Mutations in genes encoding proteins of the human dystrophin-associated glycoprotein complex (DGC) cause the Duchenne, Becker and limb-girdle muscular dystrophies. Subsets of the DGC proteins form tissue-specific complexes which are thought to play structural and signaling roles in the muscle and at the neuromuscular junction. Furthermore, mutations in the dystrophin gene that lead to Duchenne muscular dystrophy are frequently associated with cognitive and behavioral deficits, suggesting a role for dystrophin in the nervous system. Despite significant progress over the past decade, many fundamental questions about the roles played by dystrophin and the other DGC proteins in the muscle and peripheral and central nervous systems remain to be answered. Mammalian models of DGC gene function are complicated by the existence of fully or partially redundant genes whose functions can mask effects of the inactivation of a given DGC gene. The genome of the fruitfly Drosophila melanogaster encodes a single ortholog of the majority of the mammalian DGC protein subclasses, thus potentially simplifying their functional analysis. This study reports the embryonic mRNA expression patterns of the individual DGC orthologs. They are predominantly expressed in the nervous system and in muscle. Dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 1, and all three sarcoglycan orthologs are found in the brain and the ventral nerve cord, while dystrophin, dystrobrevin-like, dystroglycan-like, syntrophin-like 2, sarcoglycan alpha and sarcoglycan delta are expressed in distinct and sometimes overlapping domains of mesoderm-derived tissues, i.e., muscles of the body wall and around the gut (Dekkers, 2004).

The Drosophila ortholog of dystrophin is, like its human counterpart, one of the largest genes in the genome and encodes at least four isoforms, three of which are expressed during embryogenesis. The large embryonic isoforms, dmDLP1 and dmDLP2, are predominantly found in the gut and the mesoderm, while the small isoform, dmDp186, is present at high levels in the central nervous system. Interestingly, a small isoform of human dystrophin, Dp71, is also found to be highly expressed in the brain (Dekkers, 2004).

Using RNA in situ hybridization on whole mount embryos, the spatial and temporal expression patterns of the other DGC orthologs during embryonic development was determined. Probes were generated from sequence-verified expressed sequence tags (ESTs) and DGC gene expression patterns were examined throughout embryogenesis. The Drosophila DGC orthologs are predominantly expressed in two domains, the nervous system and muscle (Dekkers, 2004).

Seven of the eight DGC orthologs are found at high levels in the ventral nerve cord and brain: the small dystrophin isoform dmDp186, dystrobrevin-like, dystroglycan-like, syntrophin-like 1 (syn1), sarcoglycan beta (scgbeta), sarcoglycan delta (scgdelta) and sarcoglycan alpha (scgalpha). A more detailed analysis of the DGC expression domains in the ventral nerve cord shows that dystrobrevin is also expressed at low levels by a cluster of neurons at the ventral midline whose location suggests that they are the ventral unpaired midline neurons (VUMS), while scgdelta is present in superficial ventral cells throughout the ventral nerve cord. Double labeling of embryos with scgdelta RNA and anti-Repo antibody (which labels the lateral glia) reveals that these cells are not of glial origin. Instead, their number and location suggest that they are the larval neuroblasts, the precursors of the adult nervous system. At present, lineage-specific markers for larval neuroblasts are not available to confirm the identity of these cells. Dystroglycan-like and dystrobrevin-like RNA can be detected in the cap cells that attach the atonal and chordotonal sensory organs of the peripheral nervous system to the body wall as revealed by a double labeling of embryos with dystroglycan RNA and the sensory neuronal marker antibody 22C10. Dystrobrevin-like and scgbeta can also be detected in the labral sensory system, while syn1 is highly expressed in the stomatogastric nervous system. The large embryonic dystrophin isoforms dmDLP1 and dmDLP2 are expressed by the mesectodermal cells, which are derived from the presumptive ectoderm. In later stages of embryonic development mesectodermal cells give rise to midline neuronal precursors and midline glial cells (Dekkers, 2004).

The large embryonic dystrophin isoforms dmDLP1/dmDLP2, dystrobrevin-like, scgdelta and dystroglycan-like are expressed in most body wall muscle fibres. Closer examination of the expression domains of the large embryonic dystrophin isoforms during the stages at which the musculature is forming, indicates that these isoforms are expressed at very low levels throughout the muscle fibres, and accumulate at the muscle attachment sites. Scgdelta is seen at stage 14 at high levels in ventral muscle 13 and later throughout the musculature, including an unidentified dorsal triangular-shaped muscle. Syntrophin-like 2 (syn2), dystrobrevin-like, dystroglycan-like, the large dystrophin isoforms and scgalpha are expressed in the dorsal pharyngeal muscle, a large muscle in the head. Dystroglycan-like and dmDLP1/dmDLP2 are also expressed in the dorsal median cells that are of mesodermal origin and lie dorsomedially on top of the ventral cord. The dmDLP1 and dmDLP2 isoforms, dystroglycan-like and syn2 are expressed in the visceral mesoderm, the precursor of intestinal musculature. The large embryonic dystrophin isoforms, syn2 and dystroglycan-like are expressed in the pericardial cells that constitute the mesoderm-derived dorsal vessel or embryonic heart. All DGC orthologues are detected in the midgut, although only some temporally overlap (Dekkers, 2004).

No studies detailing the embryonic functions of the Drosophila DGC orthologues have been published. However, a null allele of the dystroglycan-like gene has been generated and the resulting phenotype suggests a role for this protein in establishing cellular polarity in the developing oocyte and in imaginal disc epithelial cells. dystroglycan-like and the seven other DGC orthologues in Drosophila are expressed in dynamic patterns throughout embryogenesis. Furthermore, partly or completely overlapping domains of expression are found for a subset of these genes in the ventral nerve cord and brain and in the somatic musculature of the body wall and in a number of smaller domains in nervous and mesoderm tissues and gut. These data suggest that, as in mammals, the Drosophila DGC members may form tissue-specific complexes. Further molecular and genetic studies will provide a better understanding of the biological roles of the different members of the Drosophila DGC complex in the muscle and the nervous system (Dekkers, 2004).

Dystrophin isoforms DLP1, DLP2, and Dp186 are expressed during all stages of development, whereas DLP3 is only expressed in the adult fly (Neuman, 2001; Dekkers, 2004). During embryogenesis, the DLP1 mRNA is present in the visceral mesoderm, whereas DLP2 is expressed in the visceral mesoderm, in muscle attachment sites, throughout muscle fibers, in the mesectodermal cells at the midline, and the gut (Dekkers, 2004). Dp186 is highly expressed in the embryonic CNS but absent from the musculature. In larvae, the large dystrophin isoform DLP2 mRNA, but not DLP1, is found in all muscle fibers. The large isoforms are not detectably expressed in the larval brain or neuropil. In contrast, Dp186 mRNA is found most predominantly throughout the larval neuropil and brain and in the eye-antennal discs (van der Plas, 2006).

Three region-specific antibodies were generated that recognize (1) the known large Dystrophin isoforms DLP1, DLP2, and DLP3 (anti-Dyslarge), (2) the CNS-specific isoform (anti-Dp186), and (3) the C-terminal region common to all Dystrophin isoforms (anti-DysCO₂H). Western analyses of embryonic extracts using these antibodies show that the anti-Dyslarge antibodies recognizes a protein of the expected size for the large isoforms (~400 kDa), whose level is strongly reduced in the dystrophin mutant dys^GE20705 and increased when the large isoform DLP2 is overexpressed. The anti-Dp186 antibody recognizes a protein species of the appropriate size (~190 kDa), and the anti-DysCO₂Hantibody predominantly recognizes the large isoforms, but also Dp186, when overexpressed (van der Plas, 2006).

The region-specific antibodies were used to determine the protein expression domains of the Dystrophin isoforms. The anti-DysCO₂H and anti-Dyslarge antibodies label synaptic and extrasynaptic sites of expression in third-instar larval body walls. To determine whether the Dystrophin protein is present at the presynaptic or the postsynaptic side of the NMJ, larval body walls were colabeled for the presynaptically localized HRP protein and the Dystrophin protein. No overlap in staining was apparent, indicating that the Dystrophin protein is postsynaptically localized at the larval NMJ. This result is consistent with the finding that the large dystrophin mRNAs are absent from the neuropil in which the motoneuron cell bodies are located. The extrasynaptic Dystrophin protein colocalizes with actin at the I-band of sarcomeres. Synaptic and extrasynaptic Dystrophin expression levels are severely reduced in the dystrophin mutant dys^GE20705, indicating the specificity of the antibody. When the DLP2 large Dystrophin isoform is overexpressed in muscle using the Gal4-UAS yeast transcriptional activation system, Dystrophin protein accumulates highly at the postsynaptic regions of the NMJ. These results indicate that the large Dystrophin isoform DLP2 is localized to both the postsynaptic region of the NMJ and extrasynaptic regions, in which it colocalizes with actin (van der Plas, 2006).

Effects of Mutation or Deletion

Lack of postsynaptic Dystrophin results in an increase in neurotransmitter release

To investigate the roles of dystrophin during Drosophila development, stocks were generated by P-element excision mutagenesis that lack, or have severely reduced, expression of the large Dystrophin isoforms. The mutant dys^E6 has a deletion of 2.7 kb; dys^E31 is a precise excision control of the EP3397 P-element.The independently derived P-element line dys^GE20705 was used in which a P-element is inserted 250 bp upstream of the Dp186 initiator codon. To determine whether, and to what extent, the mRNA levels of the specific Dystrophin isoforms in these mutants are affected, semiquantitative RT-PCR was performed on total RNA derived from mutant larval tissues using primers specific for each of the isoforms. Dystrophin isoform proteins were proteolytically degraded during the preparation of larval extracts, thus precluding analysis of their expression levels by Western blotting. RT-PCR analyses revealed that dys^E6 expresses DLP1 mRNA at wild-type levels but lacks DLP2 mRNA, whereas dys^GE20705 shows an approximate threefold reduction in DLP1 and an approximate fourfold reduction in DLP2 mRNA levels. Dp186 mRNA levels are wild type in dys^E6 but are significantly reduced (~27-fold) in dys^GE20705. The precise excision control dys^E31 shows wild-type expression levels of all isoforms. The lack of detectable large-isoform expression in the dys^GE20705 mutant larval musculature was confirmed by staining mutant body walls with the anti-DysCO₂H antibody and by Western blot analysis of embryonic extracts using the three region-specific antibodies. The CG6255 gene, which encodes a putative testis-specific succinate-CoA ligase and is located in the intron between the DLP1 and DLP2 first exons, is deleted in dys^E6 but not in dys^GE20705. RT-PCR analyses indicate that CG6255 is not expressed in dys^E6 but is expressed in dys^GE20705 at wild-type levels (van der Plas, 2006).

Morphometric analyses of the muscle and motoneuron terminal were performed at the muscle 6/7 synapse to examine whether the dystrophin mutant NMJ or muscle displays any structural abnormalities. Wild-type and mutant body walls were labeled with anti-HRP, and muscle size, numbers of boutons, lengths of the synaptic termini, and the number of terminal branches were determined. No striking statistically significant differences were observed between dystrophin mutants and control larvae. Furthermore, no apparent changes were seen in the distribution of the postsynaptically localized glutamate receptor subunits DGluRIIA and DGluRIIB, components of the major neurotransmitter receptors at the Drosophila NMJ. Because mammalian dystrophin isoforms have been reported to be found associated with T-tubules (Watkins, 1988), invaginations of the sarcolemma containing voltage-gated channels, the distribution of the Discs-large protein, which labels the T-tubuli, was examined in the dystrophin mutant. Because Dystrophin colocalizes extrasynaptically with actin, actin localization was examined in the mutant. Both proteins exhibit wild-type expression in dys^E6, suggesting that general muscle morphology is not affected in the dystrophin mutants (van der Plas, 2006).

The lack of overt muscle degeneration in the dystrophin mutants allowed an examination of whether the large Dystrophin isoforms play a role in synaptic transmission. The depolarizations following from spontaneous and evoked neurotransmitter release were examined with an intracellular microelectrode at NMJs of muscle 6 in wild-type and dystrophin mutant female third-instar larvae in medium containing 1.5 mM Ca²⁺. Excitatory junction potential (EJP) amplitudes, evoked by nerve stimulation at 0.3 Hz, were ~45% increased in dystrophin mutants compared with the control larvae. Spontaneous mEJP amplitudes were essentially unchanged in the mutants compared with the controls. QC, the number of neurotransmitter quanta released on a nerve impulse, was calculated by dividing the mean EJP amplitude, corrected for nonlinear summation, by the mean mEJP amplitude. QC in the mutants, dys^E6 and dys^GE20705, was ~50%-65% higher than at wild-type control synapses. Calculation of QC without correction for nonlinear summation also resulted in statistically significant differences between the dystrophin mutants and wild-type controls. In addition, the frequency of spontaneous neurotransmitter release was elevated ~35%-50% in the mutants. These results indicate that postsynaptic dystrophin is required to maintain wild-type levels of neurotransmitter release (van der Plas, 2006).

In addition, the inter-allelic dys^E6/dys^GE20705 combination, the mutants each derived from an independent genetic background, showed a similar effect on EJPs, mEJPs, QC, and mEJP frequency as the homozygous mutants, further indicating that the phenotype observed was attributable to the mutation in dystrophin. Interestingly, a similar electrophysiological behavior was seen in the heterozygotes, dys^E6/+ and dys^GE20705/+, indicating that dystrophin is haplo-insufficient for normal Drosophila NMJ physiology. Semiquantitative RT-PCR analysis shows that the DLP2 levels are reduced twofold to fourfold in the heterozygote animals (van der Plas, 2006).

To determine whether the elevated neurotransmitter release in the mutant showed altered Ca²⁺ dependence, the QCs of dystrophin mutant NMJs were compared with controls measured at external Ca²⁺ concentrations ranging from 0.25 to 1.5 mM. Mutant NMJ QC was significantly higher than the controls at all concentrations tested. The slopes of the regression lines of the Ca²⁺ titration curves at the low Ca²⁺ range did not differ significantly, showing that the Ca²⁺ cooperativity at the mutant NMJ is apparently unchanged. Thus, the increased release observed in the dystrophin mutant is independent of the extracellular Ca²⁺ concentration and unlikely to be attributable to altered properties of the Ca²⁺ sensor that triggers presynaptic vesicle fusion. Further analyisis revealed that the increase in EJP amplitude is attributable to an increase in presynaptic glutamate release in the mutants (van der Plas, 2006).

Elevated QC can result from increased probability of release or increases in the size of the readily releasable vesicle pool. Because increased probability has been shown to be reflected by reduced paired-pulse facilitation, this short-term dynamic behavior of neurotransmitter release was determined in wild-type and dys^E6 larvae. The protocol resulted in ~50% facilitation in wild-type larvae but failed to do so in the dys^E6 mutant. The size of the readily releasable vesicle pool can be assessed experimentally by measuring asynchronous quantal release during incubation with hypertonic medium. Application of 0.05 M sucrose did not reveal significant differences in the elevated mEJP frequency level between the mutant and wild type. These analyses indicate that the elevation in evoked neurotransmitter release at the dystrophin-deficient NMJ resulted from increased probability of release rather than from an increased size of transmitter vesicle pool (van der Plas, 2006).

Transgenic RNA interference and rescue experiments show a postsynaptic requirement for Dystrophin

RT-PCR analyses indicated that the dys^E6 mutation reduces the expression levels of DLP2 and CG6255 and dys^GE20705 affects DLP2 and the CNS-specific isoform Dp186 but not CG6255. To further address potential roles for either CG6255 or Dp186 in the dystrophin mutant NMJ phenotype and to confirm that the postsynaptic absence of Dystrophin protein is responsible for the increased neurotransmitter release in the mutants, transgenic tissue-specific RNA interference was used to reduce Dystrophin levels specifically either presynaptically or postsynaptically. Transgenic flies that express double-stranded RNA (dsRNA) directed against dystrophin sequences present in the large dystrophin isoform mRNAs (UAS-RNAi-dysNH₂) under Gal4 control. Semiquantitative RT-PCR analysis shows a more than eightfold decrease of DLP2 expression levels in the larval musculature during expression of UAS-RNAi-dysNH2 driven by 24B-Gal4, but Dp186 and CG6255 levels remained at the levels observed in both wild-type and the dys^E31 precise excision larvae. Pan-neuronal expression of UAS-RNAi-dysNH₂, driven by Elav-Gal4, failed to suppress Dp186 expression levels, further confirming the specificity of the effects of this transgene. When UAS-RNAi-dysNH₂ was expressed in the muscle using either of two different muscle-specific drivers, 24B-Gal4 or G14-Gal4, QC increased to similar levels (~70% higher than control) as observed in the dystrophin mutants. However, when this construct is driven by the Elav-Gal4 transgene, no change in QC was observed compared with the control larvae (van der Plas, 2006).

It was then asked whether the dystrophin mutant phenotype could be rescued by increasing postsynaptic dystrophin expression. Lacking a rescue transgene, advantage of a P-element insertion, GS12472, which lies 1.9 kb upstream of the DLP2 initiator codon and bears a UAS-dependent promoter appropriately oriented to express DLP2. Driving this P-element does not overexpress DLP1 or CG6255; the P-element insertion site is downstream of the unique first exon of DLP1, and CG6255 has the opposite orientation to the dystrophin coding frames. Overexpression of DLP2 was confirmed by Western blot analysis of transgenic embryos. Postsynaptic expression of the DLP2 isoform in the heterozygous mutant background rescues QC to wild-type levels (G14-Gal4/+; GS12472/dys^E6) but not when expressed presynaptically (OK6-Gal4/+; GS12472/dys^E6). Together with the mutant analyses, these data indicate that electrophysiological phenotype at the dys^E6 and dys^GE20705 mutant NMJs is elicited by decreased expression of the postsynaptically expressed DLP2 isoform and is unlikely to be attributable to effects during CG6255, Dp186, or presynaptically expressed DLP2 (van der Plas, 2006).

The absence of postsynaptic Dystrophin results in an increase in the number of T-bars

To evaluate whether the electrophysiological changes in the mutant correlate with alterations in NMJ ultrastructure, morphometric measurements were performed on electron micrographs of mutant and control third-instar larval NMJs. The bouton area and the surface area of the subsynaptic reticulum relative to the bouton area were similar for all genotypes, but the shape of the boutons in the mutants dys^GE20705 and dys^E6 appeared slightly more elongated, as determined by the ratio of the longest and shortest diameter of a bouton section. The area of the bouton occupied by vesicles was increased in the dys^GE20705 and dys^E6 mutants compared with wild type and precise excision control dys^E31. T-bars are electron-dense presynaptic structures possibly representing a subtype of neurotransmitter release site. The number of active zones with a T-bar relative to the total number of active zones was significantly increased in both the dys^E6 and dys^GE20705 mutants by approximately two-fold, whereas the overall number of active zones did not increase. Although wild-type T-bar densities vary considerably between laboratories and their precise function is unclear, this increase in T-bars in the mutant relative to the isogenic control is an observed ultrastructural correlate of the increased neurotransmitter release in the dystrophin mutant (van der Plas, 2006).

BMP signaling at the NMJ is required for the increased neurotransmitter release in the Dystrophin mutant

The BMP pathway plays an important role in retrograde signaling at the larval NMJ and in the Drosophila CNS. To evaluate the role of BMP signaling in the increase in neurotransmitter release induced by the absence of dystrophin, electrophysiological recordings were performed at NMJs lacking both dystrophin and wit. The transgenic RNA interference approach was used to reduce postsynaptic dystrophin levels (van der Plas, 2006).

Homozygous wit mutant NMJs postsynaptically expressing dsRNA directed against the Dystrophin large isoforms (UAS-RNAi-dysNH2/+; G14-Gal4/+; wit) displayed mEJP amplitude, EJP amplitude, and QC values similar to the wit mutant alone. The homozygous wit NMJ has very low EJP amplitudes but maintains wild-type level mEJP amplitudes. Thus, the absence of dystrophin failed to elicit an increase in QC in the wit background. Wit function and, by extension, BMP signaling is therefore required at the NMJ for the increase in neurotransmitter release elicited by the absence of dystrophin (van der Plas, 2006).

To evaluate whether the absence of postsynaptic dystrophin affects a known BMP target, immunofluorescence analysis was performed of mutant and wild-type embryonic ventral nerve cords, using an anti-phospho-Mad (PMad) antibody that recognizes the activated form of the Mad downstream effector of wit signaling. No differences were observed in PMad expression levels or domains between the dystrophin mutants or individuals postsynaptically overexpressing DLP2 and controls, whereas homozygous wit mutants showed significantly decreased levels of the protein (van der Plas, 2006).

The dystrophin mutants show similar electrophysiological and morphological phenotypes as larvae postsynaptically expressing CaMKII inhibitors. Both display increased QC and an increase in the ratio of active zones with T-bars versus the total number of active zones, without additional significant changes in synaptic morphology. Furthermore, the increase in QC when CaMKII is reduced postsynaptically is dependent on wit function. Therefore, embryonic ventral nerve cords that have reduced or elevated levels of CaMKII, UAS-Ala, and UAS-T287D, respectively, were examined for PMad staining. No changes in the expression of PMad was observed. These results suggest that, although wit is required for increased QC at NMJs postsynaptically deficient for either Dystrophin or CaMKII, alteration of either Dystrophin or CaMKII levels does not result in changes in embryonic PMad expression. Thus, the interaction of dystrophin or CaMKII and wit in NMJ homeostasis is unlikely to involve regulation of PMad expression (van der Plas, 2006).

Drosophila Dystrophin is required for integrity of the musculature

Duchenne muscular dystrophy is caused by mutations in the dystrophin gene and is characterized by progressive muscle wasting. The highly conserved dystrophin gene encodes a number of protein isoforms. The Dystrophin protein is part of a large protein assembly, the Dystrophin glycoprotein complex, which stabilizes the muscle membrane during contraction and acts as a scaffold for signaling molecules. How the absence of Dystrophin results in the onset of muscular dystrophy remains unclear. This study used transgenic RNA interference to examine the roles of the Drosophila Dystrophin isoforms in muscle. Previous studies have shown that one of the Drosophila Dystrophin orthologs, the DLP2 isoform, is not required to maintain muscle integrity, but plays a role in neuromuscular homeostasis by regulating neurotransmitter release. This report shows that reduction of all Dystrophin isoform expression levels in the musculature does not apparently affect myogenesis or muscle attachment, but results in progressive muscle degeneration in larvae and adult flies. A recently identified Dystrophin isoform, Dp117, is expressed in the musculature and is required for muscle integrity. Muscle fibers with reduced levels of Dp117 display disorganized actin-myosin filaments and the cellular hallmarks of necrosis. These results indicate the existence of at least two possibly separate roles of dystrophin in muscle, maintaining synaptic homeostasis and preserving the structural stability of the muscle (van der Plas, 2007).

This study made use of transgenic RNA interference and heritable mutant alleles to address which Dystrophin isoforms play roles in maintaining the integrity of the fruitfly musculature. It was shown that expression of transgenically encoded dsRNAs targeting a region of the dystrophin gene common to the mRNAs encoding all known isoforms (RNAi-DysCO₂H) in the muscle does not apparently affect embryonic or pupal myogenesis, but results in severe, progressive muscle degeneration and premature death. The newly identified Dystrophin isoform, Dp117, is expressed in the muscle and therefore a likely candidate, in addition to DLP2, to play a role in stabilizing the muscle structure. While the muscle becomes hyperdepolarized upon evoked stimulation by the motoneuron when DLP2 expression levels are reduced, overall muscle morphology and viability are apparently unaffected during development. In contrast, reduction in Dp117 expression levels results in muscle degeneration and lethality. No apparent apoptotic features were observed in degenerating larval and adult muscles, suggesting that the morphological changes observed, namely disorganization of actin-myosin filaments and swelling of mitochondria, dyads and SR, likely reflect necrotic processes in the Dystrophin-deficient muscle (van der Plas, 2007).

Reduction of pan-Dystrophin expression levels, in which the Dp117 level was 3-fold reduced, caused more severe larval muscle degeneration than a 3-fold reduction of Dp117 alone, indicating that Dp117 is not solely responsible for maintaining muscle integrity. In contrast, further reduction of Dp117 expression by 27-fold, resulted in more severe muscle degeneration, approaching that seen when expression of all Dystrophin isoforms was reduced. When Dp117 expression was 3-fold reduced in the dys^{DLP2 E6} background, which lacks DLP2, muscle degeneration did not increase, suggesting the involvement of factors other than DLP2 in maintaining muscle stability. While the existence of more, unidentified Dystrophin isoforms in Drosophila cannot be ruled out, RT-PCR and 5' race analyses have been performed on mRNAs derived from both embryos and larvae and have not provided evidence that more Dystrophin isoforms are encoded within the Drosophila genome (van der Plas, 2007).

The renewal of the musculature at the beginning of pupation is apparently normal when dystrophin levels are reduced, however, at or near stages when the muscles are employed for eclosion from the pupal case, muscle degeneration occurs. Signs of necrosis are observed at the ultrastructural level and actin-myosin filaments are severely disorganized in adult flies that are still alive, but unable to emerge fully from the pupal cage and the approximately 10% of flies that do eclose, but which die after 1 day. These results suggest that Dystrophin is not required for myogenesis or attachment, but plays roles in maintaining muscle integrity, possibly subsequent to exercise. This hypothesis is supported by studies of DMD patients (Kimura, 2006), dystrophin-deficient mice (Mokhtarian, 1999), and C. elegans (Mariol, 2007) that indicate that contractile activity is required for the onset of muscle degeneration (van der Plas, 2007).

What is the relationship between muscle degeneration and the lethal stage in Drosophila with reduced levels of Dystrophin isoforms? It is hypothesized that the lethality occurring at differing stages of development in the different transgenic lines is not likely caused by the muscle degeneration observable at the light and electron microscopic levels in larval or adult body wall muscles. Nor, conversely, does muscle degeneration appear to be subsequent to the onset of the lethal phase. This is based on the following observations. First, the 2xRNAi-Dp117-24B-Gal4 animals die as 3rd instar larvae, although their muscle degeneration is less severe than that of RNAi-DysCO₂H/24B-Gal4 larvae, the majority of which die later as pharate adults. Second, 2xRNAi-Dp117-Mhc-Gal4 flies display no overt lethal phase but their musculature is disrupted. Finally, the majority of RNAi-DysCO₂H/DMef2-Gal4 pharate adults die without apparent muscle damage, whereas those that attempt (sometimes succeeding) to eclose do display muscle degeneration. Thus, these data indicate that the muscle degeneration and lethality associated with reduced levels of Dystrophin expression are unlikely to be causally related (van der Plas, 2007).

In comparing when 24B-Gal4 driven single and double copy RNAi-Dp117 animals died, a correlation was noted between the degree of reduction of Dp117 expression levels and time of onset of lethality. As muscle degeneration and lethality did not appear to correlate, possible vital roles of Dp117 were evaluated in other mesoderm-derived tissues by expressing the Dp117 dsRNA in the heart (dorsal vessel) or gut. No evidence was found for an essential role of Dp117 in these tissues, however, vital roles cannot be ruled out for Dp117 in other mesoderm-derived tissues for which Gal4 drivers do not exist. It also remains possible that the Dystrophin-deficient body wall muscle syncitium may be defective for vital functions that, depending on which isoforms expression levels are reduced, do not necessarily manifest themselves in visible structural changes prior to death. For example, Ca²⁺ flux may be altered in Drosophila Dystrophin-deficient muscle cells as has been observed in a number of studies of mammalian Dystrophin-deficient tissues (reviewed in Constantin, 2006). The failure of the 2xRNAi-Dp117-24B-Gal4 animals to continue feeding after the 2nd instar stage may cause their deaths or, otherwise, reflect deficits in the musculature involved in feeding or changes in their feeding behavior. Why animals with reduced levels of all Dystrophin isoforms or Dp117 alone die during development and why they die at different stages, however, remains unclear at present (van der Plas, 2007).

How do the roles of the Dystrophin isoforms in maintaining the musculature compare between Drosophila and humans? Muscle wasting in DMD patients is caused by the absence of the large, predominantly skeletal isoform, Dp427m, which precludes the formation of a functional DGC (reviewed in Muntoni, 2003). In addition to a role for the DGC in providing structural stability of the musculature through maintaining sarcolemmal integrity, muscular dystrophy has been linked to disruption of the costameric lattice (reviewed in Ervasti, 2003). The most severe form of dystrophy due to Dystrophin-deficiency (DMD), found in patients who lack all Dp427m protein expression, is usually caused by frame shift or stop mutations. Patients with small in frame deletions present a milder form of dystrophy (Beckers Muscular Dystrophy). There is no simple correlation between the size of the deletion and the severity of the disease, but the presence of the conserved carboxyterminal and actin-binding domains are apparently essential for at least partial functionality (van der Plas, 2007).

During the larval, pupal and newly eclosed adult phases of the Drosophila life cycle, no significant role was observed for DLP2, the Dp427 ortholog expressed in the musculature, in maintaining muscle integrity, but a role was observed for Dp117, which lacks an apparent actin-binding domain. It was found that extrasynaptic DLP2 in the muscle colocalizes with actin at the sarcomeres, as is observed in mammalian musculature (reviewed in Ervasti, 2003). An interaction between Dp117 and the costameric-sarcomeric lattice, if it exists, may explain the disrupted Z-lines and actin-myosin disorganization observed when Dp117 expression levels are reduced in the musculature (van der Plas, 2007).

A recent study (Shcherbata, 2007) reported that adult Drosophila with reduced levels of Dystrophin, including DLP2, display age-dependent muscle degeneration as characterized at the level of light microscopy. Thus, while it was found that a lack of DLP2 does not cause significant muscle degeneration during the larval stages, except for occasional actin-myosin disorganization observed at ultrastructural level, it apparently can result in muscle degeneration in 12 to 20 day old flies. This work showed that the Dp117 Dystrophin isoform is required for muscle integrity earlier during development than DLP2 (van der Plas, 2007).

Different Dystrophin isoforms are apparently required to maintain muscle integrity, when comparing the larval and pupal phases of the Drosophila life cycle to mammals. Furthermore, while the unique aminoterminal regions of the Drosophila isoforms are conserved between the Drosophilids, they bear little resemblance to their mammalian orthologs. Nonetheless, given the remarkable conservation of the common Dystrophin carboxyterminal region and the tissue-specific expression patterns of the Dystrophin orthologs, it remains possible that evolutionarily conserved mechanisms of Dystrophin function in the muscle exist. Three of the most prominent changes observed in Dystrophin deficient mammalian muscle fibers are a disturbance of calcium homeostasis, increased susceptibility to oxidative stress and elevations in membrane permeability. Furthermore, a recent study demonstrated that induction of irreversible Ryanodine receptor-mediated Ca²⁺ sparks act to initiate dystrophic processes in mammalian skeletal muscle (Wang, 2005). Evaluation of these factors in Dystrophin-deficient Drosophila muscle will likely shed light on whether different orthologs perform similar roles in maintaining muscle function (van der Plas, 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).

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).

The Dystrophin Dp186 isoform regulates neurotransmitter release at a central synapse in Drosophila

The Dystrophin protein is encoded by a gene that, when mutated in humans, can cause Duchenne muscular dystrophy, a disease characterized by progressive muscle wasting. A number of Duchenne patients also exhibit poorly understood mental retardation, likely associated with loss of a brain-specific isoform. Furthermore, although Dystrophin isoforms and the related Utrophin protein have long been known to localize at synapses, their functions remain essentially unknown. In Drosophila, this study finds that the CNS-specific Dp186 isoform localizes to the embryonic and larval neuropiles, regions rich in synaptic contacts. In the absence of Dp186, evoked but not spontaneous presynaptic release is significantly enhanced. Increased presynaptic release can be fully rescued to wild-type levels by expression of a Dp186 transgene in the postsynaptic motoneuron, indicating that Dp186 likely regulates a retrograde signaling pathway. Potentiation of synaptic currents in the mutant also occurs when cholinergic transmission is inhibited or in the absence of Glass Bottom Boat (Gbb) or Wishful Thinking (Wit), a TGF-β ligand and receptor, respectively, both implicated in synaptic retrograde signaling. These results are consistent with the possibility that Dp186 modulates other non-Gbb/Wit-dependent retrograde signaling pathways required to maintain normal synaptic physiology (Fradkin, 2008).

During embryogenesis, the Dp186 protein is found close to the longitudinal axon bundles of the ventral nerve cord. The presence of the presynaptic protein Bruchpilot in this domain indicates that it is rich in synaptic contacts that include the motoneuron dendrites and their interneuronal inputs. In the third instar larval neuropile, Dp186 is also in close proximity to the presynaptic Synapsin protein. Double stainings performed with anti-Dp186 and membrane-associated GFP (mCD8-GFP) expressed in motoneurons reveal colocalization, further suggesting that Dp186 is synaptically localized. The density of synaptic contacts in these regions precluded evaluation of the precise degree of colocalization between Dp186 and these presynaptic markers at the level of light microscopy; however, these data support the hypothesis that Dp186 is synaptically localized (Fradkin, 2008).

The absence of the large postsynaptically expressed Dystrophin DLP2 isoform resulted in increased presynaptic neurotransmitter release at the Drosophila. To address whether Dp186 might play a similar role at interneuronal synapses, mutant lines lacking Dp186 protein were generated, and the electrophysiology of a well characterized synapse between the aCC/RP2 motoneurons and their presynaptic cholinergic interneurons was evaluated . Unlike the NMJ motoneurons, the presynaptic cholinergic neurons cannot be directly stimulated to allow evaluation of evoked responses. However, in the preparation used, endogenous evoked responses, which form part of the motor pattern generator, occur at defined frequencies from late embryogenesis onward, allowing the recording of endogenous evoked responses (Fradkin, 2008).

Recordings of such endogenous evoked currents in Dp186 mutants reveal that they are significantly increased in amplitude, but not frequency, relative to wild-type controls and a mutant lacking DLP2. Recordings performed in the presence of TTX, which allows measurement of spontaneous mEPSC events in the absence of evoked responses, indicate that the postsynaptic AChR field is apparently unaffected by the absence of Dp186. Together with the increased frequency of mEPSC observed in the Dp186 mutant, these findings support the hypothesis that evoked presynaptic neurotransmitter release is significantly elevated in the absence of Dp186 (Fradkin, 2008).

Tissue-specific rescue experiments revealed that postsynaptic, but not presynaptic, expression of Dp186 in the dys^DP186 mutant background rescued presynaptic release to wild-type levels. Therefore, Dp186 is apparently predominantly required postsynaptically. The results from the transgenic RNAi approach are less unambiguous but do show that the largest increase in presynaptic release occurred when postsynaptic Dp186 expression levels were decreased. Presynaptic expression of Dp186-RNAi, however, also resulted in increased release, albeit to lower levels. A possible explanation is that decreased Dp186 expression in first-order interneurons that drive motoneurons might, in turn, elevate their own excitation from second-order interneurons (to which they are postsynaptic). The resultant increased activity in these first-order interneurons might manifest itself by increasing the synaptic excitation of downstream motoneurons. Whereas many details of the circuitry that form the motor pattern generator are lacking, first-order interneurons that synapse directly with motoneurons are indeed reliant on second-order interneurons for synaptic excitation. At present, markers suitable for evaluating whether Dp186 is present at these upstream synapses are not available (Fradkin, 2008).

The requirement for Dp186 in motoneurons for normal function of presynaptic cholinergic interneurons is consistent with the regulation of neurotransmitter release by a retrograde signal derived from the targets. Recent studies have shown that presynaptic release at these synapses is regulated, at least in part, by BMP signaling. Moreover, increasing expression of the BMP ortholog Gbb in postsynaptic motoneurons is sufficient to significantly increase synaptic current amplitudes, achieving levels similar to those observed in the Dp186 null mutants. Therefore the effects of eliminating Gbb signaling on the Dp186 electrophysiological phenotype were examined. In the absence of gbb, the relative increase in synaptic currents caused by the lack of expression of Dp186 was similar to that seen in the dys^Dp186 mutant alone, suggesting that Dp186 regulates presynaptic neurotransmitter release independently of gbb. The failure of the loss of gbb to suppress the increases in central synaptic currents observed after overexpression of Dp186 also indicates the apparent independence of these two pathways (Fradkin, 2008).

Previous studies have implicated wit in the proper morphological development of the NMJ synapse and retrograde signaling there and in establishing the competence of motoneurons to respond to muscle-derived retrograde signals. Potentiation of synaptic transmission at the NMJ as a result of postsynaptic loss of the DLP2 Dystrophin isoform requires wit. In the CNS, there is seemingly no corresponding requirement for wit for increased neurotransmitter release at the Dp186-deficient synapse. Furthermore, contrary to what was observed at the NMJ, wit is apparently not required for wild-type electrophysiology. Clearly then, although the absence of a specific Dystrophin isoform at either the NMJ or a central synapse results in increased neurotransmitter release, the underlying mechanisms likely differ, perhaps reflecting the use of different retrograde signaling pathways (Fradkin, 2008).

dys^Dp186-dependent potentiation of central synaptic currents persists when cholinergic neurotransmission is significantly reduced. This, again, is in contrast to the effect of postsynaptic overexpression of gbb under the same conditions. There are two implications of these data. First, they further support the hypothesis that Dp186 and gbb independently regulate central synaptic transmission. Second, these data predict the existence of two types of retrograde signaling pathways regulating presynaptic function, one activity-dependent and the other, constitutive. Indeed, the data are consistent with the possibility that Dp186 controls the ongoing release of a negative regulator of synaptic function. Conceivably, the strength of synaptic transmission will be regulated by the interplay of these and other mechanisms. The identity of the signal regulated by Dp186 is not known, but it may be one or more of the six related BMP signaling molecules present in Drosophila: Dpp, Screw, Activin, Activin-like protein, Myoglianin, and Maverick; or it is possibly unrelated to BMP/TGF-β signaling (Fradkin, 2008).

It is not currently clear by what mechanisms reduced postsynaptic expression levels of Dp186 potentiate presynaptic neurotransmitter release. The increased mEPSC frequency, but not amplitude, observed in the mutant is consistent with an increase in quantal content caused by heightened probability of release. Additional possibilities include increased synchronicity of release from multiple interneurons that drive these motoneurons. Alternatively, individual interneurons might have increased numbers of presynaptic motoneuron contacts. The latter possibility is considered less probable because evoked synaptic current amplitude, but not frequency, is affected in the Dp186 mutant. This indicates that the synaptic connectivity between interneurons and motoneurons is apparently normal in the absence of Dp186 (Fradkin, 2008).

In summary, the Drosophila Dystrophin Dp186 isoform is required predominantly postsynaptically for wild-type neurotransmitter release levels at an identified cholinergic central synapse. Study of the DLP2 isoform has revealed that its absence from the muscle increased the probability of presynaptic release at the glutamatergic NMJ. Therefore, these two postsynaptically localized Dystrophin isoforms are required for the appropriate regulation of presynaptic release at two different types of synapses, each using a different neurotransmitter. Furthering the understanding of the role of the Dystrophin isoforms in synaptic transmission in Drosophila should yield insights into evolutionarily conserved roles of dystrophin in the nervous system and perhaps shed light on the poorly understood mental retardation presented by a significant subset of DMD patients (Fradkin, 2008).

Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila

Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. This study found that either the mechanical or the signaling functions of Dystrophin are able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. The data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches (Taghli-Lamallem, 2014).

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date revised: 10 February 2014

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