BIOLOGICAL OVERVIEW

Mutations in the human dystrophin gene cause the Duchenne and Becker muscular dystrophies. The Dystrophin protein provides a structural link between the muscle cytoskeleton and extracellular matrix to maintain muscle integrity. Recently, Dystrophin has also been found to act as a scaffold for several signaling molecules, but the roles of dystrophin-mediated signaling pathways remain unknown. To further an understanding of this aspect of the function of dystrophin, Drosophila mutants that lack the large dystrophin isoforms were generated and their role in synapse function at the neuromuscular junction was analyzed. In expression and rescue studies, lack of the large dystrophin isoforms in the postsynaptic muscle cell were shown to lead to elevated evoked neurotransmitter release from the presynaptic apparatus. Overall synapse size, the size of the readily releasable vesicle pool as assessed with hypertonic shock, and the number of presynaptic neurotransmitter release sites (active zones) are not changed in the mutants. Short-term synaptic facilitation of evoked transmitter release is decreased in the mutants, suggesting that the absence of dystrophin results in increased probability of release. Absence of the large dystrophin isoforms does not lead to changes in muscle cell morphology or alterations in the postsynaptic electrical response to spontaneously released neurotransmitter. Therefore, postsynaptic glutamate receptor function does not appear to be affected. These results indicate that the postsynaptically localized scaffolding protein Dystrophin is required for appropriate control of neuromuscular synaptic homeostasis (van der Plas, 2006).

Duchenne (DMD) and Becker muscular dystrophy are caused by mutations in the dystrophin (dys) gene. Dystrophin and its partially redundant homolog Utrophin are associated with a number of other proteins, members of the Dystrophin-glycoprotein complex (DGC). The DGC links the actin cytoskeleton to the extracellular basal lamina, providing tensile strength to muscle fibers. In addition to its structural role, the DGC acts to scaffold signaling molecules (for review, see Rando, 2001). How the DGC and associated signaling partners interact with other cellular pathways remains unclear (van der Plas, 2006).

Members of the mammalian DGC complex, including Dystrophin and Utrophin, are found in the CNS and the extrasynaptic and postsynaptic regions of muscle cells (for review, see Blake, 2002). Reduction of postsynaptic junctional folds and acetylcholine receptor (AChR) clustering are observed at the neuromuscular junction (NMJ) in the dystrophin/utrophin double knock-out mouse, but electrophysiological analyses revealed a normal postsynaptic response to spontaneous neurotransmitter release (Deconinck, 1997a; Grady, 1997). These and other data suggest that dystrophin and utrophin likely play partially redundant and subtle roles at the mammalian NMJ (Lyons, 1991; Deconinck, 1997b). A single dystrophin ortholog exists in Drosophila (Greener, 2000); its isoforms are predominantly expressed in the muscle and nervous system (Neuman, 2001; Dekkers, 2004). This study investigates the role of the postsynaptically localized dystrophin-like protein 2 (DLP2) Dystrophin isoform at the Drosophila NMJ (van der Plas, 2006).

During maturation and modification of synaptic contacts, homeostatic mechanisms match neurotransmitter release levels to changing postsynaptic requirements, keeping depolarization levels within a narrow range. Studies of myasthenia gravis patient NMJs (in which autoantibodies reduce AChR number) revealed that, in addition to anterograde signaling, the homeostatic machinery encompasses retrograde signals required for the appropriate regulation of presynaptic neurotransmitter release. Similar compensatory neurotransmitter release upregulation was shown in rodents with decreased postsynaptic AChR levels and in Drosophila with reduced DGluRIIA glutamate receptor function (van der Plas, 2006 and references therein).

In Drosophila, several proteins have been implicated in NMJ retrograde signaling, including members of the bone morphogenetic protein (BMP) pathway, the presynaptic type II receptor wit (for wishful thinking), and its muscle-derived ligand gbb (for glass bottom boat). Postsynaptic calcium/calmodulin-dependent kinase (CaMKII) activity has also been reported to modulate retrograde signaling via wit (van der Plas, 2006 and references therein).

This study takes advantage of Drosophila genetics to study the role of dystrophin at the NMJ. dystrophin is shown to be required for appropriate homeostatic control of neurotransmitter release. Absence of postsynaptically localized Dystrophin results in a wit-dependent increase in neurotransmitter release, leading to enhanced muscle depolarization. Furthermore, short-term synaptic facilitation is impaired in the dystrophin mutant, indicating an increase in the probability of release. These results reveal a novel role for dystrophin in the dynamics of neurotransmitter release (van der Plas, 2006).

This study provides evidence that postsynaptic dystrophin is required to maintain appropriate levels of presynaptic neurotransmitter release. The absence of dystrophin from the muscle results in abnormally high QC as determined by three different methods. The NMJ homeostatic machinery is apparently engaged in the absence of dystrophin, but it has an inappropriate endpoint. In contrast to members of the BMP signaling pathway implicated previously in NMJ homeostasis but similarly to CaMKII, Dystrophin does not appear to regulate synaptic growth or overall structure. The requirement for dystrophin is haplo-insufficient, indicating that the Drosophila NMJ is highly sensitive to the levels of Dystrophin, possibly reflecting its interaction with rate-limiting signaling factors (van der Plas, 2006).

This study shows, in a number of experiments, that the lack of the postsynaptically localized DLP2 isoform is responsible for the electrophysiological phenotype observed in the dystrophin mutants: (1) expression analyses and semiquantitative RT-PCR studies indicate that, of the known dystrophin isoforms, only expression of DLP2 is affected by the mutation in dys^E6; (2) the mutant phenotype can be rescued by expressing DLP2 postsynaptically but not presynaptically, (3) expression of double-stranded RNA directed against sequences present in the DLP2 isoform results in increases in neurotransmitter release similar to those observed in the dys^E6 mutants, only when expressed postsynaptically, (4) RNA in situ analyses and RT-PCR assays show that DLP2 is highly expressed in larval muscle, DLP1 is not detectably expressed in the musculature, and all dystrophin large isoforms are absent from the neuropil in which the cell bodies of motoneurons are located, and (5) double labeling of larval body walls with anti-Dystrophin and anti-HRP, which labels the presynaptic membrane, reveals no overlapping expression domains (van der Plas, 2006).

Morphometric analyses and stainings were performed with several muscle- and motoneuron-specific antibodies to examine the general morphological characteristics of the synapse and musculature in the mutants. These studies indicate that muscle and synapse size are not significantly altered by the lack of Dystrophin. Because it was found that Dystrophin colocalizes extrasynaptically with actin, actin distribution was examined in the mutant muscle but no differences were observed relative to the controls. The morphology of the T-tubular network was also unaltered. That the absence of dystrophin does not grossly affect muscle morphology or glutamate receptor density/properties was reflected by the observation of unchanged electrical input resistance, mEJP amplitudes, and anti-glutamate receptor subunit antibody staining in the mutants (van der Plas, 2006).

Although DLP2 is expressed only postsynaptically and changes in synapse elaboration or muscle morphology were not observed in its absence, there are significant changes in the NMJ electrophysiology of the mutant. EJP amplitudes are increased with little or no change in the mEJPs, resulting in increased QC, i.e., the number of quanta released on stimulation, calculated by the direct, failure, or variance methods. Therefore presynaptic mechanisms were examined whose alteration might account for the increase in QC at the mutant NMJ. The increase was correlated to an increase in the probability of release as indicated by reduced EJP facilitation at the mutant synapse (van der Plas, 2006).

In addition to increased release probability, an increased size of the readily releasable vesicle pool may account for the increased QC at the dystrophin mutant NMJ. However, this seems not to be the case in the dystrophin mutant because the increase in mEJP frequency at the mutant NMJ in response to hypertonic shock, a method used to assess the readily releasable pool, did not differ from that at control NMJs. It should, however, be noted that it is not yet clear that only the readily releasable pool of vesicles is released after hypertonic shock of the Drosophila larval NMJ (van der Plas, 2006).

Increased release probability may result from increased efficacy of the translation of presynaptic Ca²⁺ influx into transmitter release by the neuroexocytotic machinery, possibly attributable to increased sensitivity of Ca²⁺ sensors. However, the observation that both the mutant and wild-type NMJs have highly similar slope values for the log[Ca²⁺] versus log[QC] argues against such an effect. An alternative explanation is that aberrant retrograde signaling in the absence of dystrophin results in altered modulation of presynaptic Ca²⁺ channel activity and thus leads to increased presynaptic Ca²⁺ influx (van der Plas, 2006).

Ultrastructural analysis of synaptic boutons showed a slight increase of the area occupied by vesicles in the mutants, which is unlikely to be sufficient to explain the increase in QC. Also, no significant changes were observed in the number and size of active zones, whose increase could explain the increased neurotransmitter release. No increase in T-bars, the presumed docking sites for synaptic vesicles, was observed in the mutant boutons. Although at present the precise function of these morphologically defined structures is unclear, an increase in T-bars has also found correlated with increased neurotransmitter release in other studies (van der Plas, 2006).

How does the absence of dystrophin affect the retrograde control of release? The abnormally high EJP amplitudes observed at the dystrophin mutant NMJ suggests the possibility that the Dystrophin-deficient muscle inappropriately signals to the motoneuron that it is inadequately depolarized. Thus, the absence of dystrophin may result in desensitization of an as yet unknown monitor of muscle function during depolarization. This raises the following questions: what is the homeostatic monitor and how does Dystrophin interact with it? Although a postsynaptic monitor regulating NMJ homeostatic pathways has been proposed, its identity has proven elusive. The ligand-gated glutamate receptor channel, which conducts Ca²⁺, has been an attractive candidate, and mammalian Dystrophin is known to scaffold a variety of postsynaptic Ca²⁺ channels (for review, see Carlson, 1998). However, no alterations were observed in either glutamate receptor field, sensitivity to spontaneous neurotransmitter release, or localization of the DGluRIIA or DGluRIIB subunits in the dystrophin mutant, suggesting that glutamate receptors are unlikely to play a key role in the homeostatic pathways affected by the lack of dystrophin (van der Plas, 2006).

Glutamate receptor-independent monitors of muscle depolarization, possibly voltage-gated L-type channels, have been shown sufficient to trigger NMJ homeostatic mechanisms. Alteration of L-type Ca²⁺ channel function or other changes in Ca²⁺ handling in the dystrophin-deficient larval muscle, which are well documented in mammals (for review, see Gailly, 2002), might inappropriately trigger or prolong the action of the homeostatic machinery, resulting in aberrantly high neurotransmitter release and muscle hyperdepolarization. CaMKII and Ca²⁺/calmodulin represent other attractive candidates to directly link Dystrophin to homeostatic pathways; Ca²⁺/calmodulin has been shown to associate with the DGC (Madhavan, 1992), but its role in dystrophin function is not yet understood. Possibly, dystrophin indirectly affects cAMP-dependent mechanisms (Johnson, 2005) that have been shown to be involved in synaptic plasticity (Zucker, 2002) and short-term dynamics of release (van der Plas, 2006).

This study finds that the presynaptically localized type II BMP receptor wit is required for the increased QC observed in the dystrophin mutant, as also shown for increases in QC induced by the postsynaptic inhibition of CaMKII or glutamate receptor function (Haghighi, 2003). Dystrophin and CaMKII are unlikely, however, to signal through the PMad-dependent BMP signaling pathway, because the expression levels and domains of PMad are unchanged when Dystrophin or CaMKII levels are either decreased or increased. The results address the question as to whether the retrograde BMP signal directly participates in homeostatic signaling ('instructive') or is required for the overall development of the synapse ('permissive'). The findings indicate that it is likely permissive, at least for the homeostatic mechanisms induced by perturbation of Dystrophin or CaMKII levels. BMP signaling may simply be required for the development of the presynaptic apparatus to a point at which it can respond to muscle-derived cues (van der Plas, 2006).

The degree of evolutionary conservation of the role of dystrophin in regulating neurotransmitter release at the NMJ and the potential role that defective NMJ homeostasis may play in the onset or progression of muscular dystrophy in humans is, at present, unclear. Differences between species in the severity of muscle wasting in the absence of dystrophin are observed. No muscle degeneration is observed when DLP2 is absent from the musculature; other isoforms may be required for muscle integrity in Drosophila. The dystrophin-deficient cholinergic Caenorhabditis elegans NMJ also shows elevated levels of neurotransmitter, but this is attributable to decreased clearance of acetylcholine subsequent to delocalization of the SNF-6 acetylcholine transporter (Kim, 2004). Recent work on retinal synapses in the mdx mouse suggests that the large Dystrophin isoforms may also influence neurotransmitter release at other types of synapses (Green, 2004). Whether the CNS-specific Dp186 isoform plays roles at Drosophila interneuronal synapses, similar to those played by DLP2 at the NMJ, remains to be evaluated. Disrupted homeostasis at Dystrophin-deficient interneuronal brain synapses might contribute to the poorly understood mental impairments associated with DMD in humans (van der Plas, 2006).

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation

How extracellular matrix participates to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. This study found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell autonomous control of F-actin fibers orientation relies on the previous BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a critical organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation (Cerqueira Campos, 2020).

Deciphering the mechanisms underlying tissue morphogenesis is crucial for fundamental understanding of development and also for regenerative medicine. Building organs generally requires the precise modeling of a basement membrane extracellular matrix (ECM), which in turn can influence tissue shape. However, the mechanisms driving the assembly of a specific basement membrane (BM) and how this BM then feeds forward on morphogenesis are still poorly understood. Drosophila oogenesis offers one of the best tractable examples in which such a morphogenetic process can be studied. Each ovarian follicle, which is composed of a germline cyst surrounded by the somatic follicular epithelium, undergoes a dramatic growth, associated with tissue elongation, starting from a little sphere and ending with an egg in which the anteroposterior (AP) axis is 3-fold longer than the mediolateral (ML) axis. This elongation is roughly linear from the early to the late stages, but can be separated in at least two mechanistically distinct phases. The first phase (from stage 3 to stage 8; hereby 'early stages') requires a double gradient of JAK-STAT pathway activity that emanates from each pole and that controls myosin II-dependent apical pulsations. In the second phase, from stage 7-8, elongation depends on the atypical cadherin Fat2 that is part of a planar cell polarity (PCP) pathway orienting the basal domain of epithelial follicle cells. Earlier during oogenesis, Fat2 gives a chirality to the basal domain cytoskeleton in the germarium, the structure from which new follicles bud. This chirality is required to set up a process of oriented collective cell migration perpendicularly to the elongation axis that induces follicle revolutions from stage 1 to stage 8. From each migrating cell, Fat2 also induces, in the rear adjacent cell, the formation of planar-polarized protrusions that are required for rotation. These rotations allow the polarized deposition of BM fibrils, which involves a Rab10-dependent secretion route targeted to the lateral domain of the cells. These BM fibrils are detectable from stage 4 onwards and persist until late developmental stages. Follicle rotation also participates in the planar cell polarization of integrin-dependent basal stress fibers that are oriented perpendicularly to the AP axis. Moreover, at stage 7-8, a gradient of matrix stiffness controlled by the JAK-STAT pathway and Fat2 contributes to elongation. Then, from stage 9, the epithelial cell basal domain undergoes anisotropic oscillations, as a result of periodic contraction of the oriented stress fibers, which also promotes follicle elongation . To explain the impact of fat2 mutations on tissue elongation, it is generally accepted that oriented stress fibers and BM fibrils act as a molecular corset that constrains follicle growth in the ML axis and promotes its elongation along the AP axis. However, the exact contribution of F-actin versus BM to this corset is still unclear, as is whether the orientations of stress fibers and of BM fibrils are causally linked (Cerqueira Campos, 2020).

This analyzed the function of Dystrophin (Dys) and Dystroglycan (Dg) during follicle elongation. Dys and Dg are the two main components of the Dystrophin-associated protein complex (DAPC), an evolutionarily conserved transmembrane complex that links the ECM (via Dg) to the F-actin cytoskeleton (via Dys). This complex is expressed in a large variety of tissues and is implicated in many congenital degenerative disorders. Loss-of-function studies in model organisms have revealed an important morphogenetic role for Dg during development, usually linked to defects in ECM secretion, assembly or remodeling . A developmental role for Dys is less clear, possibly because of the existence of several paralogs in vertebrates. As Drosophila has only one Dg and one Dys gene, it is a promising model for their functional study during development and morphogenesis (Cerqueira Campos, 2020).

Dg and Dys were found to be required for follicle elongation and proper BM fibril formation early in fly oogenesis. During these early stages, DAPC loss and hypomorphic fat2 conditions similarly delay stress fiber orientation. However, DAPC promotes this alignment more locally than Fat2. Moreover, DAPC genetically interacts with fat2 in different tissues, suggesting that they belong to a common morphogenetic network. Later in oogenesis, Dg and Dys are required for stress fiber orientation in a cell-autonomous manner. This is the period when the main elongation defect is seen in these mutants, arguing for a more determinant role for stress fibers compared with BM fibrils in the elongation process. Nonetheless, this latter function depends on the earlier DAPC function in BM fibril deposition. It is proposed that BM fibrils serve as a PCP memory for the late stages that are used as a template by the DAPC for F-actin stress fiber alignment (Cerqueira Campos, 2020).

Genetic data has already demonstrated that follicle elongation relies on at least two different and successive mechanisms. The first is controlled by JAK-STAT and involves the follicle cell apical domain, whereas the second is controlled by Fat2 and involves the basal domain and the BM. Between these phases, around stage 7-8, JAK-STAT and Fat2 seem to be integrated in a third mechanism based on a BM stiffness gradient. Interestingly, a very recent report suggests that this gradient may not directly influence tissue shape but rather do so by modifying the properties of the follicle cells underneath. This study shows that the DAPC influences elongation mainly at very late stages, suggesting the existence of a fourth mechanistic elongation phase. Of note, elongation at these late stages is also defective in fat2 mutants. This is consistent with the fact that rotation is required for polarized BM fibril deposition, and that this deposition depends on and is required for DAPC function. The existence of multiple and interconnected mechanisms to induce elongation, a process that initially appeared to be very simple, highlights the true complexity of morphogenesis, and the necessity to explore it in simple models (Cerqueira Campos, 2020).

Fat2 is clearly part of the upstream signal governing the basal planar polarization. However, how this polarization leads to tissue elongation is still debated. It has been proposed that elongation relies on a molecular corset that could be formed, non-exclusively, by BM fibrils or F-actin stress fibers. The initial observation that rotation is required for both elongation and BM fibrils favored a direct mechanical role for these structures. Recent data showing that BM fibrils are stiffer than the surrounding ECM supports this view. Moreover, increasing the BM fibril number and size can lead to hyper-elongation. Finally, addition of collagenase induces follicle rounding, at least at some stages, and genetic manipulation of the ECM protein levels also influences elongation. However, these experiments did not discriminate between the function of the fibril fraction and a general BM effect. Moreover, they do not demonstrate whether their impact on elongation is direct and mechanic or, indirect by a specific response of the epithelial cells. Fat2 and rotation are also required for the proper orientation of the stress fibers. The F-actin molecular corset is dynamic with follicle cells undergoing basal pulsations, and perturbation of both these oscillations and of the stress fiber structure affect elongation. In the DAPC mutants, a faint but significant elongation defect was observed during mid-oogenesis and a stronger one after stage 12. These defects are clearly correlated with the stress fiber orientation defects observed in the same mutants, both temporally and in terms of intensity. Moreover, although overexpression of Rab10 in a Dys loss-of-function mutant restores BM fibrils, it does not rescue elongation, indicating that stress fiber orientation is instrumental (Cerqueira Campos, 2020).

Thus, if the role of the BM fibrils as a direct mechanical corset appears limited, what is their function? One possibility could have been that they promote rotation, acting by positive feedback and explaining the speed increase over time. However, the rotation reaches the same speed in WT and DAPC mutants, excluding this possibility. Similarly, increasing the fibril fraction also has no effect on rotation speed (Cerqueira Campos, 2020).

The results strongly argue that BM fibrils act as a cue for the orientation of stress fibers, which then generate the mechanical strain for elongation. This appears clear in late stages when the function of the DAPC for stress fiber orientation is dependent on the previous BM fibril deposition. Although it is unknown why the cells lose their orientation from stages 10 to 12, the BM fibrils provide the long-term memory of the initial PCP of the tissue, allowing stress fiber reorientation. Such a mechanism appears to be a very efficient way to memorize positional cues, and could represent a general BM function in many developmental processes (Cerqueira Campos, 2020).

DAPC was found to impact the two key actors at the basal domain of the follicle cells: the BM and the stress fibers linked to the BM. All the defects of Dg null mutants were also observed in Dys mutants, demonstrating a developmental and morphogenetic role for this gene. In vertebrates, at least in some tissues, Dg presence appears to be essential for BM assembly. However, BM formation on the follicular epithelium does not require Dg, suggesting the existence of alternative platforms for its general assembly. The genetic data suggest that Rab10 is epistatic to Dg for BM fibril deposition. The usual interpretation of such a result would be that Dg is involved in the targeting of ECM secretion upstream of Rab10 rather than in ECM assembly in the extracellular space. In Caenorhabditis elegans, Dg acts as a diffusion barrier to define a precise subcellular domain for ECM remodeling. One could imagine that the DAPC has a similar function in follicle cells, by defining the position where the Rab10 secretory route is targeted. However, in DAPC loss of function, some ECM is still secreted between cells, suggesting that the lateral Rab10 route is not affected. Moreover, ECM proteins do not abnormally accumulate between cells in such mutants, suggesting that they are able to leave this localization but without forming BM fibrils. Therefore, the functional interplay between Rab10 and the DAPC is still unclear (Cerqueira Campos, 2020).

As mentioned before, Dg has often been proposed to act as a scaffold to promote BM assembly in mice. Deletion of the Dg intracellular domain is only sub-lethal in mice, whereas complete loss of this protein is lethal very early during development, indicating that the abolishment of Dg's interaction with Dys affects its function only partially. In these mice, laminin assembly can still be observed, for instance in the brain and retina. Similar results were also obtained in cultured mammary epithelial cells. Thus, despite the existence of Dys paralogs that could mask some effects on ECM and the fact that the same ECM alteration was observed in Dg or Dys mutant fly follicles, not all the Dg functions related to ECM assembly or secretion involve Dys. It is possible that Dys is required when Dg needs a very specific subcellular targeting for its function, whereas a more general role in ECM assembly would be independent of Dys. The results suggest that some specific effects of Dys on ECM could have been underestimated and this could help to explain the impact of its loss of function on tissue integrity maintenance. For instance, as it has been reported that Dg influences ECM organization in fly embryonic muscles, it would be interesting to determine whether this also involves Dys (Cerqueira Campos, 2020).

The DAPC is involved in planar polarization of the basal stress fibers and its ability to read ECM structure to orchestrate integrin-dependent adhesion could play a role in many developmental and physiological contexts. The link between the ECM and F-actin provided by this complex is likely required for this function, although this remains to be formally demonstrated. BM fibrils could provide local and oriented higher density of binding sites for Dg, and the alignment could then be transmitted to the actin cytoskeleton. Alternatively, DAPC function could rely on sensing the mechanical ECM properties. The hypothesis that the DAPC could act as a mechanosensor is a long-standing proposal, partly due to the presence of spectrin repeats in Dys. The basal domain of the follicle cells may offer an amenable model to combine genetics and cell biology approaches to decipher such function (Cerqueira Campos, 2020).

Altogether, this work provides important insights on the role of the BM during morphogenesis, by acting as a static PCP cue retaining spatial information while cells are highly dynamic. It also reveals important functions of the DAPC, including Dys, that may be broadly involved during animal development and physiology (Cerqueira Campos, 2020).

GENE STRUCTURE

The Drosophila gene encoding the dystrophin-like protein (DLP) is as complex as the mammalian dystrophin gene. Three 5' promoters and three internal promoters regulate the expression of three full-length and three truncated products, respectively. The existence of this complex gene structure in such evolutionary remote organisms suggests that both types of products have diverse important functions. The promoters of both the DLP gene and the mammalian dystrophin gene are located in very large introns. These introns contribute significantly to the large size of the genes. The possible relevance of the conservation of the large size of introns containing promoters to the regulation of promoter activity is discussed (Neuman, 2005).

Dystrophin is a large rod-shaped protein comprised of four domains: N-terminal actin-binding domain, twenty four spectrin-like triple helix repeats, a cysteine-rich domain, and a unique C-terminal domain. In muscle, dystrophin is a part of a large complex that links the actin cytoskeleton, the sarcolemma, and the extracellular matrix. Other proteins in the complex are collectively called dystrophin-associated proteins (DAPs). Absence of dystrophin or some components of the DAPs complex, results in progressive damage to muscle fibers during cycles of contraction and relaxation. Recent studies suggest that the dystrophin-DAPs complex may also be involved in signaling in muscle and other tissues (Neuman, 2005).

Dystrophin is encoded by one of the largest and most complex mammalian genes. Three 5′ promoters and four internal promoters regulate the 2.2 mega base gene. Thus, the gene expresses, in a tissue and cell-type specific manner, three full-length dystrophin isoforms, and four truncated proteins, each consisting of the cysteine-rich and C-terminal domains of dystrophin, with various extensions into the spectrin-like repeats domain. The 5′ promoters are active in muscle and glial cells (the M promoter), in neurons (the B promoter) and in Purkinji cells in the brain (the P promoter). The smaller proteins, named according to their molecular weights, include: Dp71, Dp116, Dp140, and Dp260 (Neuman, 2005).

Dystrophin is very similar in its domain structure, amino acids sequence (50% identity), and gene structure to another cytoskeletal protein, utrophin. While all vertebrates studied so far have both the dystrophin and utrophin genes, invertebrates have only one related gene, which is the orthologue of the ancestral dystrophin/utrophin gene. It is therefore suggested that the gene duplication event forming the dystrophin and utrophin genes, occurred during early evolution of vertebrates (Neuman, 2005).

The Drosophila homologue of the dystrophin/utrophin ancestral gene, the dystrophin-like protein (DLP) gene. contains three 5′ promoters, which regulate the expression of full-length products, and one internal promoter that regulates the expression of a 186 Kd protein, Dp186. This study describes two additional truncated gene products, showing that the gene is controlled by at least six promoters. Three promoters regulate the expression of full-size dystrophin-like proteins, and three regulate the expression of smaller proteins. According to the location of their first exons (in relation to the coding sequence), one of the proteins is the orthologue of mammalian and sea urchin Dp116. The other two Drosophila truncated products have no known vertebrate orthologues (Neuman, 2005).

Interestingly, the promoters of the DLP gene are situated in the largest introns of the gene. Likewise, the promoters of human and mouse dystrophin genes are situated in very large introns, whose size is conserved in these two species. These findings show that the large introns, which contain promoters, are major contributors to the unusually large size of the dystrophin gene in various organisms(Neuman, 2005).

A Drosophila transcript is described that is initiated in intron 16 of the DLP gene. The encoded protein, Dp186, consists of four spectrin-like repeats, the cysteine-rich domain, and the C-terminal domain of DLP. On the basis of its location in relation to the coding sequence, intron 16 of the DLP gene does not have a homologue in the human dystrophin gene (Neuman, 2005).

Human introns 29 and 44, which contain the promoters and first exons of Dp260 and Dp140, respectively, do not have homologous introns in the dmDLP gene. Human introns 55 and 62, which contain the promoters and first exons of Dp116 and Dp71, respectively, have homologous introns in the fly gene, introns 22 and 29, respectively. Since intron 29 is very small (66 bp) it is unlikely to accommodate a unique first exon and a promoter. Indeed, using the 5′RACE technique, no evidence was found of a transcript initiated in this intron. In contrast, intron 22 is very large (47 kb), and its homologues in sea urchin and mammals harbor internal promoters. Gene prediction analysis was performed on intron 22 of the DLP gene and several putative coding exons were identified. Using RT/PCR, it was found that one of these putative exons was spliced in frame to exon 23 of the gene. The predicted coding sequence begins with an ATG translation start site. The presence of 5′non-coding sequences upstream from the initiator ATG was predicted by computer analysis and confirmed by RT/PCR analysis. In addition, no evidence by RT/PCR analysis was found for splicing of the new exon to upstream known exons of the DLP gene. Using the 5′RACE technique, the transcription start site of the mRNA was found in an additional 5′ non-coding exon, residing in the same intron 22. The computer programs used did not predict the existence of this exon. The 5′RACE analysis also suggests the existence of a second transcription start site in the non-coding sequence of the second exon. The encoded protein consists of a unique large N-terminus, two spectrin-like repeats, hinge region no. 4, and the cysteine-rich and C-terminal domains of dmDLP. According to the location of the unique exons in relation to the protein coding sequences, the encoded protein is the evolutionary homologue of Dp116. However, unlike the unique first exon of Dp116, which encodes a short product-specific N-terminus consisting of 10 amino acids, the unique exons of its Drosophila orthologue encode a large polypeptide of 870 amino acids. The molecular weight of the encoded protein is 205 Kd (Accession No. AV875639). The protein, according to the nomenclature of internal products of the dystrophin gene family, is called Dp205. RT/PCR analysis showed that Dp205 mRNA is expressed in embryos, larvae, and adult flies (Neuman, 2005).

Potential new Drosophila coding sequences have been identified and confirmed to be expressed as mRNAs. By searching the database it was found that one of the annotated sequence (Accession No. BK002672) encodes the specific N-terminus of Dp205, with some additional amino acids at the C-terminus. However, the annotated protein lacks the entire sequence shared by Dp205 and DLP. RT/PCR and 5′RACE analysis indicate that this clone represents the second exon of Dp205. The analysis suggests that the extra 188 bps of the 3′ end of the annotated sequence BK002672 (which are not found in Dp205 mRNA) consist of an intronic sequence (Neuman, 2005).

A cDNA clone that was sequenced as part of a high throughput process to clone Drosophila gene collection encodes a protein consisting of the cysteine-rich and C-terminal domains of DLP and a unique 269 amino acids N-terminus. The calculated molecular weight of the encoded protein is 117Kd. The presence of 5′UTR, and RT/PCR analysis that was performed showed that the cDNA represents a third internal transcript of the DLP gene and not an alternative splicing form of DLP mRNA. The encoded protein is therefore named Dp117. Analysis of the genomic DNA shows that the 5′ unique sequence of the mRNA is transcribed from two exons localized in intron 26 of the DLP gene. The predicted initiator ATG is preceded by 635 bp of non-coding sequence. Interestingly, using RT/PCR analysis an alternative splicing form of the 5′ UTR of Dp117 mRNA was confirmed in which the donor site of exon 1 recognizes an acceptor site within exon 2. The promoter of this transcript resides in intron 26 of the DLP gene. Dp117 consists of a unique N-terminus (269 amino acids), hinge region no. 4 and the cysteine-rich and C-terminal domains of DLP. RT/PCR analysis indicates that Dp117 mRNA is expressed in all the developmental stages of Drosophila (Neuman, 2005).

The three internal products of the DLP gene contain long product-specific N-termini. Blast analysis of the unique amino acid sequences of the N-terminal regions of Dp186, Dp117 and Dp205 did not reveal significant similarity to known proteins. In addition, no significant similarity in amino acid sequence was detected between the specific N-termini of the three truncated gene products, suggesting that these sequences have different functions. A search for functional motifs revealed various potential sites for modifications, such as phosphorylation by several kinases, myristoyalation, as well as sequences that may be involved in protein-protein interactions. The functional significance of these motifs and of the unique N-termini of Dp186, Dp205 and Dp117 await further studies (Neuman, 2005).

Computer sequence analysis and comparison showed that the specific N- termini of Dp205 and Dp117 have been conserved in other Drosophila species. 92.5% and 96% sequence identity was found between the specific N-termini of Dp117 and Dp205 of Drosophila melanogaster and Drosophila yakuba, and 76% and 85% sequence identity between these specific N-termini of Drosophila melanogaster and Drosophila pseudoobscura. The estimated evolutionary distances between D. yakuba and D. melanogaster and between D. pseudoobscura and D. melanogaster are 13 and 55 million years, respectively. The conservation of the specific N-termini of the truncated products for at least 55 million years indicates that they have important functions (Neuman, 2005).

The DLP gene is one of the largest genes of Drosophila (about 130 kb). Introns comprise about 90% of the gene. Several introns are noticeably large and are the major contributors to the size of the gene. The four largest introns of the gene harbor internal promoters. Thus, the introns of the DLP gene, which contain promoters, comprise about 75% of the gene. In D. yakuba and D. pseudoobscura these introns comprise more than 80% of the gene (Neuman, 2005).

Also in the dystrophin genes of human and mouse, the internal promoters are situated in very large introns. Likewise, the 5′ introns that contain the muscle type and purkinji-type dystrophin promoters are very large. Unlike the Drosophila DLP gene, the mammalian dystrophin genes contain several additional large introns, which are not known to contain promoters (Neuman, 2005).

Due to an overall reduction in intron length, the chicken dystrophin gene is smaller than its mammalian orthologue -- its size is about one mega base. Nevertheless, the 5′ introns and the introns harboring the promoters of the internal products remained among the largest introns in the gene (Neuman, 2005).

Like Drosophila, Caenorhabditis elegans has only one gene encoding a dystrophin-like protein. The worm gene is only 31kb long, it does not have large introns, and there are as yet no reports describing multiple 5′ or internal promoters in the gene (Neuman, 2005).

Thus the DLP gene encodes three full-length gene products, DLP 1, 2, and 3 and a truncated protein Dp186 as well as two additional internal products, Dp117 and Dp205. These findings show that the DLP gene is as complex as the mammalian dystrophin gene. Thus, the structure of a large gene encoding several full-length products and several truncated products has been conserved for at least 600 million years, indicating that both type of products have important functions (Neuman, 2005).

Dp205 is the evolutionary orthologue of mammalian Dp116. Also sea urchin has a Dp116 orthologue. These findings, and the identification of a Dp116 homologue encoded by the utrophin gene, Up116, show that Dp116 is an old and conserved internal dystrophin gene product. Dp117 and Dp186 do not have mammalian homologues. Thus, with regards to location of the promoters in relation to the coding sequence, only one internal product identified so far, is found in both vertebrates and invertebrates. Due to the large evolutionary distance and the lack of sequence similarity it could not be determine whether the 5′ promoters of the fly gene are evolutionary related to the 5′ promoters of the mammalian gene (Neuman, 2005).

The variability in the location of the internal promoters in the dystrophin genes of various organisms raises the question of the origin of these promoters. One possibility is that the ancestral dystrophin gene contained all the currently existing promoters of the gene in vertebrates and invertebrates, and different promoters were lost in various evolutionary pathways. Alternatively, new promoters could be differentially formed, differentially inserted into existing introns, or differentially inserted with new introns into the genes. It is also possible that like introns, promoters have been shuttled from and into the gene, perhaps with the introns in which they are located. Currently available data are not sufficient to differentiate between these alternatives (Neuman, 2005).

The products of the dystrophin gene in mammals have very short (few amino acids) N-termini, which are product-specific. It is likely that the existence of multiple promoters (and the specific first exons) is related to a requirement for the cell-type specific expression of the various gene products. Alternatively, a common feature of the three internal products of the DLP gene is the large unique N-termini, consisting of hundreds of amino acids. These N-termini may have specific functions, possibly interactions with other proteins. However, since the three N-termini are not similar to other proteins in the database, biological functions to these parts of the proteins cannot be assigned (Neuman, 2005).

scaffold length - >141961 bp

Exons - 36

PROTEIN STRUCTURE

Amino Acids - 3497 (Dys-PA)

Structural Domains

Defects in the dystrophin complex (DC) underlie several human genetic disorders, but dissection of its function is complicated by potential redundancy of the multiple vertebrate isoforms of most DC components. This analysis shows that the fly retains all essential components of the DC, but with substantially less diversity. Seventeen known human components (three dystrophin-related proteins, two dystrobrevins, five sarcoglycans, five syntrophins, one dystroglycan and one sarcospan) appear to be reduced to eight in Drosophila (one, one, three, two, one and none, respectively). The simplicity of this system recommends it as a model for its human counterpart (Greener 2000).

Residing within the genomic scaffold clone AE003726, the Drosophila orthologue of dystrophin (DmDYS) consists of an unusual gene of at least 130 kb in length (the size could not established because of the uncertainty of one intron), with 31 introns ranging from 61 bp up to 48 kb. No genes were detected within the large introns using NIX to apply a range of bioinformatic gene-detecting algorithms, and the reason for the large size of this gene remains as enigmatic as the human dystrophin gene size. The intron/exon structure is virtually unrelated to that of the human gene (one coding exon is an extraordinary 3.5 kb in length). The 2.7 Mb human dystrophin gene encodes a large protein of 3685 amino acids in length, comprising four main sections: an actin-binding N-terminus, a rod-like spectrin repeat region, a cysteine-rich region (containing a WW domain, four EF-hands and a ZZ domain) and a C-terminal region. Using the highly conserved C-terminal end, a similar sequence representative of the sole Drosophila dystrophin-like protein has been isolated. The recent characterisation of the complete coding sequence for DmDYS reveals a shorter protein (3124 residues) that retains all four sections that are distinctive of its vertebrate counterpart. The cysteine-rich and C-terminal regions remain the most highly conserved part of DmDYS, with an identity of 54% between human and fly. These regions of the vertebrate protein contain sequences known to interact with β-dystroglycan, the syntrophins and dystrobrevins (Greener 2000).

An alignment of the N-terminus reveals a lower degree of conservation in the predicted actin-binding region (43% identity, 62% similarity); the C. elegans sequence is correspondingly divergent (22% identity, 43% similarity). Assuming that the N-termini of worm and fly dystrophins bind actin, it is apparent that the mode of interaction is subject to fewer evolutionary constraints than are those of the C-terminal domains. Unlike the human protein, there does not appear to be any recognisably unique basic region within the central rod domain that may represent an alternative means of F-actin-binding (Greener 2000).

Analysis of the rod domain shows a high level of degeneracy, and unlike the C. elegans DYS-1, the fly rod domain is 20% shorter than its human dystrophin counterpart (and 12% shorter than utrophin). The central region of the DmDYS rod domain departs from the canonical dystrophin-like spectrin repeat motif: its characteristic tryptophan residues show that any specific resemblance between human and fly dystrophin is replaced for much of the rod domain by a more generic adherence to a looser repeat motif. Finally, the first and last few spectrin repeats of both fly and worm dystrophins show a high degree of continuous specific similarity to human dystrophin, suggesting a more critical role for these particular repeats (Greener 2000).

The gene which is defective in Duchenne muscular dystrophy (DMD) is the largest known gene containing at least 79 introns, some of which are extremely large. The product of the gene in muscle, dystrophin, is a 427 kDa protein. This gene encodes at least two additional non-muscle full length dystrophin isoforms transcribed from different promoters located in the 5'-end region of the gene, and four smaller proteins transcribed from internal promoters located further downstream, that lack important domains of dystrophin. Several other genes, encoding evolutionarily related proteins, have been identified. To study the evolution of the DMD gene and the significance of its various products, genes encoding dystrophin-like proteins have been sought in sea urchin and in Drosophila. A sea urchin gene encodes a protein which is an evolutionary homologue of Dp116, one of the small products of the mammalian DMD gene, and the partial sequencing of a large product of the same gene is reported. The full-length product shows strong structural similarity and sequence identity to human dystrophin and utrophin. A Drosophila gene closely related to the human dystrophin gene is described. Like the human gene, the Drosophila gene encodes at least three isoforms of full length dystrophin-like proteins (dmDLP1, dmDLP2 and dmDLP3,), regulated by different promoters located at the 5' end of the gene, and a smaller product regulated by an internal promoter (dmDp186). As in mammals, dmDp186 and the dmDLPs share the same C-terminal and cysteine-rich domains which are very similar to the corresponding domains in human dystrophin and utrophin. In addition, dmDp186 contains four of the spectrin-like repeats of the dmDLPs and a unique N-terminal region of 512 amino acids encoded by a single exon. The full length products and the small product have distinct patterns of expression. Thus, the complex structure of the dystrophin gene, encoding several large dystrophin-like isoforms and smaller truncated products with different patterns of expression, existed before the divergence between the protostomes and deuterostomes (Neuman, 2001).

The Drosophila dystrophin gene encodes at least four protein isoforms bearing a number of highly conserved domains (Greener, 2000; Neuman, 2001). The three large isoforms DLP1, DLP2, and DLP3 have an N-terminal actin-binding domain, spectrin repeats, and a C-terminal cysteine-rich domain, which in mammals has been shown to interact with other DGC proteins. A shorter isoform, Dp186, has a unique N-terminal domain appended to the pan-Dystrophin C-terminal domain (van der Plas, 2006).

date revised: 18 February 2024

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