InteractiveFly: GeneBrief

Dystroglycan: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Dystroglycan

Synonyms -

Cytological map position - 52D2--15

Function - receptor

Keywords - cell polarity, oogenesis

Symbol - Dg

FlyBase ID: FBgn0034072

Genetic map position - 2-

Classification - N-terminal mucin-like domain; transmembrane domain; C-terminal region with WW-, SH2- and SH3-binding domains

Cellular location - surface transmembrane

NCBI links: Precomputed BLAST | Entrez Gene

The transmembrane protein Dystroglycan is a highly glycosylated central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin (see Drosophila Laminin A), agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Drosophila Dystroglycan (Dg) is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity (Deng, 2003).

Muscular dystrophies are genetic disorders that are characterized by progressive muscle degeneration. These diseases are caused by mutations in different members of the Dystrophin-associated glycoprotein complex (DGC), which is composed of multiple cytocortical, transmembrane and extracellular proteins (Burton, 2002; Henry, 1999; Winder, 2001). How these mutations cause the observed muscle defects is not fully understood and no cures for the diseases exist. In addition to muscle cells, the DGC is required in other cell types such as epithelial cells and neural cells (Durbeej, 1995; Durbeej, 1999; Williamson, 1997; Michele, 2002; Moore, 2002). Reduced expression of the DGC components is observed in breast and prostate cancers (Henry, 2001a). Dystroglycan (DG), a transmembrane protein, is a central player of the DGC. It acts as a receptor for the extracellular matrix (ECM) component Laminin (Ibraghimov-Beskrovnaya, 1992), and connects to the actin cytoskeleton through an actin-binding protein, Dystrophin (Deng, 2003).

The cellular function and regulation of the interactions of Dystroglycan remain elusive. Drosophila is an excellent model organism with which to study basic cellular functions of evolutionarily conserved genes, particularly human disease genes. The DG homolog in Drosophila has been analyzed and shown to be required for the establishment of cellular polarity (Deng, 2003).

Since Dg is highly expressed in the follicle cells, it was first asked whether Dg plays a role in establishing or maintaining epithelial morphology in this tissue. The follicle cell epithelium (FE) has a typical apical-basal polarity, with its apical side facing the germline cells. Since all follicle cells are derived from two to three somatic stem cells, mosaic analysis provides an excellent tool with which to study gene functions in epithelial development (Deng, 2003).

The FLP/FRT system was used to generate follicle cell clones of all three Dg alleles, and the Gal4/UAS-mediated RNAi technique was applied to silence DG expression in all follicle cells (tubP-Gal4/dsDG). Similar phenotypes are observed in these different Dg mutant backgrounds. Some mutant cells lost their epithelial appearance and formed multiple layers, a typical terminal phenotype for polarity defects in epithelial cells. Within the multi-layer groups, the mutant cells from the mosaic egg chambers were frequently excluded from the layer that contacts the germline cells. Discontinuity of the epithelium was also visible in egg chambers containing Dg follicle cell clones. These phenotypes are similar to loss-of-function phenotypes of crumbs (crb), dlt, dlg or lgl in follicle cells and suggest that DG is required for proper epithelial polarity. The mutant follicle cells eventually die off; clones 9-10 days after heat shock were rarely seen, while sister clones (twin spots) were readily observed (Deng, 2003).

To characterize the apicobasal polarity defect in more detail, the expression and distribution of molecular markers were examined in mutant cells that still maintained their columnar shape. In Dg follicle cell clones and tubP-Gal4/dsDG follicle cells, mislocalization of apical markers, Dlt and ß-Heavy-Spectrin (ßH-Spec) was observed. Instead of a strict apical localization, Dlt and ßH-Spec were present at both the apical and basal sides of the mutant epithelia. Dlg, a basolateral marker, exhibits a significant reduction of staining in the basolateral domain in Dg RNAi follicle cells. The function of Dg in apicobasal polarity formation is not restricted to the FE, since mislocalization of Dlt to the lateral and basal sides was also observed in the mutant epithelial cells in an antennal disc. Taken together, these results suggest that DG is required in different epithelial cells for proper formation or maintenance of apicobasal polarity (Deng, 2003).

To ask whether Dg, when overexpressed, is sufficient to interfere with the epithelial cell polarity two UAS constructs were used, the full-length DG-construct (UAS-DG) and the short construct with cytoplasmic and transmembrane domains (UAS-DGcyto). These constructs were expressed in the follicular epithelium (FE) and in the embryonic salivary glands. Both constructs expressed proteins of the expected sizes and were induced by the following Gal4 driver lines: daughterlessGal4 (daGal4), for maternal expression; elavGal4, for the salivary gland expression, and the flip-out Gal4 system for the FE expression. Similar defects in epithelial polarity were observed with all three drivers (Deng, 2003).

In wild-type salivary glands, Crb is localized to the apical membrane of the epithelium, facing the lumen of the gland, while Dg expression is undetectable. Embryos that overexpress Dg show strong ectopic Dg staining on both the apical and basolateral membranes of the salivary gland. In about 75% of these salivary glands, the expression of Crb was strongly reduced. Whereas Crb localization is disrupted by overexpression of full-length Dg (UAS-DG), it is unaffected by overexpression of the form of DG lacking the extracellular domain (UAS-DGcyto). These results suggest that the mislocalization of Crb was not due to nonspecific interference with the secretory apparatus but due to a defect in cell polarity. The lateral membrane domain was unaffected as assayed by the localization of Neurotactin, a lateral marker. As seen in the salivary glands, follicle cells that overexpressed Dg lose the apical markers, ßH-Spec and Bazooka (Baz), while normal apical localization of these proteins is observed in neighboring wild-type cells. Again, overexpression of the DGcyto-form does not cause any obvious defects in the follicle epithelial polarity (Deng, 2003).

Since Laminin A is required in the posterior follicle cells for proper oocyte polarity at stages 7-10, attempts were made to ask whether Dg functions in the germline cells to receive the polarity signal from the Laminin ECM by clonal analysis. Unfortunately, egg chambers bearing germline clones of all deletion alleles are arrested at previtellogenic stages, prior to the stage at which signaling could be detected between the posterior follicle cells and the oocyte. Therefore, the establishment of oocyte polarity in earlier stages, a process that is marked by a posterior movement of the MTOC, was examined. During these stages, a low-level expression of Dg is detected at the oocyte membrane (Deng, 2003).

To detect whether the early oocyte polarity is properly established in Dg germline clones, the localization was examined of two MTOC markers, Nod-ß-Galactosidase (Nod-ß-Gal) and ORB, which (in the wild type) are localized at the anterior of the oocyte at stage 1 and move to the posterior in later stages. Mislocalization of both markers was observed in the mutant germline clones [Nod-ß-Gal mislocalization: 60%; ORB mislocalization: 76% in Dg323; 60% Dg248]. In half of the mislocalization cases, the markers either remained in the anterior of the oocyte or surrounded the nuclei after stage 3. The remaining egg chambers exhibited diffuse staining. Compared with wild type, the staining was significantly reduced. Furthermore, no accumulation of alpha-tubulin was observed in the mutant oocytes, while normal posterior accumulation was detected in the control oocytes between stages 2 and 6. In conclusion, these data suggest that Dg is required in the early oocyte for the maintenance or translocation of the MTOC from the anterior to the posterior of the oocyte. This step is crucial in establishing AP polarity in the oocyte and the future embryo (Deng, 2003).

Although links between DG and MT cytoskeleton have been suggested (Lumeng, 1999), the linkage between DG and the actin cytoskeleton via dystrophin/utrophin is far more evident. Therefore the actin distribution was examined in the developing oocyte in the wild-type and Dg germline clones. Actin is known to be enriched at the cortex of early wild-type oocytes. Interestingly, this actin enrichment is disrupted in the Dg germline clones. In addition, 'spreading' of the ring canals normally observed in stage 1-2 oocyte is not detected in egg chambers that lack germline Dg (Deng, 2003).

At the basal side of the FE, actin filaments have a planar polarity that is perpendicular to the long axis, the AP axis, of the egg chamber. Integrins and receptor tyrosine phosphatase Lar are involved non-cell-autonomously in organizing this basal actin orientation. In an analysis of the ßH-Spec staining in follicle cells that express dsDG, it was noticed that ßH-Spec is mislocalized to the basal side of the FE to bind the basal actin fibers. Noticeably, the fibers decorated with ßH-Spec in different follicle cells appear to be oriented in a random fashion. To test whether this defect reflects problems in basal actin orientation, planar polarity of the actin arrays was analyzed in control egg chambers and in the mutant Dg follicle cell clones. Instead of normal perpendicular orientation to the AP axis, random misorientation was observed in the Dg mutant egg chambers. Moreover, the basal actin fibers in follicle cells adjacent to the mutant clones were also misoriented, revealing a non-cell autonomous requirement for DG function. Although the actin filaments are not organized perpendicular to the AP axis in the mutant cells, they align with the neighboring cells, suggesting that some communication of the orientation from one cell to the other still exists. These results suggest that Dg has a non-cell-autonomous role in organizing the actin cytoskeleton in the follicle cells, similar to other receptors such as Integrin and Lar. Losing any one of these receptors still allows some orientation transfer but the global direction is defective suggesting that multiple receptor-ECM interactions are required for precise orientation (Deng, 2003).

Thus, Dg is required cell-autonomously for polarizing two different cell types: epithelial cells and the oocyte. In Dg mutant epithelial cells, apicobasal polarity is disrupted, while in oocytes, anteroposterior polarity is abnormal. Loss of Dg function in follicle and disc epithelia results in expansion of apical markers to the basal side of the cells and overexpression results in a seemingly opposite phenotype, reduced localization of apical markers. Dg mutations in the germline, however, disturb the enrichment of the oocyte cortical actin and the movement of the MTOC to the posterior oocyte: a process that is the prerequisite for the establishment of all polarity within the egg chamber and embryo. In addition, Dg has a non-cell-autonomous effect on the planar polarity of basal F-actin in follicle cells. The non-cell-autonomous phenotype probably results from a lack of instructive interaction between the actin cytoskeleton and the ECM, since Dg is required for and sufficient in organizing Laminin in follicle cells (Deng, 2003).

Drosophila Dg contains most of the hallmarks of vertebrate DG, but is significantly longer than its vertebrate orthologs, due to a ~250 amino acid duplication in the extracellular domain. The N-terminal half of fly Dg harbors a mucin-like domain, similar to vertebrate DG, but is otherwise only weakly conserved. Since the mucin-like sugars have been implicated in Laminin binding it is interesting to note that splicing variants of Dg that lack exon 8, also lack most of this domain. In addition, altered glycosylation of DG is related to two forms of congenital muscular dystrophy (Brockington, 2001; Hayashi, 2001; Michele, 2002; Moore, 2002, and reduced expression of DG is observed in a mouse model (Ervasti, 1993) of Duchene's muscular dystrophy (Deng, 2003).

The C-terminal half of Drosophila DG is conserved with 31% identity (46% similarity). Especially well conserved are the protein-protein interaction sites in the cytoplasmic domain of DG, including the binding site for Dystrophin. Seven of the eight amino residues that are crucial for Dystrophin binding (Huang, 2000) are conserved in Drosophila. Recent studies demonstrate that phosphorylation of the tyrosine residue within the dystrophin/utrophin binding motif can interfere with binding to utrophin, leading to recruitment of SH2 domain proteins (Sotgia, 2001; Ilsley, 2002). The putative SH2-binding motif involved in this interaction is conserved in Drosophila. The third protein-protein interaction described for vertebrate DG is the binding of the SH2-SH3 adaptor GRB2. GRB2 helps initiate the Ras-MAP kinase signal transduction cascade and is involved in controlling cytoskeletal organization (Yang, 1995). However, the SH3-binding motif, thought to mediate GRB2 binding, is not fully conserved in Drosophila (Deng, 2003).

Reduced expression of DG is often associated with tumor formation, suggesting that DG can act as a tumor suppressor (Henry, 2001a). It is likely that loss of DG function in some cancers leads to abnormal cell-ECM interactions and thus contributes to progression to a metastatic state. Defects in epithelial interactions normally result in cell death, when associated with abnormal cell growth and division. However, such defects could induce metastasis. The analysis in Drosophila supports this hypothesis: lack of Dg function results in tumor-like structures and abnormal cell movement because of the lack of epithelial integrity and cellular polarity (Deng, 2003).

Reduction of Dg function expands the apical domain and overexpression of Dg reduces this domain in epithelial cells. In Dg loss-of-function follicle cell clones, a component of the Lgl-complex, Dlg, is mislocalized. This mislocalization could explain the expansion of apical markers in the clones, since Dlg and Scrib are each required for the lateral localization of each other and their function is essential to restrict the apical markers Crb and Dlt to the apical surface. Further experiments need to be carried out to distinguish whether mislocalization of Dlg is caused directly by lack of physical interaction with Dg or indirectly by lack of proper cytoskeletal arrangements (Deng, 2003).

Drosophila oocyte polarity is essential for morphogen localization and therefore for the formation of the major body axes. The establishment of oocyte polarity is a gradual process that involves multiple steps. Key events in the process are cytoskeletal rearrangements. Initially, the MTOC is present in the anterior region of an early oocyte. By stage 3, the first rearrangement has occurred and the MTOC is positioned in the posterior portion of the developing oocyte. By the end of stage 6, a signal from the posterior follicle cells has initiated a new MT rearrangement, the posterior MTOC disappears and a new anterior MTOC forms. Although this signaling pathway remains a mystery, several molecules including Laminin A have been shown to be involved (Riechmann, 2001; Deng, 2000). As for the first rearrangement, genes encoding the Drosophila Par3/Par6/aPKC-complex, Par-1, and Maelstrom are required. However, the mechanism for the MTOC movement or anchoring is not clear. Dg, similar to the Par proteins, is required in the germline for this first rearrangement step. Since Dg germline clones also exhibit a defect in cortical actin enrichment in the oocyte, it is possible that the cortical actin plays an important role in MTOC movement and/or anchoring. Alternatively, since DGC contains proteins that can interact with either actin or microtubular cytoskeletons, it could play a role in coordinating actin and microtubule functions in this process (Deng, 2003).

The fact that Dg is required for both epithelial and oocyte polarity re-iterates the idea that common strategies may exist for polarizing these two very different cell types. In addition to Dg, Par proteins also act in polarity formation in both cell types, suggesting that the Par proteins and Dg complex have functional similarities. Interestingly, Dg can affect localization of the Par complex, since one of the members, Baz (Par3), is mislocalized when Dg is overexpressed. In addition, both Par-proteins and the Dg-complex interact with molecules that can associate with either actin or microtubular cytoskeletons. Par-1 associates with Myosin II heavy chain and also phosphorylates a MT-associated protein. Dg can interact with actin through Dystrophin-like proteins. Furthermore, the mammalian Dystrophin-associated protein, Syntrophin, interacts with MT-associated proteins via a two-hybrid assay. It is possible that both Par proteins and the DG complex facilitate interactions between actin and microtubules and that these interactions between the two cytoskeletal systems are key regulators for establishment of polarity in both cell types (Deng, 2003).

Surprisingly, Dg mutant follicle cells generate actin defects in neighboring cells; the basal actin is misoriented in adjacent follicle cells. How would a defective Dg in one cell alter the dynamics of actin organization in the neighboring cell? It is proposed that the interaction between ECM and Dg is bi-directional: in one case, DG organizes the Laminin ECM architecture (Henry, 2001b; Deng, 2003), suggesting that a defect in Dg will be transmitted to a defect in ECM organization; in the other case, a defective Laminin lattice will extend to the surface of the neighboring cell and there this architectural information could be transmitted to the cellular actin cytoskeleton by Dg in the neighboring cell (Colognato, 1999). Three pieces of evidence support this hypothesis: (1) Drosophila Dg is capable of organizing the Laminin lattice; (2) the Laminin lattice in the basal side of follicle cells is oriented in the same orientation as the underlying basal actin lattice; (3) Laminin, similar to Dg, could also be involved in basal actin organization. Interestingly, two other Laminin receptors, Integrin and Lar, are also required for basal actin planar polarity in follicle cells. It is possible that one connector alone would not give enough rigidity or allow enough flexibility in relaying information between the ECM and the basal actin (Deng, 2003).

In summary, Drosophila Dg has two separate functions in cell polarity: cell autonomous in apical-basal and anteroposterior polarity, and non-cell-autonomous in planar polarity. Future research aims to take advantage of Drosophila as a model organism to genetically dissect the partners of DG in these two functions (Deng, 2003).


Dystroglycan down-regulation links EGF receptor signaling and anterior-posterior polarity formation in the Drosophila oocyte

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).

Drosophila miR-9a targets the ECM receptor Dystroglycan to canalize myotendinous junction formation

Establishment of intercellular interactions between various cell types of different origin is vital for organism development and tissue maintenance. Therefore, precise timing, expression pattern, and amounts of extracellular matrix (ECM) proteins must be tightly regulated. Particularly, the ECM is important for the development and function of myotendinous junctions (MTJs). This study finds that precise levels of the ECM receptor Dystroglycan (Dg) are required for MTJ formation in Drosophila and that Dg levels in this process are controlled by miR-9a. In the embryo, Dg is enriched at the termini of the growing muscles facing the tendon matrix and absent from miR-9a-expressing tendons. This gradient of Dg expression is crucial for proper muscle-tendon attachments and is adjusted by miR-9a. In addition to Dg, miR-9a regulates the expression of several other critical muscle genes, and it is therefore proposed that during embryogenesis, miR-9a specifically controls the expression of mesodermal genes to canalize MTJ morphogenesis (Yatsenko, 2014).

To achieve successful results, despite the extreme fluctuation of internal cues, genetic background, and external conditions, embryonic development must be stabilized. Coordinated transcription factor networks are prominent regulatory features of cell fate establishment during embryonic development and adult life. It is now becoming evident that, in conjunction with transcription factors, at least three epigenetic elements help to form a reciprocal regulatory circuit to maintain cell identity and differentiation: chromatin structure, DNA methylation, and microRNAs (miRNAs). miRNAs, based on their paradoxical properties, e.g., being highly evolutionarily conserved, but not essential, have been proposed to play a role in generating biological robustness as canalization factors to buffer gene expression against perturbation or variability. As canalization factors, miRNAs have previously been shown to liquidate transcripts resulting from aberrant gene expression or leaky splicing The previously described in vivo cases of miRNA-based regulation mostly are examples of simple pairs, in which one miRNA is targeting one gene. However, increasing evidence suggests that functionally related genes are clustered at the level of DNA sequence, histone modifications, chromatin loops, or chromosome territories and are under similar transcriptional control. Taking into account that, first, the gene expression in general is a noisy process that incidentally allows leaky expression of 'neighboring' genes and, second, that one miRNA can regulate multiple genes, it is logical to propose that as a canalization factor one miRNA should be capable of regulation of multiple genes that are involved in the same signaling network. Therefore, this study investigated whether this type of miRNA-based regulation, employed to confer robustness of embryonic development, actually takes place (Yatsenko, 2014).

Assembly of muscle tissue requires communication between mesoderm-derived myotubes and ectoderm-originated epidermal muscle-attachment cells or tendons. Since tendon cells invaginate into mesoderm, some mechanism that reassures the robustness of their identity must exist. Initially, the pretendon cells send signals to the myotubes and direct myotube attraction and adhesion to their target cells; subsequently, the muscle cells communicate a reciprocal signal to the epidermal muscle attachment cells, initiating their terminal differentiation into tendon-like cells. This suggests the necessity of a microenvironment that will allow for both a rapid and precise signal transduction between these ectodermally and mesodermally derived cell types (Yatsenko, 2014).

Importantly, the process of muscle guidance and attachment in Drosophila is remarkably similar to that of vertebrates, as both are greatly dependent on the extracellular matrix (ECM) gradient that is established through differential recruitment and clustering of transmembrane receptors by extracellular-presented signaling molecules. During Drosophila embryonic development, the initial determination of myoblast fate is controlled by high expression of the basic-helix-loop-helix protein Twist; after the myoblast division and fusion, multinucleated myotubes are formed. At stage 12-14, myotubes undergo a substantial transformation: not only do they continue to grow through cell fusion, but they also change their shape and form elongated filopodia at the leading edge that help to find their proper tendon cells in the epidermis. At the same time, the tendon cells also undergo a series of cell shape rearrangements, including apical constriction and apical-basal elongation, which results in the formation of epidermal furrows. When myotubes reach their targets, the surface of the myotube facing the tendon cells loses filopodia and multiple adhesion complex molecules accumulate at the muscle attachment site toward the tendon cell in order to form a stable adhesion complex. While the signaling crosstalk between these cell types has been extensively studied, it is not clear whether a genetic program exists that would aid cells that are subjected to similar spatiotemporal signaling to undergo distinct developmental programs. The role of miRNAs in this process, vital for muscle physiology, has not yet been analyzed; however, vigorous cell rearrangements and cell fate specifications that take place during establishment of the muscle attachment suggest a need for a mechanism that enhances robustness of the process by attenuating leaky transcripts (Yatsenko, 2014).

This study found that Drosophila miR-9a is involved in canalization of myotendinous junction (MTJ) assembly. Deficiency of miR-9a affects embryonic survival, a phenotype that can be rescued by specific expression of this miRNA in tendon cells. The survival of miR-9a mutants depends on the speed of embryonic development that reciprocally correlates with transcriptional noise. miR-9a is expressed in epidermally derived tendon cells, while many miR-9a predicted targets are essential muscle genes that are misregulated due to miR-9a loss and gain of function. Moreover, exogenous expression of miR-9a in mesoderm completely abolishes muscle formation. Therefore, it was hypothesized that miR-9a adjusts tendon cell differentiation by preventing misexpression of muscle genes resulting from stress or aberrant transcription. To prove this hypothesis, putative miR-9a targets were expressed in tendon cells, and ectopic heartless (htl), wishful thinking (wit), and Dystroglycan (Dg) in tendons was found to cause muscle attachment and embryonic lethality phenotypes similar to those found in miR-9a mutants. In particular, it was found that the muscular-dystrophy-associated ECM receptor, Dg, is regulated posttranscriptionally via the miRNA, miR-9a. During the early embryonic stages, Dg is present in all epidermal cells; however, for proper assembly of muscle attachment sites it is essential that Dg is eliminated from epidermally derived tendon cells, with miR-9a modulating the precision of this expression. Dg establishes a specific ECM gradient that influences muscle-tendon signaling; therefore, its differential localization is crucial for proper muscle-tendon attachments and is adjusted by miR-9a. When Dg is misexpressed in tendon cells, the composition of the tendon matrix is affected, resulting in aberrant muscle attachments and embryonic death (Yatsenko, 2014).

This study shows that the muscular-dystrophy-associated ECM receptor Dg can be posttranscriptionally regulated by miR-9a. During embryogenesis, both miR-9a and Dg have dynamic expression patterns that become mutually exclusive in the regions of muscle-tendon connections. Dg protein is present in all ectodermal cells, except for the ones that are differentiating into epithelial tendon cells and are also expressing miR-9a. The data show that the elimination of Dg from tendon precursor cells is required for accurate muscle-tendon matrix assembly. The miR-9a ensures that Dg is not misexpressed in tendon precursors due to leaky transcription, as these epidermal cells invaginate into and reside within the mesoderm (Yatsenko, 2014).

Embryonic development is an extremely dynamic process in which rapid cell specifications and rearrangements take place, features indicative of the need for stabilization. miRNAs have been implicated in stabilization of biological robustness in different animal systems. miRNAs are involved in the stabilization of the process of muscle-tendon attachment in the developing Drosophila embryo. In particular, the data imply that miR-9a acts as a backup mechanism in tendons to diminish the effects of leaky expression of a group of muscle genes. When two adjacent cells have different cell fates, evolutionarily it would make a lot of sense for a canalization factor in one cell type to regulate multiple genes critical for the differentiation of the other cell type. Apparently, many essential muscle differentiation genes are miR-9a predicted targets, and their ectopic expression in ectodermal tendon cells causes embryonic lethality and abnormal MTJs. Moreover, exogenous expression of miR-9a in the mesoderm completely abolishes muscle formation. With this in mind, it was hypothesized that miR-9a specifically acts as a guardian to prevent aberrant muscle gene expression in the epidermal tendon precursor cells (Yatsenko, 2014).

It has already been shown that mir-124 and miR-9a act to canalize nonneuronal versus neuronal fates. Genes expressed in the nervous system are highly enriched for miR-9a binding sites and the current data show that multiple muscle genes also have miR-9a target sites. Since miR-9a is ectoderm specific and genes expressed in ectodermal tissues avoid miR-9a sites, the findings insinuate that miR-9a can act as the key ectodermal canalization factor that protects ectodermal cell fate by repressing genes of the sibling tissues (such as muscle and nervous). This should reinforce the robustness of ectodermal cell differentiation. It would be interesting to find in the future if miRNAs that canalize mesodermal or endodermal fate exist and to address the question of whether there are more examples of the miRNAs that target multiple genes from the same functional network (Yatsenko, 2014).

One of the muscle genes that this study showed is a bona fide miR-9a target is the ECM receptor, Dg. The transmembrane protein Dg has a distinct expression pattern at the MTJ. It is present at the membrane of the developing muscle and is enriched at the myotube ends; however, it is absent from the tendon cell membranes. Similarly, a restricted expression pattern of Dg is required for neuromuscular junction (NMJ) establishment in vertebrates (Xenopus), with Dg being present at the entire muscle membrane and showing enrichment at the NMJ site, where it acts as a sink for the ECM component agrin, preventing its binding to muscle-specific kinase (MuSK). At the site of nerve contact, in the absence of Dg, agrin can bind to MuSK, allowing acetylcholine receptor aggregation and synaptic development Thus, due to the distinct expression patterns in cells that form connections via the ECM, Dg is able to establish the ECM gradient, which is also essential for proper formation of MTJs in the developing Drosophila embryo. It would be interesting to investigate if there is a regulatory molecule that is differentially distributed between muscle and tendon due to specific binding to Dg at the MTJ (Yatsenko, 2014).

The extracellular environment of the cell is a complex organization of ECM receptors, matrix proteins, and the regulatory molecules that reside in it. Also, it continuously changes during development and allows rapid communication between different cells to coordinate tissue formation. Therefore, changes in the composition of the ECM can have a profound effect on an organism's development. This study shows that miR-9a-based regulation of Dg is needed to adjust the ECM composition at the MTJ. Regulation of the affinity of the transmembrane adhesion receptor integrins has a key role during development as it generates strong adhesion of cells to the insoluble ECM. This study shows that, at the MTJ, Dg also acts as a receptor regulating ECM gradient at the tendon matrix, since Dg levels affect the amount of the ECM protein Lan. In addition, Dg can modulate expression of a key ECM receptor, βPS Integrin. These data are consistent with previous findings revealing a regulatory pathway between the DGC and integrin receptors and lends the idea that Dg is involved in selective regulation of integrin gene expression Moreover, integrin overexpression alleviates the development of muscular dystrophy phenotypes in mdx mice, supporting the possibility that Dg and integrin compensate for each other in mediating cell-ECM adhesion. Additionally, this study showed that this regulation can be cell nonautonomous, since abnormal Dg levels, through modification of Lan amounts, affect integrin expression in the neighboring cells. In intestinal epithelial cells, the DGC coprecipitates with β1-integrin, suggesting a possible direct interaction among these proteins where the strength of this interaction depends on the Lan type. Moreover, it has been shown that increased Lan expression can ameliorate muscular dystrophy in mice. These results, on the one hand, support the findings that alterations in Lan levels influence the expression of the ECM receptors but, on the other hand, pose an interesting question of why, depending on the animal's genetic background (dystrophic or not), the increased levels of Lan have positive or negative effects on MTJs and muscles. The beneficial influence of Lan was seen so far only when it was upregulated in dystrophic animals: muscular dystrophy mdx mouse (dystrophin deficient), dyw-/- merosin-deficient congenital muscular dystrophy mouse model (Lan deficient), and muscular dystrophy zebrafish model (Dg reduced). All above muscular-dystrophy-related components per se are required for accurate Lan localization and distribution, suggesting that restoration of Lan levels in the ECM has favorable effects on dystrophic muscle maintenance. Previous studies did not address what would happen if Lan were overexpressed in the otherwise normal background or they were differentially upregulated in muscle or tendons. Since elegant studies on the role of Lan-111 in muscle development and maintenance (Van Ry, 2014) propose Lan protein therapy as a treatment option for muscular dystrophy patients, it would be important in the future to study, using different models, the effects of differential Lan expression on muscle-tendon attachments during development and to determine the levels that can be tolerated without induction of deleterious effects on muscle maintenance and tendon attachments during adulthood (Yatsenko, 2014).

Taken together, previous studies and the current findings show that the amounts and types of the ECM receptors affect ECM constitution and govern its remodeling, and then via 'dynamic reciprocity' the ECM readjusts intracellular signaling, gene expression, and morphology of the cells and tissues. The crosstalk between tendons and muscles depends on differentially expressed ECM receptor Dg that, together with integrins, helps to establish the ECM gradient. The information about the tendon matrix composition is communicated back to the muscles and tendon cells that readjust their ECM receptor expression profiles in order to reinforce and stabilize the MTJ. Providing a link between the ECM and cytoskeleton, Dg acts as a vital signal-transducing element that allows for communication between the cell's outer environment and inner milieu. In vertebrates, Dg is implicated in multiple biological processes: for example, formation of spatiotemporally regulated microenvironments necessary for muscle fiber morphogenesis at the MTJ. In Drosophila, in addition to its function in muscle maintenance, Dg is involved in control of neuron behavior; modulation of the concentration of postsynaptic and synaptic proteins, in particular the ECM component Lan at the neuromuscular junction, and regulation of miRNA expression profiles. The currently increasing amount of research on the diverse roles of Dg during development demonstrates its critical role in multiple developmental circuits, suggesting that there is a necessity for precise and dynamic regulation of Dg levels. Despite the vast data about posttranslational regulation of Dg activity, Dg posttranscriptional regulation has not been studied. The current data show that Dg can be regulated by miRNAs and this regulation has an important functional role at the MTJ. Since the human homolog of Drosophila Dg (Dag1) also contains multiple predicted miRNA biding sites, it would be important to study if miRNAs also play a role in regulation of Dg in mammals. Even though there are numerous studies in vertebrate models indicating that MTJ assembly affects muscle development, the role of aberrant MTJ in muscle maintenance and function in muscular dystrophy patients is greatly underappreciated; therefore, understanding of the miRNA-based mechanisms controlling the ECM assembly at the MTJ may suggest new directions for muscular dystrophy research (Yatsenko, 2014).

Protein Interactions

Perlecan and Dystroglycan act at the basal side of the Drosophila follicular epithelium to maintain epithelial organization

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).

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

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).

A putative Src homology 3 domain binding motif but not the C-terminal dystrophin WW domain binding motif is required for dystroglycan function in cellular polarity in Drosophila

The conserved dystroglycan-dystrophin (Dg·Dys) complex connects the extracellular matrix to the cytoskeleton. In humans as well as Drosophila, perturbation of this complex results in muscular dystrophies and brain malformations and in some cases cellular polarity defects. However, the regulation of the Dg-Dys complex is poorly understood in any cell type. This study finds that in loss-of-function and overexpression studies more than half (34 residues) of the Dg proline-rich conserved C-terminal regions can be truncated without significantly compromising its function in regulating cellular polarity in Drosophila. Notably, the truncation eliminates the WW domain binding motif at the very C terminus of the protein thought to mediate interactions with dystrophin, suggesting that a second, internal WW binding motif can also mediate this interaction. This hypothesis was confirmed by using a sensitive fluorescence polarization assay to show that both WW domain binding sites of Dg bind to Dys in humans (Kd = 7.6 and 81 microM, respectively) and Drosophila (Kd = 16 and 46 microM, respectively). In contrast to the large deletion mentioned above, a single proline to an alanine point mutation within a predicted Src homology 3 domain (SH3) binding site abolishes Dg function in cellular polarity. This suggests that an SH3-containing protein, which has yet to be identified, functionally interacts with Dg (Yatsenko, 2007; full text of article).

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Genetic screens have been performed using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, this study has analyzed the DGC modifiers' function in the developing Drosophila brain and eye. Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, the function of previously screened components and their genetic interaction with the DGC in this tissue were determined. This study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. It was determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. These data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton (Marrone, 2011).

The roles that Dys and Dg play in disease have been apparent for some time since their disruption or misregulation has been closely linked to various MDs. Dg depletion results in congenital muscular dystrophy-like brain malformations associated with layering defects and aberrant neuron migration. These defects arise due to extracellular matrix protein affinity problems that influence neuronal communication and result in learning and memory defects. Similar to brain layer formation, the migration of R1-R6 growth cones into the lamina occurs in a similar manner where glia cells that migrate from progenitor regions into the lamina provide a termination cue to innervating axons. In Drosophila Dys and Dg are expressed in the CNS, PNS and visual system and both proteins are required for proper photoreceptor axon guidance and rhabdomere elongation. This work has identified novel components implicated in the process of eye-neuron development. Moreover, it was found that Nrk, Mbl, Cam and Capt genetically interact with Dys and/or Dg in visual system establishment (Marrone, 2011).

The proteins Mbl, Capt, Cam, Robo, Lis1 and Nrk have been shown previously to be associated with the nervous system, and this study has additionally found that mutations in chif, SP2353, CG34400 and vimar cause abnormal photoreceptor axon pathfinding and/or differentiation phenotypes. Lis1 has been shown to bind microtubules in the growth cone, and the human Lis1 homologue is important for neuronal migration and when mutated causes Lissencephaly, a severe neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. This study has found that Lis1RNAi/GMR-Gal4 mutants have abnormally formed lamina plexuses, shortened rhabdomeres, and retinal vacuoles. Chif has been shown to regulate gene expression during egg shell development and is related to a DNA replication protein in yeast. The human ortholog for SP2353 (AGRN) is involved in congenital MD development. Drosophila SP2353 is a novel agrin-like protein that contains Laminin G domains, which makes it a potential new extracellular binding partner for Dg. CG34400 encodes for a protein homologues to human DFNB31 (Deafness, autosomal recessive 31) that causes congenital hearing impairment in DFNB31 deficient people and mouse whirlin, that causes deafness in the whirler mouse. Hearing loss has been as well demonstrated in association with various forms of muscular dystrophy. Vimar has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Marrone, 2011).

Mbl is a Drosophila homologue of the human gene MBNL1. Mutations of this gene cause myotonic dystrophy and are associated with the RNA toxicity of CUG expansion diseases protein. This study shows that Mbl deficiency results in similar phenotypes to Dys and Dg loss of function, and to specifically interact with Dys in axon projections which is in accord with the Dys specific interaction seen in muscle. Dys has multiple isoforms, and the variability of DMD patients to have mental impairment has been linked in part to small Dys isoform mutations, which leads to speculation that Dys is a target for Mbl mediated splicing (Marrone, 2011).

Interestingly, Mbl isoforms have been demonstrated to regulate splicing of α-actinin, which belongs to the spectrin gene superfamily that also includes dystrophins. α-actinin and Capt, the Drosophila homologue of Cyclase-associated protein (CAP) are actin-binding proteins in the growth cone. Capt was first identified in yeast and is highly conserved throughout eukaryotic evolution. The main known function of Capt is to act in the process of actin recycling by working in conjunction with Actin Depolymerization Factor (ADF a.k.a. Cofilin) to help displace Cofilin from G-actin during depolymerization. It has already been reported that ADF/Cofilin has a role in retinal elongation. The actin cytoskeleton is a major internal structure that defines the morphology of neurons, and Capt has already been shown to be required to maintain PNS neuronal dendrite homeostasis in Drosophila via kinesin-mediated transport. Additionally, Capt has been found to lead to excessive actin filament polymerization in the eye disc and to cause premature differentiation of photoreceptors. The rate of axon projection is much slower than the rate of microtubule polymerization during axonal growth, implying that depolymerization/polymerization of actin is important during pathfinding. This study has also shown that Capt interacts with Dys and is necessary for proper projection of photoreceptor axons in the developing brain, and when absent, eyes develop with abnormal rhabdomeres. Furthermore, captRNAi mutants exhibit overgrowth of photoreceptor axons, and it is believed that a possible explanation for this is improper turnover of actin (Marrone, 2011).

Importantly, proteins that can be regulated by Ca2+ to organize actin filament bundles and to promote filament turnover include α-actinin and (ADF)/Cofilin, respectively. Cam functions as an intracellular Ca2+ sensor, and when Ca2+-Cam was selectively disrupted in a subset of neurons in Drosophila embryos, stalls in axon extension and errors in growth cone guidance resulted. Actin turnover is highly regulated by Ca2+ levels, and many proteins are Ca2+-mediated to regulate motility and axon guidance. The results and those from prior studies suggest that Cam is a major functional player of Ca2+ regulation in growth cones. Since it was shown here that mutations in Cam and capt have similar phenotypes in photoreceptor axon pathfinding and rhabdomere development, it is postulated that actin dynamics is the link between these two proteins and the phenotypes described here. Due to the importance of Cam for actin dynamics, its interaction with both Dg and Dys suggests that the DGC coordinates the actin cytoskeleton in the developing eye (Marrone, 2011).

The last gene identified in this work is Nrk. Recently various kinases, channels and other enzymes have been shown to associate with the DGC, although only a few of these interactions have been confirmed in vivo. Since Nrk is a component found to interact with Dys in photoreceptor axon pathfinding, it is most likely that it functions as a receptor to sense guidance cues rather than as a molecule affecting actin cytoskeletal rearrangement. The data here hint that Dg and Nrk could be two receptors integral to transferring signals important for neuronal layering (Marrone, 2011).

It is concluded that dynamic rearrangement of the actin cytoskeleton is crucial for the central and peripheral nervous system establishment, which depends on proper neuron migration and differentiation. This process requires not only the cell autonomous regulation of neuron motility, but also the interaction between the migrating cell and its underlying substrate. This interaction is often dependent on the signaling transduced via the ECM. The DGC and other factors are believed to be mediators of actin dynamics in growing axons and during neuronal cell morphogenesis, and this study found components that interact with Dys and/or Dg in both of these activities (see The DGC coordinates actin cytoskeleton remodeling). Additionally, disruption in gene expression of these components results in the same phenotypes seen with Dys and Dg mutants in the developing and adult eye. The data lead to the conclusion that the DGC is involved in signaling to cause cytoskeletal rearrangement and actin turnover in growth cones. Since many cases of muscular dystrophies are associated with mental retardation, it is believed that it is important to understand the role of the DGC in axon migration because understanding of this process could aid in finding an adequate therapy for this aspect of the disease's physiology. Since the human brain continues to develop well after gestation, and evidence shows that nerves maintain plasticity throughout an individual's lifespan, therapies could be devised that reverse these defects after birth (Marrone, 2011).


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).

Medioni, C., et al. (2008). Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation. J. Cell Biol. 182(2): 249-61. PubMed Citation: 18663140

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. This study analyzed the formation of the Drosophila cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. This study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, a genetic pathway is identified in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data a model is proposed for Drosophila cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. It is suggested that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis (Medioni, 2008).

The analysis provided here establishes the cellular basis of lumen formation of the Drosophila cardiac tube. The lumen of the tube is formed from the migration of two bilateral rows of polarized cardioblasts (CBs), which join at the dorsal midline. One main result of this study is the characterization of two types of cell membrane domains directly involved in lumen formation, the luminal domains (L domains) and adherent domains (J domains). Adherens junctions that are responsible for sealing the tube originate from the J domain, whereas the membrane walls of the lumen originate from the L domain (Medioni, 2008).

Remarkably, the L domain displays characteristics of basal membranes, revealed by expression of molecular markers normally associated with a basal membrane. Furthermore, specification of the L and J domains takes place very early in the tubulogenesis process, significantly before coalescence of the bilateral rows of CBs at the dorsal midline. Finally, during CB migration, membrane domains undergo remodeling, concomitant with profound cell shape changes. These two cellular processes appear to be closely connected and are probably regulated by the cellular environment of the CBs composed by the overlying dorsal ectoderm and the amnioserosa cells. These interactions will be investigated in a future work (Medioni, 2008).

The mechanism of Drosophila cardiac lumen formation reported in this study is thus notably different from the previously described mechanisms of epithelial tubulogenesis. In epithelial tubulogenesis, after receiving a polarization signal that sets apicobasal polarity, the cells or group of cells establish a basal surface and generate vesicles carrying apical membrane proteins. The vesicles are targeted to the prospective apical region, where they fuse with the existing membrane or with each other to form a lumen. Finally, continued vesicle fusion and apical secretion expand the lumen (Medioni, 2008).

In contrast, constriction of the leading edge domain during cardioblast (CB) migration, precise control of cell shape changes, and delimitation of specific membrane domains appear to be the driving forces of Drosophila cardiac lumen formation. Cells forming the dorsal vessel have the features of migrating cells. In contrast to epithelial tubulogenesis, which involves apical membrane domains, the apex of polarized CBs constricts, forms adherens junctions, and consequently does not constitute the L domain. Instead, the luminal membrane domain possesses basal membrane characteristics, as is also the case in endothelial cells. Moreover, the size of the cardiac lumen is determined by the isotropic growth of CBs, and not, as in other models, by anisotropic extension of the L domain involving apical membrane vesicles.

Finally, the genetic control of the process involves gene products of slit, robo, how, and dg, which are not known regulators of lumen formation in epithelial tubes (Medioni, 2008).

This study leads to the identification of a genetic pathway, including slit, robo, how, and dg, controlling membrane domain specification and dynamics during cardiac lumen formation. Within this pathway, Slit appears to play a central role and a previously unrecognized function in cell morphogenesis (Medioni, 2008).

Several studies have shown that Slit-Robo function is essential for cardiac tube formation by controlling the proper migration, cohesion, and alignment of the two rows of CBs. The results reported in this study show that Slit is also involved in the correct specification of the L domain and its distinct features with respect to the adjacent J domains. Activation of Slit-Robo signaling determines the respective size of these two types of domains (Medioni, 2008).

The data suggest that activation of this pathway inhibits the formation of adherens junctions. This possibility is supported by recent findings in chick retina cells, where activation of the Slit-Robo pathway leads to the inactivation of β-catenin (Arm in Drosophila), resulting in the dissociation of N-cadherin from the junctional complex and preventing the formation of adherens junctions. Consistent with these observations, DE-Cad (Shg) is expressed in the J domains of CBs and is required for cardiac tube morphogenesis. Moreover, slit and shg show genetic interaction in cardiac tube morphogenesis. In the absence of slit function, the size of the L domain is strongly reduced, suggesting that Slit-Robo signaling prevents the formation of Arm/DE-Cad-mediated adherens junctions in the L domain (Medioni, 2008). How encodes an RNA-binding protein involved in mRNA metabolism, and given its exclusive nuclear localization at this stage of development, How may regulate slit splicing. In the absence of the How protein, the gene splicing could be affected, producing a Slit protein unable to correctly localize at the L domain. This hypothesis is consistent with the fact that expression of wild-type Slit in CBs can suppress the effect of how18 mutation on Slit localization and lumen formation. How has also recently been shown to regulate the splicing of neuronal membrane proteins such as neurexin. Moreover, How is expressed in the midline glia with Slit and Dg, suggesting that interaction among these three genes is part of a general mechanism by which junctions and lumen formation are controlled (Medioni, 2008).

A model is preposed for the genetic control of lumen formation in the cardiac tube. According to this model, How could directly regulate Slit by controlling its splicing and targeting the luminal compartment. Consequently, Slit binds to Robo activating the signaling pathway, which in turn inhibits Arm/DE-Cad-mediated adherens junction formation in the luminal compartment, leading then to the specification of distinct J and L domains. Parallel to this, activation of Slit-Robo signaling modulates the actin cytoskeleton and triggers CB cell shape remodeling required for lumen formation and growth. As How is able to act on many targets, it could also directly control the actin cytoskeleton by targeting an actin-binding molecule. Concerning Dg, it was shown that dg and slit genetically interact; however, overexpression of Slit does not rescue the lumen phenotype observed in dg mutants, contrasting with how mutations. Thus, it is proposes that Dg could regulate Slit localization at the L domain by its function in the specification and differentiation of the L domain, and therefore acts parallel to slit for lumen formation, behaving, for example, as a coreceptor of Robo. In addition, Dg could control actin cytoskeleton dynamics via its interaction with Dystrophin (Medioni, 2008).

The data clearly show that cardiac tube formation in Drosophila differs substantially from all other described mechanisms of tubulogenesis. Is this mechanism of tubulogenesis unique or is it shared with other organs and/or other organisms? Primary vasculogenesis in vertebrates leads to the formation of large median vessels, the dorsal aorta and the cardinal vein. These vessels arise from migrating mesenchymal cells of the lateral mesoderm, termed angioblasts, that are organized in bilateral groups of cells. Angioblasts migrate toward the midline as a cohort of cells, coalesce, and form a lumen. At this stage, as in flies, cells around the lumen show a crescentlike shape and an extracellular matrix is deposited at the internal face of luminal membranes. Similar cellular events are also observed during the formation of the primitive cardiac tube in vertebrates, suggesting that a common mechanism of tubulogenesis might exist for all tubes that arise from the coalescence of migrating bilateral mesenchymal cells (Medioni, 2008).

The Drosophila cardiac tube, or dorsal vessel, shares many similarities with the cardiovascular system of vertebrates. A significant fraction of genes expressed in the Drosophila cardiac tube are also annotated to be expressed in vertebrate blood vessels, suggesting that vasculogenesis and dorsal vessel morphogenesis might share common genetic regulators (Medioni, 2008).

Finally, components of the genetic pathway controlling cardiac lumen formation that are described in this study have potentially similar functions in vertebrates. It has been shown that numerous proteins involved in axon guidance are expressed in vertebrate blood vessels. In particular, the Slit-Robo signaling pathway has been involved in promoting tumor vascularization, hSlit2 being expressed in tumor cells and hRobo1 in endothelial cells. Moreover, mSlit3 has been implicated in mammalian cardiogenesis, and Quaking, the mouse homologue of How, is required for vasculogenesis and expressed in the developing heart (Medioni, 2008).

In conclusion, analysis of CB morphogenesis during development of the Drosophila cardiovascular system provides evidence for a new model of biological tube formation. It is proposed that this mechanism might also be used for the formation of the large median vessels and primitive heart tube in vertebrates (Medioni, 2008).


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).

Dystroglycan and protein O-mannosyltransferases 1 and 2 are required to maintain integrity of Drosophila larval muscles

In vertebrates, mutations in Protein O-mannosyltransferase1 (POMT1) or POMT2 are associated with muscular dystrophy due to a requirement for O-linked mannose glycans on the Dystroglycan (Dg) protein. This study examined larval body wall muscles of Drosophila mutant for Dg, or RNA interference knockdown for Dg and find defects in muscle attachment, altered muscle contraction, and a change in muscle membrane resistance. To determine if POMTs are required for Dg function in Drosophila , larvae mutant for genes encoding POMT1 or POMT2 were studied. Larvae mutant for either POMT, or doubly mutant for both, show muscle attachment and muscle contraction phenotypes identical to those associated with reduced Dg function, consistent with a requirement for O-linked mannose on Drosophila Dg. Together these data establish a central role for Dg in maintaining integrity in Drosophila larval muscles and demonstrate the importance of glycosylation to Dg function in Drosophila. This study opens the possibility of using Drosophila to investigate muscular dystrophy (Haines, 2007).

The dystrophin glycoprotein complex (DGC) is known to play a central role in maintaining integrity in a variety of different muscle types. This study established that Dg is required in Drosophila larval muscles to maintain integrity. Changes were identified in muscle contraction associated with reduced Dg function. Second instar Dg248 larvae had short and wide muscles, and a decrease in sarcomere size, together indicating that the muscles are hypercontracted. Among larvae null for Dg it was found that sarcomere size was more variable between individuals, with some muscles having smaller sarcomeres and others larger. Among 3rd instar larvae knockdown for Dg changes were observed in sarcomere size. When an RNAi construct against Dg was driven with the ubiquitous driver P-tub-Gal4, it was found that sarcomeres were consistently smaller than controls. Driving the RNAi construct with the mesoderm driver 24B-Gal4 resulted in a variable phenotype, with both small and large sarcomere being observed. This phenotype shifted to exclusively larger sarcomeres when these larvae developed at 30°C, likely due to a greater decrease of Dg protein. Because the large sarcomere phenotype was seen in null Dg larvae and with strong RNAi knockdown compared with smaller sarcomeres in the Dg248 individuals and with weaker RNAi knockdown of Dg, it is concluded that the large sarcomere phenotype is related to more severe loss of Dg than with hypercontraction (Haines, 2007).

Because changes were observed in muscle sarcomere size with RNAi driven with the ubiquitous driver and the mesoderm driver but not with the pan-ectodermal driver, it is concluded that the sarcomere size changes are due to loss of Dg in muscles. Together these results demonstrate that reduced levels of Dg alter muscle contraction and that Dg plays a role in Drosophila muscles (Haines, 2007).

Consistent with these findings is that both hypercontraction and overstretching of sarcomeres is seen in vertebrate dystrophic muscles before they progress to a more advanced degeneration phenotype. A tendency to hypercontract is also associated with disruption of DGC proteins in Caenorhabditis elegans. These findings suggest that contractile changes are a common early response to lack of Dg function in different types of muscle. Further studies will focus on understanding whether these changes in muscle contraction stem from developmental changes in the muscles or are the result of early changes associated with muscle degeneration (Haines, 2007).

In vertebrate muscles lacking DGC components, the mechanisms leading to muscle dystrophy remain unclear; however, membrane fragility is likely involved. Mechanical stress from muscle contraction is thought to lead to membrane microlesions and compromised muscle membrane function. Electrophysiological analysis showed an increase in membrane resistance and greater EJP amplitude in 24B-Gal4::UAS-Dg-i muscles. The increase in passive membrane resistance could be due to down-regulation of the leakage channel activity by the muscle. The increased resistance would help to maintain excitation-contraction coupling. Interestingly, a similar increase in muscle membrane resistance has been reported in mice mutant for dystrophin (Haines, 2007).

In vertebrate cardiac muscle T-tubule-associated Dys, Dgβ and Lam have been reported. This location differs from the sarcolemma localized DGC found in vertebrate skeletal muscle. The inherited muscular dystrophies are associated with dilated cardiomyopthy, and differences in the degree of dysfunction between cardiac and skeletal muscle in individuals with muscular dystrophy has lead to the suggestion that the DGC may have alternative cellular roles in these muscle types. The finding that Dg and Laminin are T-tubule associated in Drosophila larval muscles suggests these Drosophila muscles may provide a good model for investigating the cellular function of T-tubule-associated DGC. Studying the role of the DGC in different types of muscle will increase overall understanding of how this complex functions at both a molecular and cell biological level (Haines, 2007).

Laminin and adhesion molecules such as intergrins play important roles in muscle attachment in Drosophila . Muscles detach from the epidermis and round up or stick nonspecifically to other muscles or the ECM in Drosophila carrying mutant alleles of these genes. Given the role of Dg in ECM adhesion in other cellular contexts and the role of Lam in muscle attachment in Drosophila , the muscle attachment phenotypes observed in the Dg mutants most likely result from weakening of the connection between muscle and epidermal tendon cell to which it connects or these cells and the ECM. Such a weakness could explain the random nature of the phenotypes. Failure of a muscle to maintain its connection with the tendon cell could result in loss of the muscle or result in the muscle forming a link with another muscle or tendon resulting in a mis-attachment phenotype (Haines, 2007).

In vertebrates, Dg function is regulated by glycosylation. Changes in sarcomere size and defects in muscle attachment in Drosophila mutant for genes encoding POMT1 and POMT2. It was also found that both these mutants interact with Dg mutant alleles in transheterozygous combinations. The similarity of the rt and tw muscle phenotypes to those associated with reduced Dg function and the interactions between mutant alleles of rt, tw, and Dg provide strong evidence that rt and tw are required for Dg-dependent processes in Drosophila . Given the O-mannosyltransferase activity of the rt and tw gene products toward Dg (Ichimiya, 2004) and the genetic evidence from vertebrates that loss of POMTs results in hypoglycosylation of Dg and subsequent disruption to Dg function, these data provide compelling evidence supporting a functional requirement for O-mannose glycans on Dg in Drosophila . The adult abdomen in tw and rt mutant flies is rotated. This phenotype was not observed in current experiments with Gal4-driven Dg RNAi knockdown. Possibly the abdominal rotation is due to loss of Dg function in these mutants and the lack of this phenotype in the RNAi flies is due to insufficient knockdown of Dg. It also remains possible that rt and tw have another substrate, and loss of glycan structures on this protein results in the rotated abdomen phenotype (Haines, 2007).

Altogether the phenotypes identified by manipulating Dg, tw, and rt demonstrate that Dg plays a central role in maintaining cell integrity in the Drosophila larval muscles and that glycosylation of Dg is important to its function. This report therefore opens the possibility of using genetic analysis of the highly accessible neuromuscular system available in this model organism to analyze the mechanisms by which loss of DGC function leads to muscle dystrophy (Haines, 2007).

The detached locus encodes Drosophila Dystrophin, which acts with other components of the Dystrophin Associated Protein Complex to influence intercellular signalling in developing wing veins

Dystrophin and Dystroglycan are the two central components of the multimeric Dystrophin Associated Protein Complex, or DAPC, that is thought to provide a mechanical link between the extracellular matrix and the actin cytoskeleton, disruption of which leads to muscular dystrophy in humans. This paper presents the characterization of the Drosophila 'crossveinless' mutation detached (det); the gene encodes the fly ortholog of Dystrophin. Genetic analysis shows that, in flies, Dystrophin is a non-essential gene, and the sole overt morphological defect associated with null mutations in the locus is the variable loss of the posterior crossvein that has been described for alleles of det. Null mutations in Drosophila Dystroglycan (Dg) are similarly viable and exhibit this crossvein defect, indicating that both of the central DAPC components have been co-opted for this atypical function of the complex. In the developing wing, the Drosophila DAPC affects the intercellular signalling pathways involved in vein specification. In det and Dg mutant wings, the early BMP signalling that initiates crossvein specification is not maintained, particularly in the pro-vein territories adjacent to the longitudinal veins, and this results in the production of a crossvein fragment in the intervein between the two longitudinal veins. Genetic interaction studies suggest that the DAPC may exert this effect indirectly by down-regulating Notch signalling in pro-vein territories, leading to enhanced BMP signalling in the intervein by diffusion of BMP ligands from the longitudinal veins (Christoforou, 2008).

The discovery that the Drosophila DAPC plays a role in vein development is striking as it is the first instance where the DAPC has been implicated in a developmental process that bears no obvious relationship to the DAPC functions ascribed to the complex in mammals. In the mouse, knock-outs of DAPC components lead to muscular dystrophy, defects in the post-synaptic membrane of the neuromuscular junction (NMJ), central nervous system and retinal abnormalities, reduced nNOS levels in the sarcolemma, and, for Dystroglycan, defects in embryonic basement membrane assembly. In zebrafish, the Dystroglycan mutant results in detachment of somitic muscles during embryogenesis. In the invertebrates, mutations in the Caenorhabditis elegans Dystrophin ortholog result in a decrease in acetylcholinesterase activity at the NMJ, but have no effect on muscle integrity, and in Drosophila , defects associated with the neuromuscular junction, neuronal migration, muscle integrity, and epithelial polarity have been described based on analyses of classical mutants and RNAi knock-downs. Despite this wide range of mutant studies in a variety of model systems, the DAPC has not previously been implicated in more general developmental processes. Yet, in flies, the DAPC clearly plays a role in vein specification, a process that has no relation to either muscles or neurons, and this raises the question of whether this function is specific to Drosophila or represents a more general function for the DAPC in insects and in other phyla (Christoforou, 2008).

A second important point about the results in this report is that they are not entirely in agreement with the previously published work on Drosophila Dystrophin and Dystroglycan. Previous studies identified two Dystroglycan alleles Dg248 and Dg323 which were isolated by imprecise excision of the P-element insertion EP(2)2241. These mutations were reported to be lethal, and mutant clones in ovarian germline and follicle cells give rise to defects in oocyte and epithelial polarity, respectively. These results have been confirmed in other reports using the same alleles. By contrast, the alleles reported in this study are at least semi-viable, both as homozygotes and hemizygotes, and show no evidence of the polarity defects that have been reported for the other alleles. These differences may be accounted for, at some level, in light of the different types of lesions associated with the alleles. The two alleles described previously are small deletions affecting the first non-coding exon of Dg and adjacent cis-regulatory sequences, whereas the alleles reported in this study are all located within the Dg coding region. Given that the currently studied alleles are molecular nulls, it is possible that the more severe phenotypes associated with Dg248 and Dg323 are in fact due to these deletions affecting either adjacent or nearby genes or their cis-regulatory sequences. Further work will need to be done to verify this possibility (Christoforou, 2008).

In the case of Dystrophin, two independent reports using RNAi to knock-down the function of all protein isoforms have claimed that loss of Dystrophin results in either lethality or age-dependent muscle degeneration. It is noteworthy that these studies are not entirely in agreement with one another. Shcherbata (2007) claims that Dg-RNAi or Dys-RNAi, when expressed ubiquitously with Tubulin-Gal4 or in muscles with 24B-Gal4, are adult viable, and the animals exhibit mobility defects and chronic muscle degeneration. van der Plas (2007), in contrast, claims that Dys-RNAi driven by 24B-Gal4 is predominantly pharate lethal with a few escapers that die shortly after eclosion. The results presented in this study lie between these two extremes. Df(3R)Exel6184 homozygotes are semi-viable, and the majority of animals that do not survive are pharate lethal. The surviving flies have a somewhat shorter lifespan than other genotypes tested, but easily survive to 40 days after eclosion, and show no evidence of mobility defects or muscle degeneration. With regard to the lethality, the differences observed in these studies could be a consequence of the different genetic backgrounds and insertions that were used. It is more difficult to account for the differences observed in the muscle degeneration phenotype. One possibility is that the experiments reported here were performed at the normal temperature of 25°C whereas the RNAi experiments of van der Plas (2007), at least, were all done at 29°C. It is possible that the elevated temperature exacerbates the degeneration phenotype leading to the reported results. Again, further experiments will need to be done to resolve these differences (Christoforou, 2008).

Since DAPC mutations in mammals give rise to muscular dystrophies, the primary role assigned to the complex has been a structural one: to maintain the integrity of the sarcolemma by forming a bridge between the ECM and the Actin cytoskeleton. In the developing crossvein, whatever the mechanism of action, the point of DAPC function appears to be to affect the activity of signalling pathways that govern crossvein specification, and thus, the function is not merely structural. The data suggest a mechanism whereby the DAPC augments BMP signalling in the pro-crossvein territory by down-regulating the activity of the Notch pathway in the pro-vein territory flanking L4 and L5 at the junction with the prospective crossvein. This down-regulation would allow diffusion of BMP ligands from the longitudinal veins into the crossvein territory and thus, indirectly augment BMP signalling in the crossvein (Christoforou, 2008).

This model can be reasoned as follows. Since P-Mad accumulation in the crossvein territory precedes DAPC function, the DAPC-dependent augmentation is presumably a consequence of the initial BMP signalling event. This would place the BMP and DAPC pathways as two sequential elements in a feedback loop: the initial BMP signal activates the DAPC which then signals back, either directly or indirectly, to augment the activity of BMP signalling. As both Dystrophin and Dystroglycan appear to be expressed uniformly throughout the pupal wing, these DAPC components are presumably activated by BMP signalling rather than being transcriptional targets of the pathway (Christoforou, 2008).

One of the consequences of DAPC activation is the anchoring of haemocytes in the pro-crossvein territory. It is clear that BMP signalling is sufficient to recruit haemocytes to the region, as evidenced by the persistent haemocytes observed in det and Dg mutant wings, but BMP signalling alone is not able to anchor them there, which accounts for the lack of haemocyte accumulation in the pro-crossvein in det and Dg mutants despite the relatively normal early accumulation of P-Mad. The persistence of haemocytes in the vein fragment that eventually arises simply reflects the continued recruitment of haemocytes to the site of highest BMP signalling, which, in the mutant wings, is half way between the two longitudinal veins. Whether the haemocytes themselves are essential for normal vein development cannot be determined at present, but the presence of a complete crossvein in Df(3R)ED5492/Df(3R)Exel6184 wings indicates that in wings with compromised Notch signalling, neither the DAPC nor the accumulation of haemocytes is necessary for crossvein formation (Christoforou, 2008).

While the defects in P-Mad accumulation that are observed in det and Dg mutant wings indicate that the ultimate effect of DAPC function in the wing is augmentation of BMP signalling, the results of genetic interaction studies suggest that this effect may be indirect. A direct effect on BMP signalling is not consistent with the failure to recover interactions between BMP components and det or Dg. While, in principle, this negative result does not rule out the possibility that the two pathways intersect (as they may be sufficiently robust so as not to show interactions under the conditions that were created), it is unexpected given the striking sensitivity of det and Dg mutations to genetic background. This sensitivity suggests that the DAPC phenotypes are on or near a threshold that would be susceptible to enhancement. A possible explanation for this result can be found in the dramatic suppression of the det phenotype by Delta. Taking into account this result, it is possible that the effect of the DAPC on BMP signalling may be indirect, resulting from an effect on Notch signalling. Thus, the initial BMP signal may activate the DAPC, but the DAPC feeds back on BMP signalling by down-regulating the activity of the Notch pathway in the pro-vein regions. In this case, the DAPC augments BMP signalling by down-regulating a BMP antagonist (Christoforou, 2008).

It has previously been suggested that the source of BMP signalling that gives rise to the early broad accumulation of P-Mad in the crossvein territory is dependent on the diffusion of Dpp:Gbb heterodimers from the longitudinal veins. This model, while accounting for some of the phenotypes produced by somatic clones of BMP pathway components, does not account for how these heterodimers are able to overcome the repression of BMP diffusion that is due to up-regulation of tkv by Notch signalling in the pro-vein territory. The proposal that the DAPC may function as a link between the initial Gbb-dependent BMP signal and the down-regulation of Notch signalling in the pro-veins would reconcile this problem. In this scenario, the initial BMP signal activates the DAPC in the crossvein territory. The DAPC then down-regulates Notch signalling which opens the vein regions of L4 and L5 to the intervein allowing the diffusion of BMP ligands into the presumptive crossvein territory. An interesting corollary of this model is that, since a vein fragment forms in DAPC mutant wings, the specification of the crossvein in the intervein territory does not require either the DAPC or diffusion of BMP ligands from the longitudinal veins. Moreover, as the resulting crossvein fragment is of normal thickness and morphology, refinement and sharpening are also independent of the diffusion of BMP heterodimers from the longitudinal veins (Christoforou, 2008).

Given this model, it remains to be determined first, how the initial Gbb-dependent BMP signalling activates the DAPC, and second, how the DAPC exerts its effect on Notch signalling. With regard to the first point, it is possible that the BMP signal affects DAPC function via Rho. There is precedent for a direct effect of TGF-β signalling on Rho leading to reorganization of the Actin cytoskeleton, and this type of mechanism is consistent with the involvement of cv-c, a Rho-GAP, in vein development. With regard to the second point, the effect of the DAPC on Notch signalling could occur directly with a DAPC-dependent reorganization of the basal plasma membrane of pro-vein cells that interferes with cell signalling events at the plasma membrane. Alternatively, the effect could be due to overriding the antagonistic effect of Notch activity by creating an extracellular environment that allows BMP diffusion from the longitudinal veins independent of Notch activity. Further research will be required to determine the precise mechanisms involved in this process (Christoforou, 2008).

Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction

The Dystrophin-glycoprotein complex (DGC) comprises dystrophin, dystroglycan, sarcoglycan, dystrobrevin and syntrophin subunits. In muscle fibers, it is thought to provide an essential mechanical link between the intracellular cytoskeleton and the extracellular matrix and to protect the sarcolemma during muscle contraction. Mutations affecting the DGC cause muscular dystrophies. Most members of the DGC are also concentrated at the neuromuscular junction (NMJ), where their deficiency is often associated with NMJ structural defects. Hence, synaptic dysfunction may also intervene in the pathology of dystrophic muscles. Dystroglycan is a central component of the DGC because it establishes a link between the extracellular matrix and Dystrophin. This study focused on the synaptic role of Dystroglycan (Dg) in Drosophila. Dg is concentrated postsynaptically at the glutamatergic NMJ, where, like in vertebrates, it controls the concentration of synaptic Laminin and Dystrophin homologues. Synaptic Dg controls the amount of postsynaptic 4.1 protein Coracle and alpha-Spectrin, as well as the relative subunit composition of glutamate receptors. In addition, both Dystrophin and Coracle and required for normal Dg concentration at the synapse. In electrophysiological recordings, loss of postsynaptic Dg did not affect postsynaptic response, but, surprisingly, led to a decrease in glutamate release from the presynaptic site. Altogether, this study illustrates a conservation of DGC composition and interactions between Drosophila and vertebrates at the synapse, highlights new proteins associated with this complex and suggests an unsuspected trans-synaptic function of Dg (Bogdanik, 2008).

The widely accepted hypothesis about the function of the DGC complex is its protective role in the sarcolemma against muscle contraction induced size changes. This study analyzed the synaptic function of a core member of the DGC, Dystroglycan. Drosophila Dg is concentrated at the NMJ, and most Dg immunoreactivity at the NMJ is postsynaptic. A proportion of synaptic Dg contained the mucin-like domain (MLD), which is the most heavily glycosylated domain in vertebrate Dg. Haines (2007) has shown that the MLD containing Drosophila Dg isoform is indeed glycosylated. Thus, like the vertebrate cholinergic NMJ, the Drosophila NMJ is enriched in Dg, and notably in glycosylated forms of this protein. These data are in accordance with concentration of Dystrophin at the Drosophila NMJ, suggesting the presence of all DGC members at the postsynapse (Bogdanik, 2008).

It is possible that the NMJ defects observed in the dg mutants used in this study are a consequence of a general muscle dysfunction, due to the loss of Dg at extrasynaptic sites. Indeed, muscle dysfunction has been observed in dg null mutants that are lethal at the embryonic and first instar larval stage. However, the mutants analyzed in this study are hypomorphs and the allelic combination used, dge01554/dg323, is viable. The larvae crawl, pupate and give rise to fertile adults, which do not show any wing position phenotype corresponding to flight muscle degeneration. Although it cannot be ruled out that there are some subtle muscle defects at extrasynaptic sites, the data illustrate that synaptic electrophysiological and morphological defects are already present in these mild loss of function conditions (Bogdanik, 2008).

The lanA gene, encoding a Laminin A subunit, stabilizes the initial motoneuron/muscle contact during synaptogenesis. This study shows that Laminin is still present during late larval stages, and that it is concentrated around synapses in varicosities. The data indicate that, like in mice where Dg is required for synaptic Utrophin, Laminin alpha5 and Laminin alpha1 concentration, Drosophila Dg controls synaptic Laminin and Dystrophin concentration. In addition, Dystrophin is required for Dg sarcolemmal localization in vertebrate muscles, and both Dystrophin and Utrophin account for part of the clustering of Dg at the NMJ. This study shows that Dystrophin also controls synaptic Dg concentration. Thus the interdependence between Laminin, Dg and Dystrophin at the NMJ seems to be conserved phylogenetically. Importantly, in dystrophin/utrophin double mutants, a significant amount of Dg remains at the synapse, indicating that other proteins control, in parallel, its synaptic localization. The current observations indicate that, similarly, the Utrophin-Dystrophin homologue in flies does not account for the whole synaptic localization of DG, and Coracle was identified as a new, additional synaptic anchor for Dg (Bogdanik, 2008).

Looking for any new potential partners of Dg, Cora localization was studied in late larval stages at the NMJ. Cora has a function in early larval stages, but no clear synaptic localization of Cora was seen in late larval stages, as seen using a monoclonal antibody recognizing all Cora isoforms. Instead, a strong immunoreactivity in NMJ associated glial cells has been reported. A polyclonal antibody was used that recognized only the large Cora isoform. With this antibody, no immunoreactivity was detected in any NMJ associated glial cell, but a postsynaptic concentration of Cora, which partially disappeared in a cora hypomorph mutant, was easily detected and increased when Cora was overexpressed in the muscle. These data indicated that the observed staining was indeed Cora. The Localization of protein 4.1 members in vertebrate muscle fibers is not well documented. It has been shown that protein 4.1R isoforms were indeed present in the muscle cells, notably at the cell periphery (probably the sarcolemma). Interestingly, in DMD patients, the peripheral localization of protein 4.1R isoforms is lost, although the sub-sarcolemmal spectrin cytoskeleton is still present. This set of data already indicates that protein 4.1 sarcolemmal localization is dependent on the DGC complex. The current data show that this is the case at the NMJ, and that Dg is the principal component involved in Cora localization, since loss of postsynaptic Dys gives much weaker phenotypes compared to loss of postsynaptic Dg. In addition, Cora was shown to co-immunoprecipitate with Dg, indicating the presence of the two proteins in the same complex, although further biochemical analysis will be required to assess whether they interact directly or indirectly (Bogdanik, 2008).

Unexpectedly, it was observed that Cora was required for the normal postsynaptic localization of Dg and, to a lesser extent, of Dys. This result was observed using a hypomorph cora mutant in which the C-terminal domain is partially deleted. In this mutant, synaptic amount of Cora was strongly reduced. Further structure-function studies will be required to understand 1) which domain of Cora is required for its synaptic localization and for its interaction with Dg, 2) which part of Dg C-terminal tail is involved in Cora interaction. Previous studies have shown that the juxtamembrane region of the C-terminal Dg tail interacts with Ezrin, a protein containing a FERM domain, like Cora. It is possible that the same Dg domain interacts with Cora (Bogdanik, 2008).

Since Cora controls synaptic GluRIIA abundance, an expected consequence of the loss of synaptic Cora in dg mutant NMJ was a reduction in the amount of GluRIIA subunit at the NMJ. Such a reduction was found, but to a mild degree. This small effect may be due to the fact that dg-induced reduction of synaptic Cora is not as strong as a complete cora loss of function, which was the situation analyzed originally. The small effect observed on DGluRIIA probably explains why there was no change in the amplitude of mEJCs in dg loss of functions. Indeed, DGluRIIA is the dominant subunit compared to DGluRIIB and a significant loss of DGluRIIA should lead to a decrease in mini amplitude (Bogdanik, 2008).

Loss of synaptic Dystroglycan resulted in a clear decrease in postsynaptic spectrin cytoskeleton, as assessed with alpha-Spectrin immunoreactivity. Although the spectrin defect may be a consequence of the loss of synaptic Cora, a more direct interaction between Dg and the spectrin cytoskeleton remains a possibility. Hence, the link between Dg, Cora and spectrin cytoskeleton remains to be further defined. The postsynaptic spectrin cytoskeleton has been shown to play a role in the repartition of postsynaptic receptor fields. Indeed, loss of postsynaptic immunoreactivity for both alpha and beta-Spectrin leads to a disorganization of postsynaptic receptor fields. Such a defect was sught in the dystroglycan loss of function conditions, but but none was found. This is probably due to the fact that the loss of spectrin immunoreactivity in these mutants was not complete (Bogdanik, 2008).

This study demonstrated that Dg plays a functional role in neuromuscular synaptic transmission. Indeed, glutamate release was decreased by approximately 40% in absence of muscle Dg. The main specificity of the insect NMJ, compared to the vertebrate NMJ is the presence of glutamate as a neurotransmitter instead of acetylcholine. Hence, these synapses are not only NMJ models, but also models of glutamatergic synapses, which are by far the most frequent synapses found in the vertebrate brain. Study of Dg function in mammalian brain synapses has illustrated an alteration of LTP in DG-CNS mice, but no modification of the amplitude of synaptic responses evoked by low frequency stimulation of Schaeffer collaterals, and no changes in paired-pulse facilitation. In the current study, a reduced synaptic response was detected at low frequency, indicating a function of Dg in basal glutamatergic synaptic transmission (Bogdanik, 2008).

One surprising result in the electrophysiology experiments was the fact that defects in quantal content of the dg mutant are also present, with the same intensity in flies expressing a 24B Gal4 driven dg-RNAi. This indicated that loss of postsynaptic Dg leads to a functional change in the other synaptic compartment, the presynapse. Such a presynaptic effect associated with postsynaptic modifications is not new, since the NMJ function displays homeostasis, and decrease in postsynaptic responsiveness is often associated to increase in neurotransmitter release and vice-versa in order to maintain constant EJCs. The molecular mechanisms involved in this homeostatic control are largely unknown. In this study, in dg mutants, homeostatic control is likely absent since mini amplitude (receptor field) is not altered in absence of postsynaptic Dg, but glutamate release is modified. This suggests that Dg-deficient muscles inappropriately signals to the presynaptic release machinery. Previous studies have observed a similar trans-synaptic effect of loss of muscle Dystrophin onto presynaptic quantal content has been observed. What can be the mechanisms involved? One possibility is that postsynaptic Dg directly controls the levels of synaptic ECM molecules such as Laminin. These proteins, by interacting with presynaptic receptors, would affect the structure of the presynapse, e.g. the amount, size or molecular composition of active or periactives zones. This hypothesis is strongly supported by the finding in mouse, that a synaptic Laminin-calcium channel interaction organizes active zones in motor nerve terminals. Another presynaptic Laminin receptor could be the synaptic vesicle protein SV2. Looking for modifications at the presynapse, no obvious change was detected in the number and size of active zones, using Bruchpilot immunoreactivity as a marker and no modification was detected in the immunoreactivity of the periactive zone marker Fas2. Still, the regulation of synaptic Laminin by Dg, together with the observed presynaptic electrophysiological phenotype, make the hypothesis of Laminin bridging postsynaptic Dg and the presynapse, at least in periactive zones, very likely (Bogdanik, 2008).

These findings, i.e. the new components of a Dystroglycan complex, as well as the unexpected trans-synaptic role of Dg pave the way for understanding the role of the DGC in the formation, maintenance and plasticity of glutamatergic synapses (Bogdanik, 2008).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

The screens described in this paper revealed some expected interactors, Dys, Cam and Khc. Calmodulin, a calcium binding protein required for muscle and neuronal functions has previously been shown to interact with mammalian the Dg-Dys complex. However, whether reduction of Cam activities suppresses or enhances the muscular dystrophy phenotype is not totally clear. Targeted inhibition of Cam signaling exacerbates the dystrophic phenotype in mdx mouse muscle while genetic disruption of Calcineurin improves skeletal muscle pathology and cardiac disease in ä-sarcoglycan null mice. Since reduction of Cam showed suppression of the phenotypes caused by reduction of the long forms of dystrophin in the Drosophila wing, it will be interesting to analyze whether reduction of Cam will suppress the Drosophila Dys muscle phenotype as well. Khc involvement in Dg-Dys complex is also expected since work in mammalian system has shown that Khc can bind Dystrobrevin, a component of Dg-Dys complex. It will be interesting to test in the future whether Drosophila Dystrobrevin can similarly bind Khc and what the functional significance of this interaction is in muscles and neurons. In oocyte development Khc is required as early as is Dys and Dg. It is, therefore, interesting to test the potential requirement of dystrobrevin in this process and to further dissect the Khc function in this complex during early polarity formation (Kucherenko, 2008).

An unexpected new interactor was identified in these screens, the homeodomain interacting protein kinase, HIPK. In mammalian systems HIPK is involved in the cell death pathway by phosphorylating p53. Recently Drosophila HIPK has shown to be involved in a communal form of cell death, sudden, coordinated death among a community of cells without final engulfment step (Link, 2007). It remains to be seen whether this HIPK communal death pathway will utilize p53 phosphorylation. However, it is tempting to speculate that the cell death observed in muscular dystrophies use the newly described HIPK dependent communal death pathway. HIPK is shown to interact with a WD40-protein in mammalian system. Since three WD40 proteins were identified in these screens, it will be interesting to test whether any of these interact with Drosophila HIPK (Kucherenko, 2008).

Another interactor that might shed light in the pathways utilized by the Dg-Dys complex is an SH3-domain adapter-protein, POSH. Structure-function analysis of Dg protein has revealed that a potential SH3-domain binding site in Dg C-terminus is essential for Dg function. However, the critical SH3-domain protein in this complex is still at large. The present screen revealed that POSH can interact with the Dg-Dys complex in the wing vein assay. It will now be interesting to determine whether POSH is the missing critical SH3-domain protein that interacts with Dg-Dys complex in Drosophila (Kucherenko, 2008).

There are only a few examples of signaling pathways that have been shown to transmit information from outside the cell that results in cytoskeletal rearrangements inside the cell. Slit-Robo, Netrin-Frazzled and Semaphorin-Plexin pathways are examples of such activity. Dg-Dys complex appears also regulate the cytoskeleton based on extracellular information. Interestingly, the interaction screens described in this paper show that these aforementioned pathways are much more intricately connected than previously thought. The Robo and Netrin Receptor (DCC) pathways have previously been shown to interact, now this study reports that Dg-Dys complex interacts with these pathways as well (Kucherenko, 2008).

The interactions seen in wing development involving the Drosophila DGC and the genes that affect neuronal guidance (sli, robo, fra, sema-2a, sema-1a, Sdc) might be explained by their possible role in hemocyte (insect blood cell) migration. Analysis done in Drosophila shows that known axon guidance genes (sli, robo) are also implicated in hemocyte migration during development of the central nervous system. Similar findings have been reported in mammals, where blood vessel migration is linked to the same molecular processes as axon guidance. Both sli and robo have been implicated in the vascularization system in vertebrates. A recent study demonstrated that proper hemocyte localization is dependent upon Dys and Dg function in pupa wings. Mutations in these genes result in hemocyte migration defects during development of the posterior crossvein. Hence, it is speculated that the neuronal guidance genes that were found may interact with the DGC in wing veins by having a role in the migration process (Kucherenko, 2008).

Similar to sli and robo, the Dys and Dg mutants also affect photoreceptor axon pathfinding in Drosophila larvae. It is therefore possible that this group of modifiers will interact with the DGC in axon pathfinding and other processes. Supportive of that notion is the fact that mammalian Syndecan-3 and Syndecan-4 are essential for skeletal muscle development and regeneration. In addition slit-Dg interaction has previously been observed in cardiac cell alignment. Sequence analysis of slit reveals that it possesses a laminin G-like domain at its C-terminus. Dystroglycan's extracellular domain has laminin G domain binding sites and has been shown to bind 2 of the five laminin G domains in laminin. It is therefore possible that slit, through its laminin G-like domain, binds to Dystroglycan and that Dystroglycan is a slit receptor. It will be informative to reveal the mechanisms and nature of these interactions (Kucherenko, 2008).

The establishment and formation of oocyte polarity during development is a complex multistep process. In the anterior part of the germarium each stem cell undergoes asymmetric cell division to give rise to another stem cell and a cystoblast. The cystoblasts divide four times with incomplete cytokinesis to form a 16 cell cyst. The oocyte fate is determined when the cyst reaches the end of the germarium. At this point, BicD protein, Orb protein, the microtubule organizing center (MTOC) and the centrioles move from the anterior to the posterior of the oocyte. These events mark the first sign of polarity in the oocyte. Subsequent Gurken signaling induces posterior follicle cells to signal back to the oocyte which repolarizes the microtubule cytoskeleton. This signal appears to require an intact extracellular matrix since Laminin A mutants do not undergo repolarization. The outcome of the repolarization results in the disassembly of the MTOC at the posterior, nucleation of microtubules anteriorly and subsequent migration of the oocyte nucleus to an antero-lateral position (Kucherenko, 2008 and references therein).

Germ line clones that lack Dg show developmental arrest and mislocalization of the oocyte polarity marker Orb which is usually diffused or absent in the oocyte. This phenotype might be due to Dg affecting the localization of the MTOC. But how exactly Dg is involved in this process is not clear. One possible explanation is that Dg is required for extracellular matrix (ECM) integrity since it is known to bind Laminin. Such a structural conduit may be necessary for proper signaling from the posterior follicle cells to the oocyte. This is supported by the fact that Dg loss-of-function mutants show defects in Actin accumulation. Another possibility is that Dg may be involved in mircrotubule organization. Since the regulation of actin- and microtubule-cytoskeleton are connected, these two models are not mutually exclusive (Kucherenko, 2008).

Interestingly, in the genetic screens several genes were found that showed phenotypes in oocyte development. One of these genes is kek1, a transmembrane protein of the Drosophila Kekkon family that has been reported to be a negative regulator of the EGFR receptor. It has been shown that EGFR signaling regulates the expression pattern of Dystroglycan to establish anterior-posterior polarity of oocyte (Poulton, 2006). Further study is required to determine if kek1, as an EGFR regulator controls Dg expression in the germ line (Kucherenko, 2008).

Another interesting gene found in the screens is POSH (Plenty of SH3 domains), a Drosophila homologue of human SH3MD2 protein. Interestingly POSH is a multidomain scaffold protein that can interact with Rho related GTPase - Rac1 and promotes the activation of the JNK pathway. POSH has also shown to regulate POSH-MLK-MKK-JNK complex (Figueroa, 2003). A defect in this complex can affect brain function. Furthermore, POSH and JNK-mediated cell death pathway is thought to play an important role in Parkinson's disease. With many SH3 domains, POSH has the potential to bind Dg that has a predicted SH3-domain binding site and has been shown to be necessary for the establishment of oocyte polarity (Kucherenko, 2008).

In addition, interactions were found with Khc, Lis-1 and Dmn, three genes known to be part of the Dynein-Dynactin complex which in addition to Kinesin microtubule motor activity have been shown to be necessary for establishment of intracellular polarity within the Drosophila oocyte. In mid-oogenesis dynein, dynactin and kinesin are thought to act cooperatively in cargo transport. Since these genes interact with Dys and show similar phenotypes in Orb localization, it will be interesting to dissect their potential functional interactions with Dys in early oocyte development. Furthermore, since mammalian Dystrobrevin physically interacts with Khc, it is plausible, that the Dynein-, Dynactin-, Kinesin-complex will utilize localization cues set-up by Dg-Dys Complex (Kucherenko, 2008).

In addition to the interactions with microtubular motor-complexes, interactions were also found with a Drosophila Formin homologue, FHOS. Mammalian FHOS directly binds to F-actin and promotes actin fiber formation. Drosophila actin nucleators, Capu and Spire have shown to assemble a cytoplasmic actin mesh that maintains microtubular organization in the middle of oogenesis. Therefore, it will be important to determine whether the actin nucleator, FHOS is also involved in actin nucleation that regulates microtubular activity in early oocyte development. Further study of these cytoskeletal genes will result in a more detailed understanding of how Dg and Dys function to ensure proper oocyte polarity during oogenesis (Kucherenko, 2008).

Similar to microtubule and actin interplay in the regulation of oocyte polarity, the dynamic actin-microtubule interactions regulate growth cone steering at the growing axons. It is therefore possible that similar mode of function for Dg-Dys interactions with these cytoskeletal modules is used in various cell types. Furthermore the axon pathfinding and oocyte polarity formation processes are similar in that they are controlled by extracellular information which is transmitted to the cell resulting in cytoskeletal rearrangement (Kucherenko, 2008).

At the basal side of follicle epithelium, actin filaments exhibit a planar cell polarity that is perpendicular to the long axis, the AP axis, of the egg chamber. In Dg follicle cell clones the basal actin array is disrupted non-cell-autonomously. Integrins and the receptor tyrosine phosphatase Lar are also involved in basal actin orientation. It is unclear whether Dg and the other genes involved in basal actin polarity act together with the canonical planar cell polarity pathway or function independently of this pathway. Interestingly, strong interactions were found between the DGC and grainy head (grh) a transcription factor which is required for several different processes during the differentiation including the function of the frizzled dependent tissue polarity pathway, epidermal hair morphogenesis and wing vein specification. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of Stan (or Flamingo) protein are dramatically decreased. The interactions seen with stan (Fla) and wg in wing veins supports the hypothesis that Dg might act together with the frizzled-dependent tissue polarity pathway in coordinating the polarity of cells in epithelial sheets (Kucherenko, 2008).

By screening for alterations of a dominant wing vein phenotype modifiers of the DGC were found that are involved in cytoskeletal organization. Initial characterization of some of these genes revealed that they have phenotypes also in other tissues, in which the DGC is known to function. These tissue/cell types include the oocyte, the brain and the indirect flight muscles. This argues strongly that the identified interactors may be involved globally in DGC function. Further study is required to determine mechanistically how these modifiers work in the context of the Dg-Dys complex. However a common theme, already arising is that the identified interactors appear to regulate cytoskeletal rearrangement. Mechanistic understanding of how the new interactors might regulate Dg-Dys communication with cytoskeleton of muscle cells may serve as a basis for the development of novel therapeutic approaches that might improve the quality of life of individuals afflicted with muscular dystrophy (Kucherenko, 2008).

Synaptic defects in a Drosophila model of congenital muscular dystrophy

The congenital muscular dystrophies present in infancy with muscle weakness and are often associated with mental retardation. Many of these inherited disorders share a common etiology: defective O-glycosylation of α-dystroglycan, a component of the dystrophin complex. Protein-O-mannosyl transferase 1 (POMT1) is the first enzyme required for the glycosylation of α-dystroglycan, and mutations in the POMT1 gene can lead to both Walker-Warburg syndrome (WWS) and limb girdle muscular dystrophy type 2K (LGMD2K). WWS is associated with severe mental retardation and major structural abnormalities in the brain; however, LGMD2K patients display a more mild retardation with no obvious structural defects in the brain. In a screen for synaptic mutants in Drosophila, mutations were identified in the Drosophila ortholog of POMT1, dPOMT1. Because synaptic defects are a plausible cause of mental retardation, the molecular and physiological defects associated with loss of dPOMT1 were investigated in Drosophila. In dPOMT1 mutants, there is a decrease in the efficacy of synaptic transmission and a change in the subunit composition of the postsynaptic glutamate receptors at the neuromuscular junction. dPOMT1 is required to glycosylate the Drosophila dystroglycan ortholog Dg in vivo, and this is the likely cause of these synaptic defects because (1) mutations in Dg lead to similar synaptic defects and (2) genetic interaction studies suggest that dPOMT1 and Dg function in the same pathway. These results are consistent with the model that dPOMT1-dependent glycosylation of Dg is necessary for proper synaptic function and raise the possibility that similar synaptic defects occur in the congenital muscular dystrophies (Wairkar, 2008).

POMT1 is required for the O-glycosylation of dystroglycan, and mutations in POMT1 can lead to two variants of congenital muscular dystrophy, WWS and LGMD2K. Both diseases are associated with mental retardation; however, for the milder LGMD2 no apparent structural abnormalities are present in the brain that would explain the onset of mental retardation. In this study, a Drosophila model of POMT1 deficiency was characterized. As in vertebrates, Drosophila POMT1 is required for glycosylation of dystroglycan. In Drosophila, the inability to glycosylate dystroglycan, or the genetic disruption of dystroglycan, does not lead to gross structural abnormalities at the neuromuscular junction, but rather disrupts presynaptic glutamate release and alters the subunit composition of postsynaptic glutamate receptors. Similar synaptic changes at vertebrate central synapses are a potential cause of mental retardation in CMD patients (Wairkar, 2008).

The glycosylation of dystroglycan is affected in many forms of CMD. Glycosylation of dystroglycan is required for its binding to components of the extracellular matrix. In addition, loss of glycosylation can lead to a decrease in the levels of dystroglycan. Hence, glycosylation enzymes such as POMT1 may be required for both the activity and stability of dystroglycan. In Drosophila, dPOMT1 promotes the glycosylation of Dg in vitro, and the loss of dPOMT1 in cultured Drosophila SF21 cells results in hypoglycosylation of Dg. This study demonstrates that dPOMT1 is required in vivo for the normal glycosylation of Dg. In the absence of dPOMT1, the total levels of dystroglycan are decreased, because the glycosylated band is lost with no commensurate increase in the nonglycosylated band. The failure to observe more nonglycosylated Dg suggests that glycosylation may be important for the stability of Dg (Wairkar, 2008).

Drosophila perlecan binds to Dg that lacks the mucin-rich O-glycosylation domain, so it is plausible that the nonglycosylated Dg could retain some function. The ability to manipulate Dg and dPOMT1 independently allowed test of whether the decrease in total Dg was the major cause of the dPOMT1 phenotypes. Uncreasing the levels of dystroglycan in a dPOMT1 mutant leads to lethality. This suggests that too much nonglycosylated Dg may be toxic, and is consistent with the model that glycosylation is required for Dg function (Wairkar, 2008).

Previous analysis of dPOMT1 mutants demonstrated that loss of dPOMT1 leads to a rotated abdomen phenotype and disrupted muscle structure (Ichimiya, 2004; Lyalin, 2006; Haines, 2007). This analysis adds a second major phenotype: a severe impairment in the ability to release neurotransmitter. This study investigated the mechanism underlying this synaptic phenotype. No change was detected in the number of anatomically defined neurotransmitter release sites (n), suggesting that probability of release (p) is impaired in the mutant. Consistent with this hypothesis, when transmitter is released in low calcium conditions, there is an increase in short-term facilitation, which usually varies inversely with release probability. In addition, high external calcium, which saturates release probability, rescues the defects in evoked transmitter release in the dPOMT1 mutant. These data demonstrate that the defect in synaptic transmission in the dPOMT1 mutant is attributable to a reduction in release probability rather than a reduction in the number of release sites (Wairkar, 2008).

What might be the molecular cause of this decrease in probability of release? The data suggest that the proximate cause is probably the loss of glycosylated dystroglycan. Mutations in Dg also have a decrease in p, and the strong genetic interactions between dPOMT1 and Dg heterozygotes are consistent with the genes working in the same pathway to promote transmitter release. Why then would the loss of glycosylated dystroglycan impair transmitter release? The answer is not known, but it is speculated that dystroglycan, via its interactions with the extracellular matrix, is an important part of a transsynaptic complex that plays a structural and/or functional role at the synapse to promote normal synaptic function. Indeed, components of the extracellular matrix and dystrophin regulate synaptic function at the Drosophila NMJ. However, the reduced synaptic function in dPOMT1 and Dg mutants is unlikely to be caused by the reduction in levels of postsynaptic dystrophin, because mutations in dystrophin lead to an increase, rather than decrease, in evoked transmitter release (Wairkar, 2008).

In which cells does glycosylated dystroglycan function to promote transmitter release? A functional dPOMT1 transgene was generated whose ubiquitous expression rescues the rotated abdomen phenotype. The spatial requirement for dPOMT1 in synaptic function was investigated by driving the transgene at the NMJ using neuronal, muscle, ubiquitous, and neuronal/muscle synaptic Gal4 driver lines. It was found that the synaptic dPOMT1 phenotypes are rescued only when the transgene is driven by either the neuronal/muscle C142-Gal4 or ubiquitous actin-Gal4 driver and not when it is expressed exclusively in the presynaptic or postsynaptic cell. Therefore, dPOMT1 may be required for glycosylating dystroglycan both in neurons and muscles to maintain the normal function of the NMJ. Dystroglycan is expressed in both muscles and brain in Drosophila, and the results are consistent with the model that it functions in both neurons and muscles at the NMJ (Wairkar, 2008).

One of the intriguing findings of this study is the specific reduction in the DGluRIIB subunit of the glutamate receptor in dPOMT1 and dg mutants. At the Drosophila NMJ, postsynaptic glutamate receptors are comprised of three essential subunits as well as either of two nonessential subunits, DGluRIIA and DGluRIIB. Receptors with these alternate subunits are differentially localized opposite the terminals of distinct motoneurons that synapse with the same muscle cell, leading to the suggestion that presynaptic activity may shape glutamate receptor subunit composition. Although extensive studies have been done in vertebrate AMPA-type receptor subunit composition and trafficking, mechanisms that describe such subunit-specific regulation of glutamate receptors are not well understood at the Drosophila NMJ. Recently, the actin/spectrin-binding protein Coracle was shown to regulate the subunit composition of glutamate receptors at the Drosophila NMJ. Mutations in Coracle lead to a specific loss in the DGluRIIA subunit, demonstrating that distinct molecular pathways can control subunit composition. The results indicate that dPOMT1 via Dg also regulates the subunit composition of glutamate receptors at the Drosophila NMJ. It is tempting to speculate that dystroglycan, which participates in clustering acetylcholine receptors in vertebrates, could be involved in the clustering of DGluRIIB subunit of glutamate receptors. However, it is also plausible that the changes in DGluRIIB levels are secondary to the changes in synaptic function and do not reflect a direct function of dystroglycan in receptor localization. These findings open a new path for understanding the molecular and/or activity-dependent cues that control the localization of specific glutamate receptor subunits at the Drosophila NMJ (Wairkar, 2008).

Dystroglycan and perlecan provide a basal cue required for epithelial polarity during energetic stress

Dystroglycan localizes to the basal domain of epithelial cells and has been reported to play a role in apical-basal polarity. This study shows that Dystroglycan null mutant follicle cells have normal apical-basal polarity, but lose the planar polarity of their basal actin stress fibers, a phenotype it shares with Dystrophin mutants. However, unlike Dystrophin mutants, mutants in Dystroglycan or in its extracellular matrix ligand Perlecan lose polarity under energetic stress. The maintenance of epithelial polarity under energetic stress requires the activation of Myosin II by the cellular energy sensor AMPK. Starved Dystroglycan or Perlecan null cells activate AMPK normally, but do not activate Myosin II. Thus, Perlecan signaling through Dystroglycan may determine where Myosin II can be activated by AMPK, thereby providing the basal polarity cue for the low-energy epithelial polarity pathway. Since Dystroglycan is often downregulated in tumors, loss of this pathway may play a role in cancer progression (Mirouse, 2009).

Clones of Dg null mutations have no effect on apical-basal polarity under normal conditions, but disrupt the planar cell polarity (pcp) of the basal actin stress fibers. The loss of this organization allows the oocyte to grow in all directions, leading the short, round-egg phenotype of Dg null homozygotes. A very similar phenotype is seen in mutants in the receptor tyrosine phosphatase DLar and in the α and β subunits of integrin. Since DLar and integrins are also receptors for the components of the ECM, three different ECM receptors are required nonredundantly for the pcp of the actin stress fibers. However, mutants in Dg, dys, and DLar have no effect on other well-characterized examples of pcp in Drosophila, such as the orientation of the apical trichomes on the wing blade. This indicates that pcp on the basal side of the cell has different requirements than apical pcp, and it would therefore be interesting to examine whether the classical pcp pathways that regulate apical planar polarity are involved in the orientation of the basal actin stress fibers (Mirouse, 2009).

A newly identified null allele in dys also gives rise to short, round eggs and causes an identical defect in the orientation of the basal actin stress fibers. Since Dys binds to the intracellular domain of Dg and to F-actin, it may provide a direct link between the two to transmit the planar polarity of the ECM to the basal stress fibers. Dg and Dys also function as links between the ECM and the actin cytoskeleton in muscle cells, where they play an important role in allowing the cell surface to withstand the mechanical forces caused by contraction, thereby preventing muscular dystrophy. The results in epithelial cells indicate that the DAPC does more than just create a physical link between the ECM and actin, raising the possibility that it also plays a role in organizing the cortical actin network in muscle (Mirouse, 2009).

The data contradict previous reports that Dg is required for the apical-basal polarity of epithelial cells and for the initial anterior-posterior polarity of the oocyte (Deng, 2003). This discrepancy can be explained by the fact that null alleles of Dg were used, whereas the earlier studies used deletions in the 5′ end of the Dg locus that also remove mRpL34, an essential gene that encodes a mitochondrial ribosomal protein. More importantly, the apical-basal polarity defects of the Dg deletion alleles can be rescued by transgenes expressing either mRpL34 or Dg, indicating that this phenotype is caused by the concomitant loss of both genes. Furthermore, the nonsense alleles of Dg give an identical polarity phenotype to the deletion alleles when the flies are cultured on food without glucose. Thus, Dg is required for epithelial polarity only under conditions of energetic stress, and the Dg deletion alleles give a polarity phenotype under normal conditions, because the loss of mRpL34 disrupts mitochondrial function, thereby reducing cellular energy (Mirouse, 2009).

Although the energetic stress caused by disruption of mRpL34 can explain the epithelial polarity phenotypes of the Dg deletion alleles, the Dg nonsense mutations have no effect on oocyte polarity even in starved flies. The early defects in oocyte polarity observed with the deletion alleles may therefore be due to loss of mRpL34 alone. It has also been reported that loss of pcan disrupts epithelial polarity and the basal localization of Dg under normal conditions (Schneider, 2006). Using the same allele, this study found that pcan null clones show normal apical-basal polarity on standard food, but show similar polarity defects to Dg mutants under energetic stress conditions, and this discrepancy may be due to differences in fly food composition in different laboratories (Mirouse, 2009).

Like Dg and Pcan, LKB1 and AMPK are required for epithelial polarity only under conditions of energetic stress (Mirouse, 2007). Indeed, the polarity phenotype of Dg or pcan mutant clones is indistinguishable from that of ampk and lkb1 mutants under glucose starvation. Apical (Crb, aPKC) and lateral (Dlg) markers are no longer localized at the cortex, whereas markers for the adherens junctions (Arm, DECad) are more stable, but eventually disappear in large mutant clones. Interestingly, the Crb complex component Patj remains apically localized in small mutant clones like the adherens junctions components. Since all other apical markers are disrupted, Patj cannot be targeted apically solely through its interaction with Sdt and Crb, suggesting that it may also interact with junctional proteins. This is consistent with the observation that Patj is still properly localized in crb mutant cells. Starved Dg and pcan mutant clones do not accumulate phosphorylated Sqh and show a reduction of basal actin and an increase in apical actin, just like ampk and lkb1 clones. As well as these polarity phenotypes, mutations in all four proteins upregulate Arm under conditions of energetic stress, whereas starved pcan, ampk, and lkb1 clones show a dramatic increase in Dg levels. Thus, mutants in these proteins have no effect on polarity under normal conditions and cause the same spectrum of phenotypes under conditions of energetic stress, strongly suggesting that they are all essential components of a low-energy polarity pathway (Mirouse, 2009).

The principal function of LKB1 and AMPK in epithelial polarity under low-energy conditions is to activate Myosin II through the direct phosphorylation of its regulatory light chain, Sqh, by AMPK, since a phosphomimetic form of Sqh rescues all of the polarity defects of starved ampk or lkb1 null cells (Lee, 2007). The current results show that Pcan and Dg are also required for the activation of Myosin II under conditions of energetic stress, but their polarity phenotypes cannot be rescued by the constitutively active forms of either AMPK or Sqh. This leads to two important conclusions. (1) Pcan and Dg are not required for the activation of AMPK, and the loss of localized, phosphorylated Sqh must therefore be due to some other defect. (2) The failure of phosphomimetic Sqh to rescue the polarity defects of starved Dg clones indicates that Dg must have another function in addition to its role in Myosin activation (Mirouse, 2009).

Sqh is mislocalized to the basal cortex of starved Dg clones, and this could account for both the failure of AMPK to phosphorylate it and the inability of phosphomimetic Sqh to rescue the polarity phenotype. Phospho-AMPK is uniformly distributed, however, and should be able to phosphorylate Sqh anywhere in the cell. In addition, Sqh and the Myosin II heavy chain still colocalize in Dg mutant cells, strongly suggesting that the lack of rescue by phosphomimetic Sqh is not caused by its failure to interact with and activate the heavy chain. An alternative possibility is that loss of Dg disrupts Myosin activation and localization indirectly, perhaps by altering the arrangement of F-actin. Phosphomimetic Sqh does not rescue normal actin organization in starved Dg clones, demonstrating that this phenotype is not caused solely by the loss of Myosin activity, and this suggests that Dg plays a myosin-independent role in the polarized organization of the actin cytoskeleton. If Myosin II activation is regulated by its actin-dependent localization and/or its binding to actin, the failure to phosphorylate Sqh in Dg clones could be a secondary consequence of a polarity defect that disrupts the actin cytoskeleton (Mirouse, 2009).

LKB1 or AMPK activation, glucose deprivation, or the expression of phosphomimetic Sqh are sufficient to induce apical-basal polarity in isolated human intestinal cells in culture, indicating that this pathway is conserved in humans (Lee, 2007 Baas, 2004). In order to polarize single cells de novo, there must be a polarity cue that provides the positional information to generate cellular asymmetries. This cannot be provided by LKB1 or AMPK, since activated P-AMPK is not spatially restricted, and its function can be bypassed by providing a constitutively active myosin (Mirouse, 2007; Lee, 2007). Cell-cell adhesion is also unlikely to act as the polarity cue, because the low-energy pathway can polarize single mammalian cells in culture in the absence of any contacts with their neighbors. The only remaining asymmetry under these conditions is cell adhesion to the ECM on the substrate. Since Pcan is a component of the basal ECM and Dg is an ECM receptor, and they are both required for polarity under energetic stress, it is attractive to propose that the adhesion of Pcan to Dg provides the basal cue for epithelial polarity under low-energy conditions (Mirouse, 2009).

In all organisms, the intracellular domain of Dg has two conserved features: a WW domain-binding motif that interacts with Dys, and a PXXP motif that can function as a SH3 domain-binding site. A null mutant in the single Dys/Utrophin homolog in the Drosophila genome has no effect on epithelial organization under low-energy conditions, suggesting that Dg does not regulate polarity through binding to Dys. In support of this view, the overexpression of full-length Dg or Dg with a mutated Dys-binding domain disrupts follicle cell polarity, whereas a construct that lacks the SH3 domain-binding site does not (Yatsenko, 2007; Deng, 2003). Thus, it seems most likely that the binding of Pcan to Dg controls epithelial polarity under low-energy conditions by signaling through the SH3-binding domain, and it will be important to identify the SH3 protein responsible (Mirouse, 2009).

LKB1 is mutated in both familial and spontaneous tumors of epithelial origin, suggesting that disruption of the low-energy polarity pathway may play a role in tumor progression. Most tumor cells undergo a metabolic switch, called the Warburg effect, in which they take up about five times more glucose than normal cells because they are generating ATP from glycolysis, which is much less efficient than oxidative phosphorylation (Brahimi-Horn, 2007). Moreover, tumor cells often have reduced access to nutrients and oxygen as the tumor outgrows the local blood supply. Thus, the cells are likely to be subjected to energetic stress, both because they are inefficient at generating ATP and because they are hypoxic. In this context, it is interesting to note that Dg is downregulated in a wide variety of tumors, with low levels of expression correlating with a poor prognosis. Furthermore, when Dg is reintroduced into breast cancer cell lines that no longer express it, it restores epithelial polarity and reduces tumorogenicity. These results suggest that Dg is required to maintain epithelial organization in tumor cells under energetic stress, and that its downregulation leads to overproliferation and a loss of polarity that contribute to metastasis (Mirouse, 2009).


Dystroglycan in C. elegans and Zebrafish

Dystrophin is the product of the gene mutated in Duchenne muscular dystrophy (DMD). Neither the function of dystrophin nor the physiopathology of the disease have been clearly established so far. In mammals, the dystrophin-glycoprotein complex (DGC) includes dystrophin, as well as transmembrane and cytoplasmic proteins. Since Caenorhabditis elegans possesses a dystrophin-like gene (dys-1), whether homologs of the DGC members could also be found in the C. elegans genome was also investigated. Conserved homologs were found for dystroglycan, delta/gamma-sarcoglycan and syntrophin. Divergent but related proteins were found for alpha- and beta-sarcoglycans. No sarcospan counterpart was found. The expression of the conserved homologs was inactivated using the RNA interference technique. Phenotypes similar to that of dys-1 were obtained, both in the wild-type background and in combination with other mutations. These results strongly suggest that a protein complex comprising functional analogies with the DGC exists in C. elegans (Grisoni, 2002).

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular alpha subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane beta subunit. The function of dystroglycan has been removed in zebrafish embryos. In contrast to mouse, where dystroglycan mutations led to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organization. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organization. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis (Parsons, 2002).

Cloning and developmental expression of mammalian Dystroglycan

The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibers more susceptible to necrosis (Ibraghimov-Beskrovnaya, 2002).

The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. During mouse development dystroglycan is expressed in epithelium of nonmuscle tissues. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two has been shown for laminin alpha 1 chain. Dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturb development of epithelium in kidney organ culture. It is suggested that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1 (Durbeej, 1995).

Characterization of dystroglycan complexes

Dystroglycan is a widely expressed extracellular matrix receptor that plays a critical role in basement membrane formation, epithelial development, and synaptogenesis. Dystroglycan was originally characterized in skeletal muscle as an integral component of the dystrophin glycoprotein complex, which is critical for muscle cell viability. Although the dystroglycan complex has been well characterized in skeletal muscle, there is little information on the structural composition of the dystroglycan complex outside skeletal muscle. The dystroglycan complex in lung and kidney has been biochemically characterized in this study. The presence of sarcoglycans and sarcospan in lung reflects association with dystroglycan in the smooth muscle. The smooth muscle dystroglycan complex in lung, composed of dystroglycan, dystrophin/utrophin, beta-, delta-, epsilon-sarcoglycan, and sarcospan, can be biochemically separated from epithelial dystroglycan, which is not associated with any of the known sarcoglycans or sarcospan. Similarly, dystroglycan in kidney epithelial cells is not associated with any of the sarcoglycans or sarcospan. Thus, the data demonstrate that there are distinct dystroglycan complexes in non-skeletal muscle organs as follows: one from smooth muscle, which is associated with sarcoglycans forming a similar complex as in skeletal muscle, and one from epithelial cells (Durbeej, 1999).

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. The N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. A region of 36 amino acids, from position 550-585, was found to be required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. This Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM) was measured. These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications (Sciandra, 2001).

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly (Talts, 1999).

Dystroglycan interaction with Dystrophin

Dystrophin and beta-dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of beta-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of beta-dystroglycan. The crystal structure of this portion of dystrophin is reported in complex with the proline-rich binding site in beta-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The beta-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains (Huang, 2000).

Glycosylation of Dystroglycan

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. A new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3, has been identified. The genomic organization of the FKRP gene is reported as well as its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals have a secondary deficiency of laminin alpha2 expression. In addition, they have both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on Western blot analysis. It is suggested that these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C (Brockington, 2001).

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids. To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain, immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCM. A selective deficiency of highly glycosylated alpha-dystroglycan, but not beta-dystroglycan, was found on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for alpha-dystroglycan, but were positive for beta-dystroglycan in FCMD in skeletal and cardiac muscles. These findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, alpha-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD (Hayashi, 2001).

Muscle eye brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations. The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase. In both MEB and FCMD patients, that alpha-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. This post-translational biochemical and functional disruption of alpha-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan-ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities (Michele, 2002).

Reduced ligand binding activity of alpha-dystroglycan is associated with muscle and central nervous system pathogenesis in a growing number of muscular dystrophies. Posttranslational processing of alpha-dystroglycan is generally accepted to be critical for the expression of functional dystroglycan. This study shows that both the N-terminal domain and a portion of the mucin-like domain of alpha-dystroglycan are essential for high-affinity laminin-receptor function. Posttranslational modification of alpha-dystroglycan by glycosyltransferase, LARGE, occurs within the mucin-like domain, but the N-terminal domain interacts with LARGE, defining an intracellular enzyme-substrate recognition motif necessary to initiate functional glycosylation. Gene replacement in dystroglycan-deficient muscle demonstrates that the dystroglycan C-terminal domain is sufficient only for dystrophin-glycoprotein complex assembly, but to prevent muscle degeneration the expression of a functional dystroglycan through LARGE recognition and glycosylation is required. Therefore, molecular recognition of dystroglycan by LARGE is a key determinant in the biosynthetic pathway to produce mature and functional dystroglycan (Kanagawa, 2004).

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. This study reports reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy (Kanagawa, 2005).

Dystroglycan interaction with laminin

The transition of laminin from a monomeric to a polymerized state is thought to be a crucial step in the development of basement membranes and in the case of skeletal muscle, mutations in laminin can result in severe muscular dystrophies with basement membrane defects. Laminin polymer and receptor interactions have been evaluated to determine the requirements for laminin assembly on a cell surface and what cellular responses might be mediated by this transition have been investigated. In muscle cell surfaces, laminins preferentially polymerize while bound to receptors that included dystroglycan and alpha7beta1 integrin. These receptor interactions are mediated through laminin COOH-terminal domains that are spatially and functionally distinct from NH2-terminal polymer binding sites. This receptor-facilitated self-assembly drives rearrangement of laminin into a cell-associated polygonal network, a process that also requires actin reorganization and tyrosine phosphorylation. As a result, dystroglycan and integrin redistribute into a reciprocal network as do cortical cytoskeleton components vinculin and dystrophin. Cytoskeletal and receptor reorganization is dependent on laminin polymerization and fails in response to receptor occupancy alone (nonpolymerizing laminin). Preferential polymerization of laminin on cell surfaces, and the resulting induction of cortical architecture, is a cooperative process requiring laminin-receptor ligation, receptor-facilitated self-assembly, actin reorganization, and signaling events (Colognato, 1999).

Laminin G-like (LG) modules in the extracellular matrix glycoproteins laminin, perlecan, and agrin mediate the binding to heparin and the cell surface receptor alpha-dystroglycan (alpha-DG). These interactions are crucial to basement membrane assembly, as well as muscle and nerve cell function. The crystal structure of the laminin alpha 2 chain LG5 module reveals a 14-stranded beta sandwich. A calcium ion is bound to one edge of the sandwich by conserved acidic residues and is surrounded by residues implicated in heparin and alpha-DG binding. A calcium-coordinated sulfate ion is suggested to mimic the binding of anionic oligosaccharides. The structure demonstrates a conserved function of the LG module in calcium-dependent lectin-like alpha-DG binding (Hohenester, 1999).

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. The role of this interaction has been examined in the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA is expressed in epithelium of developing salivary gland and lung. Immunofluorescence has demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturb epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis is seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocks interactions between alpha-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibits salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, fail to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs (Durbeej, 2001).

Dystroglycan (DG) function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, roles for other molecules in this process have been defined. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell (Henry, 2001b).

Developmental abnormalities of myelination are observed in the brains of laminin-deficient humans and mice. The mechanisms by which these defects occur remain unknown. It has been proposed that, given their central role in mediating extracellular matrix (ECM) interactions, integrin receptors are likely to be involved. However, it is a non-integrin ECM receptor, dystroglycan, that provides the key linkage between the dystrophin-glycoprotein complex (DGC) and laminin in skeletal muscle basal lamina, such that disruption of this bridge results in muscular dystrophy. In addition, the loss of dystroglycan from Schwann cells causes myelin instability and disorganization of the nodes of Ranvier. To date, it is unknown whether dystroglycan plays a role during central nervous system (CNS) myelination. This study reports that the myelinating glia of the CNS, oligodendrocytes, express and use dystroglycan receptors to regulate myelin formation. In the absence of normal dystroglycan expression, primary oligodendrocytes showed substantial deficits in their ability to differentiate and to produce normal levels of myelin-specific proteins. After blocking the function of dystroglycan receptors, oligodendrocytes failed both to produce complex myelin membrane sheets and to initiate myelinating segments when co-cultured with dorsal root ganglion neurons. By contrast, enhanced oligodendrocyte survival in response to the ECM, in conjunction with growth factors, was dependent on interactions with beta-1 integrins and did not require dystroglycan. Together, these results indicate that laminins are likely to regulate CNS myelination by interacting with both integrin receptors and dystroglycan receptors, and that oligodendrocyte dystroglycan receptors may have a specific role in regulating terminal stages of myelination, such as myelin membrane production, growth, or stability (Colognato, 2007).

A stoichiometric complex of neurexins and dystroglycan in brain

In nonneuronal cells, the cell surface protein dystroglycan links the intracellular cytoskeleton (via dystrophin or utrophin) to the extracellular matrix (via laminin, agrin, or perlecan). Impairment of this linkage is instrumental in the pathogenesis of muscular dystrophies. In brain, dystroglycan and dystrophin are expressed on neurons and astrocytes, and some muscular dystrophies cause cognitive dysfunction; however, no extracellular binding partner for neuronal dystroglycan is known. Regular components of the extracellular matrix, such as laminin, agrin, and perlecan, are not abundant in brain except in the perivascular space that is contacted by astrocytes but not by neurons, suggesting that other ligands for neuronal dystroglycan must exist. Alpha- and beta-neurexins, polymorphic neuron-specific cell surface proteins, have now been identified as neuronal dystroglycan receptors. The extracellular sequences of alpha- and beta-neurexins are largely composed of laminin-neurexin-sex hormone-binding globulin (LNS)/laminin G domains, which are also found in laminin, agrin, and perlecan, that are dystroglycan ligands. Dystroglycan binds specifically to a subset of the LNS domains of neurexins in a tight interaction that requires glycosylation of dystroglycan and is regulated by alternative splicing of neurexins. Neurexins are receptors for the excitatory neurotoxin alpha-latrotoxin; this toxin competes with dystroglycan for binding, suggesting overlapping binding sites on neurexins for dystroglycan and alpha-latrotoxin. These data indicate that dystroglycan is a physiological ligand for neurexins and that neurexins' tightly regulated interaction could mediate cell adhesion between brain cells (Sudhof, 2001).

Interaction of dystroglycan with Grb2

Dystroglycan is a novel laminin receptor that links the extracellular matrix and sarcolemma in skeletal muscle. The dystroglycan complex containing alpha- and beta-dystroglycan also serves as an agrin receptor in muscle, where it may regulate agrin-induced acetylcholine receptor clustering at the neuromuscular junction. beta-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization. Protein binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which correspond to the loss-of-function mutants in the Caenorhabditis elegans sem-5, demonstrate that the dystroglycan-Grb2 association is through beta-dystroglycan C-terminal proline-rich domains and Grb2 Src homology 3 domains. Affinity chromatography has also shown that endogenous skeletal muscle Grb2 interacts with beta-dystroglycan. Immunoprecipitation experiments have demonstrated that Grb2 associates with alpha/beta-dystroglycan in vivo in both skeletal muscle and brain. The specific dystroglycan-Grb2 interaction may play an important role in extracellular matrix-mediated signal transduction and/or cytoskeleton organization in skeletal muscle that may be essential for muscle cell viability (Yang, 1995).

Tyrosine phosphorylation of beta-dystroglycan at its WW domain binding motif, PPxY, recruits SH2 domain containing proteins

beta-Dystroglycan is a ubiquitously expressed integral membrane protein that undergoes tyrosine phosphorylation in an adhesion-dependent manner. However, it remains unknown whether tyrosine-phosphorylated beta-dystroglycan interacts with SH2 domain containing proteins. The tyrosine phosphorylation of beta-dystroglycan is shown to be constitutively elevated in v-Src transformed cells. This phosphorylation event has been reconstituted in vivo by transiently coexpressing wild-type c-Src with a fusion protein containing full-length beta-dystroglycan. Src-induced tyrosine phosphorylation of beta-dystroglycan is strictly dependent on the presence of a PPxY motif at its extreme C-terminus. In the nonphosphorylated state, this PPxY motif is normally recognized as a ligand by the WW domain; phosphorylation at this site blocks the binding of certain WW domain containing proteins. Using a GST fusion protein carrying the cytoplasmic tail of beta-dystroglycan, five SH2 domain containing proteins have been identified that interact with beta-dystroglycan in a phosphorylation-dependent manner, including c-Src, Fyn, Csk, NCK, and SHC. This binding activity has been localized to the PPxY motif by employing a panel of beta-dystroglycan-derived phosphopeptides. In addition, tyrosine phosphorylation of beta-dystroglycan in vivo results in the coimmunoprecipitation of the same SH2 domain containing proteins, and this binding event requirs the beta-dystroglycan C-terminal PPxY motif. Tyrosine phosphorylation of the PPxY motif within beta-dystroglycan may act as a regulatory switch to inhibit the binding of certain WW domain containing proteins, while recruiting SH2 domain-containing proteins (Sotgia, 2001).

Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface

In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. This study characterizes the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the alpha- and beta-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous beta-subunit is shed from the cell surface by metalloproteinases. Shedding of the beta-subunit releases the alpha-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1beta and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of alpha-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing (Herzog, 2004).

Dystroglycan and the synapse

Formation of the synaptic basal lamina at vertebrate neuromuscular junction involves the accumulation of numerous specialized extracellular matrix molecules including a specific form of acetylcholinesterase (AChE), the collagenic-tailed form. The mechanisms responsible for its localization at sites of nerve-muscle contact are not well understood. To understand synaptic AChE localization, a fluorescent conjugate of fasciculin 2, a snake alpha-neurotoxin that tightly binds to the catalytic subunit, was synthesized. Prelabeling AChE on the surface of Xenopus muscle cells revealed that preexisting AChE molecules could be recruited to form clusters that colocalize with acetylcholine receptors at sites of nerve-muscle contact. Likewise, purified avian AChE with collagen-like tail, when transplanted to Xenopus muscle cells before the addition of nerves, also accumulated at sites of nerve-muscle contact. Using exogenous avian AChE as a marker, it was shown that the collagenic-tailed form of the enzyme binds to the heparan-sulfate proteoglycan perlecan, which in turn binds to the dystroglycan complex through alpha-dystroglycan. Therefore, the dystroglycan-perlecan complex serves as a cell surface acceptor for AChE, enabling it to be clustered at the synapse by lateral migration within the plane of the membrane. A similar mechanism may underlie the initial formation of all specialized basal lamina interposed between other cell types (Peng, 1999).

In rat hippocampal cultures, pyramidal cells that receive little or no GABAergic input, mistarget alpha2-GABA(A) receptors and gephyrin to glutamatergic terminals. (Gephyrin is an ubiquitously expressed protein that, in the central nervous system, generates a protein scaffold to anchor inhibitory neurotransmitter receptors in the postsynaptic membrane). This mismatch does not occur in neurons innervated by numerous GABAergic terminals. A similar phenomenon has been reported for isolated autaptic hippocampal neurons. GABAergic synapses typically form multiple release sites apposed to GABA(A) receptor and gephyrin clusters. Remarkably, dystrophin, a protein highly abundant in skeletal muscle membranes, is extensively colocalized with alpha2-GABA(A) receptors exclusively opposite GABAergic terminals. In addition, selective apposition of syntrophin and beta-dystroglycan to GABAergic presynaptic terminals suggests that the entire dystrophin-associated protein complex (DPC) clusters at GABAergic synapses. In contrast to gephyrin and GABA(A) receptors, DPC proteins are not mistargeted to glutamatergic synapses, indicating independent clustering mechanisms. This was confirmed in hippocampal neurons cultured from GABA(A) receptor gamma2 subunit-deficient mice. Clustering of GABA(A) receptor and gephyrin in these neurons was strongly impaired, whereas clustering of dystrophin and associated proteins was unaffected by the absence of the gamma2 subunit. These results indicate that accumulation of dystrophin and DPC proteins at GABAergic synapses occurs independently of postsynaptic GABA(A) receptors and gephyrin. It is suggested that selective signaling from GABAergic terminals contributes to postsynaptic clustering of dystrophin (Brunig, 2002).

Synaptic differentiation results from an exchange of informational molecules between synaptic partners during development. At the vertebrate neuromuscular junction, agrin is one molecule presented by the presynaptic motor neuron that plays an instructive role in postsynaptic differentiation of the muscle cell, most notably in aggregation of acetylcholine receptors (AChRs). Although agrin is the best-characterized synaptogenic molecule, its mechanism of action remains uncertain, but clearly, it requires the receptor tyrosine kinase MuSK (muscle-specific kinase), the intracellular protein rapsyn, an Src-like kinase, and cytoskeletal components. In addition, the transmembrane protein dystroglycan interacts with the cytoskeleton and is implicated in agrin responsiveness. This alpha-beta heterodimer can bind agrin via its extracellular alpha subunit and associates with the membrane cytoskeleton via its beta subunit. In this study, it has been demonstrated that overexpression of the beta subunit of dystroglycan in cultured muscle cells inhibits agrin-induced AChR clustering. Deletion analysis and point mutagenesis demonstrate that the inhibition is mediated by an intracellular, juxtamembrane region composed of basic amino acids. Finally, the inhibition mediated by beta-dystroglycan extends to the minimal agrin fragment required for AChR clustering, suggesting that dystroglycan plays an important role in postsynaptic differentiation in response to agrin (Kahl, 2003).

Disruption of Dystroglycan expression

A null allele of dystroglycan (Dag1neo2) has been generated in mice. Heterozygous Dag1neo2 mice are viable and fertile. In contrast, homozygous Dag1neo2 embryos exhibit gross developmental abnormalities beginning around 6.5 days of gestation. Analysis of the mutant phenotype indicates that an early defect in the development of homozygous Dag1neo2 embryos is a disruption of Reichert's membrane, an extra-embryonic basement membrane. Consistent with the functional defects observed in Reichert's membrane, dystroglycan protein is localized in apposition to this structure in normal egg cylinder stage embryos. The localization of two critical structural elements of Reichert's membrane -- laminin and collagen IV -- are specifically disrupted in the homozygous Dag1neo2 embryos. Taken together, the data indicate that dystroglycan is required for the development of Reichert's membrane. Furthermore, these results suggest that disruption of basement membrane organization might be a common feature of muscular dystrophies linked to the DGC (Williamson, 1997).

Brain-selective deletion of dystroglycan in mice is sufficient to cause congenital muscular dystrophy-like brain malformations, including disarray of cerebral cortical layering, fusion of cerebral hemispheres and cerebellar folia, and aberrant migration of granule cells. Dystroglycan-null brain loses its high-affinity binding to the extracellular matrix protein laminin, and shows discontinuities in the pial surface basal lamina (glia limitans) that probably underlie the neuronal migration errors. Furthermore, mutant mice have severely blunted hippocampal long-term potentiation with electrophysiologic characterization indicating that dystroglycan might have a postsynaptic role in learning and memory. These data strongly support the hypothesis that defects in dystroglycan are central to the pathogenesis of structural and functional brain abnormalities seen in congenital muscular dystrophy (Moore, 2002).

Striated muscle-specific disruption of the dystroglycan (DAG1) gene results in loss of the dystrophin-glycoprotein complex in differentiated muscle and a remarkably mild muscular dystrophy with hypertrophy and without tissue fibrosis. Satellite cells, expressing dystroglycan, are found to support continued efficient regeneration of skeletal muscle along with transient expression of dystroglycan in regenerating muscle fibers. A similar phenomenon of reexpression of functional dystroglycan is demonstrated in regenerating muscle fibers in a mild form of human muscular dystrophy caused by disruption of posttranslational dystroglycan processing. Thus, maintenance of regenerative capacity by satellite cells expressing dystroglycan is likely responsible for mild disease progression in mice and possibly humans. Therefore, inadequate repair of skeletal muscle by satellite cells represents an important mechanism affecting the pathogenesis of muscular dystrophy (Cohn, 2002).

Association of the the dystrophin/utrophin network with microtubule filaments

A screen for proteins that interact with beta 2-syntrophin led to the isolation of MAST205 (microtubule-associated serine/threonine kinase-205 kD) and a newly identified homolog, SAST (syntrophin-associated serine/threonine kinase). Binding studies show that beta 2-syntrophin and MAST205/SAST associate via a PDZ-PDZ domain interaction. MAST205 colocalizes with beta 2-syntrophin and utrophin at neuromuscular junctions. SAST colocalizes with syntrophin in cerebral vasculature, spermatic acrosomes and neuronal processes. SAST and syntrophin are highly associated with purified microtubules and microtubule-associated proteins, whereas utrophin and dystrophin are only partially associated with microtubules. The data suggest that MAST205 and SAST link the dystrophin/utrophin network with microtubule filaments via the syntrophins (Lumeng, 1999).

An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis

Dystroglycan (Dg) is a transmembrane receptor for laminin that must be expressed at the right time and place in order to be involved in notochord morphogenesis. The function of Dg was examined in Xenopus laevis embryos by knockdown of Dg and overexpression and replacement of the endogenous Dg with a mutated form of the protein. This analysis revealed that Dg is required for correct laminin assembly, for cell polarization during mediolateral intercalation and for proper differentiation of vacuoles. Using mutations in the cytoplasmic domain, two sites were identified that are involved in cell polarization and are required for mediolateral cell intercalation, and a site that is required for vacuolation. Furthermore, using a proteomic analysis, the cytoskeletal non-muscle myosin IIA has been identified for the first time as a molecular link between the Dg-cytoplasmic domain and cortical actin. The data allowed identification of the adhesome laminin-Dg-myosin IIA as being required to maintain the cortical actin cytoskeleton network during vacuolation, which is crucial to maintain the shape of notochordal cells (Buisson, 2014).

Dystroglycan expression in tumors

Cellular interactions with the extracellular matrix are an important factor in the development and progression of many types of cancer. Dystroglycan is a cell surface receptor for several extracellular matrix proteins and plays a central role in the formation of basement membranes in tissues. Because abnormalities in the structure and function of basement membranes are hallmarks of metastatic disease, the status of dystroglycan expression was examined in prostate and breast tumors. In 15 cases of surgically resected prostate cancer, a noted reduced expression of dystroglycan was noted as judged by intensity of immunohistochemical staining. This reduction was most pronounced in high-grade disease. Similar results were found in 6 cases of mammary ductal adenocarcinoma, suggesting that reduced expression of dystroglycan may be a conserved feature of epithelial neoplasia. These data suggest that reduced expression of dystroglycan in prostate and breast cancers may lead to abnormal cell-extracellular matrix interactions and thus contribute to progression to metastatic disease (Henry, 2001a).

Dystroglycan (DG) is an adhesion molecule responsible for crucial interactions between extracellular matrix and cytoplasmic compartment. It is formed by two subunits, alpha-DG (extracellular) and beta-DG (transmembrane), that bind to laminin in the matrix and dystrophin in the cytoskeleton, respectively. This study evaluated by Western blot analysis the expression of DG in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon and breast cancers. Decreased expression of DG was observed in most of the cell lines and in both types of tumors and correlated with higher tumor grade and stage. Analysis of the mRNA levels suggested that expression of DG protein is likely regulated at a posttranscriptional level. Evaluation of alpha-DG expression by immunostaining in a series of archival cases of primary breast carcinomas confirmed that alpha-DG expression is lost in a significant fraction of tumors (66%). Loss of DG staining correlated with higher tumor stage, positivity for p53, and high proliferation index. A significant correlation was also observed between loss of alpha-DG and overall survival in an univariate analysis. These data indicate that DG expression is frequently lost in human malignancies and suggest that this glycoprotein might play an important role in human tumor development and progression (Sgambato, 2003).

Receptors mediating cell-basement membrane interactions are potent regulators of epithelial architecture and function, and alterations in signals from the basement membrane are implicated in the aberrant behavior of carcinoma cells. This study has investigated the role of the basement membrane receptor dystroglycan (DG) in mammary epithelial cell function, and the significance of loss of DG function in breast tumor cell lines. Nonmalignant mammary epithelial cells express a functional DG. Analysis of multiple breast carcinoma cell lines revealed that DG is expressed in all of the cell lines examined, as evidenced by beta-DG expression, but alpha-DG is functionally diminished in the majority. High levels of alpha-DG correlated strongly with the ability of cells to polarize in the presence of the basement membrane. Overexpression of the DG cDNA in HMT-3522-T4-2 cells elevated alpha-DG levels and altered responsiveness to the basement membrane; DG overexpression restored the ability of the cells to undergo cytoskeletal changes, to polarize, and to restrict growth in response to basement membrane proteins. Moreover, restoration of DG function to these cells greatly reduced their tumorigenic potential in nude mice. These data point to DG as an important mediator of normal cell responses to the basement membrane, and as a significant variable in carcinoma cells, in which its frequent loss can contribute to aberrant cell behavior (Muschler, 2002).

Alpha-dystroglycan interactions affect cerebellar granule neuron migration

The interaction of alpha-dystroglycan (-DG) with its extracellular binding partners requires glycans attached to its mucin core domain, and defects in the glycosylation of alpha-DG are associated with both muscular dystrophy and neuronal migration defects. The involvement of alpha-DG and one of its ligands, agrin, in cerebellar neuronal migration was investigated. Antibodies directed against glycosylated alpha-DG inhibited granule neuron migration in cerebellar slice cultures. alpha-DG interactions did not appear to influence neurite outgrowth in cerebellar explant cultures, but enhanced granule neuron binding was observed on cells transfected with alpha-DG. These results suggest that interactions involving alpha-DG influence the strength of attachment of granule neurons to the alpha-DG-expressing Bergmann glial cells that guide granule neuron migration in the cerebellum. Experiments using anti-agrin antibodies suggest that agrin is not involved in these interactions (Qu, 2004).

Dystroglycan suppresses Notch to regulate stem cell niche structure and function in the developing postnatal subventricular zone

While the extracellular matrix (ECM) is known to regulate neural stem cell quiescence (see Drosophila neuroblast) in the adult subventricular zone (SVZ), the function of ECM in the developing SVZ remains unknown. This study reports that the ECM receptor dystroglycan regulates a unique developmental restructuring of ECM in the early postnatal SVZ. Dystroglycan is furthermore required for ependymal cell differentiation and assembly of niche pinwheel structures, at least in part by suppressing Notch (see Drosophila Notch) activation in radial glial cells, which leads to the increased expression of MCI (see Drosophila geminin), Myb (see Drosophila Myb), and FoxJ1 (see Drosophila CG32006), transcriptional regulators necessary for acquisition of the multiciliated phenotype. Dystroglycan also regulates perinatal radial glial cell proliferation and transition into intermediate gliogenic progenitors, such that either acute or constitutive loss of function in dystroglycan results in increased oligodendrogenesis. These findings reveal a role for dystroglycan in orchestrating both the assembly and function of the SVZ neural stem cell niche (McClenahan, 2016).

Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling

Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, this study discovered that the invasive uterine anchor cell activates Notch signaling (see Notch in Drosophila) in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, it was found that Notch activation upregulates expression of ctg-1 (see CG13893 in Drosophila), which encodes a Sec14-GOLD protein and member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, these results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) (see Dg in Drosophila) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues (McClatchey, 2016).


Search PubMed for articles about Drosophila Dystroglycan

Andac, Z., Sasaki, T., Mann, K., Brancaccio, A., Deutzmann, R. and Timpl, R. (1999). Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis. J. Mol. Biol. 287: 253-264. Medline abstract: 10080889

Baas, A. F., et al. (2004). Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 116: 457-466. PubMed Citation: 15016379

Bogdanik, L., et al. (2008). Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction. PLoS ONE 3(4): e2084. PubMed Citation: 18446215

Brancaccio, A., Schulthess, T., Gesemann, M. and Engel, J. (1995). Electron microscopic evidence for a mucin-like region in chick muscle alpha-dystroglycan. FEBS Lett. 368: 139-142. Medline abstract: 7615068

Brockington, M., Blake, D. J., Prandini, P., Brown, S. C., Torelli, S., Benson, M. A., Ponting, C. P., Estournet, B. and Romero, N. B. et al. (2001). Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin 2 deficiency and abnormal glycosylation of Dg. Am. J. Hum. Genet. 69: 1198-1209. 11592034

Brunig, I., Suter, A, Knuesel, I, Luscher, B and Fritschy, J. M. (2002). GABAergic terminals are required for postsynaptic clustering of dystrophin but not of GABA(A) receptors and gephyrin. J. Neurosci. 22(12): 4805-13. 12077177

Burton, E. A. and Davies, K. E. (2002). Muscular dystrophy — reason for optimism? Cell 108, 5-8. 11792315 ]

Buisson, N., Sirour, C., Moreau, N., Denker, E., Le Bouffant, R., Goullancourt, A., Darribere, T. and Bello, V. (2014). An adhesome comprising laminin, dystroglycan and myosin IIA is required during notochord development in Xenopus laevis. Development 141: 4569-4579. PubMed ID: 25359726

Christoforou, C. P., Greer, C. E., Challoner, B. R., Charizanos, D. and Ray, R. P. (2008). The detached locus encodes Drosophila Dystrophin, which acts with other components of the Dystrophin Associated Protein Complex to influence intercellular signalling in developing wing veins. Dev. Biol. 313(2): 519-32. PubMed Citation: 18093579

Cohn, R. D. and Campbell, K. P. (2000). Molecular basis of muscular dystrophies. Muscle Nerve 23: 1456-1471. Medline abstract: 11003781

Cohn, R. D., et al. (2002). Disruption of DAG1 in differentiated skeletal muscle reveals a role for dystroglycan in muscle regeneration. Cell 110(5): 639-48. 12230980

Colognato, H., Winkelmann, D. A. and Yurchenco, P. D. (1999). Laminin polymerization induces a receptor-cytoskeleton network. J. Cell Biol. 145: 619-631. 10225961

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Biological Overview

date revised: 11 November 2016

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