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 dysGE20705 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 dysGE20705, 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).
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 dysE6 has a deletion of 2.7 kb; dysE31 is a precise excision control of the EP3397 P-element.The independently derived P-element line dysGE20705 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 dysE6 expresses DLP1 mRNA at wild-type levels but lacks DLP2 mRNA, whereas dysGE20705 shows an approximate threefold reduction in DLP1 and an approximate fourfold reduction in DLP2 mRNA levels. Dp186 mRNA levels are wild type in dysE6 but are significantly reduced (~27-fold) in dysGE20705. The precise excision control dysE31 shows wild-type expression levels of all isoforms. The lack of detectable large-isoform expression in the dysGE20705 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 dysE6 but not in dysGE20705. RT-PCR analyses indicate that CG6255 is not expressed in dysE6 but is expressed in dysGE20705 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 dysE6, 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, dysE6 and dysGE20705, 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 dysE6/dysGE20705 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, dysE6/+ and dysGE20705/+, 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 dysE6 larvae. The protocol resulted in ~50% facilitation in wild-type larvae but failed to do so in the dysE6 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).
RT-PCR analyses indicated that the dysE6 mutation reduces the expression levels of DLP2 and CG6255 and dysGE20705 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 dysE31 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/dysE6) but not when expressed presynaptically (OK6-Gal4/+; GS12472/dysE6). Together with the mutant analyses, these data indicate that electrophysiological phenotype at the dysE6 and dysGE20705 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).
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 dysGE20705 and dysE6 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 dysGE20705 and dysE6 mutants compared with wild type and precise excision control dysE31. 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 dysE6 and dysGE20705 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).
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).
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 dysDLP2 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).
Reference names in red indicate recommended papers.
Baker, P. E., et al. (2006). Analysis of gene expression differences between utrophin/dystrophin-deficient vs mdx skeletal muscles reveals a specific upregulation of slow muscle genes in limb muscles. Neurogenetics 7(2):81-91. 16525850
Bessou, C., et al. (1998). Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission. Neurogenetics 2(1): 61-72. 9933302
Blake, D. J., Weir, A., Newey, S. E. and Davies, K. E. (2002). Function and genetics of dystrophin and dystrophin-related proteins in muscle. Physiol. Rev. 82: 291-329. 11917091
Carlson, C. G. (1998). The dystrophinopathies: an alternative to the structural hypothesis. Neurobiol Dis 5: 3-15. 9702783
Carre-Pierrat, M., et al. (2006). The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy. J. Mol. Biol. 358(2): 387-95. 16527307
Constantin, B., Sebille, S. and Cognard, C. (2006). New insights in the regulation of calcium transfers by muscle dystrophin-based cytoskeleton: implications in DMD. J. Muscle Res. Cell. Motil. 27: 275-286. PubMed citation: 16897576
Courdier-Fruh, I. and Briguet, A. (2006). Utrophin is a calpain substrate in muscle cells. Muscle Nerve [Epub ahead of print]. 16598790
Deconinck, A. E., et al. (1997a). Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90: 717-727. 9288751
Deconinck, A. E., et al. (1997b). Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice. J. Cell Biol. 136: 883-894. 9049253
Dekkers, L. C., et al. (2004). Embryonic expression patterns of the Drosophila dystrophin-associated glycoprotein complex orthologs. Gene Expr. Patterns 4: 153-159. 15161095
Denti, M. A., et al. (2006). Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model. Proc. Natl. Acad. Sci. 103(10): 3758-63. 16501048
Ervasti, J. M. (2003). Costameres: the Achilles' heel of Herculean muscle., J. Biol. Chem. 278: 13591-13594. PubMed citation: 12556452
Gailly, P. (2002). New aspects of calcium signaling in skeletal muscle cells: implications in Duchenne muscular dystrophy. Biochim. Biophys. Acta 1600: 38-44. 12445457
Gieseler, K., Grisoni, K. and Segalat, L. (2000). Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans. Curr. Biol. 10(18): 1092-7. 10996789
Gieseler, K., et al. (2001). Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans. J. Mol. Biol. 307(1): 107-17. 11243807
Gieseler, K., Grisoni, K., Mariol, M. C. and Segalat, L. (2002). Overexpression of dystrobrevin delays locomotion defects and muscle degeneration in a dystrophin-deficient Caenorhabditis elegans. Neuromuscul. Disord. 12(4): 371-7. 12062255
Giugia, J., Gieseler, K., Arpagaus, M. and Segalat, L. (1999). Mutations in the dystrophin-like dys-1 gene of Caenorhabditis elegans result in reduced acetylcholinesterase activity. FEBS Lett. 463(3): 270-2. 10606735
Goyenvalle, A., et al. (2004). Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306(5702): 1796-9. 15528407
Grady, R. M., et al. (1997). Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell 90: 729-738. 9288752
Grady, R. M., Wozniak, D. F., Ohlemiller, K. K. and Sanes, J. R. (2006). Cerebellar synaptic defects and abnormal motor behavior in mice lacking alpha- and beta-dystrobrevin. J. Neurosci. 26(11): 2841-51. 16540561
Green, D. G., Guo, H. and Pillers, D. A (2004). Normal photoresponses and altered b-wave responses to APB in the mdxCv3 mouse isolated retina ERG supports role for dystrophin in synaptic transmission. Vis. Neurosci. 21: 739-747. 15683561
Greener, M. J. and Roberts, R. G. (2000). Conservation of components of the dystrophin complex in Drosophila. FEBS Lett. 482: 13-18. 11018515
Grisoni, K., et al. (2002). Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans. Gene 294(1-2): 77-86. 12234669
Grisoni, K., et al. (2003). The stn-1 syntrophin gene of C.elegans is functionally related to dystrophin and dystrobrevin. J. Mol. Biol. 332(5): 1037-46. 14499607
Haghighi, A. P., McCabe, B. D., Fetter, R. D., Palmer, J. E., Hom, S. and Goodman, C. S. (2003). Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction. Neuron 39: 255-267. 12873383
Hanft, L. M., et al. (2006). Cytoplasmic gamma-actin contributes to a compensatory remodeling response in dystrophin-deficient muscle. Proc. Natl. Acad. Sci. 103(14): 5385-90. 16565216
Johnson, B. D., Scheuer, T. and Catterall, W. A. (2005). Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. 102: 4191-4196. 15753322
Johnson, R. P., Kang, S. H. and Kramer, J. M. (2006). C. elegans dystroglycan DGN-1 functions in epithelia and neurons, but not muscle, and independently of dystrophin. Development 133(10): 1911-21. 16611689
Judge, L. M., Haraguchiln, M., Chamberlain, J. S. (2006). Dissecting the signaling and mechanical functions of the dystrophin-glycoprotein complex. J. Cell Sci. 119(Pt 8): 1537-46. 16569668
Kanagawa, M., et al. (2004). Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 117(7): 953-64. 15210115
Kim, H., Rogers, M. J., Richmond, J. E. and McIntire, S. L. (2004). SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430(7002): 891-6. 15318222
Kimura, S., Ikezawa, M., Nomura, K., Ito, K., Ozasa, S., Ueno, H., Yoshioka, K., Yano, S., Yamashita, T., Matuskura M. and Miike, T. (2006). Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy. Brain Dev. 28: 473-476. PubMed citation: 16516424
Krag, T. O., et al. (2004). Heregulin ameliorates the dystrophic phenotype in mdx mice. Proc. Natl. Acad. Sci. 101(38): 13856-60. Epub 2004 Sep 13. 15365169
Lowe, D. A., et al. (2006). Molecular and cellular contractile dysfunction of dystrophic muscle from young mice. Muscle Nerve[Epub ahead of print]. 16634063
Lu, Q. L., et al. (2003). Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat. Med. 9(8): 1009-14. 12847521
Lyons, P. R. and Slater, C. R. (1991). Structure and function of the neuromuscular junction in young adult mdx mice. J Neurocytol 20: 969-981. 1686056
Madhavan, R., Massom, L. R. and Jarrett, H. W. (1992). Calmodulin specifically binds three proteins of the dystrophin-glycoprotein complex. Biochem. Biophys. Res. Commun. 185: 753-759. 1610366
Mann, C. J., et al. (2001). Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc. Natl. Acad. Sci. 98(1): 42-7. 11120883
Mariol, M. C., Martin, E., Chambonnier, L. and Segalat, L. (2007). Dystrophin-dependent muscle degeneration requires a fully functional contractile machinery to occur in C. elegans. Neuromuscul. Disord. 17: 56-60. PubMed citation: 17134897
Mokhtarian, A. et al., (1999). Hindlimb immobilization applied to 21-day-old mdx mice prevents the occurrence of muscle degeneration. J. Appl. Physiol. 86: 924-931. PubMed citation: 10066706
Montanaro, F. and Carbonetto, S. (2003). Targeting dystroglycan in the brain. Neuron 37: 193-196. Medline abstract: 12546815
Muntoni, F., Torelli, S. and Ferlini, A. (2003). Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol. 2: 731-740. PubMed citation: 14636778
Neuman, S., Kaban, A., Volk, T., Yaffe, D. and Nudel, U. (2001). The dystrophin/utrophin homologues in Drosophila and in sea urchin. Gene 263: 17-29. 11223239
Neuman, S., Kovalio, M., Yaffe, D. and Nudel, U. (2005). The Drosophila homologue of the dystrophin gene - introns containing promoters are the major contributors to the large size of the gene. FEBS Lett. 579(24): 5365-71. 16198353
Nico, B., et al. (2006). Increased matrix-metalloproteinase-2 and matrix-metalloproteinase-9 expression in the brain of dystrophic mdx mouse. Neuroscience [Epub ahead of print]. 16650610
Qu, Q. and Smith, F. I. (2004). Alpha-dystroglycan interactions affect cerebellar granule neuron migration. J. Neurosci. Res. 76: 771-782. Medline abstract: 15160389
Rando, T. A. (2001). The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve 24: 1575-1594. 11745966
Rooney, J. E., et al. (2006). Severe muscular dystrophy in mice that lack dystrophin and {alpha}7 integrin. J. Cell Sci. [Epub ahead of print]. 16684813
Shcherbata, H. R., et al. (2007). Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26(2): 481-93. Medline abstract: 17215867
Stone, M. R., et al. (2005). Specific interaction of the actin-binding domain of dystrophin with intermediate filaments containing keratin 19. Mol. Biol. Cell 16(9): 4280-93. 16000376
van der Plas, M. C., Pilgram, G. S., Plomp, J. J., de Jong, A., Fradkin, L. G. and Noordermeer, J. N. (2006). Dystrophin is required for appropriate retrograde control of neurotransmitter release at the Drosophila neuromuscular junction. J. Neurosci. 26(1): 333-44. 16399704
van der Plas, M. C., et al. (2007). Drosophila Dystrophin is required for integrity of the musculature. Mech. Dev. 124(7-8): 617-30. PubMed citation: 17543506
Wang, X., Weisleder, N., Collet, C., Zhou, J., Chu, Y., Hirata, Y., Zhao, X., Pan, Z., Brotto, M., Cheng, H. and Ma, J. (2005). Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle. Nat. Cell Biol. 7: 525-530. PubMed citation: 15834406
Watkins, S. C., Hoffman, E. P., Slayter, H. S. and Kunkel, L. M. (1988). Immunoelectron microscopic localization of dystrophin in myofibres. Nature 333(6176): 863-6. 3290684
Zucker, R. S. and Regehr, W. G. (2002). Short-term synaptic plasticity. Annu. Rev. Physiol. 64: 355-405. 11826273
date revised: 20 July 2008
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.