RNA in situ hybridizations to wild-type embryos first reveal duf RNA at very low levels at gastrulation in the invaginating mesoderm. This expression is transient and rapidly disappears. Expression is reinitiated at stage 11 in mesodermal patches that quickly resolve into single cells. These cells were identified by position and coexpression of specific markers as the progenitors of the somatic, pharyngeal, and visceral muscles. At this stage, duf is also expressed in the garland cells. In somatic mesoderm, duf expression is closely linked to the process of fusion. It starts in the muscle progenitors and is maintained in the sibling founder cells resulting from their division and in the syncytial muscle precursors but is lost from the progenitors of adult muscles (which do not fuse during embryogenesis). Interestingly, duf RNA can be detected in muscle precursors as long as they are increasing in size by the incorporation of new myoblasts to the syncytia. Thus, its expression fades from different precursors at different times, being lost from the smaller muscles first. By stage 14, duf is undetectable in visceral mesoderm, and at stage 15, it is no longer expressed in any muscles. duf is also expressed on the midline of the CNS from stage 14 and in additional neurons from stage 15 until the end of embryogenesis (Rúiz-Gomez, 2000).
The mechanisms underlying the setting of myotubes and choice of myotube number in adult Drosophila were examined. The pattern of adult myotubes is prefigured by a pattern of duf-lacZ-expressing myoblasts at appropriate locations. Selective expression of duf-lacZ in single myoblasts emerges from generalized, low-level expression in all adult myoblasts during the third larval instar. The number of founders, thus chosen, corresponds to the number of fibers in a muscle. In contrast to the embryo, the selection of individual adult founder cells during myogenesis does not depend on Notch-mediated lateral inhibition. These results suggest a general mechanism by which multi-fiber muscles can be patterned (Dutta, 2004).
This study examines the formation of multi-fiber muscles, i.e. muscles that function together as a contractile unit, in the adult fly. In the mesothorax of the adult, the most prominent muscles are the indirect flight muscles (IFMs); their development has been charted in some detail. The IFMs consist of the dorsal longitudinal muscles (DLMs), an array of six large fibers, and three groups of dorsoventral muscles: DVM-I (three fibers), DVM-II (two fibers) and DVM-III (two fibers). The mesothorax contains another large muscle, namely the tergal depressor of the trochanter or jump muscle, which consists of many fibers bundled together as a unit. The dorsal thorax also contains the direct flight muscles (DFMs), involved in changing the orientation of the wing. Each of these muscles is a multi-fiber contractile unit. The muscles in the adult abdomen are also arranged as well-defined sets of fibers, which form dorsally, laterally and ventrally in each segment (Dutta, 2004).
This investigation of myotube formation in the adult began by looking at a prominent set of muscles, the DVMs, which form de novo from groups of aggregating myoblasts in the adult thorax. How the three different DV muscles are formed from these myoblasts and how each muscle attains its typical fiber number were examined. It could be that, as in the embryo, each fiber is seeded by the formation of a single, specialized founder cell. Alternatively, myoblasts might be selected in some other way; for example, as a group that would fuse to form a single myotube. To distinguish between these two alternatives, a P-lacZ insertion (rp298) in the gene dumbfounded was used; this resulted in the expression ofß-galactosidase in the nuclei of cells that express duf. At 7 hours after puparium formation (APF), at each epidermal location where a DVM fiber will develop, a single prominent myoblast is seen that expresses duf-lacZ. Thus, three, two and two duf-lacZ expressing cells are seen, corresponding, respectively, to the three, two and two fibers of the future DVM I, DVM II and DVM III muscles. These nuclei continue to express duf-lacZ at high levels as each of the syncitial fibers in the DVMs form. In addition, the fused adult myoblast nuclei within the syncitia show low levels of duf-lacZ expression. By 36 hours APF, the level of ß-galactosidase in all DVM nuclei begins to fall and disappears by 70 hours APF (Dutta, 2004).
These findings suggest that, just as in the embryo, the formation of myotubes in the adult may be initiated by the selection of single founder myoblasts that are identifiable by their expression of duf-lacZ. Other myoblasts would be recruited to these founders and fuse with them and (again, as in the embryo) these fusing cells would themselves be induced to express duf-lacZ, albeit at a lower level. If this view of adult fiber formation is correct, then it should be generally true for all cases in the adult where fibers form de novo from groups of aggregating myoblasts. With this in mind, the regular arrays of fibers that form dorsally and laterally in the adult abdomen were examined. There are many such fibers laid out in a well-organized pattern and they are derived from myoblasts that, unlike the DVM cells, come not from the discs but from pools of cells associated with the abdominal nerves (Dutta, 2004).
The formation of the syncitial muscles in the abdomen begins at about 28 hours APF. A stage prior to this was examined to see whether single duf-lacZ-expressing cells appear before fibers form. Once again, a striking correspondence was observed between forming fibers and duf-lacZ expression in the abdomen, with every fiber preceded by a single duf-lacZ-expressing nucleus at the appropriate position. At 24 hours APF, an array of duf-lacZ-expressing cells was seen in each of the dorsal hemisegments. By 28 hours APF, these cells are in positions where the future muscle fibers will form. By 50 hours APF, when the formation of the syncitial fibers is largely complete, one nucleus in each fiber was observed that expresses duf-lacZ at higher levels than the rest. A similar pattern of duf-lacZ expression was observed in the developing lateral muscles. At positions where the future lateral muscles will form there are single duf-lacZ-expressing cells that are also 22C10 positive. These mononucleate cells develop into multinucleate fibers, which each contain several duf-lacZ-expressing nuclei (Dutta, 2004).
On the face of it, the development of the DLMs follows a different scheme. Here, the muscles assemble on a set of preexisting templates provided by a small set of persistent larval fibers, the three larval oblique muscles (LOMs). Subsequently, the three templates split to form the six fibers of the DLMs. Despite this novel form of myogenesis, it has been suggested that the larval fibers serve a founder-like function in organizing the development of the DLMs, and it is certainly true that the adult myoblasts aggregate on the larval fibers and fuse with them to form syncitial myotubes. With this in mind, the expression of duf-lacZ was examined during DLM development. duf-lacZ is indeed expressed in the founder analogues, the larval templates of the adult muscles. duf-lacZ expression begins in the three larval templates at 6.5 hours APF. All of the nuclei in each of the three templates appear to express duf-lacZ. The expression of duf-lacZ continues as the templates split and form the six fibers of the DLMs. By 36 hours APF, the intensity of ß-galactosidase staining starts to decrease, and by 70 hours APF the staining has disappeared. The duf-lacZ-expressing nuclei in the templates could be larval nuclei or the nuclei of adult myoblasts that have already fused with the templates by 6.5 hours APF. Larval nuclei are large and easily distinguishable from the smaller nuclei of the adult myoblasts. The transcription factor Erect wing (Ewg) is expressed in the nuclei of the larval muscles and this expression persists as the larval muscles are transformed to templates. Ewg is also expressed in adult myoblasts as they migrate over the templates to fuse with them. These two subsets of nuclei, both expressing Ewg, can be easily distinguished on the basis of their size. A double label using antibodies against ß-galactosidase and Ewg reveals that duf-lacZ-expressing nuclei co-localise with Ewg-expressing nuclei, which by their size are larval in origin (Dutta, 2004).
If duf-lacZ expressing founder myoblasts are, as in the embryo, a decisive factor in controlling the spatial pattern of adult myogenesis, then it is necessary to understand how this pattern of expression develops and is controlled. The emergence of duf-lacZ expression in the myoblasts of the abdomen was examined. The abdominal muscles develop from pools of muscle-forming cells that are associated with the nerves that innervate the larval muscle field. These cells in turn are derived from single adult muscle progenitors that arise in the embryo as the siblings of embryonic muscle founder cells. Unlike their embryonic founder cell siblings, the adult precursors maintain twist expression and proliferate during larval life to form pools of nerve-associated, twist-expressing myoblasts. The adult precursors do not express duf-lacZ in the embryo. These myoblasts then migrate, aggregate and fuse to form the muscles of the adult abdomen. When the expression of duf-lacZ was examined in these precursor cell populations in late third instar larvae, all the cells were found to express duf-lacZ at a low level. This expression persists during early pupal stages (13-14 hours APF) but, by 16 hours APF, levels of ß-galactosidase in the myoblasts have declined and, at 20 hours APF, only a subset of Twist-positive myoblasts remains ß-galactosidase positive. By 24 hours APF, however, there is a clear upregulation of duf-lacZ expression in particular nuclei, and by 28 hours APF, these duf-lacZ-expressing cells are positioned at the sites where individual muscle fibers will now form. These are the same nuclei that are present in 22C10-stained cells and that continue to express high levels of duf-lacZ within the fully formed fibers. A similar sequence of duf-lacZ expression is seen in the lateral clusters of myoblasts. Expression does not persist into the adult and begins to diminish by 70 hours APF (Dutta, 2004).
A similar sequence of expression is found in the myoblasts of the wing disc that give rise to the DLMs and DVMs. In late third instar larvae, duf-lacZ expression can be detected at low levels in all of these cells. To follow this pattern of expression in the pupa, duf-lacZ pupae were double-labeled with markers for adult myoblasts, using either UAS-GFP driven by 1151-GAL4, which is expressed in all adult myoblasts, or antibodies to the transcription factor Vestigial (Vg), which is expressed in most of the myoblasts on the wing disc. These stainings reveal that the generalized low level of duf-lacZ expression had disappeared by 12 hours APF and had been replaced by expression in specific cells. It is concluded that, as in the abdomen, the duf-lacZ-expressing founders of the thoracic muscles other than the DLMs are derived from the pool of adult myoblasts, all of which initially express duf-lacZ at low levels (Dutta, 2004).
The decisive function of muscle founder cells in myotube formation is revealed in the embryo by their unique capacity to form muscles in mutants where myoblast fusion is compromised. Thus in embryos that are defective in the machinery of myoblast fusion, founder myoblasts differentiate to form thin mononucleate fibers at appropriate positions and with molecular characteristics similar to those of normal wild-type muscles. Other myoblasts aggregate on the differentiating founders but cannot fuse with them and do not succeed in differentiating to form muscle fibers themselves. These observations show that, in the embryo, myoblasts are of two kinds: founders, which are competent to complete myogenesis in the absence of fusion, and feeders, which can only contribute to myogenesis after fusion with a founder (Dutta, 2004).
To show whether there is a similar division of myoblasts during adult myogenesis, a fusion-defective phenotype was generated during adult myogenesis, by overexpressing a dominant-negative form of the protein Rac1, a member of the small GTPase superfamily involved in the process of fusion. Overexpression of the dominant-negative Rac1 (Rac1N17) in the embryonic mesoderm severely delays the fusion process and results in abnormal fusion in the later stages. Overexpression of Rac1N17 in the adult myoblast pool severely reduces myoblast fusion, the effect being most dramatic in the lateral muscles of the abdomen and, to a lesser extent, in the thoracic muscles. Nevertheless, duf-lacZ-expressing myoblasts are present in the correct number at the correct positions. In the absence of fusion, each putative founder cell begins to express myosin at the appropriate stage, elongates and differentiates into a thin myotube. In the absence of fusion, the founders eventually develop into mononucleate, myosin-expressing fibers, like the mononucleate muscles observed in the embryos of fusion mutants (Dutta, 2004).
The putative founders of the DVMs in the thorax of 1151GAL4/UAS-Rac1N17 pupae are also present in a wild-type pattern and initiate fiber formation. Some fusion does occur, but to a lesser extent than normal. The DVM II fibers are not mononucleate but have fewer nuclei than wild-type fibers of the same stage. These fibers ultimately give rise to muscles, albeit thin, at the correct position and with the correct number of fibers. These results suggest that where myoblast fusion is prevented during adult myogenesis, a population of duf-lacZ-expressing myoblasts segregates normally, as in the embryo, and that, like the founders in the embryo, these cells uniquely have the capacity to complete differentiation to form muscles. They also demonstrate that, as in the embryo, by the onset of fiber formation adult myoblasts are of two classes: fusion-competent cells that do not express duf-lacZ and founders that do express duf-lacZ. It is these latter cells that have the capacity to complete myogenic differentiation even when fusion is blocked or reduced (Dutta, 2004).
In the Drosophila embryo, the diversification of muscle forming mesoderm into founders and fusion-competent cells occurs through a process of lateral inhibition mediated by Notch. Since single duf-lacZ-expressing cells are selected and appear to act as founder myoblasts during adult myogenesis, it is important to show whether, as in the embryo, a Notch-dependent lateral inhibition pathway mediates this selection process. To test whether Notch has a function in selecting specific myoblasts for duf-lacZ expression, a dominant-negative and a constitutively active form of Notch was used. It was reasoned that if lateral inhibition were involved, then overexpression of a dominant-negative form of Notch (dnNotch) in adult myoblasts would lead to an increase in the number of duf-lacZ-expressing founders, whereas overexpression of the active form (Nintra) should suppress duf-lacZ expression altogether (Dutta, 2004).
In fact, the results of these experiments appear to be contradictory: thus, expression of UAS-dnNotch caused no change in the number of DVM founders and flies of the genotype 1151GAL4;UAS-dnNotch had the correct number of DLM and DVM fibers. This conclusion was verified by reducing Notch function in two additional ways. Function in the Notch signalling pathway in myoblasts was reduced by overexpressing truncated forms of the protein Mastermind (Mam), an essential component of the Notch signalling pathway. Mam interacts with the intracellular domain of Notch and with Suppressor of Hairless, and forms a transcriptional activation complex. Two truncated versions of Mam, MamH and MamN, when overexpressed by the GAL4-UAS system behave as dominant-negative proteins and elicit Notch loss-of-function phenotypes. Overexpression of either UASMamN or UAS-MamH in myoblasts using 1151-GAL4 had no effect on the number of DVM founders. The role of Notch was further examined by using a conditional allele, Nts1. Because of the close proximity of the duf and Notch loci, duf-lacZ, Nts recombinants could not be generated and hence 22C10 was used as the marker for founder cells in the abdomen. The earliest time at which myoblasts expressing high levels of duf-lacZ are also labeled with 22C10 is at 24 hours APF. Notch function was removed for different periods (2 hours, 4 hours, 6 hours and 8 hours) before this stage by raising Nts animals to the non-permissive temperature, and the number of 22C10-stained cells associated with the abdominal nerves was determined. The numbers of 22C10-expressing cells in the dorsal or lateral segments of the abdomen were determined and shown to be unaffected in these experiments. It is known that all three approaches -- whether using the dominant-negative Notch or mastermind constructs, or using Nts animals -- are effective, since they all can reduce the levels of Twist expression in adult myoblasts, a known consequence of Notch reduction in adult myoblasts (Dutta, 2004).
Taken together, these results suggest that Notch is not required for the selection of duf-lacZ-expressing myoblasts. However, in the converse experiment, expression of Nintra in the myoblasts does suppress the formation of founders, as would be expected for a selection mechanism based on lateral inhibition. How can these apparently contradictory findings be reconciled? Earlier studies have shownthat Notch acts as a positive regulator of Twist in the myoblast population. Thus, Nintra expression in adult myoblasts leads to maintained expression of Twist in these cells and to a failure of muscle differentiation. If the absence of founders that was observe is a consequence of this sustained expression of Twist in the myoblasts, then it would be expected that simply expressing Twist constitutively in the myoblasts would mimic the activated-Notch phenotype. Using 1151-GAL4 to drive Twist expression in the adult myoblasts, it was found that at 12 hours APF there were no DVM founders, suggesting that a decline in Twist expression, which is antagonized by the action of Nintra, is a requirement for elevated duf-lacZ expression. Indeed, founders are the first cells in the myoblast pool to show declining levels of Twist expression, with the result that the duf-lacZ-expressing founders and Twist-expressing myoblasts are mutually exclusive cell populations (Dutta, 2004).
A set of GFP markers and confocal microscopy has been used to analyse Drosophila leg muscle development, and all the muscles and tendons present in the adult leg are described. Importantly, evidence is provided for tendons located internally within leg segments. By visualising muscle and tendon precursors, it was demonstrated that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg (Soler, 2004).
A common feature of all Drosophila muscles is that they arise from twi-expressing non-differentiated cells. Leg muscles originate from a restricted subpopulation of such cells (5-10 myoblasts) associated with the embryonic leg disc primordia. These cells start to proliferate in the second instar larvae to form a population of about 500 myoblasts that are randomly deployed on the disc epithelium and also are known as adepithelial cells. Unlike the embryonic promuscular cells, they do not seem to be organised into clusters of cells from which progenitors of individual muscles segregate, but rather they follow the segmental subdivision of the leg disc within the proximodistal axis. This leads to the early loss of twi expression in adepithelial cells from the tarsal segments. The main feature of all Drosophila muscles that form de novo, including the larval body wall and the adult direct flight muscles, is that they develop from the specialised myoblasts named muscle founder cells. The leg muscles belong to this category of muscle, and this study shows that their formation is preceded by the specification of cells expressing the muscle founder marker duf-lacZ. How the duf-lacZ-expressing cells segregate from the population of adepithelial cells and how they become muscle founders remains unclear, but their association with sr-positive tendon progenitors suggests that interactions between these two cell types may promote their differentiation (Soler, 2004).
Interestingly, in third instar leg discs, duf-lacZ cells segregate in around only one out of five sr-expressing epithelial domains. This domain, termed the 'a' domain, is located in the dorsal Dpp-dependent portion of the disc, suggesting that Dpp signalling may be involved in eliciting this group of presumptive founders. Similar to the leg tendon precursors described in this study, sr-expressing domains have been reported in the notum of the third instar wing discs. These sr-positive domains have been reported to be involved in flight muscle patterning (Soler, 2004).
In spite of all the similarities, marked differences in appendicular versus flight and larval body wall musculatures exist that can be explained by the specific properties of leg tendons. As demonstrated by analyses of Stripe-GFP-expressing leg discs, at the end of third instar, concomitant with disc evagination, the epithelial domains of tendon progenitors start to invaginate inside the disc. This leads to the formation of internal tendons that have not been described in other body parts of the adult fly. Importantly, the presumptive founder cells associated with the invaginating tendon precursors are vectored and deployed throughout the proximodistal axis of the leg segments. Such a system provides an effective way to generate multifibre muscles in an invertebrate leg devoid of internal skeleton (Soler, 2004).
The mechanisms governing the formation of internal tendons remain to be elucidated; however, the co-expression of sr with odd in invaginating tendon precursors suggests a potential involvement of Notch. odd was previously described as an important element of the Notch-dependent cascade that controls the invagination of segmental joints. Thus, it is possible that a similar set of genes controls the different epithelial invagination events that occur in the developing leg (Soler, 2004).
Using transgenic lines that express GFP in tendon precursors (Stripe-GFP), in myoblasts and in tendons (1151-GFP), and in developing myotubes (MHC-tauGFP), it was possible to monitor appendicular myogenesis during pupa metamorphosis. At 20 hours APF, a large number of myoblasts are associated with the internal tendons, suggesting that the founder cells that are initially linked to tendons have attracted fusion-competent myoblasts to form prefusion complexes. Five hours later muscle precursors can be discerned composed of 5 to 10 nuclei, indicating that the first wave of fusion takes place between 20 and 25 hours APF. Shortly after, at 35 hours APF, the second fusion wave occurs, giving rise to the multinucleated myotubes that are attached on one side to the internal tendons. The timing of the observed fusion events is comparable to that reported previously for the de novo forming direct flight muscles. The next myogenic steps, including myotube growth, recognition of cognate sr-expressing epithelial attachment sites and induction of expression of myofibrillar proteins, are similar to the previously described events that lead to the formation of the flight and body wall muscles. The most important, unique, feature of leg muscle fibres that makes them different from other Drosophila muscles is their association with the internal tendons (Soler, 2004).
The appendicular muscle pattern revealed by this study consists of two principal muscles (levator and depressor) in each leg segment. The organisation of the muscle fibres composing levators and depressors reveals that they are attached to internal tendons. The long tendon of the tarsus extends to the femur and harbours two previously undescribed muscles, which have been designated ltm1 and ltm2 (Soler, 2004).
Overall, the computer-assisted reconstruction of the leg musculature enabled all the appendicular muscles and tendons to be identified, their anteroposterior, dorsoventral and proximodistal positions to be defined, and the number of muscle fibres that compose the individual muscles to be determined. Since this is the first reported systematic analysis of the Drosophila leg musculature, designations and their corresponding abbreviations have been proposed for all the identified muscles and tendons. In general, the proposed designations reflect the muscle and tendon functions. For example, muscles located in the femur that ensure movements of the adjacent tibia are named tibia levator (tilm) and tibia depressor (tidm) muscle (Soler, 2004).
The observations also indicate that the general pattern of appendicular muscles is invariant in males and females. However, muscle fibres that contribute to depressors and levators display distinct characteristics, suggesting differences in the genetic programme that ensures their specification. Most specifically, they differ at the ultrastructural level, displaying variations in sarcomere size and number of mitochondria. As determined by the analyses of dissected appendicular muscles, the number of nuclei that contribute to the mature fibres differs in the different types of muscle, but is relatively invariant when the same muscles from two different legs are compared. This suggests a precise control mechanism that sets up the complex events of appendicular myogenesis in Drosophila (Soler, 2004).
The association of muscle and tendon precursors in the imaginal leg discs of Drosophila reported here resembles the temporally and spatially linked development of avian tendons and muscles described in the chick hind limb, the specification of tendon progenitors in vertebrate embryos takes place very early in development, in a compartment immediately adjacent to the myotome. Thus it seems that conserved mechanisms may control the co-ordinated development of muscles and tendons in both the Drosophila leg and vertebrate embryos. An attractive possibility is that the muscle and tendon progenitors mutually promote each other's specification. The existence of such a mechanism could be easily tested in the future using Drosophila as a model system (Soler, 2004).
Motoneurons directly influence the differentiation of muscle fibers, regulating features such as muscle fiber type and receptor development. Less well understood is whether motoneurons direct earlier events, such as the patterning of the musculature. In Drosophila, the denervation of indirect flight muscles results in a diminished myoblast population and smaller or missing muscle fibers. Whether the neuron-dependent control of myoblast number is due to regulation of cell division, motoneuron-dependent apoptosis, or nerve-dependent localization and migration of myoblasts, was examined. Denervation results in a reduced rate of cell division, as revealed by BrDU incorporation. There is no change in the frequency of apoptotic myoblasts following denervation. Using time lapse imaging of GFP-expressing myoblasts in vivo in pupae, it was observed that despite denervation, the migration and localization of myoblasts remains unchanged. In addition to reducing myoblast proliferation, denervation also alters the segregation of myoblasts into the de novo arising dorso-ventral muscles (DVMs). To address this effect on muscle patterning, the expression of the founder-cell marker Dumbfounded/Kirre (Duf) in imaginal pioneer cells was examined. There is a strong correspondence between cells that express Dumbfounded/Kirre and the number of DVM fibers, consistent with a role for these cells in establishing adult muscles. In the absence of innervation the Duf-positive cells are no longer detected, and muscle patterning is severely disrupted. These results support a model where specialized founder cells prefigure the adult muscle fibers under the control of the nervous system (Fernandes, 2005).
The motoneuron exerts a mitogenic influence on IFM myoblasts. Following unilateral denervation, the BrDU birthdating experiments revealed a significant decline in the rate of proliferation. This decline is likely sufficient to account for the reduced myoblast population observed in denervated hemisegments (previously quantified by morphometry; Fernandes, 1998). The smaller muscles that result are thus likely due to the smaller number of available myoblasts, and possibly to the absence of neuromuscular excitation following denervation (Fernandes, 2005).
Two alternate mechanisms were ruled out: there was no evidence for a change in the rate of myoblast cell death following denervation, indicating that the motoneuron does not provide an essential survival factor for the cells. Also, no significant change was observed in the migratory behavior of myoblasts following unilateral denervation, when examined at either the single cell or population level. This indicates that the reduced population size was not the result of myoblast emigration from denervated sites (Fernandes, 2005).
The second major effect of denervation was the gross disruption of normal muscle formation in the de novo arising DVMs. Normally, myoblasts coalesced into discrete primordia that prefigure the three sets of DVM muscle fibers. When denervated, the DVM myoblasts remain unpatterned, and the muscles fail to form. This may be due to a direct effect of the motoneuron on the myoblasts. However, denervation also disrupts the behavior of a potential intermediary player, the imaginal pioneer cells, that are thought to prefigure the DVM fibers as myoblast fusion targets (Fernandes, 2005).
BrDU birthdating experiments reveal a significant rise in the rate of DLM myoblast proliferation that normally occurs at 18-24 APF. In denervated regions, myoblast proliferation remains unchanged, holding steady at the earlier, basal level seen prior to the onset of myoblast fusion (at 12-16 h APF). Thus, DLM myoblast proliferation involves two components: a basal and nerve-independent phase (at 12-16 h APF) and a later incremental nerve-dependent phase (at 18-24 h APF). It is proposed that the nerve-dependent increase in myoblast proliferation regulates the number of myoblasts available for fusion, and thus is a way for motoneurons to control muscle size (Fernandes, 2005).
The nerve-dependent rise in DLM myoblast proliferation correlates with the expansion of motoneuronal terminal arbors on the muscle fiber surface. While it is possible that the expansion of motoneuron terminals and the change in myoblast proliferation are independent responses to exogenous hormone signals, the data argue that motoneurons strongly influence myoblast cell division, since proliferation is reduced following denervation. It is proposed that the growing motoneuron terminal either releases a factor that influences myoblast cell division, or alternatively potentiates myoblast responsiveness to available growth factors and mitogens. In either case, the nerve-dependent control of myoblast proliferation would in turn influence the growth of the muscle fiber (Fernandes, 2005).
The rise in myoblast proliferation observed during the nerve-dependent phase of DLM development resembles a feature seen during photoreceptor development in the Drosophila eye. During differentiation of the neuroepithelium, there is a rise in cell proliferation, referred to as the second mitotic wave (SMW), which produces additional precursors that are recruited to eventually form a complete ommatidium. It is likely that the rise in DLM myoblast proliferation similarly serves to maintain the size of the myoblast pool, so that cells can be continuously drawn from the pool until the desired muscle size is achieved (Fernandes, 2005).
That the two phases of DLM myogenesis differ in their nerve-dependence resembles events associated with vertebrate myogenesis. Mammalian skeletal muscles form in two waves: primary myotubes form first, and serve as scaffolds for secondary myotube formation. Primary myogenesis is independent of innervation, while secondary myogenesis is nerve dependent. This is due to the presence of a nerve-dependent population of myoblasts essential for secondary myotube formation (Fernandes, 2005).
The DVMs develop from the de novo fusion of myoblasts, and critically depend on the motoneuron for muscle fiber formation (Fernandes, 1998 and Fernandes, 1999). Denervation also results in a failure of myoblasts to segregate into distinct DVM primordia. Myoblast patterning and fiber development for the DVMs have been proposed to depend on specialized imaginal pioneer cells. The 'imaginal pioneer' (IP) cells lie in close association with motoneuron arbors, as demonstrated by EM analysis, and are thus potentially dependent on neurons for their normal function or survival. The IP cells are thought to serve as myoblast fusion targets, and thus to prefigure the mature muscle fibers. There have been, however, no reported molecular markers for these cells (Fernandes, 2005).
In the Drosophila embryo the mesodermal cells that generate the somatic muscles are critically dependent on specialized founder cells. Each embryonic founder cell is the precursor of a specific muscle fiber, and is the target of fusion-competent myoblasts. The founder cells each express Dumbfounded/Kirre, a key component of the cell fusion machinery. In loss of function duf mutations myoblast fusion is disrupted (Fernandes, 2005).
Intriguingly, it was found that there are Duf-positive cells within the DVM myoblast pool. Their location, number, and size indicate that they are likely to be the IP cells previously described. The Duf-positive cells are present in the DVM I and II primordia in direct correspondence to the final number of DVM fibers, as is the case for the IP cells. Duf-positive cells have also been reported for other pupal muscles, and a correlation exists between Duf-positive cells and the numbers of both IFM and abdominal muscle fibers. Significantly, it was found that denervation affects the Duf cells of the DVM primordia. Although Duf-positive cells are initially present in the denervated hemisegments in the normal pattern and number (at 12 h APF), following denervation they are no longer reliably observed by 18-20 h APF. By 24 h APF, when control hemisegments possess well patterned Duf-positive DVMs, Duf-positive cells on the denervated side are rarely observed (Fernandes, 2005).
These observations support a model where Duf-positive IP cells in the pupa serve as fusion targets of myoblasts, as is the case for the Duf-positive founder cells in the embryo. Since the Duf molecule is an essential component of the cell fusion machinery, its disappearance following denervation suggests that fusion events are severely disrupted and may explain the associated muscle patterning defects. It cannot as yet be determine whether the loss of Duf labeling is due to a loss of Duf expression in the IP cells, or due to apoptosis. Distinguishing between these scenarios will require in situ time lapse imaging of vitally labeled IP cells in denervated hemisegments (Fernandes, 2005).
DLM fibers arise from larval muscles that persist into the pupal stage. Like all embryonically established somatic muscle fibers, the persistent larval fibers also do not depend on the motoneuron for their formation or maintenance. When denervated, the larval fibers persist and DLM fibers still form, albeit at a slower rate (Fernandes, 1998; Fernandes. 1999). Duf expression is also detected in the persistent larval fibers, consistent with the fact that they function as myoblast fusion targets. However, unlike the DVMs, denervation does not result in a loss of Duf expression in the developing DLMs. The reason for this independence remains uncharacterized, but likely reflects the distinct origin of these cells from larval precursor muscles (Fernandes, 2005).
A dependence on motoneurons for the regulation of muscle size and patterning has been observed for several insect systems. When abdominal Drosophila muscles are denervated, the adult fibers are significantly reduced in mass. The most prominent effect involves the male-specific muscle (MSM) of the fifth abdominal segment of the adult. This muscle is larger than other body wall muscle fibers, a difference attributed to the enhanced recruitment of myoblasts from a common myoblast pool. When the abdominal myoblast pool is reduced experimentally through hydroxyurea treatment, a smaller muscle is present at the MSM location in segment A5. A BrDU labeling analysis remains to be performed to confirm the role of myoblast proliferation on MSM development (Fernandes, 2005).
Denervation studies in Manduca have similarly shown that proliferation of myonuclei is reduced in leg, abdominal, and DLM muscles. At the onset of metamorphosis, muscle precursors appear in the region of the future adult muscles and become associated with tendons (leg muscles) or persistent larval muscles (DLM). This accumulation is then followed by the appearance of proliferating 'myonuclei' within the developing primordia. By contrast, in the case of Drosophila DLMs, BrDU incorporation is restricted to myoblasts present outside the primordia, and there is no evidence of nuclear division within the muscle fibers (Fernandes, 2005).
In conclusion, it is proposed that the motoneuron critically influences the size of the myoblast pool through a direct effect on myoblast cell division, and that this helps regulate the final size of adult muscle fibers. The motoneuron has a second role in regulating the development of de novo forming fibers, where it is essential for the partitioning of myoblasts into muscle primordia. Moreover, continued Duf labeling within the primordia depends on the motoneuron's presence. Thus, the motoneuron influences both the number of cells available for fusion, as well as potentially regulates the fusion events themselves. This is an elegant mechanism for controlling muscle fiber differentiation during myogenesis, and may have evolved as a way to ensure that muscle primordia develop into muscles that meet the diverse demands placed on them by the nervous system (Fernandes, 2005).
The Immunoglobulin superfamily (IgSF) proteins Neph1 and Nephrin are co-expressed within podocytes in the kidney glomerulus, where they localize to the slit diaphragm (SD) and contribute to filtration between blood and urine. Their Drosophila orthologs Kirre (Duf) and Sns are co-expressed within binucleate garland cell nephrocytes (GCNs) that contribute to detoxification of the insect hemolymph by uptake of molecules through an SD-like nephrocyte diaphragm (ND) into labyrinthine channels that are active sites of endocytosis. The functions of Kirre and Sns in the embryonic musculature, to mediate adhesion and fusion between myoblasts to form multinucleate muscle fibers, have been conserved in the GCNs, where they contribute to adhesion of GCNs in the 'garland' and to their fusion into binucleate cells. Sns and Kirre proteins localize to the ND at the entry point into the labyrinthine channels and, like their vertebrate counterparts, are essential for its formation. Knockdown of Kirre or Sns drastically reduces the number of NDs at the cell surface. These defects are associated with a decrease in uptake of large proteins, suggesting that the ND distinguishes molecules of different sizes and controls access to the channels. Moreover, mutations in the Sns fibronectin-binding or immunoglobulin domains lead to morphologically abnormal NDs and to reduced passage of proteins into the labyrinthine channels for uptake by endocytosis, suggesting a crucial and direct role for Sns in ND formation and function. These data reveal significant similarities between the insect ND and the SD in mammalian podocytes at the level of structure and function (Zhuang, 2009).
In Drosophila, the Immunoglobulin superfamily (IgSF) proteins encoded by kin of irre [kirre; also known as dumbfounded (duf)], roughest (rst), sticks and stones (sns) and hibris (hbs) function as ligand-receptor pairs on the surface of founder cells and fusion competent myoblasts. These proteins mediate recognition, adhesion and fusion to form multinucleate syncitia through direct interaction at sites of myoblast contact. However, neither their action nor their expression is exclusive to the musculature, and previous studies noted their role in cell recognition and adhesion in the Drosophila eye. Moreover, multiple studies have confirmed the presence of the kirre transcript and sns transcript in the binucleate garland cell nephrocytes (GCNs). These nephrocytes possess a structure visible by transmission electron microscopy (TEM) reminiscent of the slit diaphragm (SD) in the vertebrate kidney, and process waste products from the hemolymph. It is therefore compelling that the fly detoxification machinery may have similarities to that in mammals, and that Sns and Kirre play roles similar to those of their vertebrate counterparts (Zhuang, 2009).
Removal of waste products from the closed circulatory system of vertebrates takes place in the kidney glomerulus. Podocytes, kidney epithelial cells that surround the capillary blood vessels, extend foot processes that contact the surface of these vessels. Filtration then occurs as molecules flow out of the bloodstream through slits between adjacent foot processes into the urine. Neph1, vertebrate orthologs of the above Drosophila IgSF proteins, localize to this filter and appear to be an important determinant of glomerular permeability (Hamano, 2002; Liu, 2003). Mutations in nephrin and neph1 are associated with congenital nephrotic syndrome as a consequence of defects in this filtration diaphragm. Lack of either nephrin or neph1 leads to podocyte foot process effacement and detachment of podocytes from the glomerular basement membrane, loss of SDs, and proteinuria (Donoviel, 2001; Putaala, 2001; Zhuang, 2009 and references therein).
In addition to their high degree of homology, Nephrin and Neph1 have other features in common with Sns and Kirre. Heterophilic interactions occur in trans between the extracellular domains of Nephrin and Neph1, and Sns and Kirre. Studies have suggested that, in addition to serving as a scaffold onto which other proteins in the SD assemble, Nephrin and Neph1 function as signaling molecules to direct downstream cytoplasmic events (Benzing, 2004). They cooperate to transduce a signal that directs actin polymerization (Garg, 2007), and activation of this pathway occurs through interaction of phosphorylated tyrosines in the cytoplasmic domains of Nephrin and Neph1 to adaptor proteins (Jones, 2006; Verma, 2006). These adaptor proteins recruit components of the actin polymerization machinery that include N-WASp and Arp2/3. Similar phosphotyrosine modifications are important for Sns function and studies have shown that the WASp and Arp2/3 actin polymerization machinery functions in Drosophila myoblast fusion, probably downstream of Sns and Kirre (Zhuang, 2009).
The pericardial cells and garland cells comprise two subpopulations of Drosophila nephrocytes that, along with Malpighian tubules, form the excretory system. Approximately 25-30 tightly associated binucleate GCNs encircle the anterior end of the proventriculus in a 'garland' at its junction with the esophagus. The cortical region of the cytoplasm includes elaborate channels that are generated by invagination of the plasma membrane during embryogenesis and early larval instar stages. The initial invagination is associated with formation of a junction between two sites on the plasma membrane that are visible by TEM. Through a mechanism that is not entirely clear, this initial invagination expands into an extensive array of labyrinthine channels by the third-instar larval stage. The GCNs are very active in endocytosis via coated vesicles at sites deep within these labyrinthine channels. Thus, molecules to be eliminated must gain access to the endocytic machinery deep in these channels. These studies also identified a thin bridge spanning the channel opening that is visually similar to the vertebrate SD. The presence of Sns and Kirre and a slit diaphragm-like structure in these binucleate cells raised the possibility that these IgSF proteins might function in GCN fusion and/or in formation of this structure (Zhuang, 2009).
This study, along with that of Weavers (2009) demonstrates that Sns and Kirre are present in, and crucial for, the nephrocyte diaphragm (ND). Knockdown of Kirre or Sns results in a severely diminished number of NDs and smoothening of ND-associated furrows on the GCN surface, implicating Sns and Kirre in their formation. Mutations in the extracellular domain of Sns cause major perturbations in the ND, establishing that Sns also dictates fundamental aspects of its structure. Similar smoothening of the GCN surface occurs upon knockdown of Polychaetoid (Pyd), the Drosophila ortholog of the zonula occludens (ZO-1) tight junction protein that interacts with Neph1, providing strong support for functional conservation of these molecules. The ND controls access of molecules to the labyrinthine channels for uptake by endocytosis, and can discriminate between molecules of different sizes in a rate-dependent manner. Finally, in contrast to that reported by Weavers (2009) and reminiscent of their action in the embryonic musculature, Sns and Kirre contribute to the adhesion of the GCNs into an organized garland and their fusion into binucleate cells (Zhuang, 2009).
These data those of Weavers (2009) demonstrate that the GCNs have significant structural and functional similarities to podocytes in the mammalian kidney. Sns and Kirre are instrumental in directing and/or stabilizing interactions at sites of membrane invagination that become the NDs. These proteins parallel the role of their mammalian orthologs Nephrin and Neph1 in the SD that forms between podocyte foot processes in the kidney glomerulus. In addition, Sns and Kirre mediate tight adhesion between GCNs in the embryo, and, in contrast to Weavers this study notes that these proteins also direct GCN fusion. Both proteins are expressed during larval life and significant cell death occurs in their absence. Sns clearly plays a specific structural role in the ND that is perturbed by mutations in its extracellular domain. Finally, the SD and ND both mediate the flow of molecules between the circulatory system and the excretory system, and appear to discriminate between molecules on the basis of size and rate of passage (Zhuang, 2009).
The GCNs are thought to process waste material and detoxify the insect hemolymph, its open circulatory system, through a process of endocytosis and degradation. Endocytosis occurs from sites deep within labyrinthine channels that form by invagination of the plasma membrane, and proteins associated with endocytosis localize to the cortical region of the cytoplasm in membranes associated within these channels. The channels and associated membranes expand in mutants that block endocytosis, and compounds such as horseradish peroxidase, dye-conjugated BSA or avidin, and various dextrans, readily pass through the plasma membrane into these channels. Access appears to occur through a structure that is reminiscent of the SD in vertebrates. This study has shown that this nephrocyte diaphragm is dependent on the presence of Sns and Kirre, and that perturbation of the Sns extracellular domain causes obvious defects in the ND. Thus, IgSF homologs appear to be a structural component of this access point in both insects and vertebrates (Zhuang, 2009).
The number of NDs decreases significantly upon knockdown of Sns or Kirre, but a small number still remain. The uptake of large molecular tracers is severely diminished under these conditions, suggesting that the NDs are a major route of access to the endocytic machinery within the labryinthine channels. Perhaps more revealing relative to the initial findings of Weavers, it was found that the uptake of small molecules is slower under conditions of Sns or Kirre knockdown but ultimately achieves normal levels. Thus, like the SD, the ND appears to be more permeable to small molecules. Interestingly, studies in vertebrates have addressed the relative contributions of the podocyte basement membrane and the slit diaphragm to glomerular permeability, and Nephrin and Neph1 were found to be crucial. Moreover, electron tomography has identified Nephrin as a decisive determinant for filtration of molecules larger than BSA (Zhuang, 2009).
Nephrin and Neph1 are capable of forming both homodimers and heterodimers, and these abilities could reflect interactions that occur in vivo in cis and/or in trans. The diameter of the vertebrate SD is consistent with a model in which this distance could be spanned by homophilic interaction of Nephrin or heterophilic interaction between Neph1 and Nephrin in trans. The similar diameter of the Drosophila ND therefore supports a model in which interactions between the Kirre and Sns ectodomains determine this distance. The exact molecular interactions remain to be determined, however, and may differ in vertebrates and Drosophila. For example, Nephrin is capable of homophilic interactions in trans, a property that Sns does not appear to have. Thus, it seems unlikely that Sns spans this distance, as suggested for Nephrin. Homophilic interactions of Kirre, which can occur, could serve this purpose. One might then predict the spacing to be decreased from the observed 30-35 nm due to the shorter extracellular domain of Kirre. Of note, kinetic studies in Drosophila S2 cells indicate a strong preference for interaction with Sns. Moreover significant levels of Sns or Kirre remain in GCNs from second instar larvae upon knockdown of the corresponding partner, yet the number of NDs is diminished. Localization of each protein by immunoEM analysis under these conditions may prove to be illustrative in this regard. Given the above interaction studies and fact that both proteins are continuously present in the GCN, a model is favoed in which heterotypic interactions are preferred as in the embryonic musculature. One fundamental difference between Sns and Kirre in the embryonic musculature and the GCNs is that they are expressed in different myoblast cell types but co-expressed within individual garland cells. However, their co-expression in GCNs is another feature in common with Nephrin and Neph1 in vertebrate podocytes (Zhuang, 2009).
It is unclear whether Sns and Kirre function through interactions with signal transduction components that parallel those of Nephrin and Neph1 in the GCNs. Signaling molecules thought to be downstream of Sns and/or Kirre in the musculature, and known to be downstream of Nephrin, include N-WASp and components of the Arp2/3 pathway. One other functional parallel between the SD and ND is that of the tight junction protein Pyd, which contributes to formation of ND-associated furrows on the surface of the GCN. Although Pyd interacts biochemically with two different forms of Kirre, it remains to be shown whether this interaction occurs through postsynaptic density-95/disks large/zonula occludens-1 (PDZ)-binding sites in Kirre, as observed for binding of its vertebrate counterpart ZO-1 to Neph1 (Zhuang, 2009).
GCNs become binucleate before or immediately after their assimilation into the garland of cells that surrounds the esophagus at its junction with the proventriculus. This binucleate nature seems almost invariant, with cells rarely remaining mononucleate or having more than two nuclei. Although an explanation for this invariance is not apparent, the cell appears to accommodate multiple processes to ensure it. Quantitation of cells and nuclei over time, the absence of dying GCNs, and time-lapse imaging suggest that cell fusion is the primary mechanism utilized by wild-type GCNs, and that the IgSF proteins contribute to this process. Some mutant cells are still binucleate, but the possibility cannot be eliminate that other molecules contribute to GCN fusion or that these IgSF proteins function in yet more redundant ways to drive this fusion. Perhaps a drive to become binucleate has forced the cell to compensate for defects in fusion in other ways, such as cell division without cytokinesis. Although all efforts to address such a mechanism have yielded negative results, behavior of this type may be another common property between insect garland cell nephrocytes and mammalian podocytes (Zhuang, 2009).
The requirement for duf was analyzed in Df(1)w67k30 embryos. Although this deficiency also removes roughest (rst), it is clear that duf is responsible for the lethal phenotype observed in the deficient embryos and that rst does not contribute to it. The reasons for this are as follows: (1) the expression patterns of rst and duf are entirely different. It is therefore highly unlikely that rst could substitute for the absence of duf. (2) Deficiencies that remove rst are homozygous viable. (3) The phenotype of Df(1)w67k30 embryos can be rescued by reintroducing Duf into the mesoderm. (4) There is no gene in the region apart from rst that has any sequence similarity to duf (Rúiz-Gomez, 2000).
Antibody staining against muscle myosin shows that myoblast fusion fails completely in Df(1)w67k30 embryos. During normal development, myosin expression in the somatic mesoderm starts at stage 13 in muscle precursors and is maintained in all mature muscles. It is also expressed in the visceral and pharyngeal muscles and in the cardioblasts, and it is an excellent marker for muscle morphology and pattern. Although in wild-type embryos fusion-competent myoblasts do not express myosin before they are incorporated into syncytia, in Df(1)w67k30 all myoblasts express myosin by late stage 13. At later stages, the founders elongate to form mononucleate muscles that span the territory that they would have occupied as syncytial fibers in wild-type conditions. Fusion-competent myoblasts, in contrast, die and are eliminated by macrophages. In all cases tested, founders maintain characteristic patterns of gene expression (e.g., Krüppel). In addition, antibodies to Connectin and Fasciclin II reveal a normal pattern of innervation by motorneurons. Thus, it is concluded that the specification of individual founders is unaffected by the lack of duf function (Rúiz-Gomez, 2000).
To this extent, the phenotype of Df(1)w67k30 embryos resembles the phenotype of embryos mutant for genes essential for fusion such as myoblast city (mbc) and singles-bar. However, closer examination of the embryos reveals important differences between the behavior of fusion-competent myoblasts in Df(1)w67k30 embryos and in embryos mutant for known genes that affect the fusion process. In a stage 13 singles-bar embryo, where the fusion process is arrested, myosin staining reveals fusion-competent myoblasts clustered around the founders, with filopodia extending toward them. This clustering of myoblasts on founders is never detected in Df(1)w67k30 embryos. Instead, the founders and fusion-competent myoblasts remain at different levels in the mesoderm, with the founders in close contact with the ectoderm, whereas the rest of the myoblasts are more internal. This separation is not caused by the fusion-competent myoblasts failing to produce filopodia -- they do indeed extend filopodia, but these are randomly oriented and show no sign of being attracted preferentially toward the founders. These observations indicate that duf functions before any of the known fusion genes and that it is required for the attraction of fusion-competent myoblasts to the vicinity of the founders (Rúiz-Gomez, 2000).
In addition, there is an early defect in the formation of visceral mesoderm in Df(1)w67k30 embryos. Instead of two bands of tightly packed visceral muscles, several gaps are seen. These gaps are not the result of a reduction in the precursors; instead, they might be a consequence of improper alignment or adhesion of the visceral muscles (Rúiz-Gomez, 2000).
The mutant phenotype of Df(1)w67k30 embryos suggests that a gene removed by the deficiency is specifically required in the founders to aggregate myoblasts before fusion begins. Furthermore, duf is expressed in founders and is maintained in muscle precursors as fusion is taking place. To show whether lack of duf is responsible for nonfusion in Df(1)w67k30, attempts were made to rescue the phenotype by supplying duf function in the mesoderm of Df(1)w67k30 embryos, using the GAL4 expression system (Rúiz-Gomez, 2000).
When duf was provided early in the whole mesoderm using a Twi-GAL4 driver, fusion was restored in every muscle. The rescued embryos have an almost wild-type muscle pattern, though with some smaller muscles and scattered unfused myoblasts. There is also an accumulation of unfused myoblasts around the hindgut and at other locations where twi is normally expressed at high levels. The reduced size of some of the rescued muscles and the concentration of unfused myoblasts around places of high twi expression may be a consequence of using Twi-GAL4 as a driver. Twi-GAL4 drives duf expression in every founder, but this expression is unlikely to be maintained long enough to attract the full complement of myoblasts to the forming muscles. At the same time, if Duf is a signal for aggregation, then myoblasts would be free to move toward sites of ectopic duf expression such as the hindgut visceral mesoderm and might accumulate at these novel locations (Rúiz-Gomez, 2000).
In a second set of experiments, duf expression was limited to a subset of muscle founders using the Apterous (Ap)-GAL4 driver. Ap-GAL4 drives expression from stage 13 onward (after the normal onset of fusion) at variable levels in the LT1-4 and VA1-2 founders in every segment. This late and somewhat erratic expression of duf restors fusion exclusively in LT and VA muscles. These results show that reintroduction of duf expression in the mesoderm of Df(1)w67k30 embryos is sufficient to rescue the fusion phenotype (Rúiz-Gomez, 2000).
The expression pattern of duf, the failure of myoblast aggregation in Df(1)w67k30 embryos, and the relocation of myoblasts to sites of high twi expression when duf expression is driven by Twi-GAL4 suggests that Duf might act as a signal causing myoblasts to aggregate on founder cells during normal myogenesis. To test this idea, ectodermal GAL4 drivers such as Wingless (Wg) or Distal-less (Dll) were used to explore the ability of ectopically expressed duf to redirect myoblast migration and aggregation (Rúiz-Gomez, 2000).
The polynucleate myotubes of vertebrates and invertebrates form by fusion of myoblasts. Drosophila Roughest (Rst) protein is a new membrane-spanning component in this process. Rst is strongly expressed in mesodermal tissues during embryogenesis, but rst null mutants display only subtle embryonic phenotypes. Evidence is presented that this is due to functional redundancy between Rst and its paralogue Kirre. Both are highly related single-pass transmembrane proteins with five extracellular immunoglobulin domains and three conserved motifs in the intracellular domain. The expression patterns of kirre and rst overlap during embryonic development in muscle founder cells. Simultaneous deletion of both genes causes an almost complete failure of fusion between muscle founder cells and fusion-competent myoblasts. This defect can be rescued by one copy of either gene. Moreover, Rst, like Kirre, is a myoblast attractant (Strünkelnberg, 2001).
The kirre locus maps cytogenetically to region 3C6 and lies 3 kb distal to Notch. The rst and kirre loci are separated by 127 kb and are transcribed from opposite strands with their 5' flanking regions towards each other. The kirre cDNA consists of 3295 residues and contains a single long open reading frame encoding a protein of 959 amino acids. A signal peptide sequence (amino acids 7-31) has been identified and one putative transmembrane region (amino acids 575-597) (Strünkelnberg, 2001).
The conceptual Kirre sequence shows an overall similarity of 45% to Rst. Like Rst, the predicted extracellular portion of the Kirre protein displays an array of five immunoglobulin (Ig) domains. Stretches of high conservation with Rst reside primarily in the region of the five Ig domains. Within these domains, the degree of conservation successively decreases from the N terminus to the transmembrane domain. Both proteins contain stretches of amino acids with short side chains at differing positions: Rst contains a stretch of glycines between the second and third immunoglobulin-domain and Kirre harbours an array of 18 serines interrupted by a single glycine residue at the N terminus (Strünkelnberg, 2001).
The intracellular domain of Kirre is considerably longer than that of Rst and displays only low overall homology with the intracellular domain of Rst. However, three highly conserved motifs have been detected: one is located close to the transmembrane domain consisting of the sequence PADVI. The second motif, R[Y/F]SAIYGNPYLR(S)[S/T]NSSLLPP, corresponds to the consensus sequence of autophosphorylation domains of receptor tyrosine kinases. The third motif, T[A/H]V, resides at the C terminus of both sequences and corresponds to the consensus sequence of the PDZ-binding motif ([T/S]XV). In addition to the site contained in the putative autophosphorylation domain, one putative tyrosine and one putative serine phosphorylation site are conserved between Rst and Kirre. A conspicuous difference between the Kirre and Rst proteins is the lack of the opa-like repeat of Rst in Kirre (Strünkelnberg, 2001).
Similarity searches using the BLAST algorithm have shown that the four N-terminal Ig domains of Kirre, Rst, Sticks and stones and Hibris are closely related. Sns and Hibris (Bour, 2000) have been shown to be involved in muscle development (Strünkelnberg, 2001).
Expression of rst mRNA can be detected in embryonic stages 4 to 14. During stage 12, the rst transcript is detected in the majority of mesodermal cells. During stages 13 to 14 mesodermal expression of rst is detected close to the epidermis at positions where muscle founder cells reside, as well as immediately interior of the founder cells where fusion-competent myoblasts can be found. Unlike for kirre, individual muscle precursors could not be detected based on rst labelling (Strünkelnberg, 2001).
In comparison with rst, the expression of kirre is more restricted and switched on later during development. The kirre mRNA is detected from stage 11 through to stage 16. During stages 12-13, the kirre probe labels segmental clusters of mesodermal cells close to the epidermis. Based on position and morphology, this suggests that kirre is expressed in muscle founder cells. During stages 13 to 14, kirre labelled outgrowing founder cells and muscle precursors (Strünkelnberg, 2001).
A monoclonal antibody against Rst was used to address protein expression in more detail. To determine the myogenic cell types expressing Rst, the muscle founder cell-specific enhancer trap line rP298-lacZ was used. During embryonic stages 13 to 14, all cells expressing ß-galactosidase also showed Rst staining in their periphery, indicating that Rst is expressed by muscle founder cells. As predicted by in situ hybridization, Rst was also detected in mesodermal cells that did not express ß-galactosidase. Morphology and position of these cells suggest that they are fusion-competent myoblasts. The localization of Rst within the membranes of myogenic cells is restricted to discrete spots (Strünkelnberg, 2001).
In rP298-lacZ embryos, fusion-competent myoblasts that have started to fuse with founder cells begin to express ß-galactosidase. This complicates the distinction between the two cell types. To determine whether Rst is expressed in isolated founder cells, rP298-lacZ was crossed into a mbcC1 genetic background. In mbcC1 embryos, myoblast fusion is almost completely blocked and by stage 16 these embryos display a pattern of isolated, globular, fusion-competent myoblasts and stretched out, fibrous muscle founder cells. By stages 13 to 14, antibody staining for ß-galactosidase and Rst reveals a pattern comparable with staining in a wild-type background. However, during stages 15 to 16, Rst expression on fusion-competent myoblasts almost completely disappears, while labelling is pronounced on the cytoplasmic extensions of founder cells. Moreover, since rP298lacZ mirrors kirre expression, it follows that the expression patterns of rst and kirre overlap (Strünkelnberg, 2001).
Muscles attach at specific sites in the epidermis, the apodemes. Rst is also expressed in the apodemes, as shown by immunodetection of Rst in embryos of the apodeme-specific lacZ-reporter Wß1HI-lacZ (Strünkelnberg, 2001).
The deficiency Df(1)w67k30 causes embryonic lethality and displays an almost complete lack of myoblast fusion. The genomic interval removed by Df(1)w67k30 extends from white to kirre. As yet, there is no single embryonic lethal locus known within this region. Hence, the Df(1)w67k30 phenotype could be caused by the removal of two or several loci. Kirre has been shown to be a myoblast attractant expressed on founder cells and reintroduction of kirre can partially rescue the Df(1)w67k30 phenotype. Therefore, removal of kirre is partly responsible for the Df(1)w67k30 phenotype. However embryos deficient for a smaller genomic region including kirre do not show a defect in myoblast fusion. Therefore, removal of kirre alone cannot be responsible for the Df(1)w67k30 phenotype. Since the situation for rst is similar -- rst is involved in but not essential for myoblast fusion -- it is concluded that the phenotype of Df(1)w67k30 is caused by the simultaneous removal of the rst and kirre loci (Strünkelnberg, 2001).
Although the rst gene is not essential for muscle fusion, small defects, such as thinner and missing muscles can be detected in rst6 and rstirreC1 individuals, indicating the involvement of rst in muscle development. Overexpression of a secretable, extracellular version of Rst during stages when myoblast fusion occurs (stages 12-15) leads to embryonic lethality and defects in myoblast fusion. Mechanistically, the extracellular part of the protein may compete with endogenous Rst for an as yet unknown extracellular ligand or, since the Rst protein has been shown to mediate homophilic cell adhesion, the extracellular domain could also bind to endogenous Rst and thereby disturb its function (Strünkelnberg, 2001).
Ubiquitous overexpression of the full-length Rst protein also causes embryonic lethality and a severe muscle fusion phenotype. Ectodermal overexpression of Rst does not cause defects in the muscle pattern but ectopic localization and prolonged occurrence of myoblasts at sites of ectopic Rst expression. Mesodermal expression does not induce any detectable phenotype. The reason why global misexpression of Rst differs from misexpression in the mesoderm alone (in most of which Rst is expressed anyway) and from misexpression in the ectoderm alone appears to be the increase of Rst expressing sticky surfaces: the withdrawal of fusion-competent myoblasts from recruiting founders and precursors may considerably lower the probability for these cell types to contact each other (Strünkelnberg, 2001).
Some of the defects observed in rst mutants concern muscles in ectopic positions. Even though Rst is expressed in the apodemes, the data do not point to an essential role for kirre and/or rst in myotube guidance or attachment: analysis of the subcellular localization shows accumulation of Rst primarily around the apical borders of the apodemes, rather than basally, where outgrowing muscles would be expected to make contact. Moreover, apodeme specification is also not blocked in individuals lacking ectodermal Rst and Kirre, as judged by the muscle pattern. Hence, a putative function of Rst in apodeme specification would be redundantly safeguarded by additional as yet unknown factors. Apodeme specification is also not disrupted in da-Gal4/+;UAS-rst/+ embryos, as revealed by antibody staining against the signalosome component Alien. This clearly rules out the possibility that the strong muscle phenotype observed in these embryos is due to defects in specification of the muscle attachment sites, and argues that restricted expression of Rst is not essential for normal apodeme specification to occur. This is underlined by the fact that 69B-Gal4/+;UAS-rst/+ embryos that express Rst only in the ectoderm do not show attachment defects (Strünkelnberg, 2001).
Given the overlapping mesodermal expression patterns of rst and kirre, and the significant structural similarity between the two proteins, it is concluded that rst and kirre have at least partially redundant functions during muscle development. Rst expression in fusion-competent myoblasts is not essential for their attraction towards ectopic Kirre or Rst: myoblasts can be attracted to ectopic sites in a Df(1)w67k30 background, where Rst is only present at ectopic sites and not in fusion-competent myoblasts -- this strongly suggests a heterophilic trans-interaction. However, as Rst has been shown to mediate homophilic cell adhesion, a homophilic trans-interaction of Rst may also contribute to the fusion process (Strünkelnberg, 2001).
At present, the data do not allow a prediction of the molecular mechanisms in which Rst and Kirre take part; however, it is conceivable that they include the related cell adhesion molecules Sns and Hbs that are expressed on fusion-competent myoblasts. A model of the fusion machinery may include assembly of adhesion molecules within heteromeric complexes with differing compositions on the side of the fusion-competent myoblasts (including Sns, Hbs and Rst) and on the founder cells (including Kirre and Rst). These complexes may still function after loss of single components. It will need further analysis and binding assays to elucidate how these membrane proteins play together and how they are connected to the other components of the fusion machinery (Strünkelnberg, 2001).
The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. The circular muscles are organized as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, two classes of myoblasts are detected within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks and stones (SNS). During the process of cell fusion at stage 12, SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies have proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, cell lineage analyses as well as morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganized while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. These results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes (Klapper, 2002).
The gene mbc, a homolog of human DOCK180, is expressed in all somatic myoblasts during the fusion processes. In embryos mutant for mbc, syncytia within somatic muscles are almost absent, presumably due to defects in the rearrangement of the cytoskeleton during preceding myoblast fusion. Mbc is also expressed in the visceral mesoderm from stage 12 onward. Embryos mutant for mbc not only exhibit defects in the formation of midgut constrictions but also show severe abnormalities in the formation of visceral muscles. At stage 12 the visceral band is randomly interrupted and the elongated FAS III-expressing cells seem to be disoriented. During further development, parts of the visceral band either stretch out in the dorsoventral direction, as in the wild type, or form disarranged patches. The status of the founder cells in an mbc mutant background was analyzed by expressing rP298-LacZ in the strain mbcC1. At stage 14 the number of rP298-LacZ-expressing cells within the remaining population of myoblasts appears to be reduced and large gaps within the visceral band are visible that are not present in the wild type. Many globular cells are still observed at the margins of the visceral band that appear to be unfused myoblasts. Antibody staining reveals a prolonged expression of SNS within a corresponding subpopulation of myoblasts. As described for duf and sns mutant embryos, no myoblast fusions were detectable. While the circular musculature shows severe defects and is apparently reduced, the longitudinal musculature is completely absent at the end of embryogenesis (Klapper, 2002).
By using the GAL4/UAS transplantation system for cell lineage analyses of the mesoderm anlage, syncytia were detected not only within somatic muscles but also in the visceral musculature. Since the visceral muscles have classically been described as mononuclear, this surprising observation led an examination of syncytia formation within this tissue. Evidence is provided that the circular visceral muscles of the midgut are likewise organized as syncytia. The first signs of GFP expression (driven by daGAL4) within these muscles were observed in embryos at stage 15. Since there is a considerable delay of about 2-4 h between the activation of the UAS-GFP construct and the formation of the fluorescent product, it is assumed that the formation of syncytia begins at stage 12. This is consistent with the observation of the first fusion processes within the visceral band. Using GFP expression as an in vivo marker, individual syncytia could be followed throughout development. In contrast to longitudinal muscle fibers, which have been found to contain up to six nuclei, the circular muscles of hindgut as well as midgut always comprise two nuclei each. Thus, fusion processes within this tissue stop after the formation of binucleate minimal syncytia that each cover one-half of the gut tube. This is a curious finding since in other muscle types of Drosophila many nuclei share a common cytoplasm to generate a large structural and functional unit (Klapper, 2002).
Following labelled syncytia through all stages of development, it has been shown that the visceral musculature is not replaced by a newly formed imaginal tissue but persists through metamorphosis. Since the visceral musculature plays a crucial role for the proper formation of the midgut during embryonic development, the persisting visceral musculature might again serve as a template for remodelling of the gut tube during metamorphosis (Klapper, 2002).
Since myoblast fusions were discovered within the visceral musculature, whether these syncytia are also formed by Duf-expressing founder and Sns-expressing fusion-competent cells was examined. duf can be detected in muscle precursors as long as they incorporate further fusion-competent cells into the syncytia. The expression of Duf and Sns within the visceral mesoderm suggests a function of specifying founder and fusion-competent cells similar to that observed for the somatic musculature. To clarify whether these genes indeed play a functional role in visceral myoblast fusion, the phenotypes of sns and duf mutant embryos was examined with respect to syncytia formation within the developing visceral musculature. In wild type embryos the ventrally located cells of the visceral band exhibit all characteristics of muscle founder cells, in that they express Duf, become elongated and fuse specifically with Sns-expressing myoblasts. Without duf function no signs of visceral muscle fusion are detectable. It is therefore proposed that duf is the key component in the visceral muscle founder cells. This observation is in agreement with the postulated role of Duf as an attractant for fusion-competent cells in the somatic musculature (Klapper, 2002).
These results indicate that loss of duf function leads to a complete loss of fusion even though the two populations of founders and fusion-competent myoblasts are already aligned within the visceral band at stage 11. Therefore, in the visceral mesoderm, Duf expression seems not only to attract the fusion-competent cells but also to play a crucial role in the fusion process itself. Probably the gaps within the visceral band of duf mutant embryos may indicate an early Duf adhesion function necessary for a proper anterior-posterior alignment of the visceral myoblasts. Furthermore, despite initial cell alignment at stage 11, this contact between founder cells and fusion-competent myoblasts is lost during further development, so that additional gaps appear between these cell layers. In sns mutant embryos, again, no myoblast fusions were detectable. The phenotype closely resembles that of duf mutant embryos, since the two populations of founders and fusion-competent cells seem to reject one another. However, the large gaps within the visceral band of duf embryos were never observed in sns mutants. Whereas in the anterior part of the midgut severe defects of the visceral musculature are obvious at later stages of embryogenesis, the posterior part looks quite normal. This regional difference is somewhat difficult to explain, since at earlier stages no such regional distinctions were observed. It is thought that either in the posterior visceral band sns function can be mimicked or bypassed later on by another spatially restricted gene product, or that even unfused myoblasts can form a seemingly normal musculature in the posterior part. The stronger phenotype at later stages in duf mutant embryos may indicate that the founder cells play a more crucial role in visceral myogenesis than the fusion-competent cells. In somatic myogenesis, founder and fusion-competent cells play specific roles in the recognition process, while in mbc mutants the fundamental capability to fuse is lost in both cell types. Because here intracellular components of the fusion apparatus are affected, fusion-competent cells still aggregate around founders, but the tight membrane junctions are not formed and fusion does not occur. As is consistent with the somatic phenotype, in visceral myogenesis, fusion is totally blocked although founder and fusion-competent cells are in direct contact and still express Sns and Duf (Klapper, 2002).
Taken together, these results provide evidence that the founder-cell hypothesis also applies to visceral myogenesis employing the same genetic components as used in the somatic myoblast fusion processes. Thus, the specification of myoblasts as either founder or fusion-competent cells might be a fundamental step preceding syncytia formation (Klapper, 2002).
Previous analyses have provided evidence that the primordium for the longitudinal midgut musculature is located at the posteriormost tip of the mesoderm anlage. In byn mutant embryos the hindgut as well as the longitudinal musculature are absent. The longitudinal musculature can be rescued, if byn is ectopically expressed exclusively within the posterior tip of the mesoderm anlage, leading to the conclusion that the entire anlage of the longitudinal musculature is located within this posterior region. However, after transplantation within the central region of the trunk, mesoderm labelled longitudinal muscles were frequently obtained. Taking into account that the founder-cell hypothesis is also valid for this tissue, this seeming contradiction can now be solved. The founder cells of the longitudinal musculature that comprise the genetic information for the specific tissue identity arise from the posterior tip of the mesoderm anlage, while the fusion-competent cells originate from the entire trunk region as indicated by confocal analysis. During the fusion processes it is possible to distinguish between two different events of syncytia formation. (1) At stage 12 all the ventrally located founder cells simultaneously become extended dorsally. Each of them fuse with a single SNS-expressing fusion-competent cell, eventually differentiating into a binucleate circular muscle. (2) The remaining SNS-expressing cells migrate to both margins of the visceral band and successively fuse with the longitudinal founder cells that invade from the posterior tip of the mesoderm. It is thus reasonable that in byn mutant embryos the entire longitudinal musculature is missing, since the fusion-competent cells alone are not capable of differentiating longitudinal muscles (Klapper, 2002).
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date revised: 20 February 2010
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