DV boundary, thereby inhibiting Wg signaling. However, the extract mechanism of Dlp-mediated Wg inhibition needs to be further determined (Han, 2005).

Previous studies have identified Notum as a secreted inhibitor for Wg signaling. Notum is expressed at the DV boundary and has been proposed to downregulate Wg signaling by modulating Dlp activity (Giraldez, 2002). Kreuger (2004) and Selleck (Kirkpatrick, 2004) proposed that Notum negatively regulates Wg signaling by shedding of Dlp, which converts Dlp from a membrane-tethered co-receptor to a secreted antagonist. Their conclusions are mainly based on two lines of experimental data: (1) biochemical experiments clearly demonstrated that Notum can modify Dlp in a manner that resembles cleavage of the GPI anchor (Kreuger, 2004); (2) Kirkpatrick (2004) showed that transheterozygous dlp/notum flies produced ectopic mechanosensory bristles which are not seen in dlp+/- or notum+/- alone, indicating that Dlp and Notum genetically collaborate in downregulating Wg signaling (Han, 2005).

However, on the basis of the current data, it is suggested that Notum inhibits Wg signaling mainly by modifying Dally in the wing disc. (1) Genetic interaction data shown by Kirkpatrick (2004) cannot distinguish whether Dlp and Notum work in the same pathway or in two independent pathways to downregulate Wg signaling at the DV boundary (Kirkpatrick, 2004). If Dlp is indeed the main substrate for Notum, it would be expected that ectopic Wg signaling activity in dlp-notum should be similar to that in dlp mutant. However, loss-of-function analysis demonstrates that ectopic Wg signaling in dlp-notum is similar to that in notum mutant, but much stronger than that in dlp mutant. However, dally-notum clones exhibit loss of Wg signaling activity that is similar to dally mutant. (2) Dlp expression is strikingly repressed by Wg signaling and this reduction is independent of Notum. Low/absent expression of Dlp is not consistent with the view that Dlp is the main substrate for Notum. (3) It is important to mention that Notum can reduce the amount of Dally when they are co-expressed in Drosophila S2 cells (Giraldez, 2002), suggesting that Notum can modify Dally as well. Although Notum can shed Dlp, whether shed Dlp acts as a Wg inhibitor needs to be further determined. Therefore, further experiments are necessary to define the mechanism(s) of Notum-mediated Wg inhibition (Han, 2005).

One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).

It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).

Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).

Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).

The endocytic pathway and formation of the Wingless morphogen gradient

Controlling the spread of morphogens is crucial for pattern formation during development. In the Drosophila wing disc, Wingless secreted at the dorsal-ventral compartment boundary forms a concentration gradient in receiving tissue, where it activates short- and long-range target genes. The glypican Dally-like promotes Wingless spreading by unknown mechanisms, while Dynamin-dependent endocytosis is thought to restrict Wingless spread. Short-term expression of dominant negative Rab proteins was used to examine the polarity of endocytic trafficking of Wingless and its receptors and to determine the relative contributions of endocytosis, degradation and recycling to the establishment of the Wingless gradient. The results show that Wingless is internalized via two spatially distinct routes: one on the apical, and one on the basal, side of the disc. Both restrict the spread of Wingless, with little contribution from subsequent degradation or recycling. As has been shown for Frizzled receptors, depleting Arrow does not prevent Wingless from entering endosomes. Both Frizzled and Arrow are internalized mainly from the apical membrane. Thus, the basal Wingless internalization route must be independent of these proteins. Dally-like is not required for Wingless spread when endocytosis is blocked, and it is proposed that Dally-like promotes the spread of Wingless by directing it to lateral membranes, where its endocytosis is less efficient. Thus, subcellular localization of Wingless along the apical-basal axis of receiving cells may be instrumental in shaping the Wingless gradient (Marois, 2006).

The data suggest that the spread of Wg is controlled by restrictive clearance. Preventing Rab5-dependent internalization increases the spread of Wg through disc tissue. While this may also elevate extracellular Wg levels by indirect mechanisms (for example by modulating extracellular Wg proteolysis or release from disc tissue), the possibility is favored that the gradient is shaped by internalization of Wg itself, for several reasons. First, Wg is actually found in Rab5- and Rab7-positive endosomes. Second, treating living discs with protease inhibitors does not cause Wg to accumulate. Finally, differential centrifugation experiments suggest that only about 6% of Wg is not membrane-associated (Marois, 2006).

Some ligands signal from endocytic compartments after internalization. Therefore, it was initially of interest to discover whether the high levels of Wg accumulating after dominant negative Rab5SN expression could increase signal transduction. While Rab5SN-expressing cells both accumulate Armadillo and reduce Senseless expression, whether internalization of Wg is required for signaling was not further investigated because of the striking and unexpected transcriptional changes caused by blocking Rab5 activity. For example, transcription of both Fz2 and Dlp increases and that of Arrow plummets within a few hours of initiating Rab5SN expression - any of these changes by themselves could alter Wg signaling. Although this phenomenon are not yet understood, one might imagine that coupling transcriptional regulation of endocytic receptors to their actual endocytosis and/or degradation would be an effective homeostatic mechanism (Marois, 2006).

Studies in tissue culture cells have shown that inhibiting Rab7-dependent lysosomal degradation can increase recycling of some proteins to the cell surface. In imaginal discs, Rab7TN expression increases the abundance of Rab11 recycling endosomes, and inhibiting recycling via Rab11SN enlarges the Rab7-positive degradative compartment. Thus, the recycling and degradative pathways may compete for some cargo in discs as they do in cultured cells. This raises the possibility that changing the balance of degradation and recycling might affect the pool of extracellular Wingless available for spreading. Indeed, Wg appears to be recycled in embryos, and inhibiting lysosomal degradation in the embryonic ectoderm extends its range. However, the data suggest that the increase in the range of Wg caused by inhibiting degradation (at least in imaginal discs) is not the result of increased recycling and extracellular spread. Although Wg protein is detected in endosomes over a broader range in imaginal disc tissue expressing Rab7TN, there is no increase in the range of extracellular Wg. Furthermore, neither extracellular nor intracellular Wg distribution is affected by inhibiting the Rab4- or Rab11-dependent recycling pathways. Inhibiting degradation probably extends the apparent range of Wg by stabilizing internalized protein, raising its levels above the threshold of detectability in more distant cells (Marois, 2006).

The idea that apical-basal polarity of epithelial cells might play a role in regulating morphogen trafficking has been suggested by the observation that wg mRNA is enriched apically in the embryonic ectoderm. Changing mRNA localization alters the distribution of Wg protein in receiving tissue, raising the intriguing possibility that Wg might be trafficked differently depending on whether it is secreted apically or basal-laterally. In support of this idea, the data show that Wingless is internalized specifically from the apical and basal (but not lateral) surfaces of the disc epithelium. Indeed, the distribution of Rab5- and Rab7-positive endosomes in general suggests that the apical and basal surfaces are more endocytically active than other regions. The apical and basal internalization mechanisms may be distinct; the known receptors for Wingless, Fz2 and Arrow are internalized mainly from the apical surface (despite their steady-state basal-lateral localization), suggesting that basal Wingless endocytosis must be independent of these proteins. One possibility is that membrane association of Wg via Palmitate is sufficient to allow its endocytosis -- perhaps by mechanisms similar to those used by gpi-linked proteins. Alternatively, Wg bound to Lipoprotein particles might be internalized via Lipoprotein receptors (Marois, 2006).

It was surprising to observe that the Wg accumulating on the basal side of disc epithelial cells after Rab5SN expression does not spread onto the lateral membrane, since no barrier to diffusion has been identified between these domains. Three possible explanations are suggested. (1) There may indeed be a 'fence' separating the lateral and basal sides of disc epithelial cells. Neurons have a fence that prevents diffusion of lipid and lipid-linked proteins between the axon and the cell body, although it does not resemble a classical intercellular junction. (2) It may be that the receptor(s) that normally internalize Wg basally are linked to cytoskeletal components, or interact with extracellular matrix (ECM), and are not free to diffuse. If these Wg receptors were of sufficiently high affinity, they might trap Wg before it could move laterally. (3) Perhaps Wg itself interacts efficiently with basal ECM components (Marois, 2006).

While it seems that endocytosis restricts the spread of Wg on the apical and basal surfaces, it is not yet clear which receptors might be responsible. A simple model would predict that removing such a receptor should produce phenotypes similar to Rab5SN expression, i.e. increased and more extensive extracellular Wg, and less Wg in endosomes. Conversely, overexpression might be expected to compress the range of Wg distribution and decrease extracellular Wg. None of the known receptors behaves in this way. Previous studies showed that at least a fraction of Wg is still internalized in the absence of both Fz1 and Fz2. Furthermore, overexpression of Fz2 causes extracellular Wg accumulation over longer distances. This study has shown that loss of Arrow actually increases the amount of Wg present in Rab5- and Rab7-positive endosomes - more consistent with a role in Wg degradation after endocytosis. The complexity of these observations may reflect different mechanisms of Wg endocytosis on the apical and basal sides of the cell - understanding both these pathways and their interplay will be necessary to understanding how the Wg gradient forms (Marois, 2006).

While internalization limits the range of Wg accumulation, the glypican Dlp extends it. It has been proposed that Dlp allows Wg to interact with disc cells, increasing local Wg concentration and restricting its diffusion to the epithelium. The data, however, show that disc cells can accumulate high levels of Wg on their surface in the absence of Dlp as long as Rab5-dependent internalization is blocked. This observation is not consistent with a model in which Dlp traps Wg on the cell surface or helps it transfer from cell to cell. Instead, it suggests that Dlp normally stabilizes Wg at the cell surface by antagonizing the effects of Rab5-dependent internalization. While Wg is normally internalized from the apical- and basal-most surfaces of disc cells, Dlp overexpression recruits Wg to the lateral cell surface. This raises the possibility that Dlp stabilizes Wg and increases its range by changing its subcellular localization to protect it from endocytosis. Polarizing the distribution of morphogens within an epithelium may have a key regulatory role in the trafficking events leading to gradient formation (Marois, 2006).

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. The Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function (Johnson, 2006).

Converging lines of evidence suggest that membrane-associated HSPGs serve an important purpose in the assembly, function and plasticity of excitatory synapses. The ancient HSPG families of syndecans and glypicans are necessary for Drosophila to regulate distinct aspects of synaptic morphogenesis. Genetic and biochemical data indicate that Sdc and Dlp interact with LAR to control presynaptic properties. Because LAR-family RPTPs have been shown to control the formation of excitatory synapses in Drosophila, C. elegans, and mammals, these findings may represent a more general mechanism for regulating synaptic morphogenesis and function. Despite the importance of LAR-family RPTPs during cellular morphogenesis inside and outside of the nervous system, the lack of physiologically relevant extracellular binding partners has made it challenging to study this well-conserved group of receptors (Johnson, 2006).

The data show that Sdc promotes the formation of presynaptic boutons. This Sdc function appears to be mediated by LAR, as supported by parallel phenotypes, direct binding, in vivo colocalization, and three types of double mutant analysis between Sdc and LAR. Despite the fact that Sdc exhibits gain-of-function activity and endogenous expression on both sides of the synapse, neuronal and muscle-specific rescue experiments show that Sdc function is mainly presynaptic. Because LAR is required only in neurons to promote synapse growth, these findings support a model in which Sdc acts as a neuronal cell-autonomous agonist of LAR. This is somewhat surprising, given the fact that soluble forms of Sdc bind to LAR and that endogenous Sdc appears to be released from the presynaptic membrane to fill the subsynaptic reticulum. One way for Sdc to act presynaptically would be to bind to LAR even before the two proteins are presented on the neuronal surface. Because Dlp has a competitive advantage over Sdc for binding to LAR, a prebound complex of Sdc and LAR would have the ability to stimulate synapse growth before the phosphatase could be inhibited by Dlp. Such a mechanism could provide a time- and/or HSPG concentration-dependent switch from bouton addition to active zone assembly (Johnson, 2006).

Sdc could promote LAR activity in collaboration with an additional cell-type-specific membrane protein. Data from a parallel study has also identified Sdc-LAR interactions during embryonic motor axon guidance. However, in contrast to CNS pathfinding, complete loss of Sdc alone has no significant effect on motor pathfinding, suggesting that additional LAR ligands exist in the early embryo. Recent experiments with the vertebrate LAR ortholog PTP-s suggest that non-HSPG ligands may regulate its ability to promote retinal axon outgrowth. Although it remains a formal possibility, an additional ligand may not be needed to account for the NMJ growth-promoting activity of LAR because the larval synaptic phenotype in Sdc mutants is nearly as strong as the growth defect in LAR mutants (Johnson, 2006).

Active zone assembly is vital for neurotransmission at the synapse and has been proposed as a means to modulate synaptic function over time. Analysis of Dlp reveals that synaptic glypicans are required to regulate active zone morphology and function. Moreover, Dlp is limiting for active zone morphogenesis, consistent with an instructive role. Because the activities of Dlp appear opposite to those of LAR, it is proposed that the high affinity binding of Dlp to LAR induces an inhibition of receptor function. This hypothesis is supported by the double RNAi experiments showing that the LAR effect on Ena phosphorylation is epistatic to the effect of Dlp, indicating that Dlp acts upstream of LAR. Because loss of Dlp at the NMJ did not induce a significant change in the number of presynaptic boutons, the results lead to a model in which Dlp is specialized for control of active zone properties. Such a function might provide a means to independently regulate and spatially distinguish LAR inhibition from LAR activation. In any case, the presence of active zone phenotypes in dlp but not in Sdc reveals specialization among synaptic HSPGs (Johnson, 2006).

LAR regulates both NMJ growth and active zone morphogenesis. Thus, LAR appears to provide a link between two important synaptic properties that are regulated by different extracellular factors. A mechanism to couple bouton growth and active zone formation would make sense because active zones appear early in the nascent bouton. Because LAR catalytic activity is necessary for bouton addition, and yet LAR inhibition by Dlp appears necessary for proper active zone formation, LAR's role at the active zone may be primarily structural. For example, LAR may simply provide an anchorage point for synaptic components like the scaffolding protein Liprin-alpha that regulates active zone formation. Alternatively, LAR may exist in distinct yet active signaling states, one of which is dependent on PTP activity (promoting synapse growth), and one of which is dependent on recruitment of signaling molecules (controlling active zone assembly). Because loss of Dlp or LAR has opposite effects on quantal content at the NMJ, it is attractive to speculate that the Dlp-LAR pathway normally provides a means to modulate the strength of neurotransmission, either during NMJ growth or during synaptic plasticity. LAR PTPs are required for normal physiology and plasticity at mammalian hippocampal synapses (Johnson, 2006).

Sdc and Dlp are both HSPGs that bind to LAR and thus might be expected to act similarly, but the results show that their functions are different. One way to account for the specificity might be a difference in the effect of soluble versus cell-surface HSPGs on LAR. Some ligand molecules such as Ephrins function when clustered at high density (e.g., on a membrane surface) but fail to activate their receptors when presented in a soluble, monomeric form. Another possibility could be that LAR binding or signaling is differentially influenced by direct protein-protein interactions with the Sdc versus Dlp core proteins. The two HSPGs have very different core structures; Sdc is a transmembrane molecule with HS modification sites near the N terminus, whereas Dlp is a GPI-anchored protein with HS sites proximal to the membrane and a large disulphide bonded globular domain located more distally. It may also be relevant that Dlp consistently binds more effectively to LAR than Sdc, with KD measurements in solution showing an affinity approximately 2-fold higher. These results suggest a competition model in which Dlp displaces Sdc, possibly to favor the stabilization of active zones after new growth at the synapse. In this model, presynaptic growth would be initially promoted by Sdc and would then be limited or halted by Dlp binding after formation of close membrane contact between the nerve and muscle. Such a mechanism could insure a transition from growth to synapse stabilization and could participate in subsequent maintenance or plasticity of the synapse (Johnson, 2006).

Of course other molecules influence synapse size, and these might include coligands or coreceptors that may bind to Sdc, Dlp, and/or LAR. Potential candidates might include bone morphogenic protein (BMP), the type II BMP receptor Wishful thinking (Wit), or the Wnt ortholog Wingless (Wg), which have all been shown to regulate NMJ morphology in Drosophila. However, in addition to significant phenotypic differences compared to the HSPGs, overexpression studies indicate that neither BMP nor Wg are limiting for NMJ morphogenesis. In contrast, Sdc and Dlp are both limiting for different aspects of synapse development, consistent with an instructive role in this context. Consistent with this notion, Syndecan-2 is sufficient to promote dendritic spine maturation during hippocampal synaptogenesis in culture. Although vertebrate Syndecan-2 has yet to be tested at the synapse by loss of function, the colocalization and parallel biology of Synecan-2 and vertebrate LAR-family receptors strongly suggest conservation in the regulation of synaptic LAR (Johnson, 2006).

The genetic and biochemical studies described in this study have identified a partnership between HSPGs and LAR in Drosophila NMJ development that sets precedents for (1) the in vivo requirement for members of the syndecan and glypican families in synapse growth and electrophysiological function, (2) the specificity of HSPG function at the synapse, with distinct actions of Sdc and Dlp, and (3) biochemical identification of Sdc and Dlp as LAR binding partners, plus genetic evidence to place them in a pathway regulating biological function at the synapse (Johnson, 2006).

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, this study characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. It was found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts (Kamimura, 2006; full text of article).

It was asked whether FGF signaling is impaired in mutant animals using an antibody that specifically recognizes the diphosphorylated form of MAP kinase (dpMAPK). In wild-type embryos, dpMAPK is detected in the tracheal placodes at stage 10, reflecting activation of Egfr. This dpMAPK signal was not diminished in the Hs2st; Hs6st embryos, showing that Egfr signaling is not affected by the double mutations. At stage 12, wild-type embryos show a strong dpMAPK signal in the migrating tip cells of each primary branch due to activation of FGF signaling. In contrast, the btl-dependent MAPK activation in the tip cells is disrupted in the Hs2st; Hs6st embryos. In situ RNA hybridization experiments revealed that bnl expression is not altered in the double mutant embryos, confirming that the branching defects observed in the double mutants are caused by disruption of FGF reception but not FGF expression. These results showed that HS with neither 2-O nor 6-O sulfate groups lost the ability to mediate Btl signaling (Kimimura, 2006).

Lipoprotein-heparan sulfate interactions in the Hh pathway; Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like

The Drosophila lipoprotein particle, Lipophorin (Retinoid- and fatty acid-binding glycoprotein), bears lipid-linked morphogens on its surface and is required for long-range signaling activity of Wingless and Hedgehog. Heparan sulfate proteoglycans are also critical for trafficking and signaling of these morphogens. Lipophorin interacts with the heparan sulfate moieties of the glypicans Dally and Dally-like. Membrane-associated glypicans can recruit Lipophorin to disc tissue, and remain associated with these particles after they are released from the membrane by cleavage of their gpi anchors. The released form of Dally colocalizes with Patched, Hedgehog, and Lipophorin in endosomes and increases Hedgehog signaling efficiency without affecting its distribution. These data suggest that heparan sulfate proteoglycans may influence lipid-linked morphogen signaling, at least in part, by binding to Lipophorin. They further suggest that the complement of proteins present on lipoprotein particles can regulate the activity of morphogens (Eugster, 2007).

Controlling the spread and signaling of secreted morphogens is of critical importance for pattern formation during development. Morphogens of the Wnt and Hedgehog families undergo covalent lipid modification; these lipid moieties are essential for normal trafficking and signaling. It has been shown that the cell biological mechanisms they influence are not completely understood. The lipid-linked morphogens Wingless (Wg) and Hedgehog (Hh) can be released from the plasma membrane on the Drosophila lipoprotein Lipophorin, and Lipophorin is important for their long-range but not short-range signaling activity (Panáková, 2005). How might Lipophorin association influence morphogen signaling activity? One function of Lipophorin may be to mobilize otherwise membrane-bound molecules for long-range movement. Furthermore, a variety of gpi-linked proteins are also found on these particles, raising the possibility that morphogen signaling could be subject to additional regulation by other particle-associated proteins (Eugster, 2007).

Heparan sulfate proteoglycans (HSPGs) are also essential for normal trafficking and signaling of morphogens. Sterol-modified Hh cannot accumulate in or move through tissue that does not synthesize heparan sulfate (HS). In contrast, truncated Hh that cannot be sterol modified spreads freely through tissue missing HS. The effects of HS on the Hh pathway are mediated at least in part by the glypicans Dally and Dally-like (Dlp). Whereas Dlp is required for Hh signaling in S2 cells, it appears to act redundantly with Dally in imaginal discs. Only clones of cells mutant for both dally and dlp show autonomous reduction in Hh signaling (Eugster, 2007).

While autonomous functions of Dally and Dlp are presumably exerted by membrane-linked forms of the proteins, Dlp can also be shed from the plasma membrane. Notum, an enzyme in the α/β hydrolase family, removes the gpi anchor from Dlp expressed in S2 cells, releasing the protein into the supernatant. Whether Dally can be detached from its gpi anchor by Notum or other enzymes is unresolved. Some gpi-anchored proteins are released from cell membranes by associating with lipoprotein via their lipid anchors (Panáková, 2005), suggesting yet another mechanism for glypican shedding. It is not yet clear how and to what extent shedding occurs in vivo. Furthermore, specific roles for shed glypicans would be difficult to detect genetically because mutant clones have access to released glypicans produced by wild-type tissue (Eugster, 2007).

This study examined the relationship between glypicans and Lipophorin in the Hh pathway. Lipophorin was found to bind to the HS moieties of glypicans and can be recruited to disc tissue by these proteins. Lipophorin remains associated with glypicans when they are released from the plasma membrane by gpi removal. The released form of Dally is found in endosomes containing Lipophorin, Hh, and the Hh receptor Patched (Ptc) and increases Hh signaling efficiency (Eugster, 2007).

One consequence of Lipophorin particles binding to the heparan sulfate moieties of glypicans is that membrane-associated Dally and Dlp can recruit Lipophorin particles to imaginal disc tissue. Indeed, HSPGs are autonomously required for Lipophorin's interaction with the basal side of the disc epithelium. This raises the possibility that HSPGs may recruit lipid-linked morphogens to receiving cells, at least in part, by binding to morphogen-bearing lipoprotein particles. Although Hh is present in both apical and basal pools in the wing disc, it accumulates much more strongly on the basal surface when endocytosis is blocked. This suggests that the majority of Hh protein may spread along the basal surface. ttv, botv mutant cells that cannot recruit Lipophorin basally may be compromised in their ability to interact with Hh-bearing Lipophorin particles. This idea is also consistent with the observation that Hh mutants that cannot be sterol modified (and are presumably not associated with lipoproteins) no longer depend on HSPGs to bind to receiving cells (Eugster, 2007).

These data suggest that Lipophorin must be transcytosed from the basal side of the disc to the apical lumen. Although membrane-associated Dally and Dlp are found on the basolateral membrane, removal of the gpi anchor results in apical secretion. It will be interesting to investigate whether glypican endocytosis and subsequent release might play a role in Lipophorin transcytosis. The presence of transcytosed particles in the disc lumen may explain why removal of HS from clones of cells only disrupts basal Lipophorin accumulation. Particles transcytosed by other cells would be available for apical internalization by HS-independent mechanisms (Eugster, 2007).

Dally and Dlp continue to associate with Lipophorin via HS moieties after cleavage of the gpi anchor. The fact that both glypicans remain bound to Lipophorin after shedding raises the possibility that released glypicans influence signaling from lipoprotein particles. This analysis of the role of released Dally supports this idea (Eugster, 2007).

The data indicate that released Dally is required for full-strength Hh signaling, but does not affect the range over which Hh spreads. Hh signaling promotes Ci-dependent target gene activation by three separable mechanisms that are differentially sensitive to Hh levels. Ci stabilization requires the lowest levels of Hh signaling, and thus occurs over the broadest range. Ci stabilization is insufficient for target gene activation, however. To activate transcription, full-length Ci must be released with Suppressor of Fused (Su[Fu]) from cytoplasmic complexes containing Fused and Costal-2 and translocate to the nucleus. This occurs over shorter distances than Ci stabilization. Still higher levels of Hh signaling induce other processes needed for full activity of the Ci-Su(Fu) heterodimer within the nucleus. These are less well understood, but may involve phosphorylation of Su(Fu). In dally mutant discs, Ci stabilization and nuclear translocation, which require lower levels of Hh signaling, are normal. Only Ci activation is impaired by loss of dally. Thus, released Dally appears to increase the quantitative output of the Hh signaling pathway without increasing the amount of Hh (Eugster, 2007).

Although the possibility cannot be ruled out that a small, non-particle-associated fraction of released Dally gives rise to the phenotypes that were see, the extensive cofractionation and colocalization of released Dally with Lipophorin suggests that Lipophorin may act by influencing the behavior of these particles. For example, interaction of Lipophorin with the HS moieties of released Dally might reduce the affinity of Lipophorin particles for cell-surface HS, promoting transfer of Hh-bearing particles to Ptc. Alternatively, Dally HS moieties on Hh-bearing lipoprotein particles might promote the formation of ligand-receptor complexes. IHog acts as a coreceptor with Ptc and, like released Dally, is necessary for full-strength Hh signaling (Yao, 2006). Adding soluble heparin induces IHog dimerization and increases IHog-Hh binding in vitro (McLellan, 2006). Presentation of Dally HS on the same particle with Hh may greatly increase the efficiency with which these complexes form by bringing HS, Hh, and IHog into close proximity in vivo. Furthermore, lipoprotein particles that carried multiple copies of Dally and Hh would have the potential to induce receptor crosslinking. In this way, lipoprotein particles may act as scaffolding platforms, bringing together specific ligands and increasing the diversity of combinatorial signals available for patterning during development (Eugster, 2007).

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

Studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. This study shows that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, the role of Dlp was analyzed in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, these results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila (Yan, 2007).

There are three main important findings in this work. First, Dlp was identified as an essential molecule required for tracheal development. Dlp is required for Btl-mediated tracheal branching during embryogenesis while both Dlp and Dally are involved in the formation of air sac tracheoblasts in the wing disc. Second, the data show that other HSPGs cannot replace Dlp for Btl signaling during embryogenesis and that both Dlp and Dally are not essential for Htl-mediated mesodermal cell migration. These data demonstrate that different FGFs may require different HSPGs to execute their effective signaling activities during development. Third and most importantly, strong evidence is provided that Dlp controls Btl signaling only in FGF-receiving cells in both embryonic and larval tracheal systems. This mechanism of HSPG activity in FGF signaling is very different from its roles in regulating the signaling activities of morphogens including Wnt, Hh and Dpp. Together, these new findings further define novel mechanisms and the specificities of HSPGs in FGF signaling during development (Yan, 2007).

Extensive biochemical and cell culture studies suggest that HSPGs are the part of the FGF/FGFR signaling complex. However, the mechanisms of HSPGs in FGF signaling during development are less known. Embryos mutant for two HSPG biosynthesis enzymes, sgl and sfl, exhibit defects in both Btl- and Htl-mediated FGF signaling. An important issue remaining to be solved is which HSPG core proteins are involved in these signaling events. The data in this work provide strong evidence that Dlp is the key molecule required for Btl signaling during embryonic tracheal development, while both Dlp and Dally are involved in the Btl mediated air sac tracheoblasts formation in the wing disc. The results provide several novel insights into the specificity of individual HSPG in FGF signaling. First, Dlp is involved in Btl signaling, but not in Htl signaling. These findings indicate that different FGF/FGFR complexes may require different HSPGs for their signaling activities. Second, Dlp is highly active and specific for Btl signaling; overexpression of the other three Drosophila HSPGs fail to rescue tracheal defects in dlp embryos. The specific activity of Dlp in Btl signaling could be due to the Dlp protein core or the HS GAG chains attached to the Dlp core protein. In this regard, it is especially surprising that Dally, which has 22% identity with Dlp and also bears a GPI anchor, cannot rescue tracheal phenotypes associated with dlp embryos. As Dlp is involved in several other signaling pathways such as Hh, it is unlikely that Dlp core protein interacts with the ligands directly. In this regard, it is worthwhile to note that ectopic expression of Dally also fails to rescue Hh signaling in dlp embryos. It is proposed that Dlp may have unique HS GAG chains that might provide high and specific activity for ligands such as Bnl and Hh (Yan, 2007).

The biosynthesis of HS GAG chains is determined by the HSPG protein core in which the GAG attachment sites and other protein parts such as the N-terminal cystenine-rich domain control both quantity and quality of the attached GAG chains. Detailed structure and functional studies of Dlp will further help to define specific requirements of the core protein or GAG attachment sites in FGF signaling. Furthermore, the unique GAG chains may be modified by specific enzymes. In this regard, it is particularly important to note that 6-O sulfation of HS is critical for Btl signaling, as Drosophila heparan sulfate 6-O-sulfotransferase is specifically expressed in embryonic tracheal system and is required for Btl signaling during embryogenesis. Recent study has shown that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts. In this regard, it is suggested that Dlp may be the optimal substrate for sulfation enzymes during embryogenesis. Therefore, the activity of Dlp in FGF signaling during embryogenesis cannot be replaced by other HSPGs including Dally, Syndecan and Perlecan (Yan, 2007).

Although Dlp is essential for Btl signaling during embryogenesis, both Dally and Dlp are involved in Btl signaling in air sac tracheoblast cells. Similarly, previous studies have shown that both Dally and Dlp are involved in regulating Wg, Hh and Dpp distribution in the wing disc. The different functions of the same HSPG in embryos and discs may reflect temporal and developmental stage dependent regulation of HSPG functions (Yan, 2007).

While it is well established that HSPGs can regulate FGF signaling by facilitating FGF/FGFR interaction, it is unknown whether HSPGs can also control FGF distribution, thereby modulating FGF signaling. This is a particularly important issue as in many developmental contexts FGF ligand is produced in one type of cell and acts on other cells to initiate its biological activity. One important finding of this work is that HSPGs control tracheal morphogenesis by regulating FGF signaling only in FGF-receiving cells, but not by regulating the secretion or distribution of FGF ligand in its producing cells and surrounding cells. Several important results support these conclusion: (1) dlp mutant embryos can suppress the phenotype of overexpressing Bnl in the tracheal cells. (2) Ectopic expression of Dlp in tracheal cells, rather than FGF expression cells, can effectively restore tracheal defects associated with dlp embryos. (3) Embryos rescued by prd-Gal4/UAS-dlp in dlp backbround is very similar to btl mutant embryos rescued by prd-Gal4/UAS-btl-GFP. (4) HSPGs are required for FGF signaling in its receiving cells in the air sac, but are dispensable in the columnar epithelial layer which includes FGF producing cells and other surrounding cells. Detailed analyses thus demonstrate the specific and distinct requirement of HSPGs in FGF signaling during tracheal development. Moreover, embryonic and larval data together suggest this is likely a general mechanism for HSPG function in FGF signaling in Drosophila (Yan, 2007).

Two major models are proposed for the role of HSPGs in FGF signaling. In one model, low affinity HS/GAG chains on the cell surface limit the diffusion of FGF ligand, thereby increasing its local concentration and the probability that it will interact with high-affinity FGFRs. In the second model, HSPGs facilitate the dimerization or oligomerization of FGF ligands thereby inducing receptor clustering and signal transduction. The experimental data cannot exclude either of these mechanisms. However, the results are in favour of the second case, since it is shown that HSPGs are not required in FGF concentration gradient in FGF producing cells, but are essential in FGF-receiving cells. Finally, a recent study showed that dynamin-mediated vesicle internalization is a crucial step to regulate FGF signaling in Drosophila tracheal system. Mutants in awd (abnormal wing disc) or shi (shibire), which encodes for a nucleoside diphosphate kinase and Drosophila dynamin, respectively, have increased levels of Btl in tracheal cell surface, increased FGF signaling activity and ectopic tracheal branching. In this regard, HSPGs may control FGF signaling by stabilizing the FGF/FGFR complex from degradation or internalization in FGF receiving cells. Further experiments using HSPG and awd/shi double mutant are needed to test this possibility (Yan, 2007).

Over the past several years, extensive studies in Drosophila and other model systems have established the essential roles of HSPGs in developmental signaling pathways including Wg, Hh and Dpp. In Drosophila embryo and wing imaginal disc, HSPGs are involved in the transport of morphogens including Wg, Hh and Dpp by a restricted diffusion mechanism. Narrow stripes of clones mutant for HSPGs can impede the movement of morphogens to further cells. However, in all of these cases, the first mutant cells adjacent to the morphogen source can still transduce signals arguing that HSPGs are not essential for morphogen signaling activity, but rather control the distributions or local concentrations of morphogens. The novel results from this work point out a major difference for a role of HSPGs in FGF signaling from their roles in morphogen signaling, as removal of HSPGs (dally-dlp or sfl) from FGF receiving cells can effectively block FGF signaling. Although the graded FGF activity may play an essential role in tracheal morphogenesis, the data from this work argue that the main function of HSPGs in FGF signaling is not to regulate the distribution of FGF ligand. Consistent with the different roles of HSPGs in FGF and morphogen signaling, it was found that Dlp acts cell-autonomously in FGF signaling while it functions non-autonomously in Hh signaling in embryos. These results suggest that Bnl transportation may be different from morphogen movement in the epithelial cells of the wing pouch. Indeed, morphogen molecules diffuse through the same layer of cells, columnar epithelial cells, while FGF is transported between different layers of tissues, from columnar epithelia to tracheoblasts. Moreover, leading air sac cells are always in close proximity with underlying columnar epithelia. They also extend multiple filopodia toward ligand gradient and presumably actively pursue the FGF ligands while wing disc morphogens including Wg, Hh and Dpp need to transport many cell diameters from their sources to reach their receiving cells. Studies in vertebrate also suggest that a graded distribution of FGF8 protein can be generated by the decay of fgf8 mRNA and this RNA gradient is translated into a protein gradient. In this case, no active transport mechanism is required to form a FGF gradient. In mammalian limb and lung development different FGFs are often expressed in different layers of cells, such as epithelium and mesenchyme, and signal through each other. It is interesting to determine whether HSPGs function similarly in these systems as in Drosophila (Yan, 2007).

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface

Decapentaplegic, a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. This study characterized activity, stability, and gradient formation of a truncated form of Dpp (DppΔN), which lacks a short domain at the N-terminus essential for its interaction with Dally. DppΔN shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, a model is proposed in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex (Akiyama, 2008)

The Dpp pathway is regulated by multiple cell surface and extracellular factors. In the developing wing, Dally is one of the key molecules that modulate Dpp signaling. It affects the shape of the Dpp ligand gradient (protein distribution) as well as its activity gradient (spatial patterns of signaling activity). Dally and Dpp expressed in S2 tissue culture cells are coimmunoprecipitated, suggesting that Dally forms a complex with Dpp. It was also observed that Dally colocalizes with Dpp and Tkv in cells. In addition, Dally enhances Dpp signaling in a cell autonomous fashion. These findings suggest that Dally acts as a Dpp co-receptor at least in some developmental contexts. Interestingly, however, in embryos and in imaginal disc cells close to Dpp-expressing cells, Dpp can mediate signaling without Dally, indicating that HS is not absolutely required for all BMP-dependent processes in vivo (Akiyama, 2008)

DppΔN, which does not bind to heparin, fails to interact with Dally. The easiest interpretation of this result is that wild-type Dpp interacts with Dally via its HS chains. However, a recent study using Surface Plasmon Resonance showed that binding of BMP4 to Dally is not fully inhibited by excess HS. Also, a mutant form of Dally, which does not undergo HS modification, is able to significantly rescue dally mutant phenotypes. These findings suggest that the BMP-glypican interaction is not entirely dependent on the HS chains. One possible explanation for the failure of DppΔN to bind to Dally is that Dpp normally binds to Dally through both the HS chains and its protein core, and DppΔN has reduced affinities for both sites (Akiyama, 2008)

Although DppΔN lacks the ability to interact with Dally, it shows normal in vitro protein stability and signaling activity. Therefore, this truncated form of Dpp provided a powerful system to gain insight into the functions of Dally in distribution and signaling of the Dpp morphogen: this molecule was used to elucidate the consequences of lacking the ability to bind HSPGs. In the wing disc, DppΔN cannot form a normal gradient: only a low level of DppΔN was detected in the Dpp-receiving cells. Notably, this pattern of DppΔN resembles the Dpp ligand and activity gradients observed in dally mutant wing discs. A series of in vitro and in vivo Dpp stability assays suggested that DppΔN forms a shallow gradient because it is remarkably unstable in the matrix and that the stability of Dpp depends on its interaction with Dally (Akiyama, 2008)

Genetic experiments revealed that Tkv and Dally have opposite effects on Dpp gradient formation during wing development. Dally and Tkv share some common properties as components of the Dpp signaling complex: they both autonomously enhance Dpp signaling and limit migration of Dpp by binding to Dpp protein. Nevertheless, tkv and dally mutually suppress one another’s pMad gradient phenotypes. Consistent with the genetic interactions observed in dally and tkv mutants, the pMad phenotype produced by overexpression of tkv was significantly restored by coexpression of dally. These observations indicate that dally antagonizes tkv in Dpp signaling. Since it has been proposed that Tkv promotes Dpp degradation by receptor-mediated endocytosis, dally may stabilize Dpp by inhibiting this process (Akiyama, 2008)

Altogether, these studies suggest that Dally serves as a co-receptor for Dpp and regulates its signaling as well as gradient formation by disrupting the degradation of the Dpp-receptor complex. In this model, the Dpp signaling complex with Dally co-receptor would remain longer on the cell surface or in the early endosomes to mediate signaling for a prolonged period of time. In contrast, in the absence of Dally, the complex would be relatively quickly degraded. This possible role of Dally can account for the shrinkage of the Dpp gradient in dally mutant wing discs. However, the possibility cannot be excluded that DppΔN is lost from the cell surface by lack of retention and further diffuses away (Akiyama, 2008)

A previous kinetic analysis of FGF degradation in cultured mammalian vascular smooth muscle cells also showed that HSPG co-receptors can enhance FGF signaling by stabilizing FGF. In these cells, the intracellular processing of FGF-2 occurred in stages: low molecular weight (LMW) intermediate fragments accumulated at the first step. Blocking HS synthesis by treatment of cells with sodium chlorate substantially reduced the half-life of these LMW intermediates, indicating that HSPGs inhibit a certain step of the intracellular degradation of FGF-2. HSPGs have also been implicated in the endocytosis and degradation of Wg. Wg protein is endocytosed from both apical and basal surfaces of the wing disc and degraded by cells to downregulate the levels of Wg protein in the extracellular space. It has been proposed that Dally-like (Dlp), the second Drosophila glypican, regulates the Wg gradient by stimulating the translocation of Wg protein from both the apical and basal membranes to the lateral side, a less active region of endocytosis, thereby inhibiting degradation of Wg protein (Akiyama, 2008)

Interestingly, DppΔN behaved differently in vivo from the previously reported mutant Xenopus BMP4 lacking the heparin-binding site. Although the action range of BMP4 is restricted to the ventral side during Xenopus embryogenesis, the truncated BMP4 migrated further in the embryo. In addition, heparitinase treatment of embryos also resulted in long-range diffusion of BMP4. These findings led to the conclusion that HSPGs trap BMP4 in the extracellular matrix to restrict its distribution in the Xenopus embryo. This activity of HSPGs seems to be opposite to that of Dally in the Dpp receiving cells of the Drosophila wing, where the major role of Dally is to stabilize Dpp protein. In general, ligands that fail to be retained on the cell surface can have any of the following fates: they may (1) migrate further and act as a ligand somewhere else, (2) be degraded by extracellular proteases, or (3) be internalized by endocytosis and degraded intracellularly. Theoretically, a mixture of all these phenomena can happen at the same time in a given tissue. However, which of these predominates may depend on cellular and extracellular environmental conditions such as concentrations of proteases in the matrix and the rate of endocytosis. Therefore, in the absence of HS, whether a major fraction of ligands is degraded or migrates further can be tissue-dependent, and the effects of HSPGs on BMP gradients will vary depending on the developmental context: a mutant BMP4 molecule moves further in a frog embryo, but DppΔN is degraded in Drosophila wing (Akiyama, 2008)

Although the results presented in this study support a role for HSPGs in Dpp stability, they do not rule out the possible involvement of HSPGs in migration of Dpp protein from cell to cell. The gradient of DppΔN is significantly narrower than that of wild-type Dpp, raising the argument that HS-binding plays a role also in normal Dpp migration in a tissue. Further studies will be required to determine whether or not HSPGs affect morphogen movement. This study also provides new insight into functional aspects of Dpp processing. Mature forms of Dpp generated by differential cleavages are likely to show different affinities for proteoglycans in the matrix. Therefore, they may have different half-lives and/or spatial distribution patterns in vivo. The biological significance of the occurrence of differently processed forms remains to be elucidated (Akiyama, 2008)

Cellular trafficking of the glypican Dally-like is required for full-strength Hedgehog signaling and Wingless transcytosis

Hedgehog (Hh) and Wingless (Wg) morphogens specify cell fate in a concentration-dependent manner in the Drosophila wing imaginal disc. Proteoglycans, components of the extracellular matrix, are involved in Hh and Wg stability, spreading, and reception. This study demonstrates that the glycosyl-phosphatidyl-inositol (GPI) anchor of the glypican Dally-like (Dlp) is required for its apical internalization and its subsequent targeting to the basolateral compartment of the epithelium. Dlp endocytosis from the apical surface of Hh-receiving cells catalyzes the internalization of Hh bound to its receptor Patched (Ptc). The cointernalization of Dlp with the Hh/Ptc complex is dynamin dependent and necessary for full-strength Hh signaling. Wg is secreted apically in the disc epithelium and apicobasal trafficking of Dlp allows Wg transcytosis to favor Wg spreading along the basolateral compartment. Thus, Dlp endocytosis is a common regulatory mechanism of both Hh and Wg morphogen action (Gallet, 2008).

Previous studies failed to observe Dlp at the apical surface of the wing disc epithelium despite the fact that it has been extensively shown in vertebrates that GPI-linked proteins are mainly targeted to the apical surface of epithelial cells. By performing extracellular labeling and kinetic experiments, this study demonstrated that Dlp is targeted to the apical surface before being endocytosed and readdressed to the basolateral compartment. Blocking endocytosis allowed demonstration that apical surface accumulation takes place at the expense of its basolateral location, showing that Dlp is first targeted to the apical domain of the epithelium before being sent to the basolateral compartment. The GPI anchor of Dlp is essential for its internalization, as Dlp tethering by a transmembrane domain (e.g., GFP-Dlp-CD2) abolishes its capacity to be internalized. It is also concluded that Dlp apical targeting followed by its rapid internalization is essential for full Hh signal transduction and for shaping the Wg gradient because when Dlp internalization is blocked, both processes are impaired (Gallet, 2008).

Interestingly, a rescue was observed of the first row of dlp mutant cells by the wild-type surrounding cells. Two mechanisms could explain this: (1) GPI-linked proteins are inserted in the outer leaflet of the plasma membrane and are exposed to the extracellular space. Dlp could interact with extracellular proteins located in nearby cells that could aid in flipping Dlp from the outer leaflet of the plasma membrane of one cell to the next, and/or (2) Dlp might be carried by argosomes, which are large extracellular particles resembling low-density lipoproteins containing lipophorins, esterified cholesterol, and triglycerides surrounded by a phospholipid monolayer. These argosomes not only bear GPI-linked proteins such as HSPGs but also morphogens such as Wg or Hh and are able to travel over several rows of cells (Gallet, 2008).

Whereas numerous data have clearly demonstrated the role of the HSPG in Hh signaling through regulation of Hh stabilization, movement, and reception, the function of Dlp in Hh signaling remained unclear. This study clearly demonstrates that Dlp is exclusively necessary in Hh-receiving cells for full-strength Hh signaling. Strong colocalization of Ptc, Hh, and Dlp is observed in endocytic vesicles in Hh-receiving cells. Ptc is probably responsible for the Hh/Dlp internalization, because it was not possible to detect Dlp-Hh-containing endocytic vesicles in the absence of Ptc. Overexpressing Dlp increased the number of Ptc-Hh-internalized vesicles, whereas absence or tethering Dlp at the cell surface (e.g., GFP-Dlp-CD2) lowered the number of Ptc/Hh vesicles. Therefore, it can be imagined that the presence of Dlp increases Ptc-Hh internalization to elicit a high level of pathway activation. Dlp could also stabilize Ptc/Hh within the intracellular compartment, allowing a stronger and/or a longer signaling. The role of Dlp is clearly different from other Hh coreceptors. Indeed, already identified Hh coreceptors such as Ihog and Brother of Ihog (Boi) (Cdo and Boc, respectively, in vertebrates) stabilize Hh at the cell surface, whereas Dlp overexpression does not increase Hh binding on receiving cells in vivo or in vitro (Gallet, 2008).

It has been demonstrated that Hh is apically secreted by wing disc epithelial cells; however, the compartment by which target cells receive Hh signal (e.g., apical, lateral, or basal) remains controversial. Nevertheless, the data suggest that endocytosis from the apical surface is necessary to sustain full-strength Hh signaling because blocking endocytosis inhibits Dlp internalization from the apical surface and decreases Hh signaling activation. Moreover, a colocalization between Hh and extracellular Dlp is observed at the apical side of Hh-receiving cells but not at the basolateral part of the cell. It has also showed previously that in ptc mutant embryos, Hh accumulates at the apical side of receiving cells. Nevertheless, although blocking endocytosis strongly stabilized Dlp at the apical cell surface, it also stabilized Ptc at both poles of the epithelial cells, raising the possibility that part of the signal transduction occurs basally. Unfortunately, it was not possible to see any Hh accumulation at either pole of the receiving cells when endocytosis was blocked. Hence, a model is favored in which Hh can trigger its signal both basally and apically, where the apical signaling is amplified by Dlp and is necessary for a full-strength Hh signal (Gallet, 2008).

Blocking endocytosis impairs Hh signaling both in embryos and in wing discs. However, it has been previously published that inhibiting endocytosis in imaginal discs using a thermosensitive allele of shi (shits) does not impaired Hh signaling: Ci and collier (an Hh target gene) are unaffected but Ptc is stabilized. How can the differences with these data be explained? It is important to note that the shi null allele is cell lethal; therefore, either a dominant-negative or a shits allele, which may not fully inhibit endocytosis, must be used. Accordingly, Ptc stabilization is observed in only 30% of discs, but each time such a stabilization is observed, a decrease of dpp expression was observed. Increasing the level of ShiDN expression increased the penetrance of the phenotype but triggered lethality, making analysis difficult (Gallet, 2008).

Wg is mainly secreted via the apical pole of producing cells. Strikingly, in those cells, Wg is strongly localized in endocytic vesicles that are abundant apically but also in multivesicular endosomes. Dlp overexpression decreases the level of Wg at the apical surface of cells while increasing Wg stability along their lateral compartment. Therefore, it is proposed that Wg is secreted apically and is then endocytosed with the help of Dlp. Once internalized, Dlp targets Wg by transcytosis to the lateral compartment, where it is stabilized and can spread farther away to activate long-range target genes (Gallet, 2008).

Intriguingly, Dlp seems to play antagonistic roles in Wg signaling. Although it inhibits Wg activity near the Wg source, it is also necessary for Wg pathway activation far from the Wg source. This functional duality is directly related to its pattern of expression. Indeed, Dlp is expressed at low levels along the Wg source (e.g., the D/V axis) owing to repression by the Wg pathway itself, whereas it is expressed at higher levels far from the Wg source. The results could explain how Dlp functions antagonistically in Wg signaling. The two Wg receptors DFrizzled2 (Dfz2) and Arrow are involved in both the signal transduction and the internalization of Wg, mainly through the apical surface to shape its gradient by targeting Wg to the lysosome. Therefore, it is proposed that low Dlp-dependent transcytosis of Wg in producing cells and neighboring ones allows a high level of Wg at the apical surface and hence a strong activation of the pathway. On the contrary, higher Dlp-dependent transcytosis of Wg in more distant cells reduces both Wg pathway activation and degradation, promoting Wg movement along the basolateral compartment. Accordingly, it is observed that, in absence of dlp, extracellular Wg is absent from the lateral compartment of distant cells (Gallet, 2008).

This model supposes that Dlp is able to internalize Wg independently of the receptors DFz2 and Arrow. Interestingly, several groups found some internalized Wg in the absence of the Wg receptors Dfz2 and Arrow. Moreover, Dlp overexpression stabilizes Wg at the cell surface at the expense of short-range signaling activity, in accord with the fact that Wg must be endocytosed by Dfz2/Arrow to promote strong signaling. The results further support the view that Wg may form two different complexes: on the one hand a Dlp-Wg complex involved in Wg transcytosis and stabilization, and on the other hand a DFz2-Arrow-Wg signaling complex that shapes the Wg morphogen gradient and signals. Interestingly, when GFP-Dlp-CD2 is overexpressed, although Wg is stabilized at the apical surface over a very long range, where it should activate its pathway, a much stronger inhibition of Wg signaling is observed and an absence of internalized Wg. Therefore, GFP-Dlp-CD2 may titrate Wg from its receptors and prevent internalization, giving rise to both the stabilization of Wg and the inhibition of the pathway. Under physiological conditions, Dlp targeting of Wg to the lateral compartment supports its stabilization and spreading at the expense of its internalization/degradation from the apical surface by its receptors (Gallet, 2008).

The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum

Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. This study shows that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. Genetic evidence is provided that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. The data suggest that several pools of Hh are perceived in epithelial tissues. Thus, it is proposed that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning (Ayers, 2010).

Morphogens form long-range concentration gradients to signal positional information to cells within a complex tissue. Within an epithelium, the exact apicobasal cellular position of long-range gradient formation is not well understood. The morphogen Hh appears to be released both apically and basolaterally; however, which pool of Hh represents the functional long-range Hh was previously unknown. These studies have provided firm evidence that the wing imaginal disc uses its apical space to form a long-range gradient of Hh responsible for target gene activation. These data also suggest that short-range activity of Hh may be regulated by Hh released into the basolateral space. This implies that morphogen-receiving cells must integrate the apicobasal value of the extracellular Hh gradient present at different planes (Ayers, 2010).

In addition, it was found that morphogenic long-range gradients are shaped by extracellular matrix proteins. In the case of Hh, the glypican Dally positively regulates apical Hh levels in the producing cells, and Dally release promotes long-range spreading in this plane. Finallywe show direct genetic evidence is shown that the enzyme Notum regulates Dally release, thereby controlling the formation of a functional long-range gradient of Hh. A model is therefore proposed in which Dally controls the apical accumulation of Hh in the Hh-producing cells, and cleavage of Dally by Notum helps to shape the long-range gradient (Ayers, 2010).

It is believed, based on various observations in other organisms, that apical release and formation of a functional long-range Hh gradient may be a conserved mechanism. First, studies in Caenorhabditis elegans and Drosophila embryos have hinted at a role of apical secretion for Hh. Indeed, active apical secretion of exosomes containing Hh-like peptides in C. elegans epithelial cells has been demonstrated. In accordance, this study shows that the protein Dispatched (Disp), which is essential for Hh release in invertebrates and vertebrates, regulates apical Hh release in Drosophila embryos. Moreover, the formation of C. elegans cuticle requires the apical secretion of exosomes by Che14, the homolog of Disp. Shh aggregates in the apical lumen of the chick neural tube have been described, whereas an endogenously tagged Shh:GFP has been used that enabled visualization of the Hh gradient in mouse embryos. At embryonic day 8.5 (E8.5), when Shh is produced solely in the notochord, Shh:GFP (in Nodal Vesicular Particles [NVPs]) was found mostly apically in the neural tube (although the notochord has direct contact with the basal membrane of the neural tube). This apically concentrated Shh:GFP forms a gradient that drops exponentially along the dorsal-ventral axis (i.e., farther from the source). Similar observations have been seen when using antibodies against Shh. Thus, although apical gradients have been described in several organisms, these gradients need to be functionally challenged, something that was developed in this study (Ayers, 2010).

A tradeoff has often been observed between the long-range and the short-range Hh pathway targets; for example, increased range of dpp is often coupled with a decreased En range. Several different explanations for this could be suggested. First, increasing the spreading of Hh apically (for example, in overexpression of Sec:Dally) may result in a lower level of apical Hh close to the source, perhaps below the level required for En expression, while increasing the range of the low level needed for dpp expression farther from the Hh source. However, other data presented in this study do not fit this model. Indeed, a secreted form of Dally (Sec:Dally) did not reduce Hh apical levels at or close to the source, but seemed to just increase and elongate the levels of spreading Hh, especially far in the A compartment, whereas basolateral levels of Hh were reduced. Also, when levels of lumenal Hh were reduced (by blocking Hh on the PPM with Ptc1130x), only a reduction in expression of long-range targets (dpp) was observed, not a change in En or Ptc expression. Furthermore, along with short-range signaling, the basolateral Hh gradient was unchanged in this genotype. Thus, two different secretion/signaling mechanisms of Hh are thought to occur from the Hh-secreting cells to the receiving cells. This tradeoff is often observed between increased dpp expression and decreased En expression when trafficking of Hh in its secreting cells is perturbed (e.g., in ShiDN expression), suggesting that apical and basolateral pools of Hh in the producing cells are not independent. Furthermore, because this tradeoff is also observed when Dally in the P compartment is modified, it seems that Dally partially controls the balance between apical and basolateral Hh gradient formation. The data also indicate that basolateral Hh activity is formed independently of Notum, as short-range signaling is not affected in mutants of this protein. Having said this, it is not ruled out that transcription of short-range targets, such as En, may be a response to reception of both apical and basolateral Hh pools (Ayers, 2010).

Although often decreased, En expression was never lost completely; it is often unaffected in the first row of cells. Therefore, it is proposed that Hh may be secreted to the basolateral membrane, and here may signal to the adjacent receiving cells by both cell-cell contact (resulting in high En expression in the first row of receiving cells) and very limited diffusion (to activate En in up to three cells away from the A-P boundary) (Ayers, 2010).

This study suggests that the Hh that forms a basolateral gradient may be loaded in a different form to that released into the apical lumen. This is because at the lateral position, the Hh trapped by expression of Ptc1130X in the disc proper illustrated a very different staining (highly membranous and nonpunctuate) than that found apically (which was highly punctuate). Also, the presence of basolateral Hh was found in only up to 2-3 cells away from the source, indicating a very limited ability to disperse; this could also be due to a different extracellular environment compared with the apical lumen, where Hh dispersal is higher. Although it may be basolaterally targeted, poorly diffusing Hh that is responsible for En expression in the three rows of cells, this is extremely difficult to prove. Indeed, little is known about polarized trafficking in wing imaginal discs. Although several methods have been described with which it was possible to modify apical Hh levels, no way has been found to specifically enrich or block basolateral Hh in the P compartment without affecting the apical Hh pool. Therefore, it has not been possible to directly test whether it is this gradient of Hh responsible for the pathway activation in the first row of cells in the A compartment (Ayers, 2010).

Evidence has been found for the involvement of the glypican Dally and the hydrolase Notum in the long-range apical spreading of Hh. Reduction of either of these proteins in Hh-producing cells reduces the range within which dpp is expressed, whereas short-range target En is untouched. Furthermore, Dally has been found to positively regulate apical Hh levels, and Dally release aids in long-range spreading of Hh, whereas Notum appears to be essential just for the latter. But what could the exact role of GPI-tethered Dally be in the Hh-secreting cells? It has been shown that the Heparan Sulfate of Glycosaminoglycan chains can bind and sequester both Hh and Lipophorins, which are thought to carry Hh. Therefore, GAG chain binding of Hh at the apical surface may work as a platform by which to stabilize and associate Hh with components necessary for its release and long-range spreading (Ayers, 2010).

In addition to regulating apical Hh accumulation, secretion of Dally appears to play a role in the long-range spreading and activity of Hh. It has been suggested that the secreted form of Dally increased dpp expression due to its mediation of Hh binding to the receptor complex. It cannot be ruled out that secreted Dally could have a role in stabilizing the Hh-receptor complex interaction and signaling, but it is believed that Dally shed from the P compartment is mainly involved in the formation of a long-range Hh gradient, due to changes seen in the apical Hh gradient profile. In addition, if secreted Dally was necessary for Hh-receptor interaction and signaling, then the absence of Dally in the P compartment should affect all Hh target genes. On the contrary, it was found that dally mutant clones in the P cells only affect the expression of the long-range target dpp. If promotion of ligand-receptor interaction was the only role of Dally released from the P compartment, then one would expect expression of secreted Dally to induce a higher level of signaling in the A compartment close to the Hh source, as Hh would be more highly sequestered to its receptors. On the contrary, a decrease is seen in En expression. Therefore, it is proposed that secreted Dally augments movement and extension of the Hh gradient, as opposed to solely increasing signaling (Ayers, 2010).

Lastly, analysis of Notum, a protein described as a GPI anchor cleaver (Traister, 2008), indicates that Notum in the P compartment promotes Hh long movement through its regulation of Dally. Therefore, it is proposed that after apical accumulation and clustering of Hh by GPI-anchored Dally, Notum cleaves and releases Dally, preparing Hh for its long apical voyage (Ayers, 2010).

The cell-surface proteins Dally-like and Ihog differentially regulate Hedgehog signaling strength and range during development

Hedgehog (Hh) acts as a morphogen in various developmental contexts to specify distinct cell fates in a concentration-dependent manner. Hh signaling is regulated by two conserved cell-surface proteins: Ig/fibronectin superfamily member Interference hedgehog (Ihog) and Dally-like (Dlp), a glypican that comprises a core protein and heparan sulfate glycosaminoglycan (GAG) chains. Dlp core protein can interact with Hh and is essential for its function in Hh signaling. In wing discs, overexpression of Dlp increases short-range Hh signaling while reducing long-range signaling. By contrast, Ihog has biphasic activity in Hh signaling in cultured cells: low levels of Ihog increase Hh signaling, whereas high levels decrease it. In wing discs, overexpression of Ihog represses high-threshold targets, while extending the range of low-threshold targets, thus showing opposite effects to Dlp. It was further shown that Ihog and its family member Boi are required to maintain Hh on the cell surface. Finally, Ihog and Dlp have complementary expression patterns in discs. These data have led to a proposal that Dlp acts as a signaling co-receptor. However, Ihog might not act as a classic co-receptor; rather, it may act as an exchange factor by retaining Hh on the cell surface, but also compete with the receptor for Hh binding (Yan, 2010).

Previous studies have shown that Dlp is specifically required for Hh signaling in cell-based assays and in embryos. However, the molecular basis of this specificity was unknown. This study shows that overexpression of Hh can restore naked cuticle in sugarless (sgl) and sulfateless (sfl) embryos, but not in dlp embryos. It was further demonstrated that the specificity of Dlp in Hh signaling results from its core protein. The Dlp core protein can restore Hh signaling autonomously in dlp embryos and in dlp-RNAi cells. In addition, the Dlp core protein interacts with Hh and promotes Hh signaling in the disc. Overexpression of Dlp increases Hh signaling strength, but reduces signaling range as well as Hh gradient range. These data suggest that the Dlp core protein could act as a classic co-receptor in Hh signaling by facilitating Hh-Ptc interaction (Yan, 2010).

Recent studies have shown that the vertebrate glypican-3 core protein directly promotes Wnt signaling in cancer cells, but inhibits sonic hedghog signaling during development. That the same glypican has opposite effects on Wnt and Hh is interesting because Dlp can inhibit high-threshold Wg signaling when overexpressed in discs. In addition to the essential role of the core protein, the attached GAG chains are important for the non-cell-autonomous functions of Dlp. The current results demonstrated that wild-type Dlp can rescue non-cell-autonomously in dlp embryos, whereas the core protein mainly acts in its expression domains. Interestingly, the CD2 forms of Dlp also lose the non-autonomous activity in dlp embryos. Several studies suggest that the GPI anchor of Dlp can be cleaved by the hydrolase Notum and that the GAG chains of Dlp can recruit lipoprotein particles. Thus, it will be interesting to determine the mechanism of Dlp non-autonomous activity (Yan, 2010).

This study suggests that the GPI anchor of Dlp is not essential for its activity in Hh signaling. Most importantly, two CD2 forms of Dlp, Dlp(-HS)-CD2 and GFP-Dlp-CD2, can effectively rescue Hh signaling in dlp embryos. In addition, CD2 forms of Dlp can also signal in cultured cells and discs. It is important to note that although the GPI, but not the CD2, form of Dlp is colocalized with Ptc in intracellular vesicles, both forms have similar activities in promoting Hh signaling in the disc. These data argue that colocalization of Dlp with Ptc in endocytic vesicles is not essential for Dlp function in Hh signaling. Consistent with this view, several studies have shown that endocytosis is not essential for Hh signaling in Drosophila wing discs. The current conclusion differs from a recent publication arguing that the GPI anchor of Dlp is required for its function in Hh signaling (Gallet, 2008). In that study, it was shown that overexpression of GFP-Dlp-CD2 can reduce Hh signaling in the wing discs. However, this study did not observe any dominant-negative effect of GFP-Dlp-CD2, which can rescue dlp mutant embryos and enhance Hh signaling strength in cells and discs. A possible explanation for the discrepancy is that expression of Dlp enhances signaling strength but also reduces signaling range. ap-Gal4 was always used, allowing use of the ventral disc as an internal control. The observed dominant-negative effect might reflect the reduced signaling range rather than signaling strength (Yan, 2010).

Previous studies in both Drosophila and vertebrates have demonstrated positive roles of the Ihog family proteins in Hh signaling. Ihog and Ptc synergize in mediating Hh binding to cells. These observations suggest that Ihog functions as a co-receptor for Hh. However, the new data argue that Ihog does not simply act as a classic co-receptor that only increases the binding of ligand to the signaling receptor. By altering Ihog levels in vivo and in vitro, it was shown that Ihog has biphasic activity in Hh signaling, with too much or too little Ihog leading to reduced signaling. Overexpression of Ihog leads to the accumulation of Hh, and knockdown of Ihog results in reduced Hh levels, suggesting that one major activity of Ihog is to retain Hh on the cell surface. Moreover, knockdown of both Ihog and Boi dramatically reduces Hh levels and signaling. This reduction of Hh signaling activity is likely to be due to an absence of Hh on the cell surface of the double-mutant tissue. However, a high level of Ihog causes a large amount of Hh to accumulate on the cell surface, but also reduces signaling strength. One explanation for this result is that Ihog can compete with Ptc for Hh binding. Thus, a low level of Ihog is required to maintain Hh on the cell surface, whereas a high level of Ihog can sequester Hh from its receptor. In other words, depending on the context, Ihog can either provide Hh for the receptor by retaining Hh on the cell surface, or compete with the receptor for Hh binding. This activity of Ihog is very similar to the recently proposed 'exchange factor' model, which allows the exchange of Hh between Ptc and Ihog. A recent study has demonstrated that Ihog also interacts with Ptc and that Ihog, Ptc and Hh form a triple complex (Zheng, 2010). The close association between the Ihog and Ptc receptors may thus allow them to exchange Hh ligand. It will be important to determine whether the triple complex has a greatly reduced ability to signal, a prediction from the mathematical exchange factor model (Yan, 2010).

Although this is the first demonstration of a biphasic co-factor in Hh signaling, similar biphasic co-factors have been reported. Drosophila Cv-2 enhances BMP signaling at low concentrations, but inhibits signaling at high concentrations. Syndecan-1 shows a similar concentration-dependent activation or inhibition of BMP signaling in Xenopus. Interestingly, Dlp has biphasic activity in Wg signaling, depending on its protein levels. All these co-factors are likely to act by a similar mechanism, suggesting that the biphasic co-factor is a recurring motif in different morphogen systems (Yan, 2010).

This study has shown that Dlp and Ihog play distinct roles in Hh signaling. Expression of Dlp enhances Hh signaling strength, but reduces signaling range. By contrast, expression of Ihog reduces Hh signaling strength, but extends signaling range. In addition, the Dlp level is elevated in the high Hh signaling area, whereas the Ihog level is reduced in that region. It is important to consider from a system point of view what these two co-factors provide for the Hh morphogen. For morphogens to work, they should be able to generate sharp boundaries between target genes with different thresholds. They also need to diffuse over a certain range without being lost in the extracellular space. The positive feedback of Dlp expression in the high Hh signaling areas helps to sharpen the boundaries between high- and low-threshold target genes. The negative-feedback regulation of Ihog might ensure that a strong Hh signal is attained in the areas close to the Hh source and also allow the Hh gradient to diffuse to those areas distant from the source (Yan, 2010).

Drosophila heparan sulfate 6-O endosulfatase regulates Wingless morphogen gradient formation

Heparan sulfate proteoglycans (HSPGs) play critical roles in the distribution and signaling of growth factors, but the molecular mechanisms regulating HSPG function are poorly understood. This study characterized Sulf1, which is a Drosophila member of the HS 6-O endosulfatase class of HS modifying enzymes. Genetic and biochemical analyses show that Sulf1 acts as a novel regulator of the Wg morphogen gradient by modulating the sulfation status of HS on the cell surface in the developing wing. Sulf1 affects gradient formation by influencing the stability and distribution of Wg. It was also demonstrated that expression of Sulf1 is induced by Wg signaling itself. Thus, Sulf1 participates in a feedback loop, potentially stabilizing the shape of the Wg gradient. This study shows that the modification of HS fine structure provides a novel mechanism for the regulation of morphogen gradients (Kleinschmit, 2010).

Although roles for HSPGs in morphogen signaling and distribution have been well established, the molecular basis of these activities remains to be elucidated. A number of genetic and in vitro analyses have demonstrated the critical importance of HS moieties for HSPG function during developmental patterning. Recent reports using mutant forms of Dally and Dlp that lack all HS attachment sites have revealed the essential contribution of the core protein to regulating growth factor binding and signaling activity. Thus, the regulatory function of HSPGs is likely to be affected by a combination of both HS and core protein structures (Kleinschmit, 2010).

HS biosynthesis is a complex, multi-step process catalyzed by Golgi enzymes in a highly organized fashion. Recent studies have demonstrated that extracellular Sulfs further modify the HS fine structures in a post-synthetic manner (reviewed in Gorsi, 2007). Thus, Sulfs may contribute to generating structural diversity and modify the number of ligand binding sites on HS at the cell surface (Kleinschmit, 2010).

To better understand the importance of regulating HS sulfation during development, this study investigated the role of Drosophila Sulf1 in patterning and morphogenesis. Sulf1 mutant wings show specific phenotypes characteristic of abnormally high levels of Wg signaling near the Wg-expressing cells. Extracellular levels of Wg protein were elevated throughout the Sulf1 mutant wing discs, and decreased in cells overexpressing Sulf1. In addition, a Sulf1 mutation caused Wg to accumulate near its source, altering the shape of the gradient. Thus, Sulf1 is a novel regulator of Wg gradient formation. Disaccharide analysis of Sulf1 mutant HS showed abnormally high levels of tri-S disaccharide units, indicating that Sulf1 regulates Wg signaling by modulating HS fine structure. Given that Sulf1 decreases local levels of Wg protein in the extracellular space, it is likely that the domain structure of HS to which the Wg ligand preferentially binds includes tri-S disaccharide unit(s) as a major component. Thus, Sulf1 may redefine the shape of the Wg gradient by removing some of the Wg-binding sites from HS on the cell surface (Kleinschmit, 2010).

It has been shown in Drosophila embryos that a significant fraction of Wg protein is retained on the expressing cells in a HSPG-dependent manner. High levels of dally expression near the DV boundary of the wing disc suggest that Wg may be also trapped by HS in the developing wing. In such a situation, Sulf1 activity could reduce the trapping of Wg by cell surface HSPGs near the expressing cells. Wg protein, thus released from HS, could have two possible fates. First, Wg ligand could be quickly internalized by nearby cells for degradation. Second, released Wg ligand could escape degradation and migrate away from the trapped site. Therefore, theoretically, Sulf1 can affect the Wg gradient through two differential activities: (1) destabilization of Wg and (2) enhancement of Wg re-distribution by facilitating Wg release from the HSPGs. This study showed that Sulf1 reduces extracellular levels of Wg protein without affecting wg expression. In addition, Wg signal intensity plots for wild-type and Sulf1 mutant discs suggested that Sulf1 affects Wg distribution near the DV boundary. Thus, these observations are consistent with the idea that Sulf1 indeed modulates the Wg gradient by influencing both Wg stability and distribution (Kleinschmit, 2010).

How can Sulf1 contribute to lateral distribution of Wg? Gallet (2008) has proposed that Dally-like (Dlp) mediates apicobasal trafficking of Wg, which is required for its long-range gradient formation. A more recent study has shown that Dlp can act in a biphasic manner to potentiate Wg long-range signaling (Yan, 2009). In this model, Dlp either competes with the receptor or provides ligand to the receptor, dependent on its ratio to Wg and the receptor. In both models, however, since dlp expression is repressed at the DV compartment border, an additional mechanism by which Wg reaches the dlp-expressing cells appears to be required. Wg secreted from cells at the DV boundary is likely to be first trapped by Dally, a glypican expressed at high levels in this region. One possible function of Sulf1 is to facilitate the short-range movement of Wg from the expressing cells to Dlp. In this model, Sulf1, which is also abundant near the source of Wg, removes 6-O sulfate groups from Dally HS chains. This enzymatic cleavage would lower the efficiency of Wg trapping by Dally, allowing it to migrate away from the DV boundary. Released Wg would now have a better chance to reach Dlp, which recaptures and facilitates further diffusion of Wg (Gallet, 2008; Yan, 2009). Thus, this study demonstrates that modification of HS fine structure provides a novel mechanism to shape morphogen gradients (Kleinschmit, 2010).

Given that vertebrate Sulfs are known to positively regulate Wnt signaling, it is surprising that Drosophila Sulf1 has an opposite effect on the Wg pathway. The results suggest that Drosophila Sulf1 has a similar biochemical activity and it is expected that a direct consequence of the function of Sulf enzymes on Wnt/Wg protein is also similar between vertebrate and invertebrate models: Sulfs release Wnt/Wg ligands from HSPGs. It is proposed that the fate of the released Wnt/Wg could be different dependent on extracellular environment. In vertebrate systems where Sulfs enhance Wnt signaling, released Wnt appears to have better chance to bind and activate receptors. In contrast, a major fraction of Wg protein detached from HSPGs may be degraded in the Drosophila wing disc (Kleinschmit, 2010).

Although Sulfs are believed to function post-synthetically in the extracellular space, the effects of Sulf1 function were observed cell autonomously. In addition, experiments using Sulf1-Golgi showed that this modified form retains the ability to decrease extracellular levels of Wg protein, indicating that Sulf1 does not have to be secreted into the extracellular space to function. Thus, Sulf1 may act in the Golgi and/or on the cell surface. If Sulf1 acts extracellularly, Sulf1 is likely to adhere to the surface of the secreting cells as has been shown in vertebrate models: previous studies reported that Sulf enzymes associate with the cell fraction and not the medium fraction of transfected cultured cells. The binding of QSulf1 to the cell fraction in CHO cells has shown to be dependent on a large hydrophilic domain. Since a similar conserved hydrophilic domain is found in Drosophila Sulf1, it is hypothesized that Sulf1 may bind to a constituent of the ECM in close proximity to the expressing cells (Kleinschmit, 2010).

It was found that small Sulf1 clones show more severe phenotypes than large clones and Sulf1 homozygous mutant discs. This observation suggests that morphogen gradients are more severely disrupted in a developmental field with discontinuity of cell surface HS structures (e.g. discs with Sulf1 small clones) compared to one where HS sulfation is uniformly altered (e.g. Sulf1 homozygous mutant discs). However, the molecular mechanism behind this difference remains to be elucidated (Kleinschmit, 2010).

In situ hybridization showed that Sulf1 mRNA is expressed at high levels near both the AP and DV borders of the wing disc. Interestingly, this feature is similar to the expression pattern of dally in the wing disc. The DV boundary expression of dally is induced by Wg signaling. It ws shown that expression of Sulf1, like that of dally, is induced by Wg signaling. Thus, Sulf1, a negative regulator of the Wg pathway, participates in a negative feedback loop within this morphogen system. It has been previously shown that Dally is a component of the negative feedback loop for the Dpp signaling pathway, potentially stabilizing the shape of the Dpp gradient. In addition, Wg also induces Notum, which is a secreted antagonist of Wg and functions through the posttranslational cleavage of glypicans at the wing margin. Collectively, these results implicate HSPGs and HS biosynthetic machinery components as general constituents of morphogen feedback systems, supporting the stability and the robustness of morphogen signaling gradients (Kleinschmit, 2010).

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. This paper reports the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and a localized region on the Dlp surface important for mediating its function in Hh signaling was identified. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex (Kim, 2011).

Glypicans modulate the activity of multiple growth factors active during development, and defects in glypican function lead to widespread and diverse developmental malformations. Much of the activity of glypicans can be attributed to interactions between their heparan sulfate attachments and heparan-binding growth factors, but recent work has demonstrated important functional roles for the protein cores of glypicans, notably in Hh and Wnt signaling. In particular, the protein cores seem likely to mediate functions that are specific to particular glypicans. The crystal structure of the N-terminal globular region of a glypican, DlpδNCF, adopts an elongated, all α-helical fold with no evident homology to previously determined structures. The high level of sequence conservation among the N-terminal protein cores of glypicans (greater than 40% sequence identity exists between Drosophila and human glypicans in this region) indicates that the DlpδNCF structure provides a sound basis for the design and interpretation of experiments with other glypicans. The absence of any apparent active site-like cavities or sources of conformational flexibility in the DlpδNCF structure suggests that the protein regions of glypicans exert their effects by serving as binding proteins, consistent with proposed roles as coreceptors or in targeting ligands to specific subcellular compartments (Kim, 2011).

One reason Dlp has been proposed as an Hh coreceptor is the ability of Dlp lacking heparan sulfate to coimmunoprecipitate with Hh. The inability to detect a high-affinity interaction between DlpδNCF and HhN using purified proteins suggests that Dlp may interact with Hh as part of a larger complex and additional factors are needed to promote Dlp/Hh interactions. One candidate for such a factor is the Hh coreceptor Ihog, which is an essential component of the Hh receptor complex, but this study shows that purified DlpδNCF does not form a high-affinity complex with either an active fragment of Ihog (IhogFn12) or an HhN:IhogFn12 complex. This result does not rule out Ihog as important for mediating Hh:Dlp interactions but suggests that an additional factor or factors may be needed. An obvious candidate for such a factor is Patched, a key cell-surface component of the Hh signaling pathway, but assessing its role in Hh-containing complexes awaits purification of suitable amounts of this 12-pass integral membrane protein. An additional element complicating interpretation of results of studies of receptor-ligand interactions in solution arises from the absence of membrane tethering and ligand multivalency. Restricting components to a membrane surface orients them and greatly enhances their local concentration, and a multivalent ligand greatly increases the avidity of binding. Interactions between a monovalent ligand and cell-surface components may thus not be strong enough to be observed with soluble components in solution (Kim, 2011).

The inability to observe an interaction between ShhN and the N-terminal domain of glypican-3 is puzzling, however, given that the protein region of glypican-3 has been reported to bind to ShhN with nanomolar affinity. Several possibilities may explain this discrepancy: (1) ShhN may interact with the glypican- 3 stalk region, which was present in the earlier study but not in the current experiment; (2) attachment of histidine-tagged ShhN to Ni-NTA agarose may have blocked a glypican interaction site in these studies; or (3) an unidentified cofactor that promotes high-affinity interaction was present in the earlier studies but absent in the earlier studies. Calcium ions, for example, are required to promote high-affinity interactions between ShhN and CDO, the mammalian homolog of Ihog (Kim, 2011).

The ability to identify a localized region on the C lobe of the Dlp surface important for proper Dlp function in Hh signaling is further consistent with Dlp participating in Hh signaling primarily as a binding protein, although the nature and number of binding partners remains to be determined. Curiously, the identified surface is composed largely of hydrophilic residues, which is unusual for protein-protein interfaces. This surface also occurs on the opposite surface of Dlp relative to the disordered Dlp-specific insertion that follows the furin-like processing site, suggesting that interactions mediated by this region are likely independent of furin-like processing and this insertion. Glypican mutations previously associated with functional impairments, for example those in glypican-3 that cause Simpson-Gohlabi-Behmel syndrome and those in glypican-6 that cause omodysplasia, appear to result in severe truncations or complete loss of expression of the affected glypican. Whether the C-lobe region identified on Dlp is generally involved in glypican function or is specific to Dlp or positive regulators of Hh signaling is an interesting question for future investigation. The results presented in this study establish a molecular foundation to guide design and interpretation of studies investigating the molecular bases of glypican function (Kim, 2011).

The role of glypicans in Wnt inhibitory factor-1 activity and the structural basis of Wif1's effects on Wnt and Hedgehog signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, this study reports on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding 'EGF-like' domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the 'WIF' domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling (Avanesov, 2012; full text of article). The 'WIF' domain of WIF1 does not bind HS sidechains, but is sufficient for Wnt binding; the 'EGF-like' domains show only weak binding to Wnts on their own, but appear to strengthen Wnt binding to the 'WIF' domain. But while the Drososphila WIF1 homolog Shf contains both 'WIF' and 'EGF-like' domains, it does not inhibit Wg signaling; instead, it increases the levels or range of Hh signaling. This study found that a construct containing Shf's 'WIF' domain and the zebrafish Wif1's 'EGF-like' domains also cannot inhibit Wnt signaling, while the reciprocal construct with Wif1's 'WIF' domain and Shf's 'EGF-like' domain can. Similar results have been obtained with constructs made from Shifted and human WIF1. Thus, the ability to inhibit Wg activity, and likely to bind significant levels of Wg, resides in the different 'WIF' domains of Wif1 and Shf (Avanesov, 2012).

Surprisingly, Shf did show a weak ability to improve Wg signaling in sensitized backgrounds expressing either Wif1 or the dominant negative DFz2-GPI construct. While no obvious effect was ever detected of Shf on ex-Wg levels, it may weakly interact with Wg in a manner that reduces the levels bound to Wif1 or DFz2-GPI and increases the levels available for the Wg receptors. Consistent with this interpretation, UAS-shf did not alleviate margin defects caused by expression of UAS-wg RNAi, even though UAS-Dfz2-GPI and UAS-wg RNAi show a very comparable impact on Wg activity. Alternatively, Shf's effect on Wnt signaling might be due to interactions with the Wnt4 or Wnt6 expressed along the wing margin, which may have redundant roles in wing margin development that are only obvious in a sensitized background. Indirect effects via Hh signaling are unlikely, as Shf overexpression does not further increase Hh signaling (Avanesov, 2012).

The situation with Hh signaling is more complex. First, vertebrate WIF1's are not known to regulate vertebrate Hh signaling, but this study found that zebrafish Wif1 can weakly affect the reduced movement or accumulation of Hh normally observed in shf mutant wing discs. The Hh-GFP accumulation is abnormal, however, appearing more punctuate than in normal wing discs, perhaps accounting for its ability to reduce the expression of Hh targets (Avanesov, 2012).

Placing WIF domain of zebrafish Wif1 in the context of Shf's 'EGF-like' domains in a chimeric WIFWif1-EGFShf construct almost fully rescues loss of shf function, something not observed after expression of the Shf 'EGF-like' domains alone. Together, these data suggest that the 'WIF' domains of both Shf and zebrafish Wif1 are capable of interacting with Hh. Like Wnts, Hh is palmitoylated, and it has been suggested that these palmitates might bind a hydrophobic pocket found in the WIF domain, although this has been recently questioned. The activity of 'WIF' domains in Hh signaling may also vary between different vertebrates, since unlike the WIFWif1-EGFShf construct made using zebrafish 'WIF' domains, a similar construct made using the 'WIF' domain from human WIF1 does not rescue loss of shf function (Avanesov, 2012).

The Shf 'EGF-like' domains are necessary to confer a Shf-like level of Hh-promoting activity to the 'WIF' domains of zebrafish Wif1. The Hh-promoting activity of Wif1's 'WIF' domain is increased by placing it in the context of Shf's 'EGF-like' domains, and the low Hh-promoting activity of Shf's 'WIF' domain is not changed by placing it in the context of Wif1's 'EGF-like' domains. It is unlikely that the 'EGF-like' domains of Shf and Wif1 differ significantly in their HSPG-binding activities, since Wif1 and WIFWif1-EGFShf differ only slightly in their ability to inhibit Wnt signaling and interact genetically with Dlp. Therefore the alternative hypothesis is favored that Shf's 'EGF-like' domains contribute to Hh signaling through a mechanism independent of glypican binding. While the Shf 'EGF-like' domains alone (ShfδWIF) cannot increase Hh signaling, it was found that they can increase the levels of extracellular Hh, suggesting that they contribute to Hh binding, much as the 'EGF-like' domains of WIF1 do to Wnt binding (Avanesov, 2012).

Since Wif1 can alter Hh distribution and, more weakly, signaling in Drosophila, an important question is whether it can also do so in vertebrates. Because of its strong effects on Wnt signaling, vertebrate WIF1 family proteins have rarely been assayed for their effects on other pathways, so a weak modulation of one of the vertebrate Hhs remains a possibility (Avanesov, 2012).

Dispatched mediates Hedgehog basolateral release to form the long-range morphogenetic gradient in the Drosophila wing disk epithelium

Hedgehog (Hh) moves from the producing cells to regulate the growth and development of distant cells in a variety of tissues. This study has investigated the mechanism of Hh release from the producing cells to form a morphogenetic gradient in the Drosophila wing imaginal disk epithelium. Hh reaches both apical and basolateral plasma membranes, but the apical Hh is subsequently internalized in the producing cells and routed to the basolateral surface, where Hh is released to form a long-range gradient. Functional analysis of the 12-transmembrane protein Dispatched, the glypican Dally-like (Dlp) protein, and the Ig-like and FNNIII domains of protein Interference Hh (Ihog) revealed that Dispatched could be involved in the regulation of vesicular trafficking necessary for basolateral release of Hh, Dlp, and Ihog. It was also shown that Dlp is needed in Hh-producing cells to allow for Hh release and that Ihog, which has been previously described as an Hh coreceptor, anchors Hh to the basolateral part of the disk epithelium (Callejo, 2011).

By using shits1 mutant disks, it is possible to freeze Hh internalization and visualize on which side of the anterior compartment (A) wing disk epithelium, apical or basal, Hh gradient is being formed. Thus, an extended basolateral accumulation of Hh was observed in receiving cells, whereas, at the apical plane, Hh accumulation was only evident in the first row of A cells, indicating that the long-range Hh gradient is formed mainly basolaterally. Accordingly, Ptc accumulated in shits1 disks equally apically and basolaterally but mainly colocalized with Hh at the basolateral sections, suggesting a specific mechanism to deliver Hh to Ptc in this surface of the disk epithelium. To analyze the mechanism of Hh release in the P cells, an apical accumulation of Hh was noticed in the producing cells in shits1 mutant disks that was also observed in P shits1 clones and in Rab5DN ectopic clones, suggesting that the apically secreted Hh is also internalized in P cells and probably is recycling to other membranes. Accordingly, when blocking recycling endosomes (using the dominant-negative form of Rab8 or Rab4), Hh accumulation could be detected in producing cells. This Hh recycling in P cells is probably necessary to form a proper Hh gradient in the receiving cells. In agreement with the above, Hh signaling is compromised when endocytosis is blocked by expressing either Rab5DN or ShiDN in the P compartment. Interestingly, and in agreement with these results, Ayers (2011) suggested that apical Hh internalization in the Hh-producing cells is necessary to process or route Hh to activate the responses that require high levels of Hh. However, in contrast to the interpretation provided in this study, Ayers also proposed that the apical Hh pool is responsible for the long-range Hh gradient formation. It is not necessary to envision two Hh gradients, an apical long-range Hh and another basolateral short-range Hh, when all responses can be produced by means of a single gradient. Because only the recycled Hh is capable of activating the high-threshold Hh targets, there is no reason to believe that this processed Hh would not be efficient enough to induce the low-threshold targets basolaterally (Callejo, 2011).

Also in contrast to a previous report proposing a function for Disp in regulating the apical secretion of Hh in Drosophila epithelia, this study demonstrates that Disp is required for the basolateral release of Hh in the wing imaginal disk epithelium. The subcellular localization of Disp, and its function in the basolateral release of Hh, is in agreement with a recent report in vertebrates (Etheridge, 2010). The cellular phenotype of the loss of Disp function, such as the increase in the amount of Hh found in endocytic vesicles, which are supernumerary and disorganized, can be interpreted as a failure in Hh trafficking that subsequently affects its proper release. In this sorting process, Hh would interact with Disp either in the recycling endosome or in MVBs. Disp, a member of the RND family of proton-driven transporters, is likely to function only in compartments where a transmembrane proton gradient exists, such as in early and late endosomes, trans-Golgi, and MVBs. In support of this view, this study showed by confocal and EM studies that Disp protein is located not only at the basolateral plasma membrane but in vesicles and MVBs, where it colocalizes with Hh. Based on the disp-/- phenotypes and the localization of Disp protein in MVBs, it is proposed that Disp might have a function in redirecting the apically internalized Hh toward the basal domain. Interestingly, and in agreement with the above, a form of Disp, mutant for the proton-driven transporter function (DispAAA), does not localize at the basolateral plasma membrane but in supernumerary cytoplasmic vesicles that do not colocalize with Hh puncta, implying that DispAAA may not participate in Hh vesicular trafficking to the basolateral plasma membrane (Callejo, 2011).

In noticeable contrast but also supporting the above, after freezing Hh internalization in shits1 mutant disks, Hh accumulates apically in the first row of A compartment cells, suggesting that paracrine signaling could also occur through the apical plasma membrane. Interestingly, in P mutant cells for Dlp, Hh is able to signal to the abutting A cells but long-range signaling does not occur. A suggestive possibility is that the capacity for apical signaling is not affected in these mutant conditions; however, for long-range signaling to occur, a basolateral release implicating the coordinated actions of Dlp and Ihog together with Disp would be required. During Hh sorting in the producing cells, Disp may interact with Dlp and Ihog; in fact, the interaction of Disp with Dlp and Ihog may be important for the apical-to-basal transcytosis of these proteins, because the ectopic expression of Disp but not of mutant DispAAA increases Dlp and Ihog levels at the basolateral membranes. As in disp-/- cells, dlp-/- cells in the P compartment showed an accumulation of Hh at both the apical and basolateral plasma membranes, suggesting that Dlp might cooperate with Disp during Hh release. In agreement with the proposed mechanism, transcytosis of Dlp has previously been suggested to be important for Wingless (Wg) release and spreadin (Callejo, 2011).

Although the data cannot support that the total amount of synthesized Hh has to undergo this apical-to-basal transcytosis, an intriguing question is why Hh is placed and internalized apically and then shuttled to the basolateral part of the cell. One possibility is that the newly synthesized Hh protein, because of its unusual modifications with cholesterol and palmitate, uses the apical surface, which is enriched in cholesterol and glycosphingolipids, for primarily plasma membrane localization. Alternatively, it is also possible that the apical internalization of Hh allows its interaction with Disp, glypicans, and Ihog. Therefore, Hh that reaches the apical plasma membrane needs to be internalized to recycle to the basolateral plasma membrane, where the machinery for secretion and gradient formation is found. As has already been discussed, transcytosis of Dlp and Ihog together with Hh from the apical membrane to the basolateral membrane may also occur. Cholesterol and triglycerides also undergo apical-to-basolateral transcytosis across intestinal epithelial barriers to reach the blood. Cholesterol and palmitic acid modifications could attribute lipid-like properties to the Hh protein, such as the ability to be anchored to the plasma membrane, and could thus affect Hh intracellular trafficking. In agreement with the above, it has been described that lipid-unmodified Hh in the wing disk epithelium is not able to form a proper Hh gradien. As previously reported, it was observed that Hh mutant forms that lack lipid modifications are released and do not accumulate in disp-/- clones. Interestingly, this study shows that lipid-unmodified Hh does not colocalize with Ihog-labeled basal cell extensions, indicating that lipid modifications are necessary to interact with Disp, Dlp, and Ihog, and therefore for proper Hh trafficking from the apical to basolateral plasma membrane regulated by Disp function. Reinforcing these findings, it has been reported that disp and ttv functions are not required for either release or transport of lipid-unmodified Hh, strongly suggesting that Disp and glypicans are needed for the appropriate basolateral release of Hh (Callejo, 2011).

This work shows that Hh has a more complicated mechanism for release than has been previously anticipated. The finding of a basolateral route for Hh release and gradient formation will help to understand Hh interaction with different Hh pathway components, such as Disp, Dally, Dlp, Ihog, Boi, and Ptc, during the process of Hh gradient formation. Related to this issue, it is quite intriguing to find Disp, Dlp, Ihog, and Hh decorating long basal cellular extensions in disk cells expressing Ihog ectopically. Some of the long filaments labeled with IhogYFP extend up to several cell diameters and are reminiscent of the 'cytonemes', with a function in the transport of morphogens. In contrast to the previously described apical cytonemes, the extensions this study visualize are mainly found at the basal part of the disk epithelium. Interestingly, in the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between nonneighboring cells. However, further investigation will be necessary to demonstrate the implication of cytonemes in Hh gradient formation (Callejo, 2011).

A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling

A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product,functionally paired heparan sulfate (HS) 6-O-sulfotransferase (hs6st) and sulfatase (sulf1), which bidirectionally control HS proteoglycan (HSPG) sulfation, were characterized. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st) and increased (sulf1) neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg) and BMP (Glass Bottom Boat; Gbb) ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects (Dani, 2012).

It is well known that synaptic interfaces harbor heavily-glycosylated membrane proteins, glycolipids and ECM molecules, but understanding of glycan-mediated mechanisms within this synaptomatrix is limited. A genomic screen aimed to systematically interrogate glycan roles in both structural and functional development in the genetically-tractable Drosophila NMJ synapse. 130 candidate genes were screened, classified into 8 functional families: N-glycan biosynthesis, O-glycan biosynthesis, GAG biosynthesis, glycoprotein/proteoglycan core proteins, glycan modifying/degrading enzymes, glycosyltransferases, sugar transporters and glycan-binding lectins. From this screen, 103 RNAi knockdown conditions were larval viable, whereas 27 others produced early developmental lethality. 35 genes had statistically significant effects on different measures of morphological development: 27 RNAi-mediated knockdowns increased synaptic bouton number, 9 affected synapse area (2 increased, 7 decreased) and 2 genes increased synaptic branch number. These data suggest that overall glycan mechanisms predominantly serve to limit synaptic morphogenesis. 13 genes had significant effects on the functional differentiation of the synapse, with 12 increasing transmission strength and only 1 decreasing function upon RNAi knockdown. Thus, glycan-mediated mechanisms also predominantly limit synaptic functional development. A very small fraction of tested genes (CG1597; pgant35A, CG7480; veg, CG6657; hs6st, CG4451; sulf1, CG6725 and CG11874) had effects on both morphology and function. A large percentage of genes (~30%) showed morphological defects with no corresponding effect on function, while only 7% of genes showed functional alterations without morphological defects, and <5% of all genes affect both. These results suggest that glycans have clearly separable roles in modulating morphological and functional development of the NMJ synapse (Dani, 2012).

A growing list of neurological disorders linked to the synapse are attributed to dysfunctional glycan mechanisms, including muscular dystrophies, cognitive impairment and autism spectrum disorders. Drosophila homologs of glycosylation genes implicated in neural disease states include ALG3 (CG4084), ALG6 (CG5091), DPM1 (CG10166), FUCT1 (CG9620), GCS1 (CG1597), MGAT2 (CG7921), MPDU1 (CG3792), PMI (CG33718) and PPM2 (CG12151). Two of these genes, Gfr (CG9620) and CG1597, showed synaptic morphology phenotypes in the RNAi screen. Given that connectivity defects are clearly implicated in cognitive impairment and autism spectrum disorders, it would be of interest to explore the glycan mechanism affecting synapse morphology in Drosophila models of these disease states. Glycans are well known to modulate extracellular signaling, including ligands of integrin receptors, to regulate intercellular communication. In the genetic screen, several O-glycosyltransferases mediating this mechanism were identified to show morphological (GalNAc-T2, CG6394; pgant35A, CG7480, O-fut2, CG14789; rumi, CG31152) and functional (pgant5, CG31651; pgant35A, CG7480) synaptic defects upon RNAi knockdown. These findings suggest that known integrin-mediated signaling pathways controlling NMJ synaptic structural and functional development are modulated by glycan mechanisms. The screen showed CG6657 RNAi knockdown affects functional differentiation, consistent with reports that this gene regulates peripheral nervous system development. The corroboration of the screen results with published reports underscores the utility of RNAi-mediated screening to identify glycan mechanisms, and supports use of the screen results for bioinformatic/meta-analysis to link observed phenotypes to neurophysiological/pathological disease states and to direct future glycan mechanism studies at the synapse (Dani, 2012).

From this screen, the two functionally-paired genes sulf1 and hs6st were selected for further characterization. As in the RNAi screen, null alleles of these two genes had opposite effects on synaptic functional differentiation but similar effects on synapse morphogenesis, validating the corresponding screen results. The two gene products have functionally-paired roles; Hs6st is a heparan sulfate (HS) 6-O-sulfotransferase, and Sulf1 is a HS 6-O-endosulfatase. These activities control sulfation of the same C6 on the repeated glucosamine moiety in HS GAG chains found on heparan sulfate proteoglycans (HSPGs). At the Drosophila NMJ, two HSPGs are known to regulate synapse assembly; the GPI-anchored glypican Dally-like protein (Dlp), and the transmembrane Syndecan (Sdc). In contrast, the secreted HSPG Perlecan (Trol) is not detectably enriched at the NMJ, and indeed appears to be selectively excluded from the perisynaptic domain. In other developmental contexts, the membrane HSPGs Dlp and Sdc are known to act as co-receptors for WNT and BMP ligands, regulating ligand abundance, presentation to cognate receptors and therefore signaling. Importantly, the regulation of HSPG co-receptor abundance has been shown to be dependent on sulfation state mediated by extracellular sulfatases. Consistently, upregulation of Dlp and Sdc was observed in sulf1 null synapses, whereas Dlp was reduced in hs6st null synapses. In the developing Drosophila wing disc, HSPG co-receptors increase levels of the Wg ligand due to extracellular stabilization, and the primary function of Dlp in this developmental context is to retain Wg at the cell surface. Likewise, in developing Drosophila embryos, a significant fraction of Wg ligand is retained on the cell surfaces in a HSPG-dependent manner, with the HSPG acting as an extracellular co-receptor. Syndecan also modulates ligand-dependent activation of cell-surface receptors by acting as a co-receptor. At the NMJ, regulation of both these HSPG co-receptors occurs in the closely juxtaposed region between presynaptic bouton and muscle subsynaptic reticulum, in the exact same extracellular space traversed by the secreted trans-synaptic Wg and Gbb signals. It is therefore proposed that altered Dlp and Sdc HSPG co-receptors in sulf1 and hs6st mutants differentially trap/stabilize Wg and Gbb trans-synaptic signals at the interface between motor neuron and muscle, to modulate the extent and efficacy of intercellular signaling driving synaptic development (Dani, 2012).

HS sulfation modification is linked to modulating the intercellular signaling driving neuronal differentiation . In particular, WNT and BMP ligands are both regulated via HS sulfation of their extracellular co-receptors, and both signals have multiple functions directing neuronal differentiation, including synaptogenesis. In the Drosophila wing disc, extracellular WNT (Wg) ligand abundance and distribution was recently shown to be strongly elevated in sulf1 null mutants. Moreover, sulf1 has also recently been shown to modulate BMP signaling in other cellular contexts. Consistently, this study has shown increased WNT Wg and the BMP Gbb abundance and distribution in sulf1 null NMJ synapses. The hs6st null also exhibits elevated Wg and Gbb at the synaptic interface, albeit the increase is lower and results in differential signaling consequences. In support of this contrasting effect, extracellular signaling ligands are known to bind HSPG HS chains differentially dependent on specific sulfation patterns. It is important to note that the sulf1 and hs6st modulation of trans-synaptic signals is not universal, as Jelly Belly (Jeb) ligand abundance and distribution was not altered in the sulf1 and hs6st null conditions. This indicates that discrete classes of secreted trans-synaptic molecules are modulated by distinct glycan mechanisms to control NMJ structure and function (Dani, 2012).

At the Drosophila NMJ, Wg is very well characterized as an anterograde trans-synaptic signal and Gbb is very well characterized as a retrograde trans-synaptic signal. In Wg signaling, the dFz2 receptor is internalized upon Wg binding and then cleaved so that the dFz2-C fragment is imported into muscle nuclei. In hs6st nulls, increased Wg ligand abundance at the synaptic terminal corresponds to an increase in dFz2C punctae in muscle nuclei as expected. In contrast, the increase in Wg at the sulf1 null synapse did not correspond to an increase in the dFz2C-terminus nuclear internalization, but rather a significant decrease. One explanation for this apparent discrepancy is the 'exchange factor' model based on the biphasic ability of the HSPG co-receptor Dlp to modulate Wg signaling. In the Drosophila wing disc, this model suggests that the transition of Dlp co-receptor from an activator to repressor of signaling depends on Wg cognate receptor dFz2 levels, such that a low ratio of Dlp:dFz2 potentiates Wg-dFz2 interaction, whereas a high ratio of Dlp:dFz2 prevents dFz2 from capturing Wg. In sulf1 null synapses, a very great increase was observed in Dlp abundance (~40% elevated) with no significant change in the dFz2 receptor. In contrast, at hs6st null synapses there is a decrease in Dlp abundance (15% decreased) together with a significant increase in dFz2 receptor abundance (~25% elevated). Thus, the higher Dlp:dFz2 ratio in sulf1 nulls could explain the decrease in Wg signal activation, evidenced by decreased dFz2-C terminus import into the muscle nucleus. In contrast, the Dlp:Fz2 ratio in hs6st is much lower, supporting activation of the dFz2-C terminus nuclear internalization pathway. This previously proposed competitive binding mechanism dependent on Dlp co-receptor and dFz2 receptor ratios predicts the observed synaptic Wg signaling pathway modulation in sulf1 and hs6st dependent manner (Dani, 2012).

At the Drosophila NMJ, Gbb is very well characterized as a retrograde trans-synaptic signal, with muscle-derived Gbb causing the receptor complex Wishful thinking (Wit), Thickveins (Tkv) and Saxaphone (Sax) to induce phosphorylation of the transcription factor mothers against Mothers against decapentaplegic (P-Mad). Mutation of Gbb ligand, receptors or regulators of this pathway have shown that Gbb-mediated retrograde signaling is required for proper synaptic differentiation and functional development. Further, loss of Gbb signaling results in significantly decreased levels of P-Mad in the motor neurons. This study shows that accumulation of Gbb in sulf1 and hs6st null synapses causes elevated P-Mad signaling at the synapse and P-Mad accumulation in motor neuron nuclei. Importantly, sulf1 null synapses show a significantly higher level of P-Mad signaling compared to hs6st null synapses, and this same change is proportionally found in P-Mad accumulation within the motor neuron nuclei. These findings indicate differential activation of Gbb trans-synaptic signaling dependent on the HS sulfation state is controlled by the sulf1 and hs6st mechanism, similar to the differential effect observed on Wg trans-synaptic signaling. Genetic interaction studies show that these differential effects on trans-synaptic signaling have functional consequences, and exert a causative action on the observed bi-directional functional differentiation phenotypes in sulf1 and hs6st nulls. Genetic correction of Wg and Gbb defects in the sulf1 null background restores elevated transmission back to control levels. Similarly, genetic correction of Wg and Gbb in hs6st nulls restores the decreased transmission strength back to control levels. These results demonstrate that the Wg and Gbb trans-synaptic signaling pathways are differentially regulated and, in combination, induce opposite effects on synaptic differentiation (Dani, 2012).

Both wg and gbb pathway mutants display disorganized and mislocalized presynaptic components at the active zone (e.g. Bruchpilot; Brp) and postsynaptic components including glutamate receptors (e.g. Bad reception; Brec/GluRIID). Consistently, the bi-directional effects on neurotransmission strength in sulf1 and hs6st mutants are paralleled by dysregulation of these same synaptic components. Changes in presynaptic Brp and postsynaptic GluR abundance/distribution causally explain the bi-directional effects on synaptic functional strength between sulf1 and hs6st null mutant states. Alterations in active zone Brp and postsynaptic GluRs also agree with assessment of spontaneous synaptic activity. Null sulf1 and hs6st synapses showed opposite effects on miniature evoked junctional current (mEJC) frequency (presynaptic component) and amplitude (postsynaptic component). Further, quantal content measurements also support the observation of bidirectional synaptic function in the two functionally paired nulls. Genetic correction of Wg and Gbb defects in both sulf1 and hs6st nulls restores the molecular composition of the pre- and postsynaptic compartments back to wildtype levels. When both trans-synaptic signaling pathways are considered together, these data suggest that HSPG sulfate modification under the control of functionally-paired sulf1 and hs6st jointly regulates both WNT and BMP trans-synaptic signaling pathways in a differential manner to modulate synaptic functional development on both sides of the cleft (Dani, 2012).

This paper has presented the first systematic investigation of glycan roles in the modulation of synaptic structural and functional development. A host of glycan-related genes were identified that are important for modulating neuromuscular synaptogenesis, and these genes are now available for future investigations, to determine mechanistic requirements at the synapse, and to explore links to neurological disorders. As proof for the utilization of these screen results, this study has identified extracellular heparan sulfate modification as a critical platform of the intersection for two secreted trans-synaptic signals, and differential control of their downstream signaling pathways that drive synaptic development. Other trans-synaptic signaling pathways are independent and unaffected by this mechanism, although it is of course possible that a larger assortment of signals could be modulated by this or similar mechanisms. This study supports the core hypothesis that the extracellular space of the synaptic interface, the heavily-glycosylated synaptomatrix, forms a domain where glycans coordinately mediate regulation of trans-synaptic pathways to modulate synaptogenesis and subsequent functional maturation (Dani, 2012).

Drosophila glypicans Dally and Dally-like are essential regulators for JAK/STAT signaling and Unpaired distribution in eye development

The highly conserved JAK/STAT pathway is a well-known signaling system that is involved in many biological processes. In Drosophila, this signaling cascade is activated by ligands of the Unpaired (Upd) family. Therefore, the regulation of Upd distribution is one of the key issues in controlling the JAK/STAT signaling activity and function. Heparan sulfate proteoglycans (HSPGs) are macromolecules that regulate the distribution of many ligand proteins including Wingless, Hedgehog and Decapentaplegic (Dpp). This study shows that during Drosophila eye development, HSPGs are also required in normal Upd distribution and JAK/STAT signaling activity. Loss of HSPG biosynthesis enzyme Brother of tout-velu (Botv), Sulfateless (Sfl), or glypicans Dally and Dally-like protein (Dlp) led to reduced levels of extracellular Upd and reduction in JAK/STAT signaling activity. Overexpression of dally resulted in the accumulation of Upd and up-regulation of the signaling activity. Luciferase assay also showed that Dally promotes JAK/STAT signaling activity, and is dependent on its heparin sulfate chains. These data suggest that Dally and Dlp are essential for Upd distribution and JAK/STAT signaling activity (Zhang, 2013).

Upd distribution is essential for the JAK/STAT signaling pathway, but its regulation is largely unknown. This study has shown that the glypicans Dally and Dlp are required for the normal Upd distribution and JAK/STAT signaling activity. Overexpression of dally activated the JAK/STAT signal in eye discs. Dally and Dlp also up-regulated JAK/STAT signaling activity in cell culture, and its function was dependent on their attached HS chains. Together, these data indicate that Dally and Dlp are essential regulators for JAK/STAT signaling activity (Zhang, 2013).

One of the most important findings in this work is that Dally and Dlp regulate Upd distribution and may be required for the retention of Upd on the cell surface. Previous studies of Upd mainly focused on its transcriptional regulation and function, but little is known about the regulation of Upd distribution. It is known that Upd has a long-range effect on JAK/STAT signaling but the mechanism(s) behind is unknown. This study found that the long-range effect of Upd in eye development was due to the long-range extracellular distribution of Upd. Loss of dally or dlp led to reduction of extracellular Upd levels, while overexpression of dally or dlp was able to increase the extracellular levels of Upd. Therefore, it is concluded that the long-range distribution of Upd and subsequent JAK/STAT signaling activity are controlled by Dally and Dlp in Drosophila eye discs (Zhang, 2013).

Data from cell culture experiments suggested that dally and dlp may be required for the retention of Upd on the cell surface and subsequent signaling activity. With high levels of Upd, Dally and Dlp had mild effects on JAK/STAT signaling activity. With low levels of Upd, Dally and Dlp dramatically up-regulated JAK/STAT signaling activity. These results indicate that dally and dlp help to retain Upd on the cell surface and activate JAK/STAT signaling activity (Zhang, 2013).

Although both Dally and Dlp play roles in the regulation of Upd distribution, these studies show that Dally is likely the major regulator of JAK/STAT signaling activity. First, loss of dally showed a stronger reduction in JAK/STAT signaling activity than loss of dlp. Second, expression of dally rescued the reduction of JAK/STAT signaling in dallydlp double mutant clones, but expression of dlp alone did not. Third, overexpression of dally but not dlp induced ectopic JAK/STAT signaling activity. Fourth, in cell culture, with high levels of Upd Dally but not Dlp can further up-regulate JAK/STAT signaling activity. All of these data support the view that Dally is the major regulator of JAK/STAT signaling activity in eye development. More experiments are needed to figure out why Dally has higher signaling activity in JAK/STAT pathway than Dlp (Zhang, 2013).

These studies also show that HS chains of HSPGs play important roles in the regulation of JAK/STAT signaling activity. In eye discs, the loss of botv or sfl led to the reduction in JAK/STAT signals, indicating that biosynthesis of HS chains is required for normal JAK/STAT signaling activity. Previous studies in cell culture showed that Upd may be associated with HS chains, and this association can be released by the addition of heparin. This study shows that in the presence of heparin, the activation of JAK/STAT by Dally and Dlp was compromised. These findings support the view that the activation of JAK/STAT signaling is dependent on the association of Upd with HS chains (Zhang, 2013).

Balancing Hedgehog, a retention and release equilibrium given by Dally, Ihog, Boi and shifted/DmWif

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. The mechanisms of Hh release and spread were studied using the Drosophila wing imaginal disc as a model system for polarized epithelium. The cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), was analyzed in the stability, release and spread of Hh. Dally and Boi were shown to be required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation (Bilioni, 2013).

This study has approached the functional interaction of the ECM components Shf/dWif and Dally, and of the Hh coreceptors Ihog and Boi in Hh release and/or spreading to form a gradient. Two major findings are described: one is an unpredicted role of Dally and Boi in the apical retention and subsequent internalization of Hh in producing cells, and the other the interaction of Dally and Ihog/ Boi with Shf/dWif, facilitating Hh movement in the basolateral part of the disc epithelium. Interactions between these components allow retention at the apical plasma membranes of producing cells necessary to prevent apical Hh spreading and facilitate subsequent recycling to basolateral side, as well as Hh release and movement in this side of the epithelium (Bilioni, 2013).

Apical Hh levels in the Hh producing cells are affected in both dally and boi (but not in ihog) null mutant conditions. Moreover, ectopic Dally or Boi (but not Ihog) cause an increase in Hh retention at the apical plane of the disc. Accordingly, Dally and Boi have an Ihog-independent function in maintaining Hh concentration at the apical part of the disc epithelium. As has been previously demonstrated, apically externalized Hh does not form a gradient (Callejo, 2011); thus, this might be accounted as a mechanism preventing the spread of apically externalized Hh. In agreement with an Ihog-independent role of Boi in apical Hh retention, a recently published study (Hartman, 2010) demonstrates that Boi is expressed in apical cells of the ovary and suppresses follicular stem cells (FSC) proliferation by binding to and sequestering Hh on the apical cell surface, thereby inhibiting Hh long range distribution (Bilioni, 2013).

In addition, it has been observed that the apically externalized Hh is subsequently internalized and recycled to the basolateral membranes of the wing disc epithelium (Callejo, 2011). In strong support of this Hh recycling scheme, overexpression of Dally in the P compartment was shown to enhance Hh apical retention, decreasing Hh levels in the most basal side and reducing Hh target activation in the A compartment. Therefore, it is likely that Dally and Boi not only prevent apical Hh spreading but also mediate the apical Hh internalization in the Hh producing cells. The observation that Hh, Dally and Boi accumulate in the apical surface when internalization is blocked by a dynamin mutation reinforces this possibility (Bilioni, 2013).

The increment of apical spreading of Hh in the A compartment cells caused by overexpression of a secretable form of Dally in the P compartment also supports Dally's function in Hh apical retention in the P compartment cells. Given that the enhanced apical spreading of Hh correlates with a reduced basolateral Hh gradient, it is proposed that in normal conditions recycling of the apical Hh pool results in the formation of a basolateral Hh gradient. In contrast, it has been propose that the hydrolase, Notum, is implicated in the release of Dally and the abnormal increase in the apical spreading of Hh in the A compartment cells by overexpression of a DallySec has been interpreted as a direct evidence of a long-range apical Hh gradient. However, notum mutants show a Wg (not a Hh) signaling phenotype, which argues against this hypothesis. Furthermore, the cell-autonomous requirement of wild type Dally for keeping Hh in the ECM suggests that Dally may not be released from its GPI anchor for this function. In addition, no non-autonomous effects of Dally were observed on Shf/dWif stability, which implies that Dally remains membrane-anchored (Bilioni, 2013).

In conclusion, these data show that Dally has a cell-autonomous role in Hh attachment to the ECM, with a double purpose: in the producing cells Dally facilitates Hh retention necessary to prevent Hh spreading, and in receiving cells it supports Hh presentation to the receptor. An autonomous Dally requirement for Hh signaling has been recently proposed. This cell-autonomous requirement for Dally in maintaining extracellular Hh concentration is in agreement with the previously described role of Dally in Wg and Dpp signaling (Bilioni, 2013).

It has been suggested that Shf/dWif mediates the function of the HSPGs in Hh stability in the ECM. This study finds that Shf/dWif stabilization also depends on Dally, Ihog and Boi because Shf/dWif levels vary accordingly in both loss and gain of function of these genes. In these mutants, Shf/dWif levels are reduced at the basolateral side of the disc epithelium and, as a consequence, Hh levels also decrease. Thus, Dally together with Shf/dWif, Boi and Ihog is implicated in Hh stability in the ECM. On the other hand, despite an increment in Hh levels when overexpressing Dally in a shf mutant background, the target expression remains severely impaired. Thus, an excess of Dally or Ihog/Boi can offset the effects of Shf/dWif mutation in terms of Hh concentration but not in terms of Hh movement. Taken together, these results lead to the conclusion that Shf/dWif is an ECM factor that counteracts the impact of Dally and Ihog/Boi on Hh attachment at the membranes. Interestingly, ectopic expression of Ihog, Boi or Dally stabilizes Shf/dWif mainly in the basolateral domain where most of Shf/dWif protein is located (Bilioni, 2013).

Shf/dWif is then required for Hh movement even when overexpressing Hh. Counteracting this effect, Ihog and Boi mediate the attachment of extracellular Hh to plasma membranes in Hh producing cells. In ihog and boi mutant cells Hh levels, mainly at the basolateral plane, are very low, and overexpression of Ihog or Boi not only causes Hh accumulation at the plasma membrane but also a restriction in Hh movement. Moreover, Shf/dWif can rescue the phenotype of restricted Hh movement imposed by ectopic Ihog or Boi, and is necessary to allow the increment of Hh spreading when knocking down Boi and Ihog in the P compartment, demonstrating that proper gradient formation requires equilibrium between these proteins. This is further confirmed when Ihog overexpression in the P compartment restricts Hh movement and decreases Hh signaling in the region anterior to a smo clone located at the A/P compartment border; and then again simultaneous overexpression of Shf/dWif reestablishes the equilibrium so Hh can now reach the wild type territory and signal across the clone (Bilioni, 2013).

It has been proposed that Boi and Ihog are not required in P compartment cells because double boi and ihog mutant clones had no effect on Hh signaling. However, this conclusion did not take account of Hh non-autonomy. Indeed, it has been reported that wings develop normally even with P compartments that have large hh mutant clones. This non-autonomy of Hh is also supported by the lack of an effect on Hh signaling of disp−/− clones or by long-range spreading of Hh through large smo mutant clones. Because of Hh non-autonomy, the function of Boi and Ihog in Hh-producing cells was only reveled when Boi and Ihog were knocked down in the whole P compartment. Interestingly, despite of the low Hh levels in the P compartment in the absence of Boi and Ihog functio, an increase of long-range Hh gradient was observed. It is thought is that a low Hh retention at the plasma membrane of P compartment cells causes an increase of Hh release, so the bulk of Hh that reaches the A compartment is higher than in the wild type condition. Supporting this hypothesis, it was shown that Ihog and Boi from A cells have indeed the capacity to 'capture' Hh from P compartment cells (Bilioni, 2013).

In addition, the ectopic expression of Ihog increases not only the endogenous levels of Hh but also Shf/dWif, Dally along cytonemes located at the basolateral side of the disc epithelium, as previously described for Dlp (Callejo, 2011). Some of these long filaments extend up to several cell diameters and are reminiscent of 'cytonemes'. In the context of Notch signaling, basal actin-based filopodia are important for lateral inhibition between non-neighboring cells. In Hh signaling, it was have observed that cytonemes act as vectors for Hh movement in the ECM, contributing to Hh gradient formation. Since Boi and Ihog are absolutely required for reception, Ptc, Ihog- and Boi-labeled cytonemes emanating from A compartment cells are probably essential for sequestering Hh from P cells. Although this work does not provide the molecular mechanism by which Shf/dWif, Dally, Ihog and Boi proteins affect cytoneme-mediated Hh transport, it is suggested that Shf/dWif might be responsible for maintaining the equilibrium between Hh attachment to cytonemes -- mediated by Dally, Ihog and Boi -- and Hh release or movement (Bilioni, 2013).

Previous analysis on Hh release in the wing imaginal disc epithelium indicates that although Hh is initially externalized through all plasma membranes, the apical Hh pool is internalized and recycled to basolateral plasma membranes where the long-range Hh gradient is formed (Callejo, 2011). This article has provided a compressive genetic analysis that confirms the hypothesis. A novel role is described of the glypican Dally and of the transmembrane protein Boi in the process of the apical internalization of Hh in P compartment cells, which is essential to guarantee that the bulk of Hh protein produced in the P compartment cells is redirected towards the basolateral domain. A role is also described of the Hh coreceptors Ihog and Boi, and the diffusible Shf/dWif factor at the basolateral plane of the epithelium. These proteins interact physically and together with Dally act to establish a balance between Hh attachment to membranes and movement across the ECM to promote gradient formation and signaling. Moreover, all these proteins associate to cytonemes in the basolateral part of the disc epithelium. Thus, the interplay of all these proteins creates an environment supporting Hh transport along cytonemes to shape a proper gradient (Bilioni, 2013).

Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

Fragile X syndrome (FXS), the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1) gene product (FMRP), an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1) null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs): GPI-anchored glypican Dally-like protein (Dlp) and transmembrane Syndecan (Sdc). Earlier work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg) ligand abundance and downstream Frizzled-2 (Fz2) receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb), and downstream ERK phosphorylation (dpERK) are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb) and downstream signaling via phosphorylation of the transcription factor MAD (pMAD) seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1) Wg and Jeb trans-synaptic signaling, and (2) synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state (Friedman, 2013).

FXS is widely considered a disease state arising from synaptic dysfunction, with pre- and postsynaptic defects well characterized in the Drosophila disease model. There has been much work documenting FXS phenotypes in humans as well as in animal models, but there has been less progress on mechanistic underpinnings. This study focuses on the extracellular synaptomatrix in FXS owing to identification of pharmacological and genetic interactions between FMRP and secreted MMPs, a mechanism that is conserved in mammals. Other studies have also highlighted the importance of the synaptomatrix in synaptogenesis, particularly the roles of membrane-anchored HSPGs as co-receptors regulating trans-synaptic signaling. Importantly, it has been shown that FMRP binds HSPG mRNAs, thereby presumably repressing translation. Based on these multiple lines of evidence, this study hypothesized that the FMRP-MMP-HSPG intersection provides a coordinate mechanism for the pre- and postsynaptic defects characterizing the FXS disease state, with trans-synaptic signaling orchestrating synapse maturation across the synaptic cleft (Friedman, 2013).

In testing this hypothesis, a dramatic upregulation of GPI-anchored glypican Dlp and transmembrane Sdc HSPGs was discovered at dfmr1 null NMJ synapses. Indeed, these are among the largest synaptic molecular changes reported in the Drosophila FXS disease model. Importantly, HSPGs have been shown to play key roles in synaptic development. For example, the mammalian HSPG Agrin has long been known to regulate acetylcholine receptors, interconnected with a glycan network modulating trans-synaptic signaling. In Drosophila, Dlp, Sdc and Perlecan HSPGs mediate axon guidance, synapse formation and trans-synaptic signaling. Previous work on dlp mutants reports elevated neurotransmission, paradoxically similar to the Dlp overexpression phenotype shown in this study. However, the previous study does not show Dlp overexpression electrophysiological data, although it does show increased active zone areas consistent with strengthened neurotransmission. The same study reports that Dlp overexpression decreases bouton number on muscle 6/7, which differs from finding in this study of increased bouton number on muscle 4. Because HSPG co-receptors regulate trans-synaptic signaling, dfmr1 mutants were tested for changes in three established pathways at the Drosophila NMJ. Strong alterations in both Wg and Jeb pathways were found, with anterograde signaling being downregulated in both cases. In contrast, no change was found in the retrograde BMP Gbb pathway, suggesting that FMRP plays specific roles in modulating anterograde trans-synaptic signaling during synaptogenesis (Friedman, 2013).

The defect in Jeb signaling seems to be simple to understand, with decreased synaptomatrix ligand abundance coupled to decreased dpERK nuclear localization. However, there is no known link to HSPG co-receptor regulation. It has been shown earlier that Jeb signaling is regulated by another synaptomatrix glycan mechanism, providing a clear precedent for this level of regulation. In contrast, the Wnt pathway exhibits an inverse relationship between Wg ligand abundance (elevated) and Fz2-C nuclear signaling (reduced). This apparent contradiction is explained by the dual activity of the Dlp co-receptor, which stabilizes extracellular Wg to retain it at the membrane, but also competes with the Fz2 receptor. This ‘exchange-factor mechanism’ is competitively dependent on the ratio of Dlp co-receptor to Fz2 receptor, with a higher ratio causing more Wg to be competed away from Fz2. Indeed, it has been demonstrated that the same elevated Wg surface retention couples to decreased downstream Fz2-C signaling in an independent HSPG regulative mechanism at the Drosophila NMJ. This study suggests that in the dfmr1 null synapse, highly elevated Dlp traps Wg, thereby preventing it from binding Fz2 to initiate signaling (Friedman, 2013).

Dysregulation of the Wg nuclear import pathway (FNI) provides a plausible mechanism to explain synapse development defects underlying the FXS disease state, with established roles in activity-dependent modulation of synaptic morphogenesis and neurotransmission. FXS has long been associated with defects in activity-dependent architectural modulation, including postsynaptic spine formation, synapse pruning and functional plasticity. Although it is surely not the only player, aberrant Wg signaling could play a part in these deficiencies. Importantly, it has been shown that the FNI pathway is involved in shuttling large RNA granules out of the postsynaptic nucleus, providing a potential intersection with the FMRP RNA transport mechanism. However, the Wg FNI pathway is not the only Wnt signaling at the Drosophila NMJ, with other outputs including the canonical, divergent canonical and planar cell polarity pathways, which could be dysregulated in dfmr1 nulls. For example, a divergent canonical retrograde pathway proceeds through GSK3β (Shaggy) to alter microtubule assembly, and the FXS disease state is linked to dysregulated GSK3β and microtubule stability misregulation via Drosophila Futsch/mammalian MAP1B. Moreover, it has been shown that the secreted HSPG Perlecan (Drosophila Trol) regulates bidirectional Wnt signaling to affect Drosophila NMJ structure and/or function, via anterograde FNI and retrograde divergent canonical pathways. It is also important to note that previous studies show that a reduction in the FNI pathway, due to decreased Fz2-C trafficking to the nucleus, leads to decreased NMJ bouton number. Future work is needed to fully understand connections between FMRP, HSPGs, the multiple Wnt signaling pathways and the established defects in the synaptic microtubule cytoskeleton in the FXS disease state (Friedman, 2013).

Adding to the complications of FXS trans-synaptic signaling regulation, it was shown that two trans-synaptic signaling pathways are suppressed in parallel: the Wg and Jeb pathways. Possibly even more promising for clinical relevance, it has been established that the Jeb signaling functions as a repressor of neurotransmission strength at the Drosophila NMJ, with jeb and alk mutants presenting increased evoked synaptic transmission. Consistently, loss of FMRP leads to increased EJC amplitudes, which could be due, at least partially, to misregulated Jeb-Alk signaling. Importantly, it has been shown that dfmr1 null neurotransmission defects are due to a combination of pre- and postsynaptic changes, and that there is a non-cell-autonomous requirement for FMRP in the regulation of functional changes in the synaptic vesicle (SV) cycle underlying neurotransmission strength. Additionally, jeb and alk mutants exhibit synaptic structural changes consistent with this FMRP interaction, including a larger NMJ area and synaptic bouton maturation defects, which are markedly similar to the structural overelaboration phenotypes of the FXS disease state. These data together suggest that altered Jeb-Alk trans-synaptic signaling plays a role in the synaptic dysfunction characterizing the dfmr1 null. The study proposes that Wg and Jeb signaling defects likely interact, in synergistic and/or antagonistic ways, to influence the combined pre- and postsynaptic alterations characterizing the FXS disease state (Friedman, 2013).

Although trans-synaptic signaling pathways, and in particular both Wnt and Jeb-Alk pathways, have been proposed to be involved in the manifestation of a number of neurological disorders, this study provides the first evidence that aberrant trans-synaptic signaling is causally involved in an FXS disease model. The study proposes a mechanism in which FMRP acts to regulate trans-synaptic ligands by depressing expression of membrane-anchored HSPG co-receptors. HSPG overexpression alone is sufficient to cause both synaptic structure and function defects characterizing the FXS disease state. Increasing HSPG abundance in the postsynaptic cell is enough to increase the number of presynaptic branches and synaptic boutons, as well as elevate neurotransmission. Correlation with these well-established dfmr1 null synaptic phenotypes suggests that HSPG elevation could be a causal mechanism. Conclusively, reversing HSPG overexpression in the dfmr1 null is sufficient to correct Wnt and Jeb signaling, and to restore normal synaptic structure and function. Because there is no dosage compensation, HSPG heterozygosity offsets the elevation caused by loss of dfmr1. Correcting both Dlp and Sdc HSPGs in the dfmr1 background restores Wg and Jeb signaling to control levels. Correcting Dlp levels by itself restores synaptic architecture, but both Dlp and Sdc have to be corrected to restore normal neurotransmission in dfmr1 null synapses. Taken together, these results from the Drosophila FXS disease model provide exciting new insights into the mechanisms of synaptic phenotypes caused by the loss of FMRP, and promising avenues for new therapeutic treatment strategies (Friedman, 2013).

Two matrix metalloproteinase classes reciprocally regulate synaptogenesis

Synaptogenesis requires orchestrated intercellular communication between synaptic partners, with trans-synaptic signals necessarily traversing the extracellular synaptomatrix separating presynaptic and postsynaptic cells. Extracellular matrix metalloproteinases (Mmps) regulated by secreted tissue inhibitors of metalloproteinases (Timps), cleave secreted and membrane-associated targets to sculpt the extracellular environment and modulate intercellular signaling. This study tested Mmp roles at the neuromuscular junction (NMJ) model synapse in the reductionist Drosophila system, which contains just two Mmps (secreted Mmp1 and GPI-anchored Mmp2) and one secreted Timp. All three matrix metalloproteome components co-dependently localize in the synaptomatrix. Both Mmp1 and Mmp2 independently restrict synapse morphogenesis and functional differentiation. Surprisingly, either dual knockdown or simultaneous inhibition of the two Mmp classes together restores normal synapse development, identifying a novel reciprocal suppression mechanism. The two Mmp classes co-regulate a Wnt trans-synaptic signaling pathway modulating structural and functional synaptogenesis, including the GPI-anchored heparan sulfate proteoglycan (HSPG) Wnt co-receptor Dally-like Protein (Dlp), cognate receptor Frizzled-2 and Wingless ligand. Loss of either Mmp1 or Mmp2 reciprocally misregulates Dlp at the synapse, with normal signaling restored by co-removal of both Mmp classes. Correcting Wnt co-receptor Dlp levels in both mmp mutants prevents structural and functional synaptogenic defects. Taken together, these results identify a novel Mmp mechanism that fine-tunes HSPG co-receptor function to modulate Wnt signaling to coordinate synapse structural and functional development (Dear, 2015).

A large number of Mmps are expressed in the mammalian nervous system, with roles in neurodevelopment, plasticity and neurological disease. Understanding how each Mmp individually and combinatorially functions is hindered by genetic redundancy and compensatory mechanisms. This study exploited the Drosophila system to analyze a matrix metalloproteome containing just one member of each conserved component: one secreted Mmp, one membrane-tethered Mmp and one Timp. Both Mmp classes were found to attenuate structural and functional synaptic development, with electrophysiological, ultrastructural and molecular roles in both presynaptic and postsynaptic cells. A surprising discovery is that the Mmp classes suppress each other's requirements at the synapse. From discrete activities to redundancy, cooperation and now reciprocal suppression, studies continue to reveal how Mmps interact to regulate developmental processes. This study shows that the two Mmp classes play separable yet interactive roles in sculpting NMJ development. During the writing of this manuscript, a genomic Mmp2 rescue line was produced (Wang, 2014), which will be critical in further testing this interactive mechanism. It will be interesting to determine whether the Mmp suppressive mechanism is used in other developmental contexts, other intercellular signaling pathways and in mammalian models. Mammalian Mmp9 regulates synapse architecture and also postsynaptic glutamate receptor expression and/or localization. Likewise, mammalian Mmp7 regulates both presynaptic properties and postsynaptic glutamate receptor subunits. Thus, the dual roles of Mmps in pre- and postsynaptic compartments appear to be evolutionarily conserved (Dear, 2015).

Previous work demonstrated that Mmp1 and Mmp2 both regulate motor axon pathfinding in Drosophila embryos, albeit to different degrees and in this study, double Mmp mutants still exhibited defasciculated nerve bundles that separate prematurely. Consistently, both Mmp single mutants display excessive terminal axon branching at the postembryonic NMJ, but here the defect is fully alleviated by the removal of both Mmps. Other studies have either not identified, or not tested, a similar Mmp interaction, suggesting that reciprocal suppression might be specific to synaptogenesis. However, there are numerous reports that highlight the importance of Mmp and Timp balance. Mmp:Timp ratios can influence protease activation, localization, substrate specificity and Timp signaling and are commonly used as predictive clinical correlates in disease pathology. At the Drosophila NMJ, a similar reciprocal suppression interaction between pgant glycosyltransferases involved in O-linked glycosylation regulates synaptogenesis via integrin-tenascin trans-synaptic signaling. A recent study reported that pgant activity protects substrates from Furin-mediated proteolysis, which is a protease responsible for processing or activating Drosophila Mmp1 and Mmp2. Thus, Mmp proteolytic and glycan mechanisms could converge within the NMJ synaptomatrix to regulate trans-synaptic signaling (Dear, 2015).

New antibody tools produced in this study provide the means to interrogate an entire matrix metalloproteome, and will be important for testing Mmp and Timp functions throughout Drosophila. Many Mmps are both developmentally and activity regulated, with highly context-dependent functions. Future work will temporally dissect this mechanism at the developing NMJ and investigate how activity might regulate Mmp localization and function. It will be informative to correlate synaptogenic Mmp requirements with Mmp enzymatic activity by using in situ zymography assays, although non-enzymatic roles are certainly also possible. Lack of ultrastructure defects in Mmp mutant NMJs suggests that Drosophila Mmps have primarily instructive functions at the synapse, rather than broad proteolytic roles in ECM degradation. Consistently, Drosophila Mmp2 instructs motor axon pathfinding via a BMP intercellular signaling mechanism. Conversely, Mmp2 functions permissively in basement membrane degradation while shaping dendritic arbors. Because synaptic bouton size is reduced in mmp1 mutants, Mmp1 activity might degrade a prohibitive physical barrier at the NMJ. However, the results indicate a primary Mmp role in regulating intercellular signaling during synaptic development (Dear, 2015).

HSPG co-receptors of trans-synaptic ligands are key modulators of NMJ synaptogenesis and HSPGs are also established substrates of both mammalian and Drosophila Mmps. Mmp1 and Mmp2 differentially regulate the HSPG Dlp co-receptor to restrict the Wnt Wg trans-synaptic signaling driving structural and functional NMJ development. How might both increased and decreased levels of the Dlp co-receptor yield increased FNI pathway signal transduction? Regulation of Wnt signaling interactions ligands, co-receptors and receptors is managed at many levels. The 'Wg exchange factor model' provides a mechanistic framework for understanding the suppressive interactions of Mmp. In this mechanism, a low Dlp:Frz2 ratio helps the Frz2 receptor obtain more Wg, whereas a high Dlp:Frz2 ratio prevents Frz2 from capturing Wg as Dlp competes and sequesters Wg away from Frz2. Importantly, however, Dlp exhibits a context-dependent, bimodal role as both activator and repressor. Indeed, previous studies show these mechanisms are a key driving force in Wg signal transduction at the Drosophila NMJ (Dani, 2012; Friedman, 2013). In mmp1 mutants, Wg and Dlp are both reduced, resulting in a low Dlp:Frz2 ratio and elevated FNI. In mmp2 mutants, Dlp is spatially diffuse and Frz2 is increased, similarly resulting in a low Dlp:Frz2 ratio and elevated FNI. Balance is reset with Mmp co-removal because neither form of Mmp-induced HSPG tuning occurs. In this regard, it might be predicted that Dlp reduction in mmp2 mutants would only further increase FNI and therefore structural and functional defects. It is likely that absolute Dlp levels are the important driving factor in synaptogenesis and/or that Dlp exhibits bimodal functions in synaptic development (Dear, 2015).

Interestingly, a recent mouse study showed the Mmp3 hemopexin domain promotes Wnt signaling by inhibiting a negative Wnt regulator, raising the possibility that Mmps can act as molecular switches (or in feedback loops) dictating Wnt transduction. Another study suggests that Wnt signaling can directly mediate co-regulation of heparanase and Mmps. Indeed, both neural activity and intercellular signaling can stimulate Mmp-dependent ectodomain shedding of plasma membrane target proteins, thereby directly regulating the surface abundance of HSPGs and receptors, as well as other Mmps, which thus reciprocally modulate intra- and extracellular organization. From this model, the spatial arrangement of Dlp could be affected by co-regulated sheddase activity that is differentially altered in mmp1 and mmp2 mutants. Specifically, Mmp2 could shed Dlp, resulting in an increased area of Dlp expression in mmp2 mutants and loss of Mmp2 regulation by Mmp1 could result in aberrant Dlp restriction in mmp1 mutants, with Mmp co-removal remediating the Dlp domain thereby restoring normal Wnt trans-synaptic signaling. Future work will test the reciprocal impacts of Wnt signaling on Mmp expression and/or function in the context of synaptic development (Dear, 2015).

Emerging evidence suggests HSPG glycosaminoglycan (GAG) chains function as allosteric regulators of Mmps, with GAG content or composition influencing the localization and substrate specificity of Mmp. Indeed, Wg signaling is sensitive to perturbations in HSPG chain biosynthesis and HS modifying enzymes, which modulate both NMJ structure and function. It is easy to envision how tissue- and development-stage-specific HS modifications could coordinate HSPG/Mmp-dependent functions, thereby differentially regulating diverse signaling events, which enable context-specific responses instructed by the extracellular environment. Future work will examine how dual inputs of the HSPG co-receptor function and how Mmp proteolytic cleavage coordinates Wnt trans-synaptic signaling during synaptogenesis, particularly in the context of the Fragile X syndrome (FXS) disease model. Given that both loss or inhibition Mmp and correction of HSPG elevation independently alleviate synaptic defects in the FXS disease state, the overlapping mechanism provides an exciting avenue to therapeutic interventions for FXS and, potentially, related intellectual disability and autism spectrum disorders (Dear, 2015).


DEVELOPMENTAL BIOLOGY

Dally-like protein, a new Drosophila glypican with expression overlapping with wingless

Proteoglycans, the molecules of extracellular matrix, carry a highly negative charge due to their glycosaminoglycan (GAG) chains and large volumes. They were considered to play a secondary role in activities like cell division, adhesion, blood coagulation, etc. until the importance of their sugar chains in the fibroblast growth factor (FGF) signaling was discovered. Studies of mutations in the genes sugarless (sgl) and sulfateless (sfl) have proved that the proteoglycans involved in Wg signaling contain heparan sulfate GAG chains. This has led to the attribution of specific functions to these molecules. The Glypican family of heparan sulfate proteoglycans (HSPGs) is characterized by core proteins with conserved cysteine residues and attachment to the cell surface by a glycosylphosphatidyl inositol (GPI) anchor. This may lead to endocytic pathways that are different from other HSPGs, higher lateral mobility and possible apical localization in a cell. Variations in their HS contents may effect binding properties and localization, thus specializing each member for a unique biological function. Glypicans play important roles in morphogenetic pathways, e.g. human glypican 3 (GPC3) is mutated in Simpson-Golabi-Behmel syndrome making an individual prone to tumors. Dally, the first Drosophila member of the family, is essential for the wingless and decapentaplegic signaling pathways. A new Drosophila glypican, dally-like protein (dlp) is described, having all the features of a glypican (Khare, 2000).

The sequence analysis confirms its position in the glypican family. At the N terminus, there is a hydrophobic stretch of amino acids indicative of a potential signal sequence. A second, shorter hydrophobic stretch is found at the C terminus. Such a stretch is typically observed in GPI anchored proteins and is necessary for their attachment to the anchor in endoplasmic reticulum. Closer to the C terminus there is a cluster of seven serine-glycine dipeptides surrounded by acidic residues, a structural feature found in potential GAG attachment sites. All of the 14 cysteines are conserved. The homology searches suggest that this gene is closest in sequence to GPC4. However, the evolutionary tree made using program PROTDIST/NEIGHBOR reveals another lineage closer to Dally. This may be because the Drosophila genes contain stretches of unique sequences. The gene has been mapped to chromosomal location 70E (Khare, 2000).

The members of the glypican family show diverse tissue-specific as well as developmental stage-specific expression patterns. A Northern blot analysis was performed using suitable cDNAs as probes on extracts from different developmental stages of Drosophila. It showed a 4-kb transcript, which remains constant during the embryonic stages but is undetectable during third instar larva. The mRNA is detectable again at the pupal stage and continues in the adult, but the intensity of the band is lower than in the earlier stages. In situ hybridization analysis was performed to determine the expression pattern of the gene during embryonic stages. The expression of dlp appears quite early. At stage 5, there is weak ubiquitous expression in the blastoderm, which concentrates into two strong anterior and two weak posterior bands at stage 6. At stage 3, the two anterior bands remain strong in the area which later forms the procephalic neurogenic region (pNR). This is the region that gives rise to some of the brain neuroblasts. At stage 10, a regular pattern of 15 stripes emerges. There is strong staining at pNR and the anteriormost region of clypeolabrum. The anterio-ventral region of future gnathal buds also shows strong expression. The expression pattern at this stage looks very similar to that of wg. Apart from this pattern, staining is also seen in the lining of the midgut, the hindgut and faint staining in the CNS (Khare, 2000).

To see if dlp and wg expression patterns colocalize, a double staining experiment was performed. Due to low resolution caused by the wg antibody, an engrailed (en) antibody was used instead. In a wild type embryo, the one-cell wide wg stripe lies immediately anterior to the two-cell wide en stripe. The dlp stripes are also seen to lie immediately anterior to the en stripes, but the stripes are wider than a single cell. It is concluded that dlp does overlap with the wg expressing cell, but to a great extent, also with the wg target cells. Yet, dally expression has been shown to overlap with en expression and thus, may not overlap with dlp (Khare, 2000).

Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless

Recent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Another Drosophila glypican gene, dally-like (dly) has been identified and it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception -- overexpression of DLY leads to an accumulation of extracellular Wg. It is proposed that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. It is concluded that HSPGs play an important role in organizing the extracellular distribution of Wg (Baeg, 2001).

One glypican molecule, Dally, has been implicated in Wg signaling. However, because numerous glypican genes are present in other animals, a search was carried out of the Drosophila database for additional glypican family members. One EST clone showed some sequence similarity to dally and a full length cDNA was cloned. The sequence of the cDNA revealed a potential open reading frame of 765 amino acid residues, with 22% and 35% identity to Dally and mouse K-glypican, respectively. The predicted primary structure of the molecule exhibits the hallmarks of a glypican protein. The hydrophilicity plot of the new molecule is similar to those of the other members of the glypican family, which is characterized by the presence of NH2- and COOH-terminal hydrophobic signal sequences. In addition, this molecule possesses four consensus serine/glycine dipeptide sequences for glycosaminoglycan (GAG) attachment sites, and a signal sequence for a GPI-moiety attachment site at the COOH-terminal region. Moreover, the number and position of cysteine residues, which are a unique feature of glypican family members, are almost completely conserved in the predicted protein. These results led to the identification of Dally-like. Hybridization using a dly-specific probe to polytene chromosomes from salivary glands localized the dly gene to the cytological division 70F on the third chromosome. Finally, Northern blot analysis revealed that dly encodes a single major 3.8 kb transcript (Baeg, 2001).

To discover the function of Dly, its expression in embryos was determined by in situ hybridization. dly transcripts are uniformly expressed at early embryonic stages, but by stage 8 they are enriched in stripes. Double staining for dly mRNA and Wg protein shows that dly transcripts are preferentially expressed in three to four cells anterior to the wg-expressing cells. Interestingly, this expression pattern is similar to that of both dally and frizzled 2, the Wingless receptor (Baeg, 2001).

In an attempt to assess the function of Dly during embryogenesis, the RNA interference (RNAi) method was used to perturb Dly protein synthesis. Embryos were injected with a dly double-stranded RNA (dsRNA). These embryos, referred to as dly dsRNA embryos, show the absence of naked cuticle. This phenotype is reminiscent of loss of either wg or hh gene activities. The segment polarity phenotype is also found when the activity of dally, which is required for Wg signaling in the embryo, is disrupted by RNAi, though the effect is less severe in dally dsRNA embryos than in dly dsRNA embryos. However, when compared with embryos injected with either dly dsRNA or dally dsRNA alone, embryos injected with an equimolar mixture of dly and dally dsRNAs show more severe segment polarity phenotypes. These embryos are smaller, particularly in the tail region. They also show an entire transformation of naked cuticle into cuticle with denticles, which is observed in wg or hh null mutations. Because dally does not appear to play a role in Hh signaling, the interaction between dally and dly observed in the RNAi interference experiment suggests that Dly and Dally function synergistically in Wg signaling. Altogether, these results suggest that dly is a novel segment polarity gene that potentiates Wg signaling (Baeg, 2001).

To further examine the role of Dly in Wg signaling, the function of Dly during wing imaginal disc development was examined. dly transcripts are uniformly expressed in wing discs. In the third instar imaginal disc, wg is expressed at the DV compartment border and acts over short and long ranges to pattern the wing disc. Short range Wg signaling induces the expression of the proneural gene achaete (ac) in a stripe on each side of the DV boundary, while long range Wg signaling controls the expression of Distal-less (Dll) within the wing blade. It was reasoned that overexpression of Dly might activate Wg signaling because dly dsRNA-injected embryos resemble those that have lost Wg activity. Interestingly, overexpression of dly (using the C96-Gal4 driver), which is highly expressed at the DV boundary of the wing disc, results in severe wing margin defects and loss of sensory bristles. These phenotypes are reminiscent of the phenotypes seen when Wg activity is reduced in the wing. Consistent with the adult wing phenotype, Ac expression is dramatically decreased in wing discs overexpressing Dly. Furthermore, when dly is overexpressed using the engrailed-Gal4 (en-Gal4) driver, the expression of Dll is reduced in the posterior compartment (Baeg, 2001).

A test was performed to see whether overexpression of transducers of the Wg signal can rescue the loss-of-function wg-like phenotypes associated with dly overexpression. Ectopic expression of either Wg or a gain-of-function Armadillo (Arm) can rescue the wing margin defects, and induced ectopic bristles that are characteristic of ectopic expression of the Wg pathway. Altogether, these results suggest that overexpression of Dly blocks Wg signaling in the wing disc, and that of Dly acts upstream of Arm (Baeg, 2001).

Since Dly is an extracellular GPI-linked molecule, it was reasoned that patterning defects associated with Dly overexpression might reflect the ability of Dly to sequester Wg, and thus prevent it from accessing and activating Frizzled 2. To visualize the effect of overexpressed Dly on Wg distribution, Dly was overexpressed using various Gal4 lines and the wing discs were stained with anti-Wg monoclonal antibodies. Two different staining protocols were used to detect Wg distribution. The first one, involved fixing the tissue before staining, and thus it detects mostly cytoplasmic Wg present in either secretory or internalized vesicles. In the second protocol, the tissue is incubated with the antibody prior to fixation and mostly detects extracellular Wg (Baeg, 2001).

Using the first protocol, in wild-type discs, Wg protein is found at high levels in a narrow stripe of three to five cells straddling the DV boundary. Following overexpression of Dly using either en-Gal4 or ptc-Gal4 a striking increased accumulation of Wg protein is observed. Using the second protocol, extracellular Wg is organized in a gradient at the basolateral surface of wg-expressing and nearby cells. Following overexpression of Dly, an increased accumulation of extracellular Wg protein is detected. This result indicates that Dly can affect extracellular Wg distribution (Baeg, 2001).

HSPGs are required to increase the local concentration of Wg ligand for its receptors. Considering the roles of Dally and Dly, there are at least two HSPGs at the cell surface of wing disc cells involved in Wg signaling. To generate mutant cells that lack all GAGs and determine the role of the HSPGs in Wg signaling, mutant clones of cells were generated that do not properly synthesize GAGs. sugarless (sgl) mutations cannot be used for this analysis because sgl acts in a cell non-autonomous manner, presumably because the enzyme synthesizes glucuronic acid that diffuses between cells. However, sulfateless (sfl) is involved in GAG modification and in its absence, the GAG chains are not synthesized properly. To determine whether the GAG chains of Dly are modified by sfl, Dly proteins expressed in wild-type or sfl mutant pupae were examined by Western blot analysis using anti-Dly antibodies. The predicted size for Dly is 80 kDa, and in wild-type pupae, Dly protein appears as a broad band that migrates to around 80-110 kDa, which presumably results from addition of GAG chains onto the core protein. In sfl mutant pupae, the modified form of Dly protein is significantly reduced and the sharp band of the core protein is increased. This results indicate that sfl plays a role in Dly modification. Using the staining protocol that detects cytoplasmic Wg, an alteration in the expression of intracellular Wg could not be detected in sfl mutant clones, indicating that sfl mutant cells normally transcribe wg and do not accumulate Wg (Baeg, 2001).

However, using the extracellular staining method, a dramatic decrease of extracellular Wg was detected in sfl mutant cells. Extracellular Wg has been shown to be mainly associated with the basolateral surface of cells, and GPI-anchored protein is thought to be primarily attached to the basal part of the cells. Together, these results suggest that the HSPGs are involved in extracellular Wg accumulation (Baeg, 2001).

Importantly, high accumulation of extracellular Wg can be detected on sfl mutant cells located adjacent to wild-type cells, suggesting that HSPGs act locally in a cell non-autonomous manner. Consistent with this observation, adult wing patterning in sfl mutant clones show local cell non-autonomy as well. Clones of sfl mutant cells are associated with wing margin defects, suggesting that sfl is required for Wg signaling, however some of the sfl mutant cells located near wild-type margin cells have a wild-type morphology (Baeg, 2001).

Taken together, these results suggest that HSPGs are involved in restricting Wg diffusion. Further, the local cell non-autonomy observed in sfl mutant clones may indicate that HSPGs may not be absolutely required for the binding of Wg to its transducing receptor(s) (Baeg, 2001).

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), RNA interference (RNAi) and a quantitative cultured cell assay were used to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes (Lum, 2002).

Larval

The expression of the P1 enhancer trap line, containing a P1 insertion under dally regulation, was examined through the division cycles of lamina precursor cells (LPCs) in third instar larval brains using anti-beta-gal antibody and digoxigenin-labelled DNA probes complementary to lacZ mRNA. The highest levels of beta-gal immunoreactivity are found in LPCs along the anterior segment of the lamina furrow. Cells in this region are in G2 and M phase of the first division. The lacZ mRNA shows a more limited distribution, presumably because of beta-gal protein perdurance and is restricted to the G2 and M phase domains of LPC division one. Cell cycle-dependent expression of enhancer trap insertions in this locus was obtained by staining third instar larval brains with both anti-beta-gal and anti-cyclin B antibodies. There is an overlap of expression of the enhancer trap insertion with cyclin B in several groups of dividing cells, including the inner proliferative center (IPC), suggesting that the cell cycle-restricted expression is not limited to LPCs. However, some cells of the central brain complex express the enhancer trap marker but do not show high levels of cyclin B immunoreactivity (Nakato, 1995).

The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like

The activity of different signaling pathways must be precisely regulated during development to define the final size and pattern of an organ. The Drosophila tumor suppressor genes dachsous (ds) and fat (ft) modulate organ size and pattern formation during imaginal disc development. Recent studies have proposed that Fat acts through the conserved Hippo signaling pathway to repress the expression of cycE, bantam, and diap-1. However, the combined ectopic expression of all of these target genes does not account for the hyperplasic phenotypes and patterning defects displayed by Hippo pathway mutants. This study identified the glypicans dally and dally-like as two target genes for both ft and ds acting via the Hippo pathway. Dally and Dally-like modulate organ growth and patterning by regulating the diffusion and efficiency of signaling of several morphogens such as Decapentaplegic, Hedgehog, and Wingless. These findings therefore provide significant insights into the mechanisms by which mutations in the Hippo pathway genes can simultaneously alter the activity of several signaling pathways, compromising the control of growth and pattern formation (Baena-Lopez, 2008).


EFFECTS OF MUTATION

Larvae homozygous for the P1 insertion which disrupts dally expression shows disorganization of the anterior segment of the outer proliferative center (aOPC)/lamina precursor cell (aOPC/LPC) epithelium in approximately 10% of the CNS preparations examined. Disordering of the eye and reductions or duplications of the antenna, also with low penetrance, are found in P1 homozygous adults. The low penetrance found in the P1 mutant made analysis of the cell division defect difficult. A search was carried out for more severe alleles within existing collections of P-element-induced mutants. One semi-lethal enhancer trap insertion in the region of 66D/E, sl(3)06464, shows a beta-gal-staining pattern in the larval brain like that of line P1. Homozygous sl(3)06464 adults exhibit abnormalities in several adult tissues, including reductions or complete loss of genitalia, disordering and reduction in the number of ommatidia, reductions and duplications of the antenna, and incomplete wing vein V and wing notching. P1 and sl(3)06464 enhancer trap expression patterns in other tissues (antenna, eye, leg and wing discs) and embryonic developmental stages were also coincident. The two P-element alleles are henceforth referred to as dally P1 and dally P2. Genetic complementation tests were performed with dally P1 and dally P2 alleles for the antennal phenotypes since these are easy to score and of fairly high penetrance in dally P1. For antennal defects, dally P1 and dally P2 fail to complement, supporting the conclusion that these two P-element insertions affect the same gene. To confirm that dally P1 and dally P2 are responsible for the phenotypes described above and that these phenotypes are caused by loss-of-function alleles, the P-element insert in dally P1 was mobilized. The imprecise excision class creates small deletions that can potentially effect the removal, either completely or partially, of the normal function of the targeted gene. 23 independent excision alleles failed to complement the adult phenotypes of dally P2 . These alleles, either as homozygotes or in combination with dally P2 show phenotypes with a range of expressivity and penetrance; the more severe mutants affect the same tissues and to the same degree as observed for dally P2 homozygous adults. For example, dally DP-188 shows abnormalities in the eye, antenna, genitalia and wing, similar to dally P2 (Nakato, 1995).

In the wild-type eye disc, the morphogenetic furrow (MF) serves as an anatomical marker for the assembly of the repeated sensory units, the ommatidia. As originally described, the MF is a broad indentation in the eye disc epithelium, which moves from the posterior to the anterior, marking the wave of differentiation that sweeps forward. Cells in different parts of the cell cycle occupy specific positions relative to the MF. Anti-cyclin B and propidium iodide staining were used to identify G2, and mitotic prophase, metaphase, anaphase and telophase cells in the eye disc. Cell division in the developing eye disc epithelium anterior to the MF is asynchronous as seen by the unpatterned cyclin B expression. However, an increase in the level of anti-cyclin B immunoreactivity anterior to the MF is observed, providing evidence of cell cycle synchrony beginning in G2 as cells approach the furrow. Immediately posterior to this G2 domain mitosis occurs. Mitotic cells in metaphase, anaphase and telophase are found immediately ahead of the MF. This mitotic domain reflects the cell cycle synchronization taking place just ahead of the MF. Cells complete mitosis as they enter the furrow, and become synchronized in G1 in the MF. Progression into the subsequent S phase takes place within the furrow, followed by G2. A second coordinate mitosis follows, completing the two division cycles distributed across the MF. In dally mutants, the first zone of cyclin B expression, which normally ends approximately 3-5 cell dimensions anterior to the MF, extends too far posterior, to the beginning of the MF. The M phase of this first division is also displaced toward the posterior in dally mutants. The mitotic cells found at the edge of the MF in dally P2 mutants are in earlier stages of mitosis compared to wild-type (Nakato, 1995).

The second division along the eye disc MF does take place in dally P2 mutants. The cell division defects observed for dally P2 are also found in two other dally alleles, both as homozygotes and in combination with dally P2. As is the case for the lamina, the cell division defects in the eye disc are found without an overall disruption of normal morphology. In the wild type, the neuronal marker Elav is expressed in the assembling ommatidia posterior to the MF. dally mutant discs show this pattern of Elav expression as well, indicating that the cell division defects are not secondary to a gross disruption of eye development. If dally mutations delay cell division, the time required for cells labelled in the S phase of the first division to traverse into the following G1 should be slower in dally mutants, when compared to heterozygous control larvae. In the wild-type eye disc, mitotic cells of the first division are found at the apical surface of the epithelium in front of the MF and, following M phase, nuclei migrate basally into the MF. The position of a nucleus labelled in the previous S phase can therefore be used as a measure of cell cycle progression -- the more basally located cells being further along in their progression through the cell cycle. In dally mutants 4 hours after pulse-labeling, nuclei are delayed in their descent into the furrow, as compared to labelled cells from the heterozygous control. These findings support the conclusion that the first division cycle is delayed as a consequence of compromised dally function (Nakato, 1995).

The DPP requirement for cell fate specification and cell cycle synchronization in the developing Drosophila eye was examined by determining whether cells defective for thickveins, saxophone or schnurri show abnormalities in cell division or differentiation. Clones mutant for a null allele of tkv that are anterior or posterior to the morphogenetic furrow have amounts of Cyclin B that are indistinguishable from those in surrounding cells. In contrast, tkv clones that span the MF maintain cyclin B expression in the anterior part of the furrow, even though the surrounding cells arrested in G1 have no detectable Cyclin B. Maintenance of cyclin B expression is thought to indicate a failure of cell cycle progression, as Cyclin B levels decline in M phase. Mitotic figures are not observed in clones in the anterior half of the MF. The phenotype observed in the clones is similar to defects caused by mutations in division abnormally delayed (dally), which is required for G2-M progression ahead of the furrow. Mutations in dally and dpp display genetic interactions in development of the eye, antenna, and genitalia, which suggests that dally augments Dpp function. The behavior of Dpp-receptor mutant clones supports a role for Dpp in controlling progression through G2-M as a means of synchronizing the divisions that accompany differentiation of the eye disc. Cell fate, however, is unaffected by receptor mutation, as revealed by the expression of atonal, a proneural gene required for retinal precursor cell 8 (R8) determination. Because atonal expression is maintained in tkv clones, hh must not act through dpp to induce its expression, and thus dpp mediates a subset of hh functions in the MF (Penton, 1997).

Wingless is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. The protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. Dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes (Tsuda, 1999).

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. For pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters (Fujise, 2001).

dally enhancer trap expression in the wing disc was examined. This study revealed expression at the A/P and D/V boundaries in the wing pouch. At mid- to late-third larval instar, the expression of dally enhancer trap overlaps with that of ac-sc at the D/V boundary, indicating that dally is expressed at high levels in cells flanking the cut- and wg-expressing edge cells like Delta and Serrate. N signaling in cells of the D/V boundary results in the transcriptional activation of several genes, including vg, wg, ct, and members of the Enhancer of split complex, E(Spl). Like other genes affecting assembly of wing margin structures, dally expression of the D/V boundary is sensitive to N-receptor activation mediated by Dl and Ser. Blocking Wg signaling at the D/V border cells can repress dally expression, indicating that dally expression along the margin is positively regulated by Wg signaling, which is also required for the expression of proneural AS-C genes. Further studies are required, however, to determine whether N also has a direct function in the regulation of dally expression (Fujise, 2001).

dally cooperates with Wg to form the wing margin structures. Ac expression is severely decreased in dally mutants, supporting the idea that dally serves as a component of the Wg receptor complex to induce AS-C expression at the prospective wing margin. Taken together, these observations indicate that dally is a target gene of Wg signaling pathway, and at the same time, it mediates the same signaling, suggesting that dally is involved in a positive feedback loop of Wg signaling at the D/V boundary of the wing pouch. Frizzled-2, the Wg receptor, is down-regulated by Wg signaling at the D/V boundary. Dally, a putative Wg coreceptor, may also participate in the feedback circuits of Wg signaling, as has been suggested for Fz2 (Fujise, 2001).

dally is required for the development of sensory organs in the adult wing and notum. dally has been also identified as a gene that affects the development of sensory organs by a gain-of-function screen. dally mutants show reduced numbers of sensory bristles and campaniform sensilla at the wing margin. Specific macrochaetae on the notum, DC, pSA, and pPA, are affected in dally mutants. In all cases, the expression of genes specific for proneural cell differentiation is compromised in dally-sensitive bristles, indicating that dally affects sensory organ development at the step of prepatterning (Fujise, 2001).

Wg and Dpp have been shown to affect prepatterning of sensory organs by governing the expression of proneural genes, such as ac-sc. dally has been shown to affect the signaling levels of either Wg or Dpp. Therefore, an examination was made to determine whether dally affects sensory organ formation via either Wg or Dpp signaling pathways. Genetic experiments provided evidence that, in the prospective notum region of the wing disc, dally selectively influences Wg signaling to form the pPA bristle and Dpp signaling to form the pSA and DC bristles. It is particularly intriguing that, during development of DC macrochaetae, dally genetically interacts with only Dpp signaling, while the formation of these bristles requires both Wg and Dpp activities. It has been indicated that the A/P coordinates of the DC cluster are limited by Dpp signaling. In dally homozygous wing discs, the DC cluster is apparently shorter in the A/P coordinates compared with wild-type discs, suggesting that dally regulates Dpp signaling activity to limit the A/P length of the DC cluster. What are the mechanisms that can account for the selective interactions of dally and specific growth factor signaling? One obvious interpretation of genetic experiments on DC macrochaetae is that differences in dose effects between dpp and wg are responsible for the apparent specificity. It is also possible that the ligand-specificity of Dally is controlled at the cellular level through modification of heparan sulfate structures (Fujise, 2001).

Dally, Dpp, and IRO-C genetically interact with each other during the formation of the pSA macrochaete. Although interactions between Dpp signaling and IRO-C have been suggested, evidence is provided that Dpp signaling components interact with the genes of IRO-C. Ectopic Dpp signaling using a constitutively active type I receptor, tkv, leads to an ectopic induction of the pSA macrochaete, supporting the idea that Dpp signaling is required for prepatterning for this bristle. Significant reductions in the expression of iro enhancer trap is observed in dally mutant wing discs. Expression of the iro at the lateral notum region is critical for the proneural cluster formation and bristle development in this region. Taken together, these findings suggest that dally mediates Dpp signaling to control expression of the genes of IRO-C during the formation of the pSA bristle (Fujise, 2001).

Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process

The signalling molecule Hedgehog (Hh) functions as a morphogen to pattern a field of cells in animal development. Previous studies in Drosophila have demonstrated that Tout-velu (Ttv), a heparan sulphate polymerase, is required for Hh movement across receiving cells. However, the molecular mechanism of Ttv- mediated Hh movement is poorly defined. Dally and Dally-like (Dly), two Drosophila glypican members of the heparan sulphate proteoglycan (HSPG) family, are shown to be the substrates of Ttv and are essential for Hh movement. Embryos lacking dly activity exhibit defects in Hh distribution and its subsequent signalling. However, both Dally and Dly are involved and are functionally redundant in Hh movement during wing development. Hh movement in its receiving cells is regulated by a cell-to-cell mechanism that is independent of dynamin-mediated endocytosis. It is proposed that glypicans transfer Hh along the cell membrane to pattern a field of cells (Han, 2004).

To dissect the molecular mechanism(s) by which HSPG(s) regulates Hh signalling, attempts were made to identify specific proteoglycan(s) involved in Hh signalling during embryonic patterning. During embryogenesis, Hh and Wingless (Wg) are expressed in adjacent cells and are required for patterning of epidermis. In stage 10 embryos, Hh is expressed in two rows of cells in the posterior compartment of each parasegment, while Wg is expressed in one row of cells anterior to Hh expression cells. The expression of Hh is controlled by Engrailed (En) whose expression is maintained by Wg signalling through a paracrine regulatory loop. Hh signalling in turn is required for maintaining the expression of wg whose activity controls the production of the naked cuticles. Loss of either Hh or Wg signalling leads to a loss of naked cuticle, which is defined as segment polarity phenotype (Han, 2004 and references therein).

Disruption of dly in embryos by RNA interference (RNAi) leads to a strong segment polarity defect, suggesting that Dly is likely to be involved in Hh and/or Wingless (Wg) signalling in embryonic epidermis. To explore the potential role of Dly in Hh signalling, a number of dly mutant alleles were isolated using EMS mutagenesis. dlyA187 is a null allele and is used for further analyses. Animals zygotically mutant for dly appears to have normal cuticle patterning and survive until third instar larvae. However, homozygous mutant embryos derived from females lacking maternal dly activity (referred to as dly embryos hereafter) die with a strong segment-polarity phenotype resembling those of mutants of the segment polarity genes hh and wg. In dly embryos, both En expression and wg transcription fade by stage 10, suggesting further that dly is involved in the Hh and/or Wg pathways (Han, 2004).

To further determine whether Dly activity is required for Hh signalling in embryogenesis, Hh signalling activity was examined in dly embryos during mesoderm development. Hh and Wg signalling have distinct roles in patterning embryonic mesoderm. Hh signalling activates the expression of a mesodermal specific gene bagpipe (bap) in the anterior region of each parasegment, whereas Wg signalling inhibits bap expression in the posterior region. bap expression is diminished in the hh mutant, but is expanded to the posterior parasegment in the wg mutant. Consistent with a role of Dly in Hh signalling, it was found that bap expression was strikingly reduced in dly embryos. Together with the segment polarity phenotype, these results strongly argue that Dly is required for Hh signalling during embryogenesis (Han, 2004).

The role of Dly in Hh signalling was further examined during wing development in which Hh and Wg signalling function independently of each other. In the wing disc, Hh signalling induces the expression of its target genes in a narrow stripe of tissue in the A compartment abutting the AP boundary. Hh signalling patterns the central domain of wing blade and controls the positioning of longitudinal veins L3 and L4. The roles of Dly in Hh signalling were examined by analyzing adult wing defects using 'directed mosaic' technique. Surprisingly, no detectable phenotypes were observed in adult wings bearing dly mutant clones. It was reasoned that Hh signalling may be mediated by other HSPGs in the wing. One candidate is the glypican dally that has been shown to be involved in Wg and Dpp signalling. Because available dally alleles used previously were hypomorphic, several dally null alleles were generated by P-element mediated mutagenesis. dally80 is a null allele and was used for analysis. However, similar to other dally alleles, homozygous dally80animals are viable. The wing bearing dally80 clones exhibits a partial loss of the L5 vein with a high penetrance, but no detectable defects in the central domain of wing blade. To determine whether dally and dly have overlapping roles in Hh signalling in wing development, clones mutant for both dally80 and dlyA187 (referred as dally-dly hereafter) were generated. Interestingly, the adult wings bearing clones mutant for dally-dly show L3-L4 fusion. This phenotype is typical of loss of Hh function, suggesting that Dally and Dly play redundant roles in Hh signalling in wing development (Han, 2004).

This study demonstrates that Dly is the main HSPG involved in Hh signalling during embryogenesis, at least in epidermis and mesoderm, the two tissues that were carefully examined. Three lines of evidence strongly support this conclusion. (1) Embryos lacking both maternal and zygotic dly activities develop a strong segment polarity defect and exhibit diminished expression of En and Wg. (2) Hh can be detected as punctate particles at least one cell diameter from its producing cells and these punctate particles are absent in dly-null embryos. (3) A reduced expression of bap was observed in dly mutant embryos, a phenotype specifically attributed to the Hh signalling rather than Wg signalling defect. Previously, it was shown that the punctate particles of Hh staining are absent in ttv null embryos. The formation of such Hh staining particles, referred to as large punctate structures (LPS), requires cholesterol modification, and movement of these large punctate structures across cells is dependent on Ttv activity. The current results are consistent with these observations and suggest that Dly is the main HSPG involved in the movement of these LPS across cells. It is conceivable that the punctate particles of Hh staining that were observed may represent Hh-Dly complexes. In this regard, Dly may either prevent secreted Hh from being degraded and/or facilitate Hh movement from its expression cells to adjacent receiving cells. These two mechanisms are not mutually exclusive. In the absence of Dly function, secreted Hh is either degraded or fails to move to the adjacent cells (Han, 2004).

In addition to dly, three other HSPGs, including Dally, Dsyndecan and Trol, are also expressed in various tissues during embryogenesis. In particular, dally is expressed in epidermis and has been shown to be involved in Wg signalling. Removal of Dally activity in embryos either by dally hypomorphic mutants or by RNA interference (RNAi) generates denticle fusions. Further studies demonstrate that the cuticle defect associated with dally embryos by RNAi is weaker than that of dly. The results in this study suggest that Dly plays more profound roles in embryonic patterning than Dally. It remains to be determined whether Dally and other two Drosophila HSPGs are involved in Hh signalling in other developmental processes during embryogenesis (Han, 2004).

Dally and Dly are involved and are redundant in Hh signalling in the wing disc. Consistent with this, the GAG chains of Dally and Dly are shown to be altered in the absence of Ttv activity, suggesting that both Dally and Dly are indeed the substrates for Ttv. Redundant roles of cell membrane proteins have been demonstrated in many other signalling systems. For example, both Frizzled (Fz) and Drosophila Frizzled 2 (Fz2) are redundant receptors for Wg, although Fz2 has relative high affinity in binding to Wg protein. Dly protein is distributed throughout the entire wing disc. Previous studies have demonstrated that dally is highly expressed at the AP border. Interestingly, Dally expression at the AP border is overlapped with the ptc expression domain and is under the control of Hh signalling. It is likely that both Dally and Dly are capable of binding to Hh and facilitating the movement of the Hh protein. In the absence of one of them, another member is probably sufficient to facilitate Hh movement (Han, 2004).

dally-dly double mutant clones have relatively weaker defects in Hh signalling in the wing disc than those of the ttv and sfl mutants. One possible explanation is the perdurance of Dally and Dly proteins. Alternatively, two other HSPGs, Dsyndecan and Trol, may also participate in Hh signalling in the absence of Dally and Dly in the wing disc. These issues remain to be examined using both dsyndecan and trol null mutants (Han, 2004).

Do HSPGs act as co-receptors in Hh signal transduction? Hh is a heparin-binding protein and is likely to interact with HSPGs through their HS GAG chains. In support of this, Dly was shown to colocalize with Hh punctate particles. It is conceivable that Dally and Dly could either transfer Hh to its receptor Ptc or form a Hh-Dally/Dly-Ptc ternary complex in which Dally and Dly may function to facilitate Hh-Ptc interaction or stabilize a Hh-Ptc complex. In this regard, Dally and Dly may function both in transporting Hh protein and acting as co-receptors in Hh signalling. Consistent with this view, a recent report using RNAi in tissue culture based assays identified Dly as a new component of the Hh pathway (Lum, 2003). It was shown that Dly plays a cell-autonomous role upstream or at the level of Ptc in activating the expression of Hh responsive-reporter, suggesting a role of Dly in the delivery of Hh to Ptc (Han, 2004).

It is important to note that some of results obtained from tissue culture based assays (Lum, 2003) are not consistent with in vivo results reported in this study as well as previous studies on Ttv. Cl-8 cells were originally derived from the wing disc. However, it was found that removal of dly activity alone has no detectable effect on Hh signalling in the wing disc. This apparent discrepancy may due to several factors: (1) Hh-N, instead of Hh-Np was used as a source for Hh in their work; (2) Cl-8 cell may have altered the proteoglycan expression pattern, which can be significantly different from Hh-responding wing cells in which Dally expression is upregulated by Hh signalling; (3) it is possible that Dly may have a higher capacity than Dally to bind Hh, as in the case for Wg. In this regard, removal of Dly will probably lead to more profound effects than removal of other HSPGs on binding of Hh-N to the cell surface, perhaps in the delivery of Hh-N to Ptc (Han, 2004).

Within sfl, or ttv or dally-dly mutant clones, the posterior-most cells adjacent to wild-type cells are still capable of transducing Hh signalling. It is most likely that Hh proteins bound by Dally and Dly in wild-type cells can directly interact with Ptc located on the cell surface of the adjacent mutant cells to transduce its signalling. In support of this view, a Hh-CD2 membrane fusion protein has the ability to activate Hh signalling in its adjacent cells. Furthermore, studies on Dispatched (Disp), a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells, have shown that the first row of anterior cells adjacent to posterior Hh-producing cells have significant Hh signalling activity in disp mutant wing discx, in which Hh is retained on the cell surface of Hh producing cells. Interestingly, Hh punctate particles were observed in the posterior-most HSPG mutant cells adjacent to wild-type cells. These Hh punctate particles are most likely intracellular Hh proteins internalized through Ptc mediated endocytosis process. In this regard, HSPGs may not be required for Ptc-mediated Hh internalization (Han, 2004).

Recent biochemical studies from vertebrate cells have shown that Shh-Np is secreted from cells and can be readily detected in conditioned culture medium. It was also shown that overexpression of Disp can increase the yield of Hh protein in the culture medium. These experiments suggest that Hh can be directly secreted from Hh expressing cells. Can secreted Hh proteins freely diffuse to Hh receiving cells through extracellular spaces? To address this issue, detailed analyses for Hh signalling have been carried out in the complete absence of HS GAG using sfl and ttv or absence of glypicans using dally-dly. A narrow strip (one cell diameter in width) of sulfateless (sfl) or ttv, or dally-dly mutant cells prevents the transpassing of the Hh signal. Hh staining disappears in sfl mutant clones, except at a residual level in the posterior-most row of cells. Based on these observations, a model is favored in which Hh movement is regulated by a cell-to-cell mechanism rather than by free diffusion (Han, 2004).

The results of this study further suggest that Hh movement is independent of dynamin-mediated endocytosis, which has been shown to be involved in the transportation of morphogen molecules such as Dpp and Wg. A blockage of dynamin function does not eliminate Hh movement and its subsequent signalling; instead, it leads to a striking reduction of punctate particles of Hh staining and an accumulation of cell-surface Hh protein. Expanded Ptc expression domain is observed when dynamin-mediated endocytosis is blocked. These new findings provide compelling evidence that dynamin-mediated endocytosis is not required for Hh movement and its subsequent signalling, but is involved in Ptc-mediated internalization of the Hh protein (Han, 2004).

Several mechanisms have been proposed to explain morphogen transport across a field of cells. These mechanisms include (1) free diffusion, (2) active transport by planar transcytosis, (3) cytonemes, (4) argosomes. The results of this study suggest that Hh moves through a cell-to-cell mechanism rather than free diffusion. Furthermore, dynamin-mediated endocytosis is unlikely to be involved in Hh movement. On the basis of these findings, the following model is proposed by which the HSPGs Dally and Dly may regulate the cell-to-cell movement of the Hh protein across a field of cells. In this model, Hh is released by Disp from its producing cells and is immediately captured by the GAG chains of glypicans on the cell surface. The differential concentration of Hh proteins on the surface of producing cells and receiving cells drives the unidirectional displacement of Hh from one GAG chain to another towards more distant receiving cells. Within the same cell, the transport of Hh may be facilitated by the lateral movement of glypicans on the cell membrane. On the receiving cells, glypicans may present Hh to Ptc, which then mediates the internalization of Hh. Glypican mutant cells can not relay Hh proteins further because they lack HS GAG on the surface. However, they are able to respond to the Hh signal because Ptc may contact the Hh on the membrane of the adjacent wild-type cells. Further studies are needed to determine whether other mechanism(s) including cytonemes and argosomes are also involved in Hh movement (Han, 2004).

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. This study demonstrates that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies reveal that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also show axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes are able to rescue some sdc visual-system phenotypes, but sdc+ transgenes are ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential (Rawson, 2005).

The distribution of Dlp was examined in the developing visual system by using a monoclonal antibody that specifically recognizes Dlp in tissues. In Drosophila, the adult eye is comprised of approximately 800 sensory units, or ommatidia, each with eight distinct photoreceptors, R1-R8. In the eye imaginal disc, Dlp was found on photoreceptor cell bodies and on cells within the morphogenetic furrow. Axons from R1-R6 terminate in the lamina, the first optic ganglion, whereas those from R7 and R8 project to the medulla. In the optic lobe, Dlp is present on photoreceptor axons at the boundary between the lamina and adjacent tissues and along the lamina plexus. Dlp is also observed in the medulla neuropil, medulla glia, medulla neuropil glia, neuroblasts of the proliferative centers, and in the mushroom body neuropil (Rawson, 2005).

To evaluate the function of Dlp in axon guidance, a photoreceptor-specific monoclonal antibody (24B10) was used to visualize photoreceptor projections in dlp mutants. In 50% of dlp mutant hemispheres, the lamina plexus was irregular and thickened. Additionally, 80% of dlp mutant larvae had fibers that aberrantly crossed between ommatidial bundles and/or photoreceptor process expansions outside the normal termination zone of the lamina plexus. Examination of dlp mutant pupae revealed that 80% of optic lobes contain irregularities in the R7 and R8 medulla termini. Crossover of R7 axons to neighboring medulla cartridges was observed (~50%) and misrouting of R7/R8 axons (~20%) (Rawson, 2005).

Visual-system function was assessed in dlp mutants by recording electroretinogram (ERG) profiles in adult flies. A wild-type ERG is composed of the photoreceptor response generated by a light-induced depolarization of the photoreceptor neurons and of the two transient voltage changes, the 'on-transient' and the 'off-transient', resulting from currents related to synaptic transmission during the initiation and termination of the light stimulus. dlp mutants show statistically significant defects in the photoreceptor response and in both on- and off-transients, suggesting that Dlp is required for proper photoreceptor currents and synaptic transmission (Rawson, 2005).

Axon guidance in the visual system depends on photoreceptor specification, as well as on glial-cell migration and lamina-neuron differentiation. Although dlp mutants have reduced and roughened eyes, thin sections of dlp eyes demonstrate that all photoreceptors are present and retain proper polarity in each ommatidium. Staining with several photoreceptor-specific markers confirm that dlp mutant photoreceptors differentiate properly. Likewise, glial cells and lamina neurons were found in the correct number and location in dlp mutants, indicating that patterning defects of these critical cells cannot account for the observed axon-guidance defects (Rawson, 2005).

Because Dlp is expressed in several visual-system elements and cell types, somatic mosaic studies were conducted to determine which cells require dlp for visual-system assembly. Using a method that generates clones encompassing a majority of cells in the retina, crossover of axons between ommatidial bundles and photoreceptor process expansions outside the lamina plexus was observed in 67% of animals with dlp mutant photoreceptors projecting to a heterozygous brain. Conversely, R7/R8 termination defects were absent from the medulla of 40 hr pupae with dlp mutant retinas and dlp/+ optic lobes. These results indicate that Dlp is required in the eye to specify proper axon guidance to the lamina, but not to the medulla (Rawson, 2005).

The photoreceptor-specific requirement for Dlp was further evaluated via the mosaic analysis with a repressible cell marker (MARCM) technique to visualize axons from small dlp mutant clones. dlp mutant photoreceptors display ectopic axon outgrowths directed away from their proper targets in the lamina (37% of dlp/dlp axons). Ectopic axon processes were four times more prevalent on axon bundles near the boundaries of the lamina than on those in more-central regions. This suggests that expression of Dlp on photoreceptors may be important for the detection of repellant cues that prevent aberrant axon outgrowth (Rawson, 2005).

Finally, the tissue-specific requirement was evaluated for Dlp in physiological function of the visual system. Using a mosaic strategy that generated eyes composed solely of dlp mutant photoreceptors, no see statistically significant defects were seen in ERG recordings compared to controls. These results demonstrate that the ERG defects of dlp mutants are produced by loss-of-function in the optic lobe, not in the eye (Rawson, 2005).

Drosophila Syndecan (Sdc) represents another class of HSPG, and it is required for normal axon guidance in the embryo. Antibody specific for Sdc revealed that this proteoglycan is present on photoreceptors in the retina and on photoreceptor projections to the optic lobe. Like Dlp, Sdc is enriched throughout the lamina plexus and at the boundary between the lamina and adjacent tissues. Sdc immunoreactivity was also detectable at a low level on cells just medial to the lamina plexus, the medulla glia, but was absent from some Dlp-expressing cells such as the mushroom body and the medulla neuropil glia (Rawson, 2005).

Analysis of sdc null mutant larvae revealed that 50% of optic lobes had photoreceptor-projection abnormalities and/or lamina-plexus defects including gaps (29%) and gross disorganization (21%, n = 28). Compared to dlp mutants, sdc mutants showed a low penetrance of the lamina-thickening (4%) and lamina-axon-crossover phenotypes. Additionally, some sdc mutants had R7/R8-axon misrouting in the larval stage. sdc mutant pupae showed crossover of R7 axons between medullary cartridges (100%) and defective axon pathfinding to the medulla (86%) but a low penetrance of R7/R8-termini disruption (~10%), a phenotype common in dlp mutants (80%-100%). As is the case for dlp, sdc mutants do not show defects in the specification of photoreceptors, glia, or lamina neurons, confirming that the above phenotypes are not secondary to other overt patterning deficiencies. Overall, dlp and sdc mutants share similar axon-guidance phenotypes, but each has a largely distinct level of penetrance for a given defect (Rawson, 2005).

Electrophysiological analysis revealed that sdc mutants have grossly abnormal ERGs, with defective photoreceptor depolarization and complete absence of on- and off-transients. These ERG abnormalities -- particularly the virtual loss of photoreceptor depolarization -- are distinct from those found in dlp animals. In addition, whereas dlp null mutants have a reduced and roughened eye, sdc mutants do not. These findings are consistent with distinct molecular functions for Dlp and Sdc during visual-system assembly (Rawson, 2005).

Sdc and Dlp are both heparan sulfate-modified proteoglycans, and Dlp expression is capable of limited rescue of sdc axon-guidance abnormalities in the embryo. To determine whether these two cell-surface molecules have overlapping functions in the visual system, a series of rescue experiments was performed. With or without a GAL4 driver, dlp mutant animals bearing a UAS-dlp+ construct show complete rescue of pupal axon-pathfinding defects and ERG abnormalities, suggesting that low levels of Dlp expression are sufficient to provide the function necessary for normal axon guidance and visual-system assembly. Despite this, ubiquitous expression of Sdc was unable to rescue the axon-guidance abnormalities of dlp mutants, demonstrating that Dlp and Sdc are not functionally interchangeable during photoreceptor axon guidance (Rawson, 2005).

The ability of UAS-dlp+ transgenes to rescue axon-guidance phenotypes of sdc mutant larvae and pupae was tested. Pupal sdc mutant phenotypes, including misrouting of R7/R8 photoreceptors to the medulla and crossover of R7 to neighboring medulla cartridges, are largely rescued with neuronal-directed expression of Sdc. Conversely, neuron-specific expression of Sdc in sdc mutants is not sufficient to rescue photoreceptor projection defects to the larval lamina or the ERG abnormalities in adults, suggesting that expression of Sdc in additional cell types is critical for complete restoration of axonal patterning and visual-system function. In contrast to the inability of Sdc expression to rescue dlp mutant phenotypes, neuron-specific expression of Dlp restores proper R7/R8 photoreceptor projection to the medulla of sdc mutant pupae. This rescue demonstrates that a structurally distinct HSPG can provide some of the functions normally served by Sdc (Rawson, 2005).

In vertebrates, glypicans are expressed on axons and growth cones in the developing nervous system. Whereas previous findings have established a function for glypicans in growth-factor signaling, their role in axon guidance has not been reported. Consistent with the expression pattern of Dlp on axons and glial cells of the developing visual system, dlp mutants show defects in photoreceptor projections to the lamina and medulla. Mosaic analysis demonstrates that Dlp is required on photoreceptors for some, but not all, aspects of axon guidance, serving functions in both peripheral and central components of the visual system. dlp is not required in the retina for normal electrophysiological responses to light, indicating that the synaptic-transmission defects in dlp mutant adults are due to loss of Dlp activity in the optic lobes (Rawson, 2005).

Comparison of sdc and dlp mutants as well as transgene rescue experiments demonstrates that Sdc and Dlp have some overlapping functions in visual-system assembly but others that are unique. For example, photoreceptor misrouting to the medulla in sdc mutants can be rescued by neuron-specific expression of dlp+, consistent with the capacity of dlp+ to rescue midline-crossover defects in sdc mutant embryos. In contrast to the ability of dlp+ transgenes to rescue sdc mutants, UAS-sdc+ constructs are unable to rescue any of the dlp mutant phenotypes, even under conditions where very modest levels of Dlp rescues completely. These findings show that Dlp functions cannot be readily provided by another unrelated HSPG, and they argue that the conserved sequence elements of the Dlp core protein are critical for these unique functions (Rawson, 2005).

Effects of HSPGs on axon pathfinding have been considered principally in the context of classical axon-guidance pathways, Slit-Robo signaling in particular. Although there is genetic evidence from mouse, Drosophila, and C. elegans that HSPGs affect Slit-Robo signaling, there are many other possibilities. HSPGs have been extensively characterized as molecules that affect both morphogen signaling and distributions. Recent studies demonstrating that the classical morphogens Wnt, Hh, and BMP also play a bona fide role in axon guidance suggest the possibility that HSPGs govern axon guidance by affecting morphogen function or distributions during this process (Rawson, 2005).

Glypicans shunt the Wingless signal between local signalling and further transport

The two glypicans Dally and Dally-like have been implicated in modulating the activity of Wingless, a member of the Wnt family of secreted glycoprotein. So far, the lack of null mutants has prevented a rigorous assessment of their roles. A small deletion was created in the two loci. Analysis of single and double mutant embryos suggests that both glypicans participate in normal Wingless function, although embryos lacking maternal and zygotic activity of both genes are still capable of transducing the signal from overexpressed Wingless. Genetic analysis of dally-like in wing imaginal discs leads to a model whereby, at the surface of any given cell of the epithelium, Dally-like captures Wingless but instead of presenting it to signalling receptors expressed in this cell, it passes it on to neighbouring cells, either for paracrine signalling or for further transport. In the absence of dally-like, short-range signalling is increased at the expense of long-range signalling (reported by the expression of the target gene distalless) while the reverse is caused by Dally-like overexpression. Thus, Dally-like acts as a gatekeeper, ensuring the sharing of Wingless among cells along the dorsoventral axis. This analysis suggests that the other glypican, Dally, could act as a classical co-receptor (Franch-Marro, 2005).

The fact that mutations in dally and dlp cause different phenotypes suggests that, although they both underpin Wingless function, these two glypicans could perform distinct activities. It is likely that both Dally and Dlp are able to capture Wingless at the surface of imaginal disc cells. From the point of view of a given cell in vivo, Wingless captured by Dally would be mostly destined for 'internal consumption', while Dlp-bound Wingless would be for export only. Subsequent long-range transport would occur by hopping from Dlp on one cell to Dlp on the next. Both glypicans would contribute to increasing the concentration of Wingless at the cell surface (Dally in cis and Dlp in trans). It is suggested that in the embryo too, Dlp and Dally help in the presentation and reception of Wingless, respectively. However, in this system, little Wingless transport takes place, maybe because release of Wingless from Dlp is not allowed. It is interesting that, in embryos, dlp is highly expressed in cells that secrete Wingless. Therefore, the role of Dlp would mainly be to ensure that plenty of Wingless is retained at the surface of Wingless-expressing cells thus allowing sustained short-range signalling. In both the embryonic and disc systems, the genetic redundancy between dally and dlp could be viewed as follows: reduction of capturing activity in dally mutants would be compensated by the 'presentation activity' of Dlp and vice versa. Further cell biological work will be needed to fully explore the specific activities of Dally and Dlp and also to discover how Wingless is transferred from one cell to another during transport, perhaps with the help of enzymes such as Notum/Wingful (Franch-Marro, 2005).

The glypican Dally is required in the niche for the maintenance of germline stem cells and short-range BMP signaling in the Drosophila ovary

The Drosophila ovary is an excellent system with which to study germline stem cell (GSC) biology. Two or three female GSCs are maintained in a structure called a niche at the anterior tip of the ovary. The somatic niche cells surrounding the GSCs include terminal filament cells, cap cells and escort stem cells. Mounting evidence has demonstrated that BMP-like morphogens are the immediate upstream signals to promote GSC fate by preventing the expression of Bam, a key differentiation factor. In contrast to their morphogenic long-range action in imaginal epithelia, BMP molecules in the ovarian niche specify GSC fate at single-cell resolution. How this steep gradient of BMP response is achieved remains elusive. In this study, it was found that the glypican Dally is essential for maintaining GSC identity. Dally is highly expressed in cap cells. Cell-specific Dally-RNAi, mutant clonal analysis and cell-specific rescue of the GSC-loss phenotype suggest that Dally acts in the cap cells adjacent to the GSCs. It was confirmed that Dally facilitated BMP signaling in GSCs by examining its downstream targets in various dally mutants. Conversely, when Dally was overexpressed in somatic cells outside the niche, the number of GSC-like cells was increased apparently by expanding the pro-GSC microenvironment. Furthermore, in a genetic setting a BMP-sensitivity distinction was revealed between germline and somatic cells, namely that Dally is required for short-range BMP signaling in germline but not in somatic cells. It is proposed that Dally ensures high-level BMP signaling in the ovarian niche and thus female GSC determination (Guo, 2009).

To understand how a steep gradient of BMP response is established and thus determines cell fate at single-cell resolution in the GSC niche of Drosophila ovary, genetic approaches were taken to examine the role of the glypican Dally in the process. Based on the current data, a model is proposed of how the expression pattern of Dally shapes the BMP-signaling range and consequently determines distinct cell fates in the ovarian niche (Guo, 2009).

Female GSC fate requires high BMP signaling, which is provided in the ovarian niche. Dally is highly expressed in the cap cells that contact the GSCs. Cap-cell-localized and membrane-bound Dally either stabilizes/concentrates BMP molecules, or enhances BMP sensitivity to ensure that only the germ cells in contact with cap cells become GSC. Upon removal of Dally, BMP concentration at the niche or BMP sensitivity in the germ cells adjacent to cap cells dissipates, and GSCs cannot be maintained and subsequently differentiate. Conversely, when Dally is ectopically overexpressed in the escort cells posterior to the niche, BMP signaling or sensitivity increases in all germ cells encapsulated by these escort cells, and GSC-like cells accumulate in the germarium (Guo, 2009).

Consistent with this model, the secreted form of Dally expressed from cap cells caused GSC loss, possibly by competing with endogenous membrane-anchored Dally for binding with BMP molecules or by interfering with BMP signaling. Because secreted Dally expressed from somatic cells in addition to cap cells did not cause GSC expansion as the membrane-anchored Dally did, the evidence further supports the idea that Dally's function in the GSC niche depends on the cap-cell-specific expression and membrane anchoring (Guo, 2009).

When somatic cells displace the differentiating germ cells in the niche and become close to or in contact with cap cells where the BMP morphogen is localized, these somatic cells are able to respond to BMP when Dally is lacking. Whether a cellular BMP response is Dally dependent or not distinguishes the germline and somatic cells (Guo, 2009).

It was noticed that it took 15 days for Dally overexpression in somatic cells to make all germ cells GSC-like in the germarium, although C587Gal4 was active since stage larval 3 at the latest. One possible explanation is that Dpp is limited and Dally stabilizes Dpp. This possibility is supported by a recent report demonstrating that Dally and Dpp physically interact with each other in the cultured S2 cells and that Dally stabilizes Dpp on the cell surface in the wing imaginal epithelia. Consistent with their theory of cell-surface-associated stabilization, the secreted Dally, although retaining the ability to bind Dpp, did not have the activity that the full-length Dally possesses in terms of enhancing GSC proliferation. It suggests that Dally can only stabilize Dpp at the cell surface. Additionally, secreted Dally expressed in the same cells in which the endogenous Dally is produced had a weak dominant-negative effect. By contrast, the secreted Dally expressed elsewhere did not have any detectable effect on GSC, suggesting that it did not compete with the endogenous Dally expressed from the cap cells. These results imply that the anterior tip of the germarium contains the main source of BMP molecules, which the secreted Dally from cap cells has a better chance to catch than that from elsewhere (Guo, 2009).

In the imaginal epithelia, glypican Dally and Dlp are essential for Dpp gradient formation but not for short-range Dpp signaling because one to two rows of cells in the glypican double mutant clone are able to respond to the nearby Dpp signals. Similarly, in dally mutant ovary, in which the germarium was emptied due to GSC loss, BMP response was observed in the escort cells getting close to the cap cells, where the BMP source is supposed to be. However, the germ cell surrounded by the BMP-responsive somatic cells was refractory to BMP morphogen in exactly the same circumstances. It appears that when Dally is compromised, the germline is less sensitive to BMP signaling and Dally either recruits more ligands to the adjacent germ cell or somehow enhances its response to BMP. Contrarily, the somatic cells do not require Dally to sense and respond to BMP morphogen in short range. Whether Dlp is essential for germarial somatic cells in BMP response is unclear. What accounts for the distinction in BMP sensitivity between germline and somatic cells remains to be investigated (Guo, 2009).


EVOLUTIONARY HOMOLOGS

Glypicans in C. elegans

Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. This study shown with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, axons can be cell specifically rerouted, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. These experiments provide genetic in vivo evidence for the 'HS code' hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions (Bülow, 2008).

Expression of heparan sulphate proteoglycans

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly as covalent modifications of proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family. Heparan sulfate has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. An mRNA has been identified and characterized that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression (Saunders, 1997).

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. The pattern of glypican-1 mRNA and protein expression was examined in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression are found throughout the brain and skeletal system. In the brain, glypican-1 mRNA is widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localizes strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 is found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 is also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression is observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development (Litwack, 1998).

A study was carried out of the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, it was found that a significant portion of the glypican and biglycan immunoreactivity colocalizes with nuclear staining by propidium iodide and is also seen in isolated nuclei. In certain regions, staining is selective, insofar as glypican and biglycan immunoreactivity in the nucleus is seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals; these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells is greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining is seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrates transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Therefore these data suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes (Liang, 1997).

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is an approximately 65 kDa glycosylphosphatidylinositol-linked protein that bears heparan sulfate chains. To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with the protein sequence predicted by a cDNA cloned from PC12 cells indicates that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detect the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detects glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes shows that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibit little or no hybridization over background. In most cases where glypican hybridization is observed, the signal can be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons and hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization is found in layers 2/3, 5, and 6, but is missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system (Litwack, 1994).

Heparan sulfate proteoglycans (HSPGs) are found on the surface of all adherent cells and participate in the binding of growth factors, extracellular matrix glycoproteins, cell adhesion molecules, and proteases and antiproteases. Reported here are the cloning and pattern of expression of cerebroglycan, a glycosylphosphatidylinositol (GPI)-anchored HSPG that is found in the developing rat brain. The cerebroglycan core protein has a predicted molecular mass of 58.6 kD and five potential heparan sulfate attachment sites. Together with glypican, it defines a family of integral membrane HSPGs characterized by GPI linkage and conserved structural motifs, including a pattern of 14 cysteine residues that is absolutely conserved. Unlike other known integral membrane HSPGs, including glypican and members of the syndecan family of transmembrane proteoglycans, cerebroglycan is expressed in only one tissue: the nervous system. In situ hybridization experiments at several developmental stages strongly suggest that cerebroglycan message is widely and transiently expressed by immature neurons, appearing around the time of final mitosis and disappearing after cell migration and axon outgrowth have been completed. These results suggest that cerebroglycan may fulfill a function related to the motile behaviors of developing neurons (Stipp, 1994).

The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart mRNAs for both syndecan-3 and glypican are abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA are also detected. Analysis of RNA isolated from hearts of rats of various ages reveals that the levels of both syndecan-3 and glypican mRNA increase dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans is expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA is localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA is not detected in myocytes, but is detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican reveals that it is a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies are consistent with the Northern blot data, and localize glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalizes with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also reveals colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane (Asundi, 1997).

An avian cDNA homolog of human and rat glypicans has been cloned from a stage 17 chicken heart cDNA library and used to analyze the distribution of this proteoglycan during development by Northern analysis and whole mount in situ hybridization. At stages 7-12, strong signals are detected in the cephalic region of the neural folds, rostral portion of paraxial mesoderm, and newly formed epithelial somites. At stages 20-25, strong expression is observed in the mantle zone of the telencephalon, the apical epidermal ridge and proximal region of developing limb. Transcripts also are found in the truncus arteriosus and arteriovenous-canal region of the heart, but not in the myocardium. This distribution pattern suggests that the avian glypican may be involved in the morphogenesis of limb, somite, heart, and brain. The expression of glypican also overlaps FGFs in limb bud, FGF receptors in heart and somite, and NGF receptors in forebrain. The affinity of heparan sulfate proteoglycans for growth factors and the distribution of the avian glypican are consistent with a role for this molecule in growth factor-mediated signals (Niu, 1996).

Transcripts for avian glypican are found in endocardial cushions, limb buds, somites and forebrain of early chick embryos. Since avian glypican is not well characterized, the cellular localization, regulation of expression, and possible function during cardiac development have been studied. A polyclonal antibody was raised against a 20-amino acid peptide corresponding to an antigenic sequence within avian glypican core protein. The antibody recognizes the expressed core protein in bacterial lysates and the endogenous HSPG in the proteoglycan fraction from chick forebrain. Immunolocalization studies indicate that the core protein is associated with cell membranes. The level of mRNA for avian glypican in MEQC (myc embryonic quail cardiomyocytes) grown in medium containing 10% fetal calf serum was compared to the message levels in cells grown without serum for 3 days. By Northern analysis, glypican transcripts increase markedly after serum starvation. Up-regulation of glypican transcripts by serum withdrawal is partially prevented by addition of TGFbeta-1 and bFGF, suggesting that these growth factors may regulate its expression. MEQC cells deprived of serum migrate into clumps that can be blocked by an antisense OND (oligodeoxynucleotide) to the mRNA encoding the avian glypican. The same antisense OND inhibits the migration of endothelial cells from chick tubular heart explants over the surface of collagen gels. These results indicate that avian glypican may play a role in cell migration during development of endocardial cushions (Niu, 1998).

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, the polarity of the expression of the cell surface heparan sulfate proteoglycans was examined in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identifies glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this is expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicate that sorting mechanisms, rather than differential turnover, account for this bipolar expression of glypican. Chlorate treatment does not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells express wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein result in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms have intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Inverse glycanation-apical sorting relationships in glypican may ensure near constant deliveries of HS to the apical compartment, or, alternatively, "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains (Mertens, 1996).

OCI-5, the rat homolog of human glypican 3 (GPC3), is believed to be involved in morphogenesis and growth control during development. The finding that GPC3 is mutated in patients with the Simpson-Golabi-Behmel overgrowth syndrome is consistent with this idea. In this report, using RNA in situ hybridization, expression of OCI-5 in the developing intestine is shown to be present in both endoderm- and mesenchyme-derived cells in a phased manner related to age and proximal/distal position. To investigate the mechanism of its regulation during intestinal development, OCI-5 expression was studied in the primitive rat intestinal epithelial cell line IEC-18. The expression of the OCI-5 transcript is increased in IEC-18 cells at confluence, in low calcium media, and during spheroid culture, all conditions which result in the cells acquiring a more rounded cell shape. In contrast, cytoskeletal disruption with colchicine causes cells to flatten and spread and abolishes both the confluence- and the low calcium-dependent induction of OCI-5. Treatment with vanadate, a phosphatase inhibitor, causes cells to acquire a spindle-shaped morphology and prevents OCI-5 induction in all situations. Nuclear run-on analysis demonstrates that the rate of OCI-5 transcription is increased at confluence, in low calcium media, and during spheroid culture of IEC-18, and decreased by treatment of cells with colchicine. Together, these data suggest that OCI-5 expression is regulated in IEC-18 by cell shape. The pattern of expression of OCI-5 in the developing intestine is consistent with it playing a role in epithelial-mesenchymal interactions during intestinal morphogenesis, when cell shape changes are likely to occur (Li, 1997).

Glypican is a glycosylphosphatidylinositol (GPI)-anchored heparan sulfate proteoglycan (HSPG) releasable by phosphatidylinositol-specific phospholipase C (PI-PLC) from the surface of differentiated skeletal muscle cells. The processing, location and amount of this HSPG in skeletal muscle cells has been evaluated during differentiation. Immunoprecipitation of incubation medium obtained from differentiated cells incubated with [35S]sulfate by specific antibodies against glypican isolated from Schwann cells demonstrates that the antisera precipitates an intact HSPG. Immunoblot analysis of the proteins released by PI-PLC after heparitinase treatment reveals the presence of a main band of 64 and a faint band of 62 kDa, whereas the sizes of the core proteins for glypican present in the incubation media are 62 and 59 kDa. Pulse-chase experiments indicates that glypican present in the membrane is spontaneously released into the culture medium with a t1/2 of 12 h. The level of expression of glypican was analyzed during in vitro differentiation. The specific amount of the PI-PLC releasable HSPG increases about fourfold during cell differentiation. No changes are detected in the level of the mRNA for glypican. Indirect analysis reveals that in myotubes glypican is present on the cell surface as well as associated with the extracellular matrix (ECM). These results indicate that glypican is present, at least, in two different compartments on the surface of skeletal muscle cells (Brandan, 1996).

Skeletal muscle has the remarkable capacity to regenerate new muscle fibers in the event of injury or disease. This capacity lies in the satellite cells, which are myogenic stem cells residing in adult muscle. While the signals that activate satellite cells to divide in vivo are not fully understood, satellite cells grown in culture respond to the mitogenic action of fibroblast growth factor (FGF). Satellite cells from the dystrophic mdx mouse are more sensitive to FGF in culture than satellite cells from normal mice. The basis for this heightened sensitivity of mdx satellite cells to FGF was investigated by measuring the number and affinity of protein and heparan sulphate proteoglycan (HSPG) receptors for FGF. HSPG receptors are elevated over four-fold in the mdx cells, as compared with cells from normal animals. This observation was supported by measuring the synthesis of heparan sulphate (HS) and chondroitin sulphate (CS) by satellite cells in culture. Mdx satellite cells synthesized approximately ten times more of these sulphated glycosaminoglycans (GAGs) than did normal cells. For muscle fibroblasts, however, no significant differences were found between normal and mdx cells in the number or affinity of protein or HSPG receptors, or in the amount of sulphated GAGs synthesized. It is proposed that the increase in FGF HSPG receptors is the basis for the heightened response of mdx satellite cells to FGF in culture and may reflect exposure of the cells to growth factors in the degenerating mdx muscle (Crisona, 1998).

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans. Mutations in dally, a gene encoding a Drosophila glypican, and in GPC3, the gene for human glypican-3, implicate glypicans in the control of cell growth and division. So far, five members of the glypican family have been identified in vertebrates. By sequencing expressed sequence tag clones and products of rapid amplifications of cDNA ends, a sixth member of the glypican family has been identified. The glypican-6 mRNA encodes a protein of 555 amino acids that is most homologous to glypican-4 (63% identity). Expression of this protein in Namalwa cells shows a core protein of approximately 60 kDa that is substituted with heparan sulfate only. GPC6, the gene encoding human glypican-6, contains nine exons. Like GPC5, the gene encoding glypican-5, GPC6 maps to chromosome 13q32. Clustering of the GPC5/GPC6 genes on chromosome 13q32 is strongly reminiscent of the clustering of the GPC3/GPC4 genes on chromosome Xq26 and suggests GPCs arose from a series of gene and genome duplications. Based on similarities in sequence and gene organization, glypican-1, glypican-2, glypican-4, and glypican-6 appear to define a subfamily of glypicans, differing from the subfamily that thus far comprises glypican-3 and glypican-5. Northern blottings indicate that glypican-6 mRNA is widespread, with prominent expressions in human fetal kidney and adult ovary. In situ hybridization studies localize glypican-6 to mesenchymal tissues in the developing mouse embryo. High expressions occur in smooth muscle cells lining the aorta and other major blood vessels and in mesenchymal cells of the intestine, kidney, lung, tooth, and gonad. Growth factor signaling in these tissues might be regulated, in part, by the presence of glypican-6 on the cell surface (Veugelers, 1999).

Glypican4 promotes cardiac specification and differentiation by attenuating canonical Wnt and Bmp signaling

Glypicans are heparan sulphate proteoglycans (HSPGs) attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, and interact with various extracellular growth factors and receptors. The Drosophila division abnormal delayed (dally) was the first glypican loss-of-function mutant described that displays disrupted cell divisions in the eye and morphological defects in the wing. In human, as in most vertebrates, six glypican-encoding genes have been identified (GPC1-6), and mutations in several glypican genes cause multiple malformations including congenital heart defects. To understand better the role of glypicans during heart development, the zebrafish knypek mutant, which is deficient for Gpc4, was studied. The results demonstrate that knypek/gpc4 mutant embryos display severe cardiac defects, most apparent by a strong reduction in cardiomyocyte numbers. Cell-tracing experiments, using photoconvertable fluorescent proteins and genetic labeling, demonstrate that Gpc4 'Knypek' is required for specification of cardiac progenitor cells and their differentiation into cardiomyocytes. Mechanistically, this study shows that Bmp signaling is enhanced in the anterior lateral plate mesoderm of knypek/gpc4 mutants and that genetic inhibition of Bmp signaling rescues the cardiomyocyte differentiation defect observed in knypek/gpc4 embryos. In addition, canonical Wnt signaling is upregulated in knypek/gpc4 embryos, and inhibiting canonical Wnt signaling in knypek/gpc4 embryos by overexpression of the Wnt inhibitor Dkk1 restores normal cardiomyocyte numbers. Therefore, it is concluded that Gpc4 is required to attenuate both canonical Wnt and Bmp signaling in the anterior lateral plate mesoderm to allow cardiac progenitor cells to specify and differentiate into cardiomyocytes. This provides a possible explanation for how congenital heart defects arise in glypican-deficient patients (Strate, 2015).

Glypican mutation

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked condition characterized by pre- and postnatal overgrowth with visceral and skeletal anomalies. To identify the causative gene, breakpoints in two female patients with X;autosome translocations were identified. The breakpoints occur near the 5' and 3' ends of a gene, GPC3, that spans more than 500 kilobases in Xq26; in three families, different microdeletions encompassing exons cosegregate with SGBS. GPC3 encodes a putative extracellular proteoglycan, glypican 3, that is inferred to play an important role in growth control in embryonic mesodermal tissues where it is selectively expressed. Initial western- and ligand-blotting experiments suggest that glypican 3 forms a complex with insulin-like growth factor 2 (IGF2), and might thereby modulate IGF2 action (Pilia, 1996).

The Glypican family of heparan sulfate proteoglycans regulates Wnt signaling and convergent extension (CE) in vertebrate embryos. They are predicted to be glycosylphosphatidylinositol (GPI)-tethered membrane-bound proteins, but there is no functional evidence of their regulation by the GPI synthesis complex. Down syndrome critical region protein 5 (Dscr5, also known as Pigp) is a component of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) complex, and is associated with specific features of Down syndrome. This study reports that Dscr5 regulates CE movements through the non-canonical Wnt pathway. Both dscr5 overexpression and knockdown impair convergence and extension movements. Dscr5 functionally interacted with Knypek/Glypican 4 and is required for its localization at the cell surface. Knockdown of dscr5 disrupts Knypek membrane localization and causes an enhanced Frizzled 7 receptor endocytosis in a Caveolin-dependent manner. Furthermore, dscr5 knockdown promotes specific Dishevelled degradation by the ubiquitin-proteosome pathway. These results reveal a functional link between Knypek/Glypican 4 and the GPI synthesis complex in the non-canonical Wnt pathway, and provide the new mechanistic insight that Dscr5 regulates CE in vertebrate embryos by anchoring different Wnt receptors at the cell surface and maintaining Dishevelled stability (Shao, 2009).

Proteins binding heparan sulphate proteoglycans

Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). Cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it. The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augments the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect is abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaces heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibits the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness (Bonneh-Barkay, 1997).

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, it was hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. This study show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, it is reportd that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. It is concluded that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway (Capurro, 2008).

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). The capacity of various species of heparin and heparan sulfate (HS) to promote bFGF receptor binding was investigated using both Chinese hamster ovary mutant cells deficient in cell surface HSPG and a soluble bFGF receptor-alkaline phosphatase fusion protein. Highly sulfated oligosaccharides are more effective than medium and low sulfate fractions of the same size oligosaccharide. O-Sulfation in heparin is critical for its capacity to promote binding of bFGF to its receptors. The highest level of bFGF-receptor binding is achieved in the presence of over-sulfated heparin fragments, regardless of whether the N-position is sulfated or acetylated. Unlike receptor binding of bFGF, which requires oligosaccharides containing at least 8-10 sugar units, displacement of heparin- or HS-bound bFGF is obtained by oligosaccharides containing as little as four sugar units and by an N-sulfated, O-desulfated heparin fragment. A preparation of total cell surface-derived HS induces bFGF receptor binding. A preliminary survey of several defined and affinity purified species of cell surface HSPG, including syndecan, fibroglycan, and glypican fail to identify natural HSPG that promotes high affinity receptor binding of bFGF. A similar lack of activity is observed with species of HS isolated from bovine arterial tissue and characterized for their effect on vascular smooth muscle cell proliferation. Most of these species of HS inhibit in a dose-dependent manner the restoration of bFGF-receptor binding induced by heparin or by total HSPG. These results suggest the involvement of defined heparin-like oligosaccharide sequences and unique species of cell surface and extracellular matrix HS in the regulation of bFGF receptor binding and biological activity (Aviezer, 1994).

The formation of distinctive basic FGF-heparan sulfate complexes is essential for the binding of bFGF to its cognate receptor. In previous experiments, cell-surface heparan sulfate proteoglycans extracted from human lung fibroblasts could not be shown to promote high affinity binding of bFGF when added to heparan sulfate-deficient cells that express FGF receptor-1 (FGFR1). In alternative tests to establish whether cell-surface proteoglycans can support the formation of the required complexes, K562 cells were first transfected with the IIIc splice variant of FGFR1 and then transfected with constructs coding for either syndecan-1, syndecan-2, syndecan-4 or glypican, or with an antisense syndecan-4 construct. Cells cotransfected with receptor and proteoglycan show a two- to three- fold increase in neutral salt-resistant specific 125I-bFGF binding in comparison to cells transfected with only receptor or cells cotransfected with receptor and anti-syndecan-4. Exogenous heparin enhances the specific binding and affinity cross-linking of 125I-bFGF to FGFR1 in receptor transfectants that are not cotransfected with proteoglycan, but has no effect on this binding and decreases the yield of bFGFR cross-links in cells that are cotransfected with proteoglycan. Receptor-transfectant cells show a decrease in glycophorin A expression when exposed to bFGF. This suppression is dose-dependent and obtained at significantly lower concentrations of bFGF in proteoglycan-cotransfected cells. Complementary cell-free binding assays indicate that the affinity of 125I-bFGF for an immobilized FGFR1 ectodomain is increased threefold when the syndecan-4 ectodomain is coimmobilized with receptor. Equimolar amounts of soluble syndecan-4 ectodomain, in contrast, have no effect on this binding. It is concluded that, at least in K562 cells, syndecans and glypican can support bFGF-FGFR1 interactions and signaling, and that cell-surface association may augment their effectiveness (Steinfeld, 1996).

OCI-5 encodes the rat homolog of glypican-3, a membrane-bound heparan sulfate proteoglycan that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome. OCI-5 and glypican-3 are 95% identical. It has been recently suggested that glypican-3 interacts with insulin-like growth factor-2 (IGF-2) and that this interaction regulates IGF-2 activity. OCI-5 was transfected into two different cell lines and an interaction between the OCI-5 proteoglycan produced by the transfected cells and IGF-2 could not be detected. In contrast to this, OCI-5 interacts with FGF-2, as has already been shown for glypican-1. This interaction is mediated by the heparan sulfate chains of OCI-5 because it can be inhibited by heparin or by heparitinase (Song, 1997).

A comparative study was undertaken of the interaction of the three mammalian transforming growth factor-betas (TGF-beta) with heparin and heparan sulfate. TGF-beta1 and -beta2, but not -beta3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-beta1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-beta1 by alpha2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-beta2.alpha2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-beta3 cannot be affected. Comparison of the amino acid sequences of the TGF-beta isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-beta dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain (Lyon, 1997).

The transforming growth factor-beta (TGF-beta) binding site in betaglycan, the type III TGF-beta receptor, has been variously assigned to the C-terminal half and N-terminal one-third of the extracellular domain. There are at least two TGF-beta-binding sites in betaglycan. Bacterially expressed fragments bg 1,2 and bg3, which represent the N-terminal two-thirds and C-terminal one-third of betaglycan extracellular domain, both compete for the binding of 125I-TGF-beta to mink lung epithelial cells. 125I-bg1,2 binds to immobilized TGF-beta with an affinity about 4-fold higher than does bg3. Both bg3 and bg1,2 enhance the bioactivity of TGF-beta. The whole ectodomain of betaglycan is more active than either bg3 or bg1,2 in the assays. The binding of 125I-bg3 to TGF-beta is inhibited by bg1,2 and vice versa. The binding of 125I-bg3 and 125I-bg1,2 to TGF-beta is also inhibited by the small decorin family of proteoglycans. These results indicate that there are at least two binding sites for TGF-beta in betaglycan and that these sites recognize the same or overlapping sites in TGF-beta (Kaname, 1996).

Although a number of growth factors bind cell-surface heparan sulphate proteoglycans, the role of this interaction is unclear except for fibroblast growth factor, which requires HSPG binding for signaling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signaling. To map the binding sites for HSPG and the Met receptor in HGF/SF, a number of HGF/SF mutants were engineered in which several clusters of solvent-accessible residues either in the hairpin structure of the amino-terminal domain or in kringle 2 were replaced. Two of the mutants (HP1 and HP2) show greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retain full mitogenic and motogenic activities on target cells in culture. When compared with wild-type HGF/SF, the HP1 mutant exhibits a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. These results establish the following: (1) the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; (2) high-affinity binding of HGF/SF to HSPGs is not essential for signalling; (3) one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor, and (4) HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo (Hartmann, 1998).

A novel cell surface HP/HS interacting protein (HIP) has been identified from human uterine epithelia and a variety of other human epithelial and endothelial cells and cell lines. HIP from HEC cells, a human uterine epithelial cell line, as well as recombinant HIP from a bacterial expression system were purified and characterized. HIP supports attachment of the human trophoblastic cell line, JAR, in a HS-dependent fashion. Predigestion of JAR cells with a mixture of heparitinases, but not chondroitinase AC, abolishes cell attachment to HIP. JAR cell attachment to HIP is highly sensitive to HP inhibition and much more selective for HP/HS than other glycosaminoglycans. Dermatan sulfate displays partial inhibitory activity as well, consistent with the observation that chondroitinase ABC digestion partially reduces JAR cell attachment to HIP. HIP binds labelled HP with high affinity, and HIP binds cell surface/extracellular matrix-associated HS, expressed by RL95 cells, a human uterine epithelial cell line. Anti-HIP antibody generated against a synthetic peptide derived from a putative HP/HS-binding motif resident within HIP inhibits about half of labelled HP binding to HIP, indicating that this domain is a functional HP-binding domain of HIP. Similarly, this same synthetic peptide motif of HIP can block about 50% of labelled HP binding to HIP; however, this peptide almost completely inhibits cell attachment to HIP, suggesting a critical role, in this regard. Collectively, these results suggest that HIP can function as a HP/HS-binding cell-cell/cell-matrix adhesion molecule (Liu, 1997).

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumors (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p. Most HME cases have been attributed to missense or frameshift mutations in these tumor-supressor genes, whose functions have remained obscure. EXT1 is shown to be an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing etiologic missense mutations fail to alter cell-surface glycosaminoglycans, despite retaining their ER-localization (McCormick).

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek and Frizzled coreceptor transmembrane protein LRP5/6

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/βcatenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a 'head inducer' during gastrulation. Lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/βcatenin pathway is overactivated, and dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a βcatenin independent fashion when modulating gastrulation movements. The glypican 4/6 homolog Knypek (Kny) binds to Dkk1, and they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. Indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the βcatenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. These data therefore indicate that Dkk1 regulates gastrulation movement through interaction the Frizzled coreceptor transmembrane protein LRP5/6 and Kny and coordinated modulations of Wnt/βcatenin and Wnt/PCP pathways (Caneparo, 2007).

Modification of glypican

Skin fibroblasts treated with brefeldin A produce a recycling variant of glypican (a glycosylphosphatidylinositol anchored heparan-sulfate proteoglycan) that is resistant to inositol-specific phospholipase C and incorporate sulfate and glucosamine into heparan sulfate chains. Structural modifications of recycling glypican have been examined, such as fatty acylation from [3H]palmitate, and degradation and assembly of heparan sulfate side chains. Most of the 3H-radioactivity is recovered as lipid-like material after de-esterification. To distinguish between formation of heparan sulfate at vacant sites, elongation of existing chains or degradation followed by re-elongation of chain remnants, cells were pulse-labeled with [3H]glucosamine and then chase-labeled with [14C]glucosamine. Material isolated from the cells during the chase consisted of proteoglycan and mostly [3H]-labeled heparan-sulfate degradation products (molecular mass, 20-80 kDa) showing that the side chains are degraded during recycling. The degradation products are initially glucuronate-rich, but become more iduronate-rich with time. The glypican proteoglycan formed during the chase is degraded either with alkali to release intact side chains or with heparinase to generate distally located chain fragments that are separated from the core protein, containing the proximally located, covalently attached chain remnants. All of the [14C]-radioactivity incorporated during the pulse is found in peripheral chain fragments, and the chains formed are not significantly longer than the original ones. It is therefore concluded that newly made heparan-sulfate chains are neither made on vacant sites, nor by extension of existing chains but rather by re-elongation of degraded chain remnants. The remodeled chains made during recycling appear to be more extensively modified than the original ones (Edgren, 1997).

The secreted serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo

The secreted serine protease xHtrA1, expressed in early Xenopus embryos and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected embryos. xHtrA1 mRNA leads to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 has the opposite effects. xHtrA1 activates FGF/ERK signaling and the transcription of FGF genes. Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cell-surface-bound FGF ligands and stimulates long-range FGF signaling (Hou, 2007).

HtrA1 belongs to the HtrA (High temperature requirement-A) family of serine proteases that is well conserved from bacteria to humans. HtrA1 was originally isolated as a gene downregulated in SV40-transformed human fibroblasts. Overexpression of HtrA1 in cancer cells suppresses growth and proliferation in vivo, suggesting that HtrA1 is a candidate tumor suppressor. More recently, a single nucleotide polymorphism in the HtrA1 promoter has been presented as a major risk factor for age-related macular degeneration. HtrA1 binds to and inactivates members of the TGFβ family and modulates insulin-like growth factor (IGF) signals, but its biological function is not yet known (Hou, 2007).

The Xenopus homolog of HtrA1 (xHtrA1) was identified in a direct screen for secreted proteins. xHtrA1 is a modulator of FGF signaling that participates in axial development, mesoderm formation, and neuronal differentiation. xHtrA1 is activated by FGF signals and induces ectopic FGF4 and FGF8 transcription. Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1; pure heparan sulfate and dermatan sulfate phenocopy xHtrA1 and FGF activities in Xenopus embryos. The results suggest that xHtrA1 acts as a positive regulator of FGF signals and, through proteolytic cleavage of proteoglycans, allows long-range FGF signaling in the extracellular space (Hou, 2007).

Heparan sulphate proteoglycans and lipid metabolism

High density lipoprotein (HDL) particles and HDL cholesteryl esters are taken up by both receptor-mediated and non-receptor-mediated pathways. Cell surface heparan sulfate proteoglycans (HSPG) participate in hepatic lipase (HL)- and apolipoprotein (apo) E-mediated binding and uptake of mouse and human HDL by cultured hepatocytes. The HL secreted by HL-transfected McA-RH7777 cells enhances both HDL binding at 4 degrees C (approximately 2-4-fold) and HDL uptake at 37 degrees C (approximately 2-5-fold). The enhanced binding and uptake of HDL are partially inhibited by the 39-kDa protein, an inhibitor of low density lipoprotein receptor-related protein (LRP), but are almost totally blocked by heparinase, which removes the sulfated glycosaminoglycan chains from HSPG. Therefore, HL may mediate the uptake of HDL by two pathways: an HSPG-dependent LRP pathway and an HSPG-dependent but LRP-independent pathway. The HL-mediated binding and uptake of HDL are only minimally reduced when catalytically inactive HL or LRP binding-defective HL is substituted for wild-type HL, indicating that much of the HDL uptake requires neither HL binding to the LRP nor lipolytic processing. To study the role of HL in facilitating the selective uptake of cholesteryl esters, HDL has been used into which radiolabeled cholesteryl ether had been incorporated.. HL increases the selective uptake of HDL cholesteryl ether; this enhanced uptake is reduced by more than 80% by heparinase but is unaffected by the 39-kDa protein. Like HL, apoE enhances the binding and uptake of HDL (approximately 2-fold) but has little effect on the selective uptake of HDL cholesteryl ether. In the presence of HL, apoE does not further increase the uptake of HDL, and at a high concentration apoE impairs or decreases the HL-mediated uptake of HDL. Therefore, HL and apoE may utilize similar (but not identical) binding sites to mediate HDL uptake. Although the relative importance of cell surface HSPG in the overall metabolism of HDL in vivo remains to be determined, cultured hepatocytes clearly display an HSPG-dependent pathway that mediates the binding and uptake of HDL. This study also demonstrates the importance of HL in enhancing the binding and uptake of remnant and low density lipoproteins via an HSPG-dependent pathway (Ji, 1997).

Potential mechanisms of non-low-density lipoprotein (LDL) receptor-mediated uptake of triglyceride-rich particles (TGRP) in the presence of apolipoprotein E (apo E) were explored. Human fibroblasts were incubated with model intermediate-density lipoprotein- (IDL-) sized TGRP (10-1000 microg of neutral lipid/mL) containing apo E. At low particle concentrations, almost all apo E-TGRP uptake is via the LDL receptor. At higher particle concentrations, within the physiologic range, most particle uptake and internalization is via HSPG-mediated pathways. This HSPG pathway does not involve classical lipoprotein receptors, such as LRP or the LDL receptor. These data suggest that in peripheral tissues, such as the arterial wall, apo E may act in TGRP as a ligand for uptake not only via the LDL receptor and LRP pathways but also via HSPG pathways that are receptor-independent. Thus, at physiologic particle concentrations apo E-TGRP can be bound and internalized in certain cells by relatively low affinity but high capacity HSPG-mediated pathways (Al-Haideri, 1997).

Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Glypicans regulate the activity of Wnts, Hedgehogs, bone morphogentic proteins, and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signaling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. In contrast, when glypicans are secreted to the extracellular environment they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signaling named Notum. The Wnt-inhibiting activity of Notum was associated with its ability to release dally-like protein (a Drosophila glypican) from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum, it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, whether Notum cleaves GPI-anchored proteins that are not members of the glypican family is also unknown. This study shows that mammalian Notum can cleave several mammalian glypicans. Moreover, Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of this study is that, unlike GPI-Phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which glypican-3 stimulates Wnt signaling it was shown that Notum can act as a negative regulator of this growth factor (Traister, 2008).

Dally-like (Dlp) is a glypican-type heparan sulfate proteoglycan (HSPG), containing a protein core and attached glycosaminoglycan (GAG) chains. In Drosophila wing discs, Dlp represses short-range Wingless (Wg) signaling, but activates long-range Wg signaling. This study shows that Dlp core protein has similar biphasic activity as wild-type Dlp. Dlp core protein can interact with Wg; the GAG chains enhance this interaction. Importantly, it was found that Dlp exhibits a biphasic response, regardless of whether its glycosylphosphatidylinositol linkage to the membrane can be cleaved. Rather, the transition from signaling activator to repressor is determined by the relative expression levels of Dlp and the Wg receptor, Frizzled (Fz) 2. Based on these data, it is proposed that the principal function of Dlp is to retain Wg on the cell surface. As such, it can either compete with the receptor or provide ligands to the receptor, depending on the ratios of Wg, Fz2, and Dlp (Yan, 2009).

The mechanisms controlling Wg signaling and its gradient formation are highly complex. This study provides two lines of findings for the mechanistic roles of Dlp in Wg signaling. First, it is shown that the core protein of Dlp has similar biphasic activity to wild-type Dlp in Wg signaling. Consistent with this, the Dlp core protein can interact with Wg, while the attached HS chains can enhance Dlp's affinity for Wg binding. Second, it is demonstrated that Dlp can get a biphasic response without Notum cleavage, and the ratio of Dlp:Fz2 determines its biphasic activity in cell culture and in the wing disc. While a low ratio of Dlp:Fz2 can help Fz2 obtain more Wg, a high ratio of Dlp:Fz2 prevents Fz2 from capturing Wg. It is proposed that the main activity of Dlp in Wg signaling is to retain Wg on the cell membrane rather than to act as a classic coreceptor. Dlp can mediate the exchange of Wg between receptors and itself; the net flow of the ligand depends on the ratios of the ligand, receptor, and Dlp. In support of this model, it was found that Fz2-GPI also has biphasic activity in Wg signaling (Yan, 2009).

Previous studies have demonstrated that Dlp acts as a biphasic modulator for Wg signaling in the wing disc; however, the mechanism underlying this biphasic response is not clear. One model suggests that Notum expressed at the D/V boundary can cleave Dlp and release it together with bound Wg, converting Dlp from a membrane coreceptor to a secreted antagonist. The current data suggest that this model needs to be revised. First, it was shown that expression of a GPI-deleted secreted form of Dlp (similar to the form cleaved by Notum) does not inhibit Wg signaling in the wing discs. Second, expression of CD2 forms of Dlp, which cannot be cleaved by Notum, can also inhibit sens expression similar to GPI versions of Dlp. An alternative model is that Dlp competes with Wg receptors on the cell surface, locally inhibiting signaling, but it also promotes long-range Wg gradient formation, and thus provides more Wg in the distal part of the wing disc. However, this model cannot explain how Dlp has biphasic effects in vitro, where Wg gradients do not form (Yan, 2009).

On the basis of these results, an exchange factor model is favored to explain the biphasic activity of Dlp in Wg signaling. The model is very similar to a recently published mathematical model for biphasic activity of CV-2 in BMP signaling (Serpe, 2008). In this model, Dlp might either compete with the receptor or provide ligands for the receptor, its role changing depending on the relative levels of ligand, receptor, and exchange factor. This study has shown that, in the wing discs, raising the levels of Fz2 can convert Dlp from a repressor to an activator. In S2 cells, the biphasic activity of Dlp also depends on the Dlp:Fz2 ratio, with a low level of Dlp increasing Wg signaling reporter activity and a high level of Dlp reducing its activity. Using Co-IP experiments, it was directly shown that a small amount of Dlp provides Wg for Fz2 receptor, while a large amount of Dlp sequesters the Wg ligand. Moreover, it was found that, for a constant amount of Dlp, it is more likely to repress Wg signaling at high Wg concentration, but to promote signaling at low Wg concentration. In contrast, Dlp is more likely to promote Wg signaling at high Fz2 concentration, but to repress signaling at low Fz2 concentration. Thus, this model could explain the situation in wing disc, where Dlp inhibits Wg signaling in regions close to the D/V border (high Wg and low Fz2), and promotes signaling in regions far from the D/V border (low Wg and high Fz2). These data are consistent with a previous reports showing that in vitro Dlp promotes Wg signaling when the Wg level is low, but reduces signaling when the Wg level is high. This result also fits well with the theoretical modeling data of Serpe (2008) for different ligand levels, suggesting Dlp acts similarly to CV-2 in different systems. In order to work, their model contains a tripartite complex between CV-2, BMP, and the receptor. No Dlp coprecipitated with Fz2 is detected; however, as the Serpe study proposed, the intermediate is a transient complex with very rapid on-off kinetics, and it is difficult to demonstrate the tripartite intermediate directly. Finally, in further support of the current model, it was found that Fz2-GPI, which can stabilize Wg on the cell surface and compete with Fz2 for Wg binding, also has biphasic activity in Wg signaling (Yan, 2009).

Previous studies reported that secreted Fz-related protein (sFRP), another family of Wnt-interacting proteins, can also exhibit biphasic activity in Wnt signaling, enhancing Wnt signaling at low concentration, but inhibiting it at high concentration. As mentioned above, the BMP-binding protein, CV-2, can act as a concentration-dependent, biphasic regulator for BMP signaling in the wing disc (Serpe, 2008). It is interesting to note that both sFRP and CV-2 can interact with HSPGs, and are likely to exert their function on the cell surface. In addition, another HSPG member, Xenopus Syndecan-1, shows a level-dependent activation or inhibition of BMP signaling during dorsoventral patterning of the embryonic ectoderm (Olivares, 2009). Moreover, it was found that Ihog, a recently identified Hh coreceptor (Yao, 2006), has biphasic activity in Hh morphogen signaling. Overexpression of Ihog represses high-threshold Hh target, and extends low-threshold Hh target gene expression. Together, other cell surface ligand-interacting proteins might regulate signaling by a similar mechanism. Traditionally, all cell surface ligand-binding receptors that cannot signal independently are equivocally called coreceptors, despite their diverse functions. On the basis of current results, it is proposed that some of the coreceptors may function as the exchange factors rather than the classical coreceptors, which only enhance signaling by providing ligand to the receptor (Yan, 2009).

Another important finding of this work is the demonstration that Dlp's major activity in Wg signaling depends on its core protein. Previous studies have shown that different HSPG proteins play very distinct roles in Wg signaling and distribution. However, the mechanism underlying this specificity is unknown. This study presents evidence that the specificity of Dlp in Wg signaling results from its core protein. First, the Dlp core protein has biphasic activity for short- and long-range signaling similar to that of wild-type Dlp. Second, the Dlp core protein interacts with Wg in co-IP experiment, cell-binding assay, as well as in the wing discs. Third, it is shown that the N-terminal domain of Dlp is essential for Wg binding, and that fusion of the N-terminal domain of Dlp to the Fz2 membrane and cytoplasmic domain can recapitulate Fz2 activity. These data are consistent with previous results indicating that Xenopus glypican-4 interacts with Wnt11 through its N-terminal domain. It is interesting to note that, similar to Fz2 CRD domain, the N-terminal domain of Dlp protein has 14 highly conserved cysteines, a shared feature of all glypican members (Yan, 2009).

Previously, Capurro (2005) has shown that vertebrate glypican-3 core protein is directly involved in Wnt signaling, whereas the GAG chains of glypican-3 are not required for the stimulatory effect in Wnt signaling . Moreover, recent data has shown that the glypican-3 core protein also binds to Sonic Hedgehog (Shh), but inhibits its signaling by competing with the receptor, Patched (Capurro, 2008). The opposite effects of the same glypican core protein on Wnt and Hh signaling are intriguing. Interestingly, it has also been observed that the Dlp core protein positively regulates Hh signaling in both Drosophila embryos and wing discs. Thus, the core proteins of glypican-3 and Dlp appear to have opposite roles in Wnt and Hh signaling. It is likely that different glypican cores may bear distinctive motifs to interact with Wnt and Hh proteins (Yan, 2009).

Although the Dlp core protein is able to bind Wg, it was found that the attached HS GAG chains are also important for the binding affinity between Dlp and Wg. Wild-type Dlp shows significantly stronger binding for Wg than the core protein alone. This result is consistent with previous genetic experiments showing that Wg signaling is compromised in HS-deficient mutants. In addition, biochemical studies also suggest that Wg is a heparin-binding protein. One possibility is that the Dlp core protein might have different membrane distribution than wild-type Dlp. However, this study did not observe obvious difference in the subcellular localizations between Dlp-GFP and Dlp(−HS)-GFP. It remains to be determined how the presence of HS GAG chains can enhance Dlp's ability to bind Wg (Yan, 2009).

All glypicans anchor to the cell membrane via a GPI anchor. GPI proteins are enriched in specific membrane subdomains called lipid rafts, which are suggested to promote the signaling activities of GPI-anchored proteins. Thus, one important issue is whether the GPI anchor is required for Dlp's activity in Wg signaling. The results suggest that the GPI anchor of Dlp is not essential for its activity in Wg signaling. Several lines of evidence support this view. First, Dlp(−HS)-CD2, a transmembrane form of Dlp core protein, has similar biphasic activity to that of Dlp(−HS). Second, the subcellular localizations of different forms of Dlp was analyzed, and it was found that Dlp's major activity is to bind Wg at the cell surface. Dlp-GFP, which has the strongest binding affinity for Wg, accumulates more Wg on the cell surface. In Dlp(−HS)-GFP and Dlp(−HS)-CD2-GFP-expressing discs, less Wg was found accumulated on the cell membrane and more internalized Wg vesicles were found. These results are consistent with a recent work showing that accumulating Wg on Dlp-expressing cells is less accessible to internalization. Although Dlp-GFP and Dlp(−HS)-GFP, but not Dlp(−HS)-CD2-GFP, form many endocytic vesicles due to a role of the GPI anchor in trafficking, based on these functional data, it is suggested that the GPI anchor of Dlp is not essential for Wg signaling (Yan, 2009).

Recently, it has been proposed that the GPI anchor of Dlp is required for Wg internalization and long-range signaling (Gallet, 2008). This conclusion is mainly based on the evidence that expression of GFP-Dlp-CD2 can reduce the expression of Wg long-range target gene dll. This result is apparently different from the current data showing that expression of Dlp(−HS)-CD2-GFP construct leads to expanded dll expression. To resolve this issue, the GFP-Dlp-CD2 transgenic flies used by Gallet were obtained, and the activity of GFP-Dlp-CD2 in the wing discs was examined. Different results were observed from the previous study. It was found that their GFP-Dlp-CD2 has very similar biphasic activity to Dlp-GFP when it is expressed by en-Gal4 or ap-Gal4, and the effects on dll expression were examined. One possibility for the difference is that the previous study used only ap-Gal4, which will cause expression of Dlp to reduce the size of the compartment; this may complicate comparisons of the effect of GFP-Dlp-CD2 in long-range signaling. Therefore en-Gal4, which allows the use of the A compartment as an internal control, was used. Furthermore, while the previous showed that GFP-Dlp-CD2 induces a more severe wing defect than the GFP-Dlp construct, this study found that GFP-Dlp-CD2 does not generate a more severe wing defect than than the Dlp-GFP construct. In this regard, it is important to note that the GFP-Dlp-CD2 and GFP-Dlp constructs used by Gallet employed GFP inserted at two different sites in Dlp, and that the insertion in the GFP-Dlp construct leads to reduced activity. In conclusion, the current data demonstrate that CD2 forms of Dlp have similar activity to the GPI forms of Dlp, suggesting that the GPI anchor of Dlp is not essential for its activity in Wg signaling (Yan, 2009).

Heparan sulphate proteoglycans: roles in development

The kidney of the Gpc3-/- mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Cellular and molecular mechanisms underlying this renal dysplasia have been identified. Glypican-3 (GPC3) deficiency is associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation is increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggests that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogates the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converts BMP7-dependent inhibition to stimulation, and enhances the stimulatory effects of KGF. Similar comparative differences are found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. It is proposed that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis (Grisaru, 2001).

The molecular basis for observations in ureteric bud and collecting duct cells remains to be determined. The demonstration that bFGF forms a molecular complex with cell surface heparan sulfate and the FGF cell surface receptor suggests that GPC3 may physically interact with receptors that bind BMP2, BMP7, and KGF. The opposite response of collecting duct cells to GPC3 deficiency, that is, inhibition of BMP2 activity and enhancement of KGF activity, suggests that the consequences of these interactions may differ. A second possibility is that GPC3 may function via independent signaling pathways that physically interact at the postreceptor level with BMP and KGF signaling intermediates. Alternatively, the GPC3 and growth factor-signaling pathways may interact indirectly by regulating competing or complementary gene products. Increasing evidence regarding the nature of inhibitory and stimulatory BMP-dependent signaling pathways in collecting duct cells provides a basis to determine the nature of GPC3 interactions with BMP2 and BMP7 (Grisaru, 2001).

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. The non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. The function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family, has been analyzed. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analyses with different Dsh mutants and Wnt11 demonstrate that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/ß-catenin pathway, to regulate gastrulation movements. Evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, the results suggest that Xgly4 functions is a positive regulator in non-canonical Wnt/PCP signaling during gastrulation (Ohkawara, 2003).

Heparan sulphate proteoglycans such as glypicans are essential modulators of intercellular communication during embryogenesis. In Xenopus laevis embryos, the temporal and spatial distribution of Glypican 4 (Gpc4) transcripts during gastrulation and neurulation suggests functions in early development of the central nervous system. The role of Xenopus Gpc4 has been functionally analyzed by using antisense morpholino oligonucleotides; Gpc4 is shown to be part of the signalling network that patterns the forebrain. Depletion of GPC4 protein results in a pleiotropic phenotype affecting both primary axis formation and early patterning of the anterior central nervous system. Molecular analysis shows that posterior axis elongation during gastrulation is affected in GPC4-depleted embryos, whereas head and neural induction are apparently normal. During neurulation, loss of GPC4 disrupts expression of dorsal forebrain genes, such as Emx2, whereas genes marking the ventral forebrain and posterior central nervous system continue to be expressed. This loss of GPC4 activity also causes apoptosis of forebrain progenitors during neural tube closure. Biochemical studies establish that GPC4 binds FGF2 and modulates FGF signal transduction. Inhibition of FGF signal transduction, by adding the chemical SU5402 to embryos from neural plate stages onward, phenocopies the loss of gene expression and apoptosis in the forebrain. It is proposed that GPC4 regulates dorsoventral forebrain patterning by positive modulation of FGF signalling (Gall, 2003).

Heparan sulphate proteoglycans as viral receptors

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, it has been demonstrated that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrate dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduce AAV attachment and infectivity. Mutant cell lines that do not produce heparan sulfate proteoglycans are significantly impaired for both AAV binding and infection. This is the first reported role for proteoglycan in the cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector (Summerford, 1998).

Heparan sulphate proteoglycans and Alzheimer's disease

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican binds strongly to the APP affinity column. Purified glypican binds to APP with an equilibrium dissociation constant of 2.8 nM and inhibits APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican is specific for APP, as glypican do not inhibit laminin-induced neurite outgrowth. Treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibits APP-induced neurite outgrowth but does not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP (Williamson, 1996).

Heparan sulfate proteoglycan (HSPG) has been found to be associated with amyloid deposits in a number of diseases including the cerebral amyloid plaques of Alzheimer's disease and the transmissible spongiform encephalopathies (TSEs). The role of HSPG in amyloid formation and the neurodegenerative pathology of these diseases have not been established. These questions were addressed using a scrapie mouse model, which exhibits both amyloid and nonamyloid deposition of abnormal PrP protein, the protein marker of TSE infection. The distribution of HSPG was examined throughout the course of the disease in the brains of experimentally infected mice and compared with the distribution of abnormal PrP. Abnormally high levels of HSPG are associated with most types of PrP pathology including all plaque types and diffuse neuroanatomically targeted forms. Scrapie-associated HSPG is present from 70 days after infection, the earliest time-point examined, in the same target areas as abnormal PrP. The association with amyloid plaques may indicate that HSPG is involved in amyloid plaque formation and/or persistence, but involvement with early diffuse forms of PrP suggests a more fundamental role in scrapie pathogenesis (McBride, 1998).


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