Anterior-posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. This study shows that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. These data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior-posterior polarity formation in the oocyte (Poulton, 2006; full text of article).
This study has identified DG as a gene whose expression pattern is both regulated by EGFR signaling in the PFC and necessary for oocyte polarity. These findings provide a mechanistic link between EGFR activity in the PFC and polarization of the oocyte. Furthermore, it was discovered that defects in apicobasal polarity caused by ectopic DG also are present in the PFC where EGFR signaling is disrupted, possibly due to the misexpression of DG in these cells. In addition, the findings that ectopic DG leads to mislocalizations of Lan at the apical surface of the PFC indicates a process of cell–cell communication in which EGFR-regulated DG expression in the PFC controls Lan organization in the ECM that in turn may affect localization of posterior determinants in the oocyte (Poulton, 2006).
It was reported that loss of LanA in the PFC disrupts oocyte polarity, which seems to be in conflict with the suggestion that high levels of apical Lan in the PFC perturbs oocyte polarity. However, a model in which Lan is required in early oogenesis, but must be localized basally after EGFR activation and DG down-regulation, reconciles these findings. In the previous research on loss-of-function lanA mosaic egg chambers, oocyte polarity defects observed at stage 9/10 could be generated only by larger lanA PFC clones. Because follicle cells are only mitotically active until stage 6/7 of oogenesis, these large PFC clones present at stage 9/10 would have represented sizeable lanA clones in prestage-6 follicle cells. Because Lan is present on the apical surface of these pre-PFCs, the polarity defects observed at stage 9/10 may have resulted from perturbation of some earlier Lan-dependent processes, such as organizing receptors on the facing surfaces of the oocyte or follicle cells. Consistent with this model, the addition of Lan to myotubes in culture is sufficient to organize the receptors integrin and DG, as well as their respective cytoplasmic counterparts, vinculin and dystrophin. Alternatively, it could be that the role of Lan in mediating the relationship between the PFC and oocyte is sensitive to any disruption of the ECM stemming from either the loss or misexpression of Lan, which then is sufficient to negatively affect oocyte polarity. Either of these models demonstrates the importance of the ECM in this process and ultimately may lead to a mechanistic understanding of the oocyte polarity defects caused by mutation in the putative Lan receptor Dlar (Poulton, 2006).
Precisely how ectopic DG on the surface of the PFC translates to mislocalizations of posterior polarity markers in the adjacent oocyte remains to be determined, however, several different explanations for this process can be considered. (1) DG down-regulation in the PFC may be necessary to allow the actin-based cortical anchoring of the posterior determinants in the oocyte. (2) The down-regulation of DG after EGFR activation might serve as a cue to the oocyte, which leads directly to MT reorganization and AP axis formation. In this analysis, however, DG overexpression did not result in defects in global microtubule organization or mislocalization of anterior oocyte polarity markers, phenotypes that have been reported in grk and top mutant egg chambers. Furthermore, simply reducing DG levels in non-PFCs by RNAi was not sufficient to mislocalize Stau to nonposterior regions of the oocyte. Therefore, DG down-regulation alone probably cannot serve as the signal to repolarize the microtubule network and, thus, establish oocyte polarity, but it is possible that changes in cell adhesion mediated through the DG/Lan complex could be part of a complex signal involving additional ECM receptors or even other signaling mechanisms that have yet to be identified. A similar model has been proposed for this signal in which changes in cell adhesion between the oocyte and PFCs serve as a nontraditional signal initiating AP axis formation. Alternatively, EGFR-mediated changes in DG/Lan patterns could regulate a novel mechanism that is required specifically for localization of posterior determinants at the oocyte cortex but is independent of the signal provided by the PFC to repolarize the oocyte microtubule cytoskeleton (Poulton, 2006).
(3) The apicobasal defects caused by up-regulation of DG may have led to the loss of apical targeting of the polarizing signal from the PFC, as has been proposed for oocyte polarity defects caused by Merlin mutation. This explanation does not seem likely, however, given the ability of DG RNAi to rescue the CAM phenotype even though the Ras clones still should be unable to produce the signal, because they do not take the PFC fate. Instead, a model is favored in which the apicobasal defects caused by ectopic DG results in apical accumulations of Lan, thereby modifying the ECM between the clones and oocyte so as to preclude diffusion of a secreted signal from the adjacent wild-type cells. Therefore, in the Ras rescue experiment, down-regulation of DG allows the basal restriction of Lan, facilitating diffusion of the polarizing signal from the remaining wild-type cells. The fact that the rescue of the CAM phenotype by DG RNAi in Ras clones was not complete (34% of these egg chambers continued to show some defect in Stau localization) may support this model, because the diffusion of a signal from the neighboring cells probably would not be expected to replace fully the endogenous signal absent from the clone cells in every case. Whether mutations in other genes required for both apicobasal polarity and oocyte polarity also disrupt the ECM will be interesting to discover (Poulton, 2006).
The study of axis formation in the Drosophila oocyte has demonstrated the importance of cell–cell communication in the tightly regulated patterning of the follicle cells, which ultimately leads to the establishment of those axes. The key findings presented here suggest a multifaceted role for EGFR signaling in PFC differentiation and oocyte polarization, highlighting the need for further study of EGFR activity, differentiation of the PFC, and formation of the AP axis (Poulton, 2006).
Dystroglycan (Dg) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basement membrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, Dg plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle leads to various types of muscular dystrophies. In epithelial cells, reduced expression of Dg is associated with increased invasiveness of cancer cells. Dg is required for epithelial cell polarity in Drosophila, but the mechanisms of this polarizing activity and upstream/downstream components are largely unknown. Using the Drosophila follicle-cell epithelium (FCE) as a model system, this study shows that the ECM molecule Perlecan [Pcan; encoded by terribly reduced optical lobes (trol)] is required for maintenance of epithelial-cell polarity. Follicle cells that lack Pcan develop polarity defects similar to those of Dg mutant cells. Furthermore, Dg depends on Pcan but not on Laminin A for its localization in the basal-cell membrane, and the two proteins bind in vitro. Interestingly, the Dg form that interacts with Pcan in the FCE lacks the mucin-like domain, which is thought to be essential for Dg ligand binding activity. Finally, two examples are described of how Dg promotes the differentiation of the basal membrane domain: (1) by recruiting/anchoring the cytoplasmic protein Dystrophin; and (2) by excluding the transmembrane protein Neurexin. It is suggested that the interaction of Pcan and Dg at the basal side of the epithelium promotes basal membrane differentiation and is required for maintenance of cell polarity in the FCE (Schneider, 2006).
In vertebrates, Dg is synthesized as a single polypeptide and post-translationally cleaved into the extracellular glycoprotein αDg and the transmembrane protein ßDg. The two subunits are believed to remain attached to one another through non-covalent interaction of the C-terminal region of αDg with the N-terminal region of ßDg (Sciandra, 2001). αDg shows a dumbbell-like molecular shape in which two less glycosylated globular domains are separated by the mucin-like domain (mucin-domain), a highly glycosylated serine-threonine-proline-rich region (Brancaccio, 1995). Laminin (Lam), Agrin, Perlecan (Pcan) and Neurexin (Nrx) serve as ligands for αDg, and Lam G (LG)-like domains mediate the interaction. The binding site on αDg is not known, but proper glycosylation of αDg is generally considered to be crucial for its ligand-binding activity. Recent studies have demonstrated that Oglycosylation within the mucin-domain is required for Lam (Kanagawa, 2004) and Pcan binding (Kanagawa, 2005), but it is not clear whether the sugar-chains of this domain are directly involved in the interaction or merely play a structural role in supporting the rod-like shape of this region (Schneider, 2006).
The cytoplasmic tail of ßDg interacts with Dystrophin (Dys) in muscle cells, and the Dys-homolog Utrophin (Utr) in epithelial cells. Dys/Utr in turn connect to actin filaments of the cytoskeleton. Dg therefore occupies a central position in an ECM-cytoskeleton link disruption of which leads to various types of muscular dystrophies (Cohn, 2000). In addition, Dg has been suggested to play a key role in the transduction and modulation of various signaling cascades (Schneider, 2006).
In epithelial cells, reduced expression of Dg has been associated with increased invasiveness of cancer cells (Muschler, 2002). In some malignant tumors, e.g. prostate and mammary cancer, the expression of αDg is reduced (Henry, 2001a; Muschler, 2002). Furthermore, the amount of reduction is correlated with the invasiveness of the tumor (Muschler, 2002). Recent results (Sgambato, 2005; Sgambato, 2003) suggest that the loss of αDg might be an early event in carcinogenesis rather than being a consequence of neoplastic transformation (Schneider, 2006).
Some reports have suggested that the major ligand for Dg in non-muscle cells might be Pcan, because the binding of αDg to Pcan LG-domains is five times stronger than that to the most active Lam fragment (Andac, 1999; Talts, 1999). Pcan is the major heparan sulfate proteoglycan in basement membranes (BMs) and connective tissue, and has been implicated in adhesion, proliferation, development and growth-factor binding. The Pcan core protein consists of five domains and binds to a variety of molecules, including FGF-7, Fibronectin, Heparin, Laminin 1, PDGF-B, αDg and Integrins. At the N-terminal domain I and the C-terminal domain V, glucosaminoglycan (GAG) chains are attached that interact with Laminin-1 and Collagen IV and bind to FGF-2, promoting its angiogenic and mitotic activities. Studies in transgenic mice have shown that Pcan is required for the maintenance of the functional and structural integrity of BMs in the heart, but is not needed for BM assembly per se (Schneider, 2006 and references therein).
Not much is known about the function of the interaction between Pcan and Dg. During the development of the neuromuscular junction, binding between Pcan and Dg is required for clustering of acetylcholine esterase at the postsynaptic membrane (Peng, 1999). In addition, cell culture studies with Pcan- and Laminin α2-deficient skin fibroblasts (Herzog, 2004) revealed that shedding of Dg is increased by the lack of Pcan, but not by lack of Laminin α2 (Schneider, 2006 and references therein).
Pcan, Dg and other components of the Dystrophin-glycoprotein complex are conserved in Drosophila and vertebrates. Drosophila Pcan (trol) is required for controlling proliferation of neuronal stem cells in the larval brain (Voigt, 2002). Pcan has been suggested to act in the ECM by binding, storing and sequestering external signals, including FGF and Hedgehog (Voigt, 2002). A role for Pcan in epithelial development has not been reported so far (Schneider, 2006).
Drosophila Dg plays a role in polarizing epithelial cells and the oocyte. In particular, Dg function has been investigated during the development of the follicle-cell epithelium (FCE). The FCE forms through a mesenchymal-epithelial transition and uses mechanisms operating on the apical, lateral and basal side for epithelial differentiation. Contact of follicle cells with the basement membrane and with the germline cells has been suggested to play a role in polarizing the cells. As a result, distinct basal, apical and lateral cell-membrane domains are established by accumulating protein complexes that are actively reinforcing cell-membrane polarity. Loss of Dg leads to an expansion of apical markers to the basal side of the cells and loss of lateral markers. Some Dg mutant cells lose their epithelial appearance, form multiple layers and eventually die (Schneider, 2006).
The finding that Dg is required for epithelial cell polarity is particularly interesting because of its role during the invasive behavior of cancer cells, but little is known about the molecular mechanism behind this polarizing activity. This study investigated the hypothesis that Pcan and Dg constitute a basal polarizing cue required for the differentiation of the basal membrane domain and epithelial cell polarity. The FCE was chosen as a model system for several reasons: (1) all follicle cells are derived from two to three somatic stem cells, making mosaic analysis an excellent tool with which to study gene function in epithelial development; (2) the trol gene is transcribed in follicle cells, and (3) Dg plays a role in follicle-cell polarization (Schneider, 2006).
The phenotypes caused by the loss of Dg or Pcan share many similarities, such loss of cell polarity, formation of multilayers and 'invasion' by mutant follicle cells of the spaces between germ cells. One interesting difference is the behavior of the apical marker Patj, which accumulated at the basal membrane in Dg clones, but was unaffected in trol clones. The reason for this difference is not known, but a possible explanation is that in trol mutant cells, Dg is still present and occasionally even enriched apically (Schneider, 2006).
Patj is a cytoplasmic PDZ domain protein that forms an apical complex with the transmembrane protein Crb. In contrast to Patj, Crb is frequently reduced in trol clones. A similar loss of Crb was observed in embryonic salivary gland after ectopic expression of Dg, suggesting that the apical enrichment of Dg in trol clones might cause the reduction of Crb. Furthermore, the results confirm the existence of a Crb-independent localization and retention mechanism for Patj in the FCE (Schneider, 2006).
Another difference between trol and Dg clones lies is the ability of the cells to survive. Whereas Dg clones eventually die, trol clones can survive until later stages of oogenesis. Studies of embryoid bodies deficient in Dg revealed an accelerated level of apoptosis, which has led to the proposal that Dg has a role in cell survival (Schneider, 2006).
The overall similarity of the trol- and Dg- phenotypes suggests that the two proteins act in the same 'polarity pathway'. In support of this view is the finding that, in trol clones, Dg is frequently lost from the basal-cell membrane. This effect seems to be specific because: (1) Dg is unaffected by the lack of Lam A, and (2) ßPS remains localized in the basal membranes of trol mutant cells that have lost Dg. Pcan could stabilize Dg at the basal cell surface, either by direct binding or indirectly through interaction with other cell-matrix or cell-surface proteins. Recent findings suggested a trimolecular complex of Pcan, Lam and Dg (Kanagawa, 2005). However, a role for Lam in stabilizing Dg in the FCE is unlikely, because Lam is not required for Dg localization. The findings that Pcan domain V can be co-immunoprecipitated with Dg, supports the view that Pcan stabilizes Dg at least in part by direct binding. These results suggest that direct interaction of the ECM molecule Pcan with the transmembrane protein Dg is required for the maintenance of follicle cell polarity (Schneider, 2006).
In this context, it is interesting that mouse Dg is continuously shed from the cell surface of normal cutaneous cells by proteolytic cleavage of ßDg. Cell culture studies with Pcan- and Lam α2-deficient skin fibroblasts further revealed that shedding of Dg is increased by the lack of Pcan, but not by the lack of Lam α2 (Herzog, 2004). Drosophila Dg appears not to be processed into an α and a ß subunit. The antibody used to detect Dg in trol- cells was directed against the cytoplasmic domain (anti-Dgcyto), so clearly at least the intracellular domain of Dg, and probably the whole protein, is lost from the cell membrane in these cells. One might speculate that the loss of Dg in trol clones represents an elevated turnover of Dg, thereby altering the cell-matrix interaction and activity of Dg in the FCE, as shedding of Dg might do in the vertebrate system. In both systems, Pcan, but not Lam, could function to counteract this mechanism and to stabilize Dg at the cell membrane, but the expression pattern of Pcan and Dg makes clear that other mechanisms of stabilizing Dg expression must exist during early stages of oogenesis, when Pcan is not yet present in the ECM (Schneider, 2006).
Glycosylation of Dg is widely accepted to be essential for its function, and recent results suggest an important role for Oglycosylation in the mucin-domain for binding to Lam (Kanagawa, 2004) and Pcan (Kanagawa, 2005). To date, it is unclear whether the sugar-chains in the mucin-domain are directly involved in the interaction or whether they play a primarily structural function required for proper presentation of the ligand-binding domain. The following findings suggest that, in Drosophila, binding of Pcan and Dg does not require the mucin domain: first, the form of Dg that is expressed at the basal side of the FCE and depends on Pcan for its maintained localization does not contain the mucin-like domain; second, ectopic expression of Dg leads to ectopic accumulation of Lam and Pcan independent of the presence of the mucin domain; and third, one single band of ~120 kDa was detected in embryonic protein extracts in overlay binding assays with PcanV. The size of this band corresponds to the size of the two Dg forms Dg-A and Dg-B, which lack the mucin-domain. These results suggest that the mucin-domain plays a structural role that might not be required in the specific surroundings of the FCE. Another possibility is that presence or absence of the mucin-like domain might regulate binding affinity and/or selectivity (Schneider, 2006).
This study is the first demonstrating a function for a Dg splicing variant lacking the mucin-like domain. It will be interesting to find out whether different Dg forms carry out different functions (Schneider, 2006).
Contact with the ECM is important for polarization of several epithelia, including the vertebrate kidney epithelium and the Drosophila midgut, dorsal vessel and follicular epithelia. In Madin-Darby canine kidney (MDCK) cells, contact with the ECM results in the formation of a basal membrane domain and in long-range effects on the differentiation of the non-basal domain. Similar long-range effects of ECM contact during the establishment of polarity have been observed in the Drosophila FCE (Schneider, 2006).
The current results suggest that, after the initial polarization, ECM-cell contact mediated by Pcan and Dg plays a role in the maintenance of cell polarity. The expansion of Arm and the reduction of the lateral marker Dlg in Dg and trol clones might indicate a long-range effect of Dg on cell polarity. It is generally accepted that Dlg functions by preventing invasion of apical proteins and adherens-junction components into the lateral domain, suggesting that the reduction of Dlg in Dg and trol clones is the cause for the expansion of Arm in these clones. The molecular mechanisms underlying the effect of Dg on Dlg remain unknown, but the results show two clear short-range effects of Dg on the differentiation of the basal membrane domain: (1) the recruitment and/or anchoring of the cytoplasmic protein Dystrophin and (2) the exclusion of the basolateral protein NrxIV (Schneider, 2006).
In vertebrates, the cytoplasmic tail of ßDg binds to Dys in muscle cells and its homolog Utr, in epithelial cells. Dys/Utr, in turn, connects to actin filaments of the cytoskeleton. Mutations in Dys cause a reduction of the expression of Dg in the sarcolemma. In Drosophila, Dg and Dys are interdependent for their localization in the basal membrane of the FCE and in wing imaginal discs, suggesting that the interaction between both proteins is conserved. Provided that Drosophila Dys also interacts with actin filaments, this result could explain the defects in basal actin organization that were observed in Dg clones (Schneider, 2006).
In contrast to Dg clones, an abundant cytoplasmic localization of Dys was observed in trol clones. Further experiments are required to understand the precise molecular mechanisms underlying the observed defects in protein localization (Schneider, 2006).
The results raise the issue of whether Dys is also required for cell polarity. In Dys clones, the polarity marker Baz is clearly reduced, indicating a polarity defect in these cells. The difference to Dg clones in which Baz is not affected, and trol clones, in which Baz expression is elevated, indicates that Dys might play a Dg-independent role in cell polarity and that the subcellular localization of Dys could play a role for its function (Schneider, 2006).
Like Pcan and Lam, Neurexins contain several LG-like modules and have been described as putative interaction partners for Dg in the brain. The results suggest that, in the Drosophila FCE, Dg is required to exclude NrxIV from the basal membrane domain. Whether a direct interaction between Dg and NrxIV is involved in this process remains to be seen (Schneider, 2006).
NrxIV is generally regarded as an integral component of pleated SJ. It was surprising to find that NrxIV is located basally to the region where SJ form, in a position that might correspond to the border between the lateral and basal cell membrane domains. The precise function of NrxIV during SJ development in the follicular epithelium remains to be elucidated (Schneider, 2006).
In the embryo, NrxIV forms a complex with Nrg and Cont, and all three proteins are interdependent for SJ localization. The co-localization of NrxIV, Nrg and Cont in dot-like structures, and the fact that Cont co-localizes with ectopic NrxIV in Dg clones, suggest that molecular interactions between NrxIV, Cont and Nrg also occur in the FCE (Schneider, 2006).
On the basis of the current observations, it is proposed that Pcan and Dg provide a basal 'polarizing cue' required for differentiation of the basal membrane and maintenance of epithelial cell polarity in the FCE. Binding of the ECM molecule Pcan to its receptor Dg stabilizes Dg in the basal membrane. Dg is required for stabilizing Dlg at the lateral membrane, which in turn prevents apical markers and ZA components from invading the basolateral membrane domain. In addition, Dg forms a complex with Dys at the basal membrane and exerts a function in excluding NrxIV from the basal membrane. Further investigations will be required to understand the molecular mechanisms underlying the effect of Dg on Dlg localization and the roles of Dys and NrxIV in this process. Hopefully, a better understanding of the function of Dg in epithelial cell polarity will also shed some light on its role in cancer (Schneider, 2006).
Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, a high conservation in Dg-Dys interaction between human and Drosophila is demonstrated. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR (Shcherbata, 2007).
The Dg-Dys binding interface is highly conserved in humans and Drosophila. Both proteins are required for oocyte cellular polarity and interact in this process. Futhermore, mutants of both Dg and Dys genes show symptoms observed in muscular dystrophy. Reduction of Dg and Dys proteins results in age-dependent mobility defects. Eliminating Dg and Dys specifically in mesoderm derived tissues reveals that these proteins are required for muscle maintenance in adult flies: age-dependent muscle degeneration was observed in mutant tissues. Dg-Dys complex is also required for neuron path-finding and has both cell autonomous and non-cell autonomous functions for this process. Further, in neuronal path-finding process Dg interacts with InR and an SH2/SH3-domain adapter molecule Nck/Dock (Shcherbata, 2007).
Animal models have been used efficiently in muscular dystrophy studies. Some of the models are naturally occurring mutations (mdx-mouse, muscular dystrophy dog, cat and hamster), others have been generated by gene targeting. However, the regulation and the control of Dg-Dys complex are not understood, and no successful therapeutics exist yet for muscular dystrophies. Recently developed models for muscular dystrophy exist in C. elegans and zebrafish. In C. elegans Dys mutant, the transporter snf-6 that normally participates in eliminating acetylcholine from the cholinergic synapses, is not properly localized, resulting in an increased acetylcholine concentration at the neuromuscular junction and muscle wasting (Kim, 2004). The function of Dys in neuromuscular junctions has been addressed in Drosophila. These results bring up the possibility that muscular dystrophies in humans might also at least partly be attributed to the altered kinetics of acetylcholine transmission through neuromuscular junctions (Shcherbata, 2007).
Drosophila acts as a remarkably good model for age-dependent progression of muscular dystrophy. Dg and Dys reduction in Drosophila show age-dependent muscle degeneration and lack of climbing ability. It is tempting to speculate that the common denominator between different defects observed in Dg-Dys mutants in Drosophila and C. elegans is defective cellular polarity. The defects observed in C. elegans could be due to a defect in polarization of a cell, which will generate a neuromuscular junction that leads to miss-targeted snf-6. Similarly, Drosophila Dg-Dys complex is required for cellular polarity in the oocyte. In addition, neural defects observed are plausibly due to polarity defects in the growing axon (Shcherbata, 2007).
Similar to neuronal defects observed in human muscular dystrophy patients, neuronal defects were also found in Drosophila Dg and Dys mutant brains. In vertebrate brains, Dg affects neuronal migration (Montanaro, 2003; Qu, 2004) possibly through interaction of neurons with their glial guides. The neuronal migration and process outgrowth have been shown to require supportive input from glial cells and involve the formation of adhesion junctions along the length of the soma. Also, the outgrowth of the leading process involves rapid extension and contraction over the length of the glial fiber. Disruption of the cytoskeletal organization within the neuron, either of actin filaments, has been shown to inhibit glial-mediated neuronal migration. The glial function in this process is less well studied (Shcherbata, 2007).
Drosophila photoreceptor path-finding provides an excellent system for genetic dissection of neuronal outgrowth and target recognition. During the formation of the nervous system, newly born neurons send out axons to find their targets. Each axon is led by a growth cone that responds to extracellular axon guidance cues and chooses between different extracellular substrates upon which to migrate. Recent work has also identified a variety of intracellular signaling pathways by which these cues induce cytoskeletal rearrangements, but the proteins connecting signals from cell surface receptors to actin cytoskeleton have not been clearly determined. Dg is a good candidate for linking receptor signaling to the remodeling of the actin cytoskeleton and thereby polarizing the growth cone. Perturbation of Dg-Dys complex causes phenotypes that resemble Nck/Dock-Pak-Trio axon path-finding phenotypes, suggesting that Dg may be one of the key players in Nck/Dock signaling pathway for axon guidance and target recognition in Drosophila (Shcherbata, 2007).
Interestingly, Insulin receptor-tyrosine kinase (InR) mutants also show similar phenotypes to those of Nck/Dock signaling in photoreceptor axon path-finding and these two proteins show genetic and biochemical interactions. These data have led to speculations of mammalian InR acting in conjunction with Nck/Dock pathway in learning, memory and eating behavior. The current data now add Dg-Dys complex to this pathway; similar to what is seen in the case of Dg and Dys photoreceptor mutants, InR mutants show no obvious defects in patterning of the photoreceptors. However, the guidance of photoreceptor cell axons from the retina to the brain is aberrant. Furthermore, genetic and biochemical evidence suggests that InR function in axon guidance involves the Dock-Pak pathway rather than the PI3K-Akt/PKB pathway. Independently, biochemical interaction between Nck/Dock and Dg has been reported supporting the hypothesis that InR, Dg and Nck/Dock interaction regulates Dg-Dys complex. Furthermore, Dg interacts genetically with InR and Dock in photoreceptor axon path-finding. Since Dys interacts with Dg but not with InR and Dock, it is tempting to speculate that Dg can selectively interact with either Dys or InR and Dock. One possibility is that the tyrosine kinase activity of InR could regulate the Dg-Dys interaction by tyrosine phosphorylation in the Dg-Dys binding interphase. This tyrosine phosphorylation could prohibit the Dg-Dys interaction and thereby result in rearrangements in the actin cytoskeleton. Alternatively, other components observed in Dg-Dys complex might be involved in this regulation. However, it is also possible that potential polarity defects in the Dg mutant axons result in defective InR membrane localization. Interestingly, in another cell type, the Drosophila oocyte, InR, Dg and Dys also show similar phenotypes. In addition, insulin-like growth factors (IGF) and InR are important in maintaining muscle mass in vertebrates. Further connection of InR to Dg-Dys complex comes from experiments showing that muscle specific expression of IGF counters muscle decline in mdx-mice. The work presented in this study is the first demonstration of genetic interaction between Dg and InR. Future biochemical studies should unravel the molecular mechanism of this interaction (Shcherbata, 2007).
Dg-Dys complex is required both in neural and in targeting glial cells for correct neuronal axon path-finding in Drosophila brain. These data reveal that Dg-Dys complex also has a non-cell autonomous effect on axon path-finding and suggest that Dg-Dys-controlled ECM both from neuron and glial cells regulate neuronal axon path-finding. Further experiments are required to reveal whether long-range Laminin fibers are involved in this process, as has been shown in epithelial planar polarity, or whether glial processes are observed in close proximity to the neural growth cone. Interestingly, similar phenotypes are observed with Integrin mutants, suggesting that, as in planar polarity, Integrin and Dg-Dys complex might act in concert to regulate the process of ECM organization that will regulate the cytoskeleton of the cells involved (Shcherbata, 2007).
Taken together, the phenotypes caused by Drosophila Dg and Dys mutations are remarkably similar to phenotypes observed in human muscular dystrophy patients, and therefore suggest that functional dissection of Dg-Dys complex in Drosophila should provide new insights into the origin and potential treatment of these fatal neuromuscular diseases. As a proof of principle, using Drosophila as a model, InR has now been identified as a signaling pathway that genetically interacts with Dg. Future studies are directed to unravel the molecular mechanism of Dg and InR-Dock interactions in invertebrates as well as vertebrates (Shcherbata, 2007).
To analyze the expression pattern of Dg protein, antibodies were raised against the cytoplasmic domain. Five major bands can be detected on a Western blot of wild-type embryonic extracts: 75 kDa, 105 kDa, 120 kDa, 180 kDa and 200 kDa. None of these major bands could be seen in the extracts from the deficiency embryos that completely delete the Dg locus, suggesting that all five forms are specific for Dg. Strong Dg mutants were isolated by imprecise excisions of EP(2)2241 element and by generating a transgenic line expressing a double-stranded Dg-RNA construct that destroys Dg RNA by RNAi-mechanism. In Dg248 or Dg323 mutant embryos, of the five major bands derived from the Dg locus only the 105 kDa band can be detected weakly, indicating that the level of Dg expression is highly reduced in these mutants. Furthermore, to test the specificity of the antibodies in tissue samples, the expression in the follicle cell epithelium was analyzed. A high level of Dg is observed on the basal side of the epithelium, while a lower level is detected on the apical side. This signal is absent in follicle cell clones homozygous for Dg248 or Dg323, suggesting that the signal observed with the antibody in the tissue is specific for Dg. Similarly, Dystroglycan protein level was highly reduced or patchy because of the expression of Dg-RNAi construct in follicle cells (Deng, 2003).
Laminin stripes in the basement membrane of the FE are normally organized in the same orientation as the basal actin fibers, suggesting an instructive interaction between the actin cytoskeleton and the ECM through a receptor(s). One explanation for the non-cell-autonomous role of Dg in basal actin organization is that Dg functions through organizing the Laminin ECM to affect the basal actin in the neighboring cell. To test this idea further, the orientation of Laminin stripes was examined in wild-type and the Dg mutant follicle cells. Instead of the orientation perpendicular to the AP axis seen in the wild type, overall reduction and misorganization of Laminin ECM occurs in the mutant clone and neighboring regions (Deng, 2003).
To test whether Dg is sufficient to organize the Laminin ECM, it was asked whether overexpression of Dg has any effect on Laminin localization. In stage 10 follicle cells, the majority of the Laminin staining is observed at the basal side. Noticeably, Laminin is accumulated at the lateral and apical sides of the follicle cells that overexpress Dg, which is consistent with the fact that high-level Dg expression is visible at the apical and basal surfaces of these cells. This result suggests that Dg can effectively organize the Laminin ECM in Drosophila. The dotted instead of stripe/line appearance of ectopic Laminin because of Dg overexpression is consistent with a previous report (Henry, 2001b) that Dg is required for Laminin binding, while Integrin is required for further formation of the Laminin stripe/line-like structures (Deng, 2003).
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