InteractiveFly: GeneBrief

Kirre/Dumbfounded: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Aggregation and fusion of myoblasts to form myotubes is essential for myogenesis. In Drosophila the formation of syncytial myotubes is seeded by founder myoblasts. Founders fuse with clusters of fusion-competent myoblasts. The gene kin of irre (kirre), referred to frequently as dumbfounded (duf) is required for myoblast aggregation and fusion. duf encodes a member of the immunoglobulin superfamily of proteins that is an attractant for fusion-competent myoblasts. It is expressed by founder cells and serves to attract clusters of myoblasts from which myotubes form by fusion (Rúiz-Gomez, 2000).

Roughest (Rst), a paralog of Kirre/Dumbfounded, 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 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 somatic muscles of the Drosophila larva are laid out in a complex pattern on the body wall. Like skeletal muscles in vertebrates, these muscles consist of syncytial fibers formed by fusion of myoblasts. However, in the Drosophila larva, each muscle is a single myotube, whereas in vertebrates many myotubes are bundled together to form a single muscle. The pattern of muscles in the Drosophila larva is extremely precise, consisting of 30 myotubes in each abdominal hemisegment. Each of these fibers is a unique element in the pattern, distinguishable by its position, size, orientation, and innervation. Thus, as the Drosophila embryo develops, each hemisegment reproducibly generates a set of 30 different myotubes and provides a unique opportunity to study the control of myoblast fusion and myotube diversification (Rúiz-Gomez, 2000).

The aggregation and fusion of myoblasts to form syncytial myotubes is an integral part of myogenesis in many organisms. In vertebrates, proliferating myoblasts migrate from the somites to sites of muscle formation and fuse to form primary embryonic myotubes. Secondary myotubes are added in parallel with primary myotubes, and additional growth occurs through the fusion of satellite cells. Several different kinds of myotubes contribute to the final muscle and the characteristics of these fibers can be profoundly influenced by innervation. However, primary myotubes form independently of nerves so that other regulatory factors must control myoblast fusion and the diversification of fibers that occurs during primary myogenesis (Rúiz-Gomez, 2000).

In Drosophila, both fusion and myotube differentiation are tightly controlled so that myotubes with distinctive characteristics are generated at precise locations in the developing muscle pattern. Control is exerted in two ways: (1) by the segregation of a special class of founder myoblasts at specific points in muscle-forming mesoderm (the founders seed the formation of myotubes at these points by fusing with neighboring fusion-competent cells that constitute a different class of myoblasts); (2) fusion is regulated by the fact that there is an essential asymmetry to the process so that the two classes of myoblasts (founders and fusion-competent cells) can only fuse with each other and not with themselves. The presence of founders at specific sites thus gates myogenesis and restricts it to those locations where muscles should form. At the same time, the characteristics of the myotubes formed at these locations are dictated by transcription factors expressed by individual founders (Rúiz-Gomez, 2000 and references thererin).

Like Drosophila, vertebrate embryos produce a population of myotubes during myogenesis, rather than fusion being a generalized process recruiting cells to a single expanding syncytium. In common with other insects, where myogenesis is seeded by muscle founders or pioneers, Drosophila exemplifies one solution to the problem of recruiting cells in groups to form myotubes. However, it may be that an asymmetry to the fusion process, with some myoblasts acting as seeds and others being recruited, is common to many organisms. Because the separation of myoblasts into two classes is so central to myogenesis in Drosophila and might be a general requirement for myoblast fusion in many different organisms, it is important to identify those genes that give the two types of myoblasts their unique properties. The products of these genes will include proteins that enable founders and fusion-competent myoblasts to recognize each other as suitable partners for fusion and that are responsible for the inherent polarity of the fusion process. The cloning and functional characterization of the first such gene is described in this study. Because of its loss- and gain-of-function phenotypes, the gene has been named dumbfounded. In the absence of duf, fusion fails. In muscle-forming mesoderm, duf is expressed only in founders and their progenitors. duf encodes a putative cell adhesion protein that causes myoblasts to aggregate on founder cells prior to fusion to form a myotube (Rúiz-Gomez, 2000).

The following sequence of events during myoblast fusion is proposed. Initially, Dumbfounded acts as an attractant for fusion-competent myoblasts. Through either direct or indirect interaction(s) between Duf and and cell adhesion protein Sticks-and-stones (Sns), fusion-competent myoblasts recognize and adhere to founder cells. In this process, Sns is localized to discrete sites in the membrane of fusion-competent myoblasts, presumably sites of cell adhesion. It is possible that Duf is also localized to discrete domains in the membrane of the founder cells. Next, within the founder cells, through interaction(s) between the cytoplasmic domain of Duf and linker protein Rolling pebbles (Rols), Rols is recruited to discrete cytoplasmic domains close to the membrane. Meanwhile, interaction between Rols and Mbc, and perhaps additional cytoskeleton-associated molecules, leads to changes in the cytoskeleton that are necessary for the proper alignment of founder cells with fusion-competent cells. This model predicts that in rols mutant embryos, despite a block of cell alignment, which requires the transmission of signals from Duf to the cytoskeleton, cell recognition and adhesion should take place normally. This is indeed what is observed. In rols mutant embryos, fusion-competent myoblasts extend filopodia toward their fusion targets. Such phenotypes are not observed in duf mutant embryos in which fusion is blocked at the cell recognition step (Ruiz-Gómez, 2000). Taken together, the model is favored that Rols acts as a linker molecule that relays signals from the membrane receptor Duf to changes in the cytoskeleton in the founder cells (Chen, 2001).

Dumbfounded was identified in an enhancer trap screen. ß-galactosidase expression in the enhancer trap line rP298 is confined to progenitors and founders of somatic and visceral muscles in the Drosophila embryo. The question was asked whether this expression might reflect the presence of a gene essential for the development of the mesoderm near the insertion site of the P element in rP298. The insertion site was mapped to band 3C6 on the X chromosome, between roughest (rst) and Notch (N). Although no lethal gene has been described in this interval, phenotypes of deficiencies that uncover this region were examined. Out of five embryonic lethal deficiencies tested, three [Df(1)w^258-11,Df(1)vt, and Df(1)w^67k30] were found with an interesting mutant phenotype. Df(1)w^67k30 embryos are typical, with a complete lack of fusion in the somatic mesoderm and gaps in the visceral mesoderm, whereas other mesodermal derivatives such as fat body, gonads, and heart develop normally. The phenotype is manifested in those mesodermal tissues where lacZ is expressed in rP298, suggesting that the pattern of lacZ expression reflects the requirement for a gene removed by the deficiency. To map the location of this putative gene, additional deficiencies and duplications were used. Flies are viable when either the 3C2-5 region or the 3C3-6 region (which includes rst) is deleted by combining Df(1)w^67k30 and Df(1)w^258-42 or Df(1)w^67k30 and Df(1)rst², respectively. The lethality of Df(1)w^67k30 is rescued by the duplication Dp(1;3)w^VCO, which excludes the possibility that the lethality could map outside the 3C region. In addition, Df(1)N^81k1 complements Df(1)w^67k30, whereas Df(1)N⁸ does not. Furthermore, when Df(1)N⁸ is combined with cosP479BE, which rescues all known N mutations, the resulting embryos die and show a phenotype indistinguishable from that of Df(1)w^67k30 embryos. Taken together, these results identify a novel mesodermal lethal function in 3C6-7 coinciding with the insertion point of the P element in the rP298 line. Using this line as a starting point, a transcription unit close to the insertion point of the P element was identified that corresponds to a new gene. Following the characterization of the mutant phenotype of deficiencies that remove this gene, the gene was named dumbfounded (Rúiz-Gomez, 2000).

Duf acts as an attractant for myoblast aggregation. In experiments using Dll-GAL4 as a driver in a Df(1)w^67k30 background, the migration of myoblasts is strikingly redirected toward sites of ectopic duf expression. In such embryos, there is a substantial aggregation of myoblasts in the head and on the primordia of the leg discs where duf is now expressed. When Wg-GAL4 is used as a driver, unfused myoblasts distribute themselves on the inner face of the epidermis in a segmentally repeated pattern of bands at stage 12. Myoblasts are also attracted toward a restricted region of the visceral mesoderm surrounding the midgut (parasegment 8) where wg is normally expressed. Double staining such embryos with anti-myosin and anti-Wg shows that myoblasts move toward the sources of Duf. They move internally toward ps 8 of the visceral mesoderm and outward to the exterior of the embryo close to the ectodermal bands of duf expression driven by Wg-GAL4. They leave empty spaces along the anteroposterior axis on either side of the Wg domain (Rúiz-Gomez, 2000).

It is concluded that Duf protein acts to attract myoblasts at a distance. During normal development, this leads to the aggregation of fusion-competent myoblasts on the founders with which they will fuse. In the absence of Duf, myoblasts fail to aggregate and fusion is blocked (Rúiz-Gomez, 2000).

Since duf expression is characteristic of muscle founders, it could be that it is required for the proper specification of these cells and/or to allow them to complete myogenic differentiation. However, all the evidence indicates that in the absence of duf each founder is specified normally, expresses the appropriate set of genes, and completes myogenesis to form a properly innervated, mononucleate muscle. The crucial feature that is lacking is myoblast fusion itself (Rúiz-Gomez, 2000).

Fusion is a multistep process that depends on mature fusion-competent myoblasts recognizing an appropriate target for fusion (a founder or a myotube). This recognition step is followed by adhesion and alignment of the cells along their long axes. Once cells are closely apposed, plasma membranes start fusion events at several places, allowing communication between them and culminating in the incorporation of the myoblast into the developing syncytium. Unfused myoblasts that are just about to fuse are round cells with a single process addressed toward the founder or the myotube. Several myoblasts may be in contact with the same founder or myotube at the same time, thus leading to the formation of myoblast aggregates. Such myoblast aggregates are short lived and quickly resolved by fusion and formation of myotubes. How the small aggregates that will contribute to a myotube are formed has not been clear. A prominent feature of the phenotype in some mutations that block fusion is the appearance of obvious myoblast clusters, and it has been suggested that such embryos are blocked at the recognition step. These clusters do not form in the absence of duf. Furthermore, in duf-deficient embryos the myoblasts not only fail to cluster but are located at different levels in muscle-forming mesoderm. In wild-type embryos, founder myoblasts arise in close contact with the ectoderm, while fusion-competent myoblasts are more internal, and this arrangement persists in the absence of duf. In wild-type conditions, fusion-competent myoblasts put out filopodia that are mainly oriented toward founders, whereas in deficiencies for duf, fusion-competent myoblasts still produce filopodia but do so without any preferred orientation. Thus, the data show that the aggregation of myoblasts on founder cells prior to the formation of myotubes is an active process in which founders produce an attractant and myoblasts move toward it. This is strikingly different from the alternative, namely that myoblast clusters form by a random process of collision and recognition (Rúiz-Gomez, 2000).

Duf is a transmembrane protein whose extracellular domain contains five Ig-like repeats and is highly homologous to the Drosophila protein Roughest (Rst). Duf is sufficient to rescue the fusion defect in Df(1)w^67k30 embryos when it is reintroduced into the mesoderm, allowing such embryos to form a relatively normal pattern of syncytial muscles. Furthermore Duf acts as a signal that attracts fusion-competent myoblasts when it is expressed at ectopic sites in the mesoderm or in the ectoderm. Although the behavior of myoblasts in wild-type embryos shows that they move toward sources Duf, it is not possible to distinguish between two alternative ways in which Duf might act. Either the external part of the molecule is cleaved and diffuses away from the source or, alternatively, it remains on the founder cell membrane and is detected by the random exploration of myoblast filopodia. However, there are a number of reasons why the view that Duf acts at the membrane of the founder is favored. (1) There is the question of distance: the furthest from their normal location that myoblasts are seen aggregating is on the ventral midline when Duf is expressed ectopically with the Wg-GAL4 driver. Although this is many cell diameters from their normal location, it is well within reach of cell processes such as filopodia and cytonemes. (2) There is the question of how a diffusible signal would act in normal development over the relatively short distances between alternative sources, namely neighboring founders. It is hard to envisage a diffusible molecule acting as an attractant without interference between adjacent sources leading to locally high concentrations between founders and consequent misrouting of aggregating myoblasts. (3) There is the question of the role of Duf in the process of fusion itself. There is complete absence of fusion in Df(1)w^67k30 embryos and this is not easily explained by the simple model that Duf merely acts to attract myoblasts to founders. If this were the case, it would be expected that random contacts between founders and fusion-competent cells would lead occasionally and perhaps quite commonly to fusion events in such embryos. The absence of such events in Df(1)w^67k30 embryos and the fact that fusion is restored if Duf is reintroduced into the mesoderm suggests that Duf acts both as an attractant for myoblasts and as an essential component of the fusion process that follows (Rúiz-Gomez, 2000).

As a putative cell adhesion protein, Duf may be required in the process of fusion to ensure a close adhesion between myoblasts and founders without which coalescence of membranes cannot occur. However, the possibility cannot be excluded that the specialized intracellular domain of the protein, which is highly divergent from that of Rst, allows signaling to occur between myoblast and founder and that it is this signaling that is essential for fusion to occur. In this view, the recognition event mediated by the binding of Duf to its (as yet unknown) partner on the myoblast would trigger the local cascade of events that allows fusion to proceed. In any event, although Duf is necessary for fusion, it is not sufficient. Duf under the control of Twi-GAL4 is present in both myoblasts and founders, but fusion still occurs only between founders and myoblasts and never among myoblasts themselves, even though these cells now express Duf and its partner. This suggests that the asymmetry of the fusion process depends not only on the selective expression of Duf in the founders but also on other specialized characteristics of founders that are not present in myoblasts generally. It may be that the intracellular domain of Duf interacts with components uniquely present in founders to initiate the fusion process. It should be noted, however, that once initiated, at an ultrastructural level at least, the events that accompany fusion are strikingly symmetrical between myoblast and founder cell (Rúiz-Gomez, 2000).

While the experiments reported here show that Duf acts nonspecifically to attract fusion-competent myoblasts to sites where it is expressed, the formation of myotubes in the Drosophila embryo is a highly regulated process that results in the fusion of specific numbers of cells to form muscles of different sizes. This might suggest that myoblasts are themselves specified to fuse uniquely with particular founders. However, there is no evidence for that kind of specificity in the myogenic pathway in Drosophila. Experimental manipulations of myogenesis in adult flies show that myoblasts are capable of fusing with any muscle that they encounter. In the embryo, muscles can be duplicated experimentally, and under these conditions, the number of cells contributing to each of the two fibers is the same as the number contributing to a single myotube in a normal embryo. Clearly, therefore, myoblasts that would not normally contribute to a developing fiber can be recruited to it if conditions change. If myoblasts are not set aside to fuse with a given myotube, what determines the ultimate size of a developing fiber? Although Duf expression could contribute to the control of size by regulating the length of time that any given founder remains an attractive target for fusion-competent myoblasts, it is certainly not the only determinant. Duf expression appears to decline early in small muscles and later in larger muscles, but in experiments, Duf expression in a subset of muscles (Ap-GAL4) or uniformly throughout the muscles (Twi-GAL4) does not cause marked aberrations in the size of the muscles that form. Once again, this suggests that there are special attributes to founder cells that contribute to the process of fusion and, in this case, set the number of fusions that are permitted for a particular myotube (Rúiz-Gomez, 2000).

Drosophila provides a model system with which to explore the essential features of myotube formation and patterning. So, for example, it is suspect that seeding events may be the key to understanding the recruitment of myoblasts to form myotubes in vertebrates as well as in flies. In both vertebrates and flies, the initial step in the formation of myotubes is aggregation and recognition. In muscle-forming mesoderm an attractant for myoblasts is selectively expressed by founder myoblasts. Thus, founders actively attract to themselves an aggregate of myoblasts with which the founders will fuse to form a myotube. The immediate task in the embryo of the fly is to identify the ligand of Duf that is expressed by fusion-competent cells and link it and Duf to the pathway of myoblast fusion. It may well be that this will reveal conserved elements in the two kinds of organisms that will clarify the formation of myotubes and the spatial organization of muscle development (Rúiz-Gomez, 2000).

rst transcriptional activity influences kirre mRNA concentration in the Drosophila pupal retina during the final steps of ommatidial patterning

Drosophila retinal architecture is laid down between 24-48 hours after puparium formation, when some of the still uncommitted interommatidial cells (IOCs) are recruited to become secondary and tertiary pigment cells while the remaining ones undergo apoptosis. This choice between survival and death requires the product of the roughest (rst) gene, an immunoglobulin superfamily transmembrane glycoprotein involved in a wide range of developmental processes. Both temporal misexpression of Rst and truncation of the protein intracytoplasmic domain, lead to severe defects in which IOCs either remain mostly undifferentiated and die late and erratically or, instead, differentiate into extra pigment cells. Intriguingly, mutants not expressing wild type protein often have normal or very mild rough eyes. By using quantitative real time PCR to examine rst transcriptional dynamics in the pupal retina, both in wild type and mutant alleles, it was shown that tightly regulated temporal changes in rst transcriptional rate underlie its proper function during the final steps of eye patterning. Furthermore it was demonstrated that the unexpected wild type eye phenotype of mutants with low or no rst expression correlates with an upregulation in the mRNA levels of the rst paralogue kin-of-irre (kirre), which seems able to substitute for rst function in this process, similarly to their role in myoblast fusion. This compensatory upregulation of kirre mRNA levels could be directly induced in wild type pupa upon RNAi-mediated silencing of rst, indicating that expression of both genes is also coordinately regulated in physiological conditions. These findings suggest a general mechanism by which rst and kirre expression could be fine tuned to optimize their redundant roles during development and provide a clearer picture of how the specification of survival and apoptotic fates by differential cell adhesion during the final steps of retinal morphogenesis in insects are controlled at the transcriptional level (Machado, 2011).

The ability of interommatidial cells to reorganize their apical contacts such as to maximize their membrane interactions with primary pigment cells, around 24% AFP, is a key step for generating the highly precise geometrical pattern of the adult compound eye, but it is also needed to create differences in intercellular adhesion and signaling that allow the final cell fate specification decision in the pupal retina - that between survival and programmed cell death - to correctly take place. A central involvement of Rst cell adhesion molecule in the IOC reorganization event has been inferred not only from the observation that this latter process paralleled Rst protein redistribution in IOC membranes but also from two independent sets of evidence. first, it was observed that the complete absence or truncation of rst protein product often blocked IOC sorting and led to an 'all to one' switch in cell fate, with surplus pigment cells appearing in the adult eye as consequence of lack of cell death. Second, when Rst redistribution was delayed, so was IOC sorting, producing an 'all to none' response in which the survival versus death choice is either not correctly made or not properly implemented, Thus lead to surplus cells dying later and erratically while the remaining ones failed to differentiate into proper pigment cells. These data emphasized the need for rst function not only at the right place but also at the right time, and implied very precise spatial and temporal controls of its expression. Unraveling the details of these control mechanisms is therefore an essential prerequisite for a fuller understanding of the nature and dynamics of the signals to which cells must respond to die or to differentiate in the final steps of ommatidial patterning. The quantitative analysis presented in this study showed a striking correlation between rst mRNA concentration over time and the qualitative dynamics of Rst protein localization in IOC membrane, both in wild type and in the regulatory rst^D mutant, thus implicating the temporal control of rst transcription, rather than a reshuffling of Rst protein molecules previously present in the membrane, as a main factor responsible for the critical changes in cell adhesion specificity that allow IOC sorting to take place. These findings help to shed light on a little studied and, so far, underestimated aspect of eye development, adding a new dimension to the complex process of ommatidial patterning and differentiation. Besides, the results demonstrate functionally the ability of kirre to rescue rst function in IOC sorting. Although the possibility of redundant roles for rst and kirre in retinal development had been previously suggested, supporting evidence was mainly indirect and obtained from either protein co-localization or overexpression experiments. Bao (2010) has provided convincing evidence, based on the analysis of retinal patterning at the time of IOC sorting, that Rst and Kirre function redundantly to maintain the spacing between developing ommatidial groups. This study has extended these observations by directly showing the ability of high levels of kirre expression in mutants with no or very little rst mRNA to bypass the need for rst function, including its influence in cell fate choice. The molecular mechanism underlying this redundancy, however, remains to be elucidated. Rst and Kirre have highly conserved extracellular domains, co-localize in IOCs and both appear to bind Hbs at the border with primary pigment cells (although in the case of Kirre direct evidence for this latter assertion is missing), seeming to imply that the two proteins are fully interchangeable in their interactions with other extracellular and intracellular molecules during retinal development. However their intracellular domains show very little similarity suggesting that they might have few, if any, common cytoplasmic binding partners. Genetic and biochemical studies aiming at identifying possible intracellular interactors of Kirre and Rst have been performed in different tissues, uncovering sets of directly binding proteins and potential signaling pathways that are distinct for each gene product. Since the intracellular domain of Rst is required for its function in the pupal retina, probably by interacting with actin cytoskeleton, it would be important to ascertain whether rst/kirre functional redundancy in this context results from their ability to act through the same intracellular pathways or it is a consequence of their interaction with different ensembles of intracellular targets that can nevertheless lead to the same end result (Machado, 2011).

Perhaps the most interesting finding presented in this study is the evidence for compensatory co-regulation of rst and kirre mRNA concentrations and its asymmetrical nature. A possible, although unlikely explanation for this asymmetry could reside in the much higher absolute levels of rst mRNA present during the temporal period examined, making small changes in relative concentration easier to detect with confidence. Also neither the effect of overexpression of one gene on the concentration of the other was investiged, nor whether similar coordination in mRNA expression is taking places in other developmental contexts where complementary or redundant functioning of rst and kirre seem to be required, such as myoblast fusion, salivary gland and optic lobe development. In this latter context it is worth mentioning that rst revertant RTW6, but neither RTW8 nor rst^D, shows axonal pathfinding defects in the optic lobe suggesting that at least in some developmental contexts rst and kirre might not be fully redundant (Machado, 2011).

Finally, an intriguing aspect of the compensatory response reported in this study is that it implies some kind of sensor mechanism capable to post-transcriptionally read rst expression levels and adjust those of kirre accordingly. Here again mRNA, rather than protein concentration, may play the main role, since in rst^CT, which carries a small deletion in the coding region of an otherwise normal mRNA, low levels of rst activity caused by the truncated intracellular domain and destabilization of the protein from the membrane leads only to a minimal, if any, increase of kirre mRNA levels at 24% APF. Also it is conceivable that rather than upregulating kirre transcription, a decrease in degradation could be taking place, leading to an accumulation of kirre mRNA molecules. Experiments designed to further test this possibility as well as to map rst mRNA sequences that might be relevant for this putative regulatory feedback loop between rst and kirre are currently underway. Whatever the case, these findings suggest a general mechanism by which the expression both genes could be fine tuned to optimize their redundant roles during development (Machado, 2011).

REGULATION

Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila

Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. This study demonstrated that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition (Bazigou, 2007).

These genetic studies in Drosophila provide functional evidence in vivo that Alk plays a crucial role in the developing central nervous system. This study shows that Alk and its cognate ligand Jeb form an anterograde signaling pathway in the fly visual system, which is required for target selection by R cell axons within the lamina and medulla. It is proposed that R cell axons release Jeb to activate Alk signaling in target neurons and, through direct or indirect regulation of downstream guidance molecules, contribute to creating the appropriate environment for target recognition (Bazigou, 2007).

In the visual system, R cell axons provide two known anterograde signals to the optic lobe to promote neuronal proliferation and differentiation of target neurons during the third instar larval stage. R cell-derived Hh induces mitotic divisions of lamina precursor cells (LPCs), as well as expression of the early neuronal marker Dachshund in both LPCs and postmitotic lamina neurons. Dachshund in turn is required to control the expression of the EGF receptor in lamina neurons, thus making them competent for the second anterograde R cell-derived signal Spitz that induces the next step of lamina neuron differentiation. A third so far unidentified signal controls glial cell development and migration in the optic lobe. The current findings show that R cell axons provide an unexpected fourth anterograde signal -- Jeb -- that is required to mediate target selection of R cell axons during pupal development. Unlike the Hh and Spitz signals, Jeb represents an anterograde signal delivered by R cell axons not only to the lamina but also to the medulla (Bazigou, 2007).

That Jeb and Alk form an anterograde signaling pathway in the visual system that is supported by three lines of evidence: first, Jeb and Alk are expressed in a largely complementary pattern from the third instar larval to midpupal stages. The ligand Jeb is produced in R cells, whereas the receptor Alk is specifically expressed by target neurons. Since the Jeb protein has been shown to be secreted in vitro, it is highly likely released from R cell growth cones. Second, jeb is genetically required in R cells, whereas Alk functions in target neurons. Third, in the converse experiment, removal of jeb function in the target or Alk in the eye does not produce any conspicuous targeting phenotypes (Bazigou, 2007).

It is proposed that Jeb/Alk signaling plays a role in regulating late events of target-neuron maturation to control R1-R6 axons in the lamina and R8 axons in the medulla. Consistent with this model, the data indicate that loss of Jeb/Alk signaling affects the expression of three guidance molecules, Duf/Kirre, Rst/IrreC, and Fmi, in the R8 recipient layer of the medulla, while Caps, LAR, PTP69D, and CadN appear normal at this level of resolution. Interestingly, animals lacking Jeb/Alk signaling display similar R8 projection defects as fmi and caps eye mosaics. It was further shown that loss of fmi in target neurons causes R8-targeting defects, which qualitatively resemble those observed in Jeb/Alk mosaics. As Jeb/Alk signaling acts upstream of multiple cell-adhesion molecules, loss of one factor likely results in milder targeting defects. In support of this notion, it was observed that phenotypes in jeb or Alk mosaics were more frequent in comparison to fmi knockdown or fmi ELF mosaics. Moreover, loss of fmi in the target appeared to cause one prevalent targeting defect, i.e., the fasciculation of R8 axons with processes in adjacent medulla columns. Notably, loss of sec15 in R cells, which encodes an exocyst component regulating the localization of cell-adhesion molecules to axon terminals, also causes distinct targeting errors. This is consistent with the model that regulating the precise expression of guidance molecules by Jeb/Alk signaling is indeed important for axon targeting in the visual system (Bazigou, 2007).

R cell-targeting defects occurred in both null and kinase domain mutant alleles of Alk, showing that tyrosine kinase activity is essential. Furthermore, studies of vertebrate Alk in vitro, as well as Drosophila Alk in vivo, demonstrate that this RTK drives an ERK/MAPK-mediated signaling pathway, suggesting that Alk may also act through this pathway in the visual system. There are three possible mechanisms as to how Jeb/Alk signaling could regulate downstream guidance molecules: (1) Jeb and Alk may directly regulate the expression of guidance molecules, (2) they could indirectly regulate the expression pattern of guidance molecules via the activation of transcriptional programs determining target neuron identities, or (3) they could separately control both the expression of guidance molecules and transcription factors. Such mechanisms would be analogous to what has been observed in the developing visceral mesoderm, where Jeb/Alk signaling induces the expression of both Duf/Kirre and Org-1, a transcription factor and mammalian Tbx1 homolog, to drive muscle fusion. At present, it cannot be excluded that Alk additionally modulates the activity of downstream targets (Bazigou, 2007).

Anterograde Jeb/Alk signaling would make it possible to coordinate the timing of R cell growth-cone extension with local expression of guidance factors in the target. These in turn could directly regulate afferent axon targeting. Alternatively, guidance factors may be required to shape dendritic and axonal arbors of target neurons and to mediate R cell-targeting decisions. Fmi could indeed take part in both processes, as it can control dendrite development, as well as axon guidance by afferent/afferent and afferent/target interactions. Similar to CadN or LAR eye mosaics, some R1-R6 axons lacking jeb function failed to extend from their original bundle. Extension and cartridge assembly phenotypes were also detected in jeb eye or Alk target mosaics, which qualitatively resembled those described for fmi eye mosaics. Future studies will require the identification and validation of (other) downstream guidance molecules, as well as the isolation of transcriptional regulators controlling target neuron subtype specificity in both the lamina and medulla to provide further insights into the mechanisms underlying Jeb/Alk function (Bazigou, 2007).

It was observed that ectopic expression of Jeb in the visual system strongly reduces the number of activated Caspase 3-positive cells in the medulla at 24 hr APF, when many postmitotic medulla neurons normally undergo apoptosis in wild-type. Thus, Jeb/Alk signaling may also mediate cell survival in parallel to neuronal maturation. The mechanism could be similar to the pleiotropic function of EGF-receptor signaling, which, depending on low or high level of activation regulates cell-cycle withdrawal, mitosis, cell survival, and differentiation in the developing eye imaginal disc of Drosophila (Bazigou, 2007).

Although Jeb shares some sequence similarity with proteins such as the secreted bovine glycoprotein Sco-Spondin , no Jeb homolog has been isolated so far in vertebrates. However, the growth factors Pleiotrophin and Midkine have been reported to act as ligands for Alk in vertebrates, and both have been linked to neuronal development and neurodegenerative diseases. Therefore, Alk may work with different ligands in the vertebrate nervous system. The C. elegans homolog of Alk is localized presynaptically at the neuromuscular junction and has been proposed to mediate synapse stabilization. Also, the vertebrate homologs of Alk are strongly expressed in the developing and adult nervous systems. This includes motor-neuron columns in the spinal cord and, intriguingly, also the superior colliculus, a higher-order processing center for visual information in the brain. That Alk may play a role in neuronal development in vertebrates is further supported by in vitro studies indicating that activated Alk can promote neuronal differentiation and neurite outgrowth in specific cell line. These observations suggest that the function of Alk in regulating specific aspects of neuronal development may be conserved (Bazigou, 2007).

The complex spatio-temporal regulation of the Drosophila myoblast attractant gene duf/kirre.

A key early player in the regulation of myoblast fusion is the gene dumbfounded (duf, also known as kirre). Duf must be expressed, and function, in founder cells (FCs). A fixed number of FCs are chosen from a pool of equivalent myoblasts and serve to attract fusion-competent myoblasts (FCMs) to fuse with them to form a multinucleate muscle-fibre. The spatial and temporal regulation of duf expression and function are important and play a deciding role in choice of fibre number, location and perhaps size. This study used a combination of bioinformatics and functional enhancer deletion approaches to understand the regulation of duf. By transgenic enhancer-reporter deletion analysis of the duf regulatory region, it was found that several distinct enhancer modules regulate duf expression in specific muscle founders of the embryo and the adult. In addition to existing bioinformatics tools, a new program was used for analysis of regulatory sequence, PhyloGibbs-MP, whose development was largely motivated by the requirements of this work. The results complement the deletion analysis by identifying transcription factors whose predicted binding regions match with the deletion constructs. Experimental evidence for the relevance of some of these TF binding sites comes from available ChIP-on-chip from the literature, and from an analysis of localization of myogenic transcription factors with duf enhancer reporter gene expression. The results demonstrate the complex regulation in each founder cell of a gene that is expressed in all founder cells. They provide evidence for transcriptional control--both activation and repression--as an important player in the regulation of myoblast fusion. The set of enhancer constructs generated will be valuable in identifying novel trans-acting factor-binding sites and chromatin regulation during myoblast fusion in Drosophila. The results and the bioinformatics tools developed provide a basis for the study of the transcriptional regulation of other complex genes (Guruharsha, 2009).

A gene which is expressed in all cells of one category -- duf in muscle founder cells (FCs), for example -- can be proposed to be regulated by a relatively simple mechanism, which is a consequence of the specification of that broad cell type. In fact, from earlier studies on mesodermal enhancers of eve, a network of factors -- dTCF, Mad, Pnt, Twi and Tin -- have been shown to positively and negatively regulate the specification of muscle FCs and also FCMs. Further, by using specific genetic perturbations, several genes with localized expression in FCs and FCMs have been identified (Philippakis, 2006). That study attempted to identify enhancers that contained matches to five transcription factor binding site motifs -- dTCF/Mad/Pnt/Twi/Tin -- as generalized regulators of FC gene expression, which identified a heartbroken (hbr) enhancer that drove expression in dorsal FCs - indicating that the FC gene regulation is not exclusively regulated by these five TFs. But with a smaller and more general subset of TFs, i.e., Pnt, Twi, and Tin, four new enhancers that drive FC like expression have been identified (Philippakis, 2006). It is suspected that FC gene regulation is more complex than previously proposed. Additional mechanisms and combinations of dTCF/Mad/Pnt/Twi/Tin along with other, currently unknown, motifs may regulate FC gene expression. The current results from the analysis of duf regulation during embryonic and adult myogenesis also reflects this complexity. They demonstrate the complex regulation of a gene that is expressed in all founder cells. Spatio-temporally regulated activation/repression of specific duf enhancer modules in distinct muscle types suggests an elegant mechanism of generating muscle diversity by transcriptional control of a key player in myoblast fusion (Guruharsha, 2009).

The smallest enhancer fragment, duf -1.0 kb, close to the transcriptional start site has basic elements that accurately identify mesodermal lineage (Twi) and allows expression in some of the somatic muscles. In slightly larger constructs (for example: duf -1.5 kb, -3.0 kb and -3.8 kb), the reporter expression is very well restricted to specific muscle types. Several ChIP-on-chip results indicate Twi binds between -2.4 kb to -3.8 kb of the duf enhancer. In this region, sites for Pnt, Mad and DMef2 were also found that match the published consensus. ChIP-on-chip studies indicate these Dmef2 sites are occupied during early mesodermal development. DMef-2 is a downstream target for twi, its early expression pattern modulates as the mesoderm organizes into cell groupings with distinct fates. DMef2 is expressed in the segregating primordia as well as the differentiated cells of the somatic, visceral and heart musculature. This study also found Nk2 group (Tin, bap) sites in the -1.5 to -2.4 kb using Stubb. When this fragment (duf -2.4 to -1.5 kb) was analyzed independently, it was found to be insufficient to drive any reporter expression. Interestingly, duf -2.4 kb lacZ is expressed strongly in cardioblasts and very weakly in all somatic muscles, while the smaller duf -1.5 kb construct is expressed in large subset of somatic muscle FCs (Guruharsha, 2009).

RTK signalling and Twi are critical for the somatic muscle fate. ChIP-on-chip results show that binding of Twi (-3 kb ) and Dmef2 (-3 to -4 kb region) during early mesodermal development. These results corroborate the bioinformatic predictions and enhancer deletion studies. In addition, Dmef2 binding sites are predicted in the -5 to -5.5 kb region. duf -3.8 kb lacZ and duf -5.1 kb lacZ are expressed strongly in majority of the somatic muscle FCs but not in any of the gut muscles. duf -5.1 kb lacZ is also expressed clearly in adult lateral abdominal muscles which is not seen in duf -5.3 kb lacZ and duf -5.5 kb lacZ (Guruharsha, 2009).

Body wall (somatic) muscles provide the force for the peristaltic locomotion of the larva while the gut (visceral) muscles provide the peristaltic force for movement of food during digestion. Longitudinal and circular muscles of the midgut as well as the visceral muscles of the foregut and hindgut arise from different primordia and follow diverse developmental pathways. In contrast to most other larval tissues that are histolyzed during metamorphosis, the visceral musculature persists through metamorphosis. This might be an important aspect, since in the deletion studies it was found duf enhancer fragments (-9.8 to -3.8 kb, -8.6 to -0.6 kb) express strongly in visceral muscles of the embryo and also in the persisitent larval muscles, which are FCs of the DLMs, at the onset of adult development (Guruharsha, 2009).

Visceral mesoderm development is abnormal in shn mutants: shn mediates action of Dpp on mesodermal cells by inducing bap. Phylogibbs finds couple weak Shn binding sites in this region. A stronger Shn binding sites are predicted further upstream by Stubb and Phylogibbs. Stubb predicts NK2 (which includes bap) binding sites in -1.5 kb to -2.4 kb constructs. PhyloGibbs predicts two weak binding sites in -2.4 kb and -3.0 kb constructs. New ChIP-on-Chip experiments (Furlong lab, personal communication to Guruharsha, 2009) suggest that Bap binds in this region of duf enhancer in mesodermal cells from stage 6-8 of embryonic development. duf -3.0 kb lacZ is expressed strongly in Longitudinal Visceral Muscle FCs, that originate from the caudal mesoderm but not in circular visceral FCs that arise from the midgut. Similarly, several Bin binding sites are predicted by both Stubb and PhyloGibbs between -1.0 to -3.0 kb. bin is important for maintaining the distinction between visceral and somatic mesoderm and its activity is essential for differentiation of the visceral mesoderm into midgut musculature (Guruharsha, 2009).

Deletions from the proximal end such as duf -7.9-3.8 kb Gal4, duf -9.8-3.8 kb Gal4and duf -8.2-0.6 kb Gal4 all show consistent and strong expression patterns in circular visceral muscle FCs and very few somatic FCs in the embryo. Many visceral mesoderm factor binding sites are predicted in this region. For example, several NK2 group sites are predicted by Stubb and few weak sites are predicted by PhyloGibbs between -9.8 to -3.8 kb region. Again several Bin sites are predicted by Stubb and PhyloGibbs between -9.8 to -7.8 kb. Similarly, two clusters of byn sites are predicted by Stubb between -1.0 to -3.0 kb and -5.5 to -7.2 kb (Guruharsha, 2009).

Thus, the results from this bioinformatics analysis are in very good agreement with the transgenic deletion studies, ChIP-on-chip data and the existing literature (Guruharsha, 2009).

Drosophila uses its genome to make two distinct developmental body plans: the larva and the adult. Duf is critical for myoblast fusion during both embryonic. Embryonic muscles of Drosophila are single fibres whereas adult muscles are bundles of muscle fibres - similar to those of vertebrate. This study found specific enhancer elements repsonsible for duf expression during adult myogenesis. duf -9.8-3.8 kb Gal4 and duf - 8.2 -0.6 kb Gal4 are expressed strongly in all the adult muscle FC analogs during adult myogenesis. Removal of enhancer elements close to the transcription start site appears to promote expression in circular visceral muscles of the embryo and all adult muscles (Guruharsha, 2009).

Ecdysone-induced protein 74EF (Eip74EF), has putative binding sites in the region >5 kb from the start site. PhyloGibbs predictes dMef2 sites between -7.2 to -9.5 kb. Several signaling pathway effectors also have distinct set of binding sites occuring in the proximal and distal regions of the enhancer. Several homeodomain factors (for example: Antp and Abd-B) also have several binding sites, predicted by both Stubb and PhyloGibbs predominantly in the distal region of the enhancer (Guruharsha, 2009).

Addtionally, putative binding sites for GAGA factor encoded by the Trithorax-like gene (Trl) characteristic of TREs (Trithorax Response Elements) are found in -4.0 kb from the duf start site. Sites further upstream are predicted by Stubb and PhyloGibbs, but the strongest sites occur below -4.0 kb. This region shows good embryonic expression but no expression in adult muscles. Published ChIP predictions for Trl agree well with predictions in this region. A re-analysis of raw tiling array data using MAT and a lower threshold, results in a larger region that overlaps a significant region of the current predictions (Guruharsha, 2009).

Similarly, putative binding sites for PHO (pleiohomeotic) and PHO-like polycomb group proteins (PcG) that bind to PREs (Polycomb group Response Elements) are found between -8.0 kb to -9.3 kb region. ChIP-on-chip experiments for TREs and PREs find GAF (Trl) binding upstream of duf, but only very little binding of PHO, in Sg4 tissue culture cells. However, data from similar experiments using Drosophila embryos suggest that PHOL binds upstream of the duf promoter. PcG protein binding at duf has not been detected, but a binding peak for ASH1 is usually associated with PcG target genes when they are derepressed, has been found (Vincenzo Pirrotta, personal communication to Guruharsha, 2009) (Guruharsha, 2009).

PREs and TREs in duf enhancer region appears restricted to specific regions. TRE/PRE mediated silencing may be responsible for switching between embryonic and adult specific enhancers by restricting access of TF to chromatin in appropriate tissue and time points. Experiments designed to test the presence or absence of each of these factors during embryonic or adult myogenesis would help answer this question more accurately (Guruharsha, 2009).

Taken together, this study has uncovered a complex regulatory mechanism operating to control important myoblast fusion gene such as duf during Drosophila myogenesis. A set of enhancer constructs generated for this study will be valuable reagents in identifying important trans-acting factor-binding sites and chromatin regulation during myoblast fusion. This study has described several contexts where the bioinformatics predictive tools and ChIP-on-chip approaches have great value and others where, clearly, more information is needed before predictive tools can be applied (Guruharsha, 2009).

Protein Interactions

Rolling pebbles, a multidomain protein required for myoblast fusion, recruits D-Titin in response to the myoblast attractant Dumbfounded

The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded, which brings them into close contact with other myoblasts. Rols7, a gene expressed in founders, has been identified as an essential component for fusion during myotube formation. Rols7 is a 7kb alternative splice of rolling pebbles. During fusion, Rols7 localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols7 is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols7 integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin (Menon, 2001).

dumbfounded is the only other fusion gene that is known to be restricted in expression to the founders and is absent in fusion competant myoblasts (FCM) (Ruiz-Gomez, 2000). In addition to the overlap in spatial expression, the temporal expression profiles of rols7 and duf in these tissues appear identical. These observations, the receptor-like nature of the molecule encoded by duf, and the loss of membrane-enriched Rols7 in the duf mutant led the authors to test whether Duf expression is sufficient to promote Rols7 membrane localization. This was done by overexpressing Rols7 early throughout the mesoderm and later in all muscles, either by itself or together with Duf, using the 24B-GAL4 driver. Under either of these conditions, no change was observed in the patterning of the somatic muscles. Rols7 localization was then examined late at stage 16, a time at which the expression of endogenous Rols7 and probably that of Duf is lost in wt muscles. When Rols7 is overexpressed alone, the protein appears as speckles throughout the cytosol of mature muscles. Despite its abundance, no membrane patches could be detected. In contrast, co-overexpression with Duf results in Rols7 becoming membrane enriched, with little or no protein remaining in the cytoplasm of muscles. In addition, higher levels of Rols7 are found along membranes that come in direct contact with other muscles. Muscles such as VL1, VO4, and VO6 that abut other muscles only on one side show significantly higher levels of Rols7 on the side in contact with its neighbor, whereas muscles such as VL2 and VO5, which lie between muscles, show equally high levels of Rols7 expression on either side of the membrane. This and observations in wt embryos, where Rols7 accumulates at discrete sites along the myotube membrane suggest that Duf and Rols7 may be present at specialized sites along the founder (or myotube) membrane that contact the FCM (Menon, 2001).

The protein encoded by the rols7 transcript has several distinct domains that can potentially participate in protein-protein interactions. At its N terminus, Rols7 carries a C₃HC₄ zinc finger, called the RING finger. While studies on several RING finger-containing proteins such as Cbl suggest that this domain is essential for E2-dependent ubiquitin protein ligase activity leading to protein destruction, the RING fingers in other proteins have been implicated in different modes of protein-protein interactions. Of note, the RING finger in the vertebrate muscle-specific proteins termed MURFs is essential to establish stable interaction between a specific MURF and Titin or the cytoskeletal network of microtubules (Centner, 2001; Spencer, 2000). At its C terminus, Rols7 encodes three different protein interaction motifs: a tandem array of nine ankyrin repeats followed closely by three TPR repeats and a coiled-coiled domain. Based on its overall structure, it is plausible that Rols7 could act as a focal point for the assembly of a multiprotein complex at the membrane where the Duf receptor is located, bringing the fusion machinery and directing changes in the cytoskeleton to sites where fusion would take place. In support of this, it has been shown that Rols7 is required for the enrichment of D-Titin to fusion sites in founders (or myotubes). However, this appears to be only one of the roles served by Rols7, since founders in the rols mutant either remain unfused or develop into small precursors, whereas fusion is arrested at a later stage in the D-Titin null allele (Menon, 2001).

Antisocial/Rolling peebles, an intracellular adaptor protein, interacts with Dumbfounded in the proceess of myoblast fusion in Drosophila

Somatic muscle formation in Drosophila requires fusion of muscle founder cells with fusion-competent myoblasts. In a genetic screen for genes that control muscle development, antisocial (ants; alternative name for rolling pebbles), a gene that encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein, was shown to be required for myoblast fusion. In ants mutant embryos, founder cells and fusion-competent myoblasts are properly specified and patterned, but they are unable to form myotubes. Ants, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. These findings suggest that Ants functions as an intracellular adaptor protein that relays signals from Dumbfounded to the cytoskeleton during myoblast fusion (Chen, 2001).

In order to gain insights into the function of rols during myoblast fusion, tests were conducted to determine whether Rols is present in founder cells or fusion-competent myoblasts. An antibody double-labeling experiment was performed with anti-Rols and anti-ß-galactosidase (ß-gal) antibodies using the rp298 enhancer trap line, which carries a P element insertion in the 5' promoter of the duf gene. Confocal microscopy has demonstrated that Rols is localized to the lacZ-expressing founder cells. Another founder cell-specific marker, even-skipped (eve), is also localized to the same cells as Rols. Interestingly, Rols is a cytoplasmic protein that aggregates to discrete foci. The aggregated appearance of Rols staining is reminiscent of that of Sns, the transmembrane receptor of fusion-competent myoblasts, which is localized to discrete sites associated with the cell membrane as fusion progresses (Chen, 2001).

Two transmembrane receptors, Duf and Sns, are implicated in cell recognition during myoblast fusion in Drosophila, whereas the cytoplasmic protein Mbc has been implicated in mediating changes in the cytoskeleton. It is not clear whether or how the known fusion molecules interact with each other during the fusion process. In addition, given the multistep nature of the fusion process, it is likely that additional components of the pathway(s) remain to be identified. Rols physically interacts with both Duf and Mbc. Thus, Rols could serve as a linker molecule that relays essential signals from a membrane receptor to changes in the cytoskeleton of founder cells (Chen, 2001).

Ankyrin proteins contain three domains, including a membrane binding domain at the amino terminus, a central spectrin binding domain, and a carboxy-terminal regulatory domain. The membrane binding domain, which contains multiple ankyrin repeats, binds to the cytoplasmic domains of specific integral membrane proteins, including adhesion molecules. Rols is not a conventional ankyrin protein, since its ankyrin repeats are located at the carboxy-terminal region and it lacks the central spectrin binding domain. Nevertheless, Rols can associate with the founder cell receptor Duf and the cytoplasmic protein Mbc. The conserved regions between Rols and its vertebrate orthologs, including the ankyrin repeats, are required for Rols' interaction with Duf, since a deletion construct lacking the conserved domains does not associate with Duf. The fact that a rols allele (ants^T321) that deletes the conserved region behaves as a null mutation is consistent with this region being important for the function of Rols in vivo. Preliminary results indicate that Mbc maintains the ability to interact with an Rols protein lacking the conserved carboxy-terminal region, suggesting that the amino-terminal domain of Rols is likely to interact with Mbc (Chen, 2001).

Antibody staining has shown that Rols is a cytoplasmic protein. Two other fusion molecules, Mbc and Blow, are also expressed in the cytoplasm. However, the localization of Rols is distinct from that of Mbc and Blow. While Mbc and Blow are expressed in both founder cells and fusion-competent myoblasts, Rols is only expressed in founder cells. In addition, while Mbc and Blow are expressed throughout the cytoplasm of myoblasts, Rols is localized in discrete domains in the cytoplasm. These results, together with the protein-protein interaction between Rols and Duf, raise the possibility that the Rols localization domains might correlate with the sites of cell recognition and adhesion between founder cells and fusion-competent myoblasts. The subcellular structures in which Rols is localized and how these domains might be related to the expression of Duf on the founder cell membrane remain to be determined. While the lack of Duf antibody prevents the examination of the Duf protein expression pattern on the founder cell membrane and the relative localization of Duf and Rols, the Sns protein has been shown to be clustered in discrete regions on the membrane of fusion-competent cells (Bour, 2000). It is conceivable that Duf may also be localized to specific membrane regions in founder cells during the fusion process. However, the possibility that there is an excessive amount of Duf on the founder cell membrane such that no localization of Duf is necessary during cell recognition and cell adhesion cannot be ruled out. Nevertheless, the altered Rols localization in duf mutant embryos supports the hypothesis that Duf is required to localize Rols to specific subcellular foci, presumably through the physical interaction between the two proteins (Chen, 2001).

Myoblast fusion requires not only the recognition and adhesion between founder cells and fusion-competent cells, but also subsequent cytoskeletal rearragements that lead to the proper alignment of the two populations of cells. Previous studies on the founder cell-specific receptor Duf have shown that it acts as an attractant for fusion-competent cells (Ruiz-Gómez, 2000). Although duf is necessary for myoblast fusion, it is not sufficient, since ectopic expression of duf in fusion-competent cells did not result in fusion among this population of myoblasts (Ruiz-Gómez, 2000). Based on this observation, it was suggested that besides duf, there must exist at least one additional protein that is present in founder cells but absent from fusion-competent myoblasts. This protein could interact with the intracellular domain of Duf to initiate fusion (Ruiz-Gómez, 2000). Rols may represent such a molecule: (1) Rols is expressed in founder cells just before and during the fusion process; (2) Rols physically interacts with the cytoplamic domain of Duf; (3) the Rols protein is localized in discrete regions in the cytoplasm of founder cells during the fusion process, and the specific localization of Rols is altered in duf mutant embryos, consistent with the possible interaction with a localized membrane receptor during the fusion process (Chen, 2001).

Given the conservation of numerous signaling pathways between Drosophila and vertebrates, it is possible that vertebrate homologs of genes required for Drosophila myoblast fusion might play similar roles in skeletal muscle development. However, none of the myoblast fusion genes identified in Drosophila so far have been implicated in a similar role in vertebrate skeletal muscle development. For example, the closest vertebrate homolog of Duf and Sns is the human Nephrin protein, which is essential for kidney development. The vertebrate homolog of Mbc, DOCK180, interacts with focal adhesion molecules and seems to be a general factor that regulates cytoskeletal events. Studies of two mouse orthologs of rols suggest that one of them, mants1, could be involved in skeletal muscle development in vertebrates. The temporal expression pattern of mants1 in the developing mouse embryo is reminiscent of rols expression in the Drosophila embryo. mants1 expression coincides with the early stages of mesodermal development, and its expression is dramatically reduced after skeletal muscle formation. The transient expression of mants1 in the mesoderm is consistent with a potential role in early skeletal muscle development, including myoblast fusion. Interestingly, mants1 is also expressed at the time of fusion in the C2 myoblast cell line. However, it should be pointed out that the expression of mants1 in the mouse embryo is not solely restricted to skeletal muscle precursors but rather is more broadly distributed throughout the mesoderm at E11.5. Obviously, further studies will be required to confirm if mants1 indeed plays a role in myoblast fusion in vertebrates as does rols in Drosophila (Chen, 2001).

Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion and mediates heterotypic adhesion with Duf/Kirre and IrreC-rst-expressing cells

The body wall muscles in the Drosophila larva arise from interactions between Dumbfounded/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and Sticks and stones (Sns)-expressing fusion competent myoblasts in the embryo. Sns, a member of the immunoglobulin superfamily that is essential for myoblast fusion (Bour, 2000), mediates heterotypic adhesion of S2 cells with Duf/Kirre and IrreC-rst-expressing S2 cells, and colocalizes with these proteins at points of cell contact. These properties are independent of their transmembrane and cytoplasmic domains, and are observed quite readily with GPI-anchored forms of the ectodomains. Heterotypic interactions between Duf/Kirre and Sns-expressing S2 cells occur more rapidly and to a greater extent than homotypic interactions with other Duf/Kirre-expressing cells. In addition, Duf/Kirre and Sns are present in an immunoprecipitable complex from S2 cells. In the embryo, Duf/Kirre and Sns are present at points of contact between founder and fusion competent cells. Moreover, Sns clustering on the cell surface is dependent on Duf/Kirre and/or IrreC-rst. Finally, although the cytoplasmic and transmembrane domains of Sns are expendable for interactions in culture, they are essential for fusion of embryonic myoblasts (Galletta, 2004).

The ability of Sns, Duf/Kirre and IrreC-rst to mediate cell–cell adhesion was examined using Drosophila S2 cells, which are predominantly non-adherent under normal conditions. As a prelude to examining the behavior of these molecules in combination, each was examined individually to evaluate their ability to direct homotypic aggregation. S2 cells were transiently transfected with Duf/Kirre, IrreC-rst, or Sns under the control of the copper inducible metallothionein promoter and allowed to aggregate. Following aggregation, the cells were fixed and examined by indirect immunofluorescence using anti-sera directed against specific domains or tags within each protein. As anticipated from previous studies (Dworak, 2001), Duf/Kirre-expressing S2 cells were frequently found in aggregates. Duf/Kirre protein accumulates at points of cell-cell contact in aggregates but is uniformly distributed on the surface of non-aggregated S2 cells. Similar to the behavior of Duf/Kirre, IrreC-rst mediates homotypic aggregation of S2 cells, and becomes enriched at points of cell-cell contact in the resulting cell clusters. Duf/Kirre and IrreC-rst enrichment is occasionally observed in regions where cell-cell contact is not apparent, possibly as a consequence of processes, visible by transmission electron microscopy, that extend around neighboring cells. In contrast to the behavior of Duf/Kirre or IrreC-rst, expression of Sns protein on the surface of S2 cells does not lead to homotypic cell adhesion (Dworak, 2001). A lower magnification view emphasizes the presence of many unassociated Sns-expressing cells. As anticipated, Sns is distributed uniformly on the surface in the absence of aggregation (Galletta, 2004).

To ensure that the Duf/Kirre and IrreC-rst clusters were the consequence of aggregation rather than cell division, the number of cells in aggregates of three cells or more were counted. Since cells should only divide at most once during the course of the experiment, clusters of three cells must represent those formed from adhesive events. In a survey of 4171 Duf/Kirre-expressing cells, 40% (1697) were found in aggregates of three or more. In a survey of 1002 IrreC-rst-expressing cells, 19% (192) were in aggregates of three or more cells. These results suggest that while Duf/Kirre may be more efficient in mediating homotypic aggregation than IrreC-rst, clearly both are capable of mediating such interactions. By contrast, a survey of 3275 Sns-expressing cells revealed only 26 cells in aggregates of three or more cells (Galletta, 2004).

While Sns-expressing cells do not aggregate homophilically, studies have indicated that these cells aggregate with cells expressing Duf/Kirre (Dworak, 2001). It was of interest to determine whether cells expressing Sns would interact with cells expressing IrreC-rst, and whether Sns and Duf/Kirre or IrreC-rst co-localize at points of cell-cell contact. To this end, S2 cells were independently, transiently transfected and the ability of Duf/Kirre and IrreC-rst-expressing cells to form aggregates and direct membrane co-localization of Sns in these aggregates was examined. All of these proteins were uniformly distributed on the cell surface in unaggregated cells. In contrast to their behavior in isolation, Sns-expressing cells readily associated in large clusters when combined with cells expressing Duf/Kirre. The Sns-expressing cells also associated with cells expressing IrreC-rst, with a similar efficiency. At least one of these IgSF members must be expressed on the cell surface for it to cluster, since no untransfected cells were observed in an analysis of 1109 small clusters of either Duf/Kirre:Sns or IrreC-rst:Sns-expressing cells. While the biological significance of such an interaction remains unclear, Duf/Kirre and IrreC-rst-expressing cells are capable of forming heterotypic aggregates with each other when expressed in S2 cells under similar conditions (Galletta, 2004).

Examination of individual proteins in small aggregates revealed clustering of Sns with either Duf/Kirre or IrreC-rst at points of cell contact. Thus, either Duf/Kirre or IrreC-rst can direct cells to associate with Sns-expressing cells, and co-localize with Sns at points of cell contact. Frequently much of the Sns protein in the cell accumulates at the points of cell-cell contact, leaving little if any protein on the rest of the cell surface. In rare cases, both proteins are observed in regions outside of obvious cell contacts. However, this pattern may reflect cell membranes that are extending around neighboring cells, mentioned earlier. Since Duf/Kirre and IrreC-rst serve redundant functions in the founder myoblasts, and behave similarly in the assays described above, subsequent experiments focused on Duf/Kirre (Galletta, 2004).

In some cases, the cytoplasmic domains of cell adhesion molecules play no role in their ability to direct cell interactions, while this domain can be critical in other cases. It was therefore of interest to determine whether these regions of Sns or Duf/Kirre were required for the S2 cell interactions. For these studies, the extracellular domains of Duf/Kirre and Sns were fused in frame to the GPI-anchor sequence of Fasciclin I. These constructs were separately, transiently transfected into S2 cells, and aggregation was examined. In the case of Duf/Kirre-GPI, the efficiency of homotypic aggregation was severely reduced compared to that of cells expressing full length Duf/Kirre. Since the relevance of Duf/Kirre homotypic aggregates in vivo is unclear, the role of the Duf/Kirre and Sns cytoplasmic and transmembrane domains in heterotypic aggregation was also examined. In an analysis similar to that done for Duf/Kirre homotypic aggregates, the ability of cells expressing the GPI-anchored or full length forms of Duf/Kirre to mediate heterotypic adhesion with cells expressing full-length or GPI-anchored forms of Sns was examined in pairwise comparisons. The influence of the Sns cytoplasmic and transmembrane domains was examined on adhesion with cells expressing full length Duf/Kirre. Within the limits of statistical significance, GPI-anchored Sns mediate aggregation at a level comparable to that of full length Sns. A similar analysis was carried out to examine the influence of the Sns cytoplasmic and transmembrane regions on adhesion with cells expressing Duf/Kirre-GPI. Again, Sns-GPI mediates adhesion with the Duf/Kirre-GPI-expressing cells at a level comparable to that of full length Sns. Thus, the Sns cytodomain and membrane spanning region appear to play no role in its ability to direct aggregation with Duf/Kirre-expressing cells (Galletta, 2004).

Since the Duf/Kirre-expressing cells were in excess in the above experiments, these data could not be used to determine the relative contribution of the Duf/Kirre cytodomain and transmembrane region. Therefore additional assays were carried out in which Sns or Sns-GPI-expressing cells were in a five-fold excess over either Duf/Kirre or Duf/Kirre-GPI-expressing cells to determine whether there was a requirement for the Duf/Kirre cytodomain or membrane spanning region in interactions with cells expressing Sns. These experiments were also set up as pairwise comparisons, and demonstrated that Duf/Kirre-GPI mediate aggregation at a level comparable to that of full length Duf/Kirre. These data suggest that there is no significant difference between the ability of Duf/Kirre or Duf/Kirre-GPI to aggregate with cells expressing full-length Sns. Lastly, the cytoplasmic and transmembrane domain of Duf/Kirre have a modest affect on its ability to direct aggregation with GPI-anchored Sns. However, the effect of the cytoplasmic or transmembrane domain on Duf/Kirre's ability to mediate heterotypic aggregation with Sns-GPI was not as great as its effect on the ability of Duf/Kirre to mediate homotypic cell adhesion. Of note, Duf/Kirre-GPI was enriched at points of cell-cell contact in both homotypic aggregates and in heterotypic aggregates with Sns and Sns-GPI. Thus, neither the cytoplasmic nor transmembrane domains of Sns or Duf/Kirre are essential for recruitment to cell-cell contacts (Galletta, 2004).

In summary, these results indicate that the cytoplasmic/transmembrane domains of Sns and Duf/Kirre do not influence the efficacy with which they direct heterotypic cell-cell adhesion. This observation is in contrast to that seen for Duf/Kirre, in which the cytodomain or membrane spanning region of Duf/Kirre plays a critical role in its ability to direct homotypic aggregation. One possible explanation for these results is that heterotypic association of Sns and Duf/Kirre is stronger, and does not require stabilization of the receptor through cytoplasmic or intramembrane interactions. Since the affinity of Duf/Kirre for homotypic versus heterotypic interactions could play a critical role in myoblast interactions in the embryo, the S2 cell aggregation assay was used to examine this preference (Galletta, 2004).

In the embryonic musculature, founder cells appear to fuse only with fusion competent myoblasts, and never fuse with each other. In principle, this directional fusion could be attributed to the differential expression of Duf/Kirre and Sns by these two cell types, and inability of these molecules to associate homotypically. However, results reported in this study and by Dworak (2001) demonstrate that Duf/Kirre-expressing cells do associate with each other in culture. It was therefore of interest to determine whether the affinity of Duf/Kirre-expressing cells for cells expressing Sns was greater than the affinity of Duf/Kirre-expressing cells for each other. To address this question, Duf/Kirre-expressing cells were aggregated in isolation or in the presence of an equal number of Sns-expressing cells. This analysis utilized stable cell lines in which approximately 30% of the corresponding population expressed Duf/Kirre and approximately 8% expressed Sns. Aggregation of Duf/Kirre-expressing cells was examined in three different conditions, all with the same total cell number. The goal was to ensure that any change in aggregation of Duf/Kirre cells was due to the specific addition of Sns-expressing cells rather than a consequence of doubling the number of adherent cells. For each time point, the number of Duf/Kirre-expressing cells free in solution was counted and the number that had been incorporated into aggregates. Duf/Kirre-expressing cells were incorporated into aggregates that included Sns-expressing cells at a faster rate and to a greater extent than those containing only Duf/Kirre-expressing cells. This behavior was not a simple consequence of the number of adherent cells present, since a two-fold increase in the number of Duf/Kirre-expressing cells did not have a dramatic effect on the rate or extent of aggregation. Thus, Duf/Kirre-expressing cells associate more readily into heterotypic aggregates with Sns-expressing cells than into homotypic aggregates with only Duf/Kirre-expressing cells (Galletta, 2004).

The striking colocalization of Duf/Kirre and Sns described earlier suggested the possibility that these proteins might physically associate in trans. To address this possibility, aggregates of stably transfected, Sns and Duf/Kirre-expressing cells were subjected to reversible protein cross-linking and lysed. HA-tagged Duf/Kirre was immunoprecipitated from the cell lysate using anti-HA resin, and the resulting immunoprecipitate examined by Western blot for the presence of Sns. HA-tagged Duf/Kirre was efficiently precipitated from both Duf/Kirre-only and Duf/Kirre-Sns mixed cell populations. As expected, Sns was not present in the anti-HA immunoprecipitate from cells expressing only Duf/Kirre or only Sns. However, it was clearly detected in immunoprecipitates from the mixed population of cells expressing Duf/Kirre-HA and Sns. Thus, Duf/Kirre and Sns are closely associated in an immunoprecipitable protein complex, possibly through a direct protein interaction (Galletta, 2004).

In the embryonic musculature, Duf/Kirre, IrreC-rst and Sns are necessary, either directly or indirectly, for the association of founder and fusion competent myoblasts. The striking co-localization of Sns with either Duf/Kirre or IrreC-rst in S2 cells prompted an examination of whether similar co-localization could be observed between embryonic myoblasts. First it was examined whether punctate clustering of Sns on the surface of embryonic myoblasts, previously described by Bour (2000), was dependent on the presence of Duf/Kirre or IrreC-rst. The distribution of Sns protein was examined in embryos deficient for both Duf/Kirre and IrreC-rst, and compared to that seen in wild-type embryos. As anticipated, Sns becomes localized to discrete sites in wild-type myoblasts (Bour, 2000). In contrast, Sns is distributed more uniformly on the myoblast surface in embryos lacking Duf/Kirre and IrreC-rst. Thus in embryos, as in S2 cells, the localization of Sns is dependent on the presence of Duf/Kirre or IrreC-rst (Galletta, 2004).

To determine whether Sns and Duf/Kirre co-localize in embryonic myoblasts in a manner similar to that observed in S2 cells, stage 13 embryos were examined by indirect immunofluorescence using polyclonal antisera directed against the Duf/Kirre and Sns proteins. As previously described for Sns (Bour, 2000), Duf/Kirre is expressed in a dynamic pattern that is restricted to discrete sites on the surface and in the cytoplasm of expressing cells. The pattern of Sns expression intersects that of Duf/Kirre, and is in close proximity to rP298-lacZ positive founder cell nuclei in the somatic mesoderm. Of note, punctate Sns expression is apparent at some sites in which Duf/Kirre expression is not detected. To address whether these sites might intersect points of IrreC-rst protein, which can interact with Sns-expressing cells and can substitute for Duf/Kirre in vivo, embryos were triple labeled with Duf/Kirre, IrreC-rst and Sns. IrreC-rst is readily detected at many sites of Sns enrichment that do not appear to colocalize with Duf/Kirre. In fact, examination of 204 discrete sites of Sns protein, derived from eight stage 13 embryos, revealed that 97% were colocalized with either Duf/Kirre and/or IrreC-rst (Galletta, 2004).

To determine whether sites of Duf/Kirre and Sns colocalization occur, as expected, on the cell surface, mesodermally expressed CD2 was used to visualize the cell membrane. CD2 staining revealed the surface of a growing myofiber and associated myoblasts. Duf/Kirre and Sns co-localize to points of contact between the fiber and a myoblast. Since the expression of both Duf/Kirre and Sns is dynamic and rapidly decreases upon fusion (Bour, 2000), co-localization of Duf/Kirre and Sns was examined in myoblast city (mbc) mutant embryos in which the myoblasts associate but remain unfused. By stage 14, the founder cells of these mutant embryos become morphologically distinct from the fusion competent cells, elongating and extending processes. As an apparent consequence of this fusion block, Duf/Kirre and Sns are stabilized at points of contact between the extended founder cell and several fusion competent cells. These data clearly show that Sns and Duf/Kirre co-localize in the embryo at critical contact points between founder cells and fusion competent myoblasts (Galletta, 2004).

Drosophila rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs

The rolling pebbles gene of Drosophila encodes two proteins, one of which, Rols7, is essential for myoblast fusion. In addition, Rols 7 is expressed during myofibrillogenesis and in the mature muscles. Here it overlaps with alpha- Actinin (a-Actn) and the N-terminus of D-Titin/Kettin/Zormin in the Z-line of the sarcomeres. In the attachment sites of the somatic muscles, Rols7 and the immunoglobulin superfamily protein Dumbfounded/Kin of irreC (Duf/Kirre) colocalise. As Duf/Kirre is detectable only transiently, it may be involved in establishing the first contact of the outgrowing muscle fiber to the epidermal attachment site. It is proposed that Rols7 and Duf/Kirre link the terminal Z-disc to the cell membrane by direct interaction. This is supported by the fact that in yeast two hybrid assays the tetratricopeptide repeat E (TPR E) of Rols7 shows interaction with the intracellular domain of Duf/Kirre. The colocalisation of Rols7 with a-Actn and with D-Titin/Kettin/Zormin in the Z-dics is reflected in interactions with different domains of Rols7 in this assay. In summary, these data show that besides the role in myoblast fusion, Rols7 is a scaffold protein during myofibrillogenesis and in the Z-line of the sarcomere as well as in the terminal Z-disc linking the muscle to the epidermal attachment sites (Kreiskother, 2006).

The scaffold protein Rols7 has been shown to be essential for myoblast fusion in the somatic mesoderm during Drosophila embryogenesis where it might interact with several components of the fusion machinery. Evidence is presented that Rols7 has an additional function in the establishment of the muscle attachment and the formation of the Z-discs, as well as in the Z-discs of the mature muscles (Kreiskother, 2006).

During myoblast fusion, Rols7 mRNA decays at stage 15. Antibody staining of stage 17 embryos reveal a concentration of Rols7 at the muscle ends next to the epidermal attachment sites, which are caused by new transcription in RT-PCR experiments. Later on in the mature larval muscles Rols7 is detected in the sarcomeric Z-discs (Kreiskother, 2006).

During the early stages of myogenesis, the interaction of the founder cell specific protein Duf/Kirre and the fusion competent myoblasts (fcm) specific Sns leads to the adhesion of the two cell types, which is a prerequisite for further steps of the fusion process. Besides this, Duf/Kirre transiently are concentrated at the end of the developing muscles at stage 15 and 16, while it disappears again at stage 17. This led to a hypothesis that Duf/Kirre might participate in the first contact of the outgrowing muscle to the attachment site, as does Vein. This would require an interaction partner in the extracellular matrix or at the epidermal site. Since the sns transcript is present in the muscle attachment sites at a low level at stage 17, antibody staining for Sns was performed, but a distinct signal in the attachment sites could not be detected. As well as the transcript of sns, its paralog, Hibris (Hbs), is also found in the muscle attachment sites, and, more exactly, localised to the contact site between the cells at the epidermal attachments. Thus, it could function as an interaction partner for Duf/Kirre (Kreiskother, 2006).

As a further possible interaction partner Rst/IrreC was considered, since Rst/IrreC, the paralogue of Duf/Kirre, shows expression in the epidermal tendon cells during embryonic stages. Due to the fact that Duf/Kirre and Rst/IrreC are indeed able to undergo heterophilic interaction in cell culture experiments, the conclusion is drawn that Rst/IrreC might be the candidate for an interaction partner of Duf/Kirre on the epidermal site, thus enabling an early contact of the muscle to the epidermal attachment site (Kreiskother, 2006).

Rols7, which interacts with the intracellular domain of Duf/Kirre, is also localised at the muscle ends from late stage 16 onwards shortly before Duf/Kirre disappears. It is speculated that Rols7 is brought to the membrane where it interacts with Duf/Kirre (Kreiskother, 2006).

alpha actinin (α-Actn) and D-Titin/Kettin, both found at the muscle attachment site in a similar pattern, also interact with Rols7 (at least in the yeast two hybrid assay) and participate in the establishment of the terminal Z-disc. For the flight muscle it was shown that α-Actn is essential for the formation of this structure and for obtaining a correct insertion of the myofibril to the epidermal tendon cell. Furthermore the yeast assay showed an interaction of α-Actn with Duf/Kirre^intra (Kreiskother, 2006).

kettin mutants show strong defects in terminal Z-disc function. This study proposes that, in addition to Kettin, Rols7 and α-Actn are important for the formation of this structure. The process might be connected to Muscleblind (Mbl), since in mutants for mbl, Z-discs are not assembled correctly. Unfortunately, a mutant analysis of Rols7 function in terminal Z-disc formation is difficult due to its essential function during myoblast fusion (Kreiskother, 2006).

Apart from the myoblasts and attachment sites, Rols7 is expressed in the developing sarcomeres of larval and adult muscles and localises to the Z-discs, as was shown using antibodies for α-Actn and D-Titin/Kettin as markers. Yeast interaction assays revealed that Rols7 might directly interact with α-Actn and Zormin, which, like Kettin, is an isoform derived from the sallimus (sls) gene and also localises to the Z-discs. Therefore, it is postulated that Rols7 serves as a scaffold protein that links α-Actn and Zormin in the Z-disc. Furthermore, the analyses of alpha actinin mutants showed that the presence of α-Actn is not necessary for Rols7 localisation to the Z-discs. In addition, Rols7, as well as α-Actn and D-Titin/Kettin, is present during the assembly of the sarcomere. In vertebrates it has been shown that in spreading edges of rat cardiomyocytes, dense bodies that contain Z-disc proteins assemble at the spreading membrane and align to premyofibrils in cooperation with newly formed actin filaments and small myosin filaments (Kreiskother, 2006).

Antibody staining showed protein aggregates that aligned to form kinds of premyofibrils and demonstrated that in Drosophila, the assembly of the Z-discs seems to be similar to that of vertebrates. So, Rols7 is the first protein that is essential for myoblast fusion and plays an additional role in the sarcomere assembly as well as in the Z-discs of mature muscles, where it is proposed that it links α-Actn and D-Titin/Kettin/Zormin. Δ-titin/kettin-mutants have a weaker fusion phenotype than rols7-mutants, however, D-Titin/Kettin is clearly expressed during myoblast fusion as a component of the adhesion complex between founder cell and fcm. Individual Rols7 domains serve different function in distinct processes of myogenesis (Kreiskother, 2006).

From coimmunoprecipitation experiments it was already supposed that the intracellular domain of Duf interacts with Rols. Furthermore, cell culture cotransfection assays showed colocalisation of Duf, Rols and D-Titin. In yeast interaction assays the individual domains of Rols7 were tested for interaction with potential partners that included components of the fusion machinery which might be relevant for muscle attachment or sarcomere assembly as well. Indeed, the different domains interact with different partners in different developmental contexts, and it is concluded that Rols7 is a multifunctional protein (Kreiskother, 2006).

The interaction of Rols7 with the intracellular domain of Duf/Kirre was confirmed and it was found that the interaction probably is mediated by the TPR repeats of Rols7, respectively, by the most C-terminal TPR E repeat and the R1 fragment that contains the RING finger and an additional part of 321 amino acids downstream. In contrast α-Actn interacts with the R1 domain and with both TPR repeats, the TPR E and the TPR X, whereas the N-terminal part of Zormin interacts only with the R1 domain in this assay. No interaction was detected for the N-terminal part of Kettin and the Rols7 domains. These results, together with the rescue capability of truncated Rols7 versions, led to the proposal of certain functions to individual Rols7 domains. Either the RING finger domain, the TPR repeats or the ankyrin repeats and the TPR repeats have been deleated and the remaining parts of Rols7 were examined for their competence to rescue the rols fusion defect. A deletion of the RING finger domain does not affect the rescue of the rols fusion phenotype, whereas a deletion of the TPR repeats leads to a partial rescue and a deletion of ankyrin repeats and TPR repeats together does not rescue fusion at all. The Rols7 version without the RING finger rescues the fusion phenotype. This RING finger is included in the R1 fragment which interacts with Duf/Kirre^intra, Blow, Zormin and α-Actn. Thus, it is proposed that the R1 domain is a candidate to mediate the transient interaction of Duf/Kirreinttra at the muscle attachment sites. The R1 domain of Rols7 could then mediate the interaction with Zormin in the Z-discs in all larval muscles. R1 is the only Rols7 fragment that interacts with Zormin. R1 and TPR E as well as TPR X have the capability to interact with α-Actn. It cannot be decide whether both domains of Rols7 interact with α-Actn in the Z-discs. The interaction of Duf/Kirre^intra with the TPR E repeat indicates a function of the TPR E repeat during myoblast fusion, since its deletion only leads to a partial rescue of the rols fusion phenotype. The ankyrin repeats did not interact with any of the proteins which have been tested in the yeast assay and which are characteristic for sarcomere assembly and muscle attachment. Taking this together with the fact that a deletion of this domain, in addition to a deletion of the TPR repeats, prevents the rescue of the fusion defect, indicates that the ankyrin repeats predominantly function during myoblast fusion (Kreiskother, 2006).

Rols7 is a scaffold protein which contains distinct domains characteristic of protein-protein interaction. It is proposed that the interaction of the appropriate domain with certain proteins is specific for the process of myogenesis, myoblast fusion, muscle attachment or sarcomere assembly (Kreiskother, 2006).

WIP/WASp-based actin-polymerization machinery interacts Dumbfounded and Sticks and Stones to facilitate myoblast fusion in Drosophila

Formation of syncytial muscle fibers involves repeated rounds of cell fusion between growing myotubes and neighboring myoblasts. Wsp, the Drosophila homolog of the WASp family of microfilament nucleation-promoting factors, is an essential facilitator of myoblast fusion in Drosophila embryos. D-WIP (termed Verprolin 1 in FlyBase), a homolog of the conserved Verprolin/WASp Interacting Protein family of WASp-binding proteins, performs a key mediating role in this context. D-WIP, which is expressed specifically in myoblasts, associates with both the WASp-Arp2/3 system and with the myoblast adhesion molecules Dumbfounded and Sticks and Stones, thereby recruiting the actin-polymerization machinery to sites of myoblast attachment and fusion. This analysis demonstrates that D-WIP recruitment is normally required late in the fusion process, for enlargement of nascent fusion pores and breakdown of the apposed cell membranes. These observations identify cellular and developmental roles for the WASp-Arp2/3 pathway, and provide a link between force-generating actin polymerization and cell fusion (Massarwa, 2007).

The evolutionarily conserved Arp2/3 protein complex is the primary microfilament-nucleating machinery in eukaryotic cells. To perform its diverse cellular roles, the complex must first be activated by nucleation-promoting factors (NPFs), such as members of the WASp and WAVE/SCAR protein families. These elements serve as essential mediators, linking signal-transduction pathways and Arp2/3-based actin polymerization. Actin polymerization triggered by this system is translated into forces that drive a variety of key cellular functions, including cell locomotion, motility of membrane-bound particles within cells, and formation of endocytic vesicles (Massarwa, 2007).

A major challenge in the field is the assignment of physiological roles to this potent cellular machinery during the development of multicellular organisms. While genetic approaches in model organisms have shown promise in this regard, the numerous and sometimes overlapping roles assigned to the Arp2/3 system often prove difficult to separate. Previous work has shown that Wsp, the Drosophila WASp homolog, acts as an Arp2/3 activator in restricted developmental contexts, thus allowing for characterization of Arp2/3 function in vivo. This approach was used to reveal an unexpected involvement of the WASp-Arp2/3 system in myogenesis. Specifically, this system is shown to play a distinct role in myoblast fusion during Drosophila embryogenesis (Massarwa, 2007).

Somatic muscle fibers in the mature Drosophila embryo are comprised of multinucleated cells that form by multiple rounds of fusion between two distinct myoblast subpopulations. After the initial specification of the mesoderm, each embryonic trunk hemi-segment contains ~30 'founder cell' myoblasts, which will direct muscle formation and differentiation, and a large number of fusion-competent myoblasts (FCMs). Founder cells possess the information necessary for determining the identity and size of the individual somatic muscles, while the FCMs serve as a repository that will add cytoplasmic bulk to each muscle fiber (Massarwa, 2007).

Recognition and association of founder cells and FCMs are based on heterotypic interactions between differentially expressed immunoglobulin superfamily cell-surface proteins. Founder cells express Dumbfounded (Duf) and the closely related Roughest (Rst), which serve as attractants for FCMs. Physical association between Duf/Rst and the FCM-specific protein Sticks and Stones (SNS) provides a key step in myoblast adhesion and alignment of the myoblast cell membranes. Founder cells initially fuse with one or two FCMs, leading to the formation of bi-/trinuclear muscle precursors. A second, major phase of muscle growth then ensues, in which the precursor myotubes undergo successive rounds of fusion with multiple FCMs. In addition to the cell-adhesion molecules, genetic approaches have revealed a number of elements that contribute to various steps of the fusion process, including transcription factors, signaling molecules, and cytoskeleton-associated proteins (Massarwa, 2007).

This study demonstrates that function of the WASp-Arp2/3 system is essential for the second phase of myoblast fusions, between maturing myotubes and FCMs, and acts after formation of fusion pores in the double membrane of the apposed cells. Recruitment of the WASp-Arp2/3 system to founder cell-FCM attachment sites is achieved via D-WIP, a Drosophila homolog of the Verprolin/WASp Interacting Protein (Vrp/WIP) family. Functional associations with members of this protein family constitute an evolutionarily conserved feature of WASp activity. D-WIP is specifically expressed in myoblasts and associates with the cell-surface proteins that mediate adhesion between founder cells and FCMs, thereby establishing a critical link between the cellular machineries that govern fusion and microfilament dynamics. These findings present a novel tissue context for the involvement of the Arp2/3 system in physiological events and extend the functional applications of the forces generated by actin polymerization to a central process of tissue morphogenesis (Massarwa, 2007).

This study has identified an exceptional and highly cell-type-specific mode for regulating the Arp2/3 system. Functional selectivity in this system is usually achieved via spatial and temporal control over the operation of signal-transduction pathways and the resulting production of potent activating elements for the relevant Arp2/3 nucleation-promoting factor. In contrast, it is the restricted expression of D-WIP in the FCMs that confines Wsp-mediated triggering of Arp2/3 activity to the fusing myoblasts of Drosophila embryos. Transcriptional control over D-WIP expression, governed directly or indirectly by the Lame Duck (Lmd) transcription factor, thus provides a means for translating embryonic patterning schemes into distinct and specific cellular activities, which can profoundly influence cell morphology (Massarwa, 2007).

The structural basis for the interaction between D-WIP and Wsp is consistent with the established principles of Vrp/WIP-WASp protein association, which rely on an interaction between an ~25 residue long peptide from the extreme C-terminal region of Vrp/WIP proteins and the WH1/EVH1 N-terminal region of WASp proteins. Most critical residues within these domains are conserved in the Drosophila homologs. Moreover, genetic data and S2 cell localization observations strongly implicate these domains in mediating physical association between the two proteins (Massarwa, 2007).

By virtue of its association with the cell-surface adhesion proteins Duf and SNS, expressed in founder cells and FCMs, respectively, D-WIP may impose a common functionality on these distinct myoblast types. Yet to be determined, however, is the nature of the interaction between D-WIP and the myoblast-attachment machinery, and whether this interaction is constitutive or is dependent upon founder cell-FCM contact. Colocalization in both developing embryonic muscles and aggregated S2 cells, as well as the coimmunoprecipitation of D-WIP and Duf, underlies the suggestion of a physical association, but whether this association is direct requires further investigation (Massarwa, 2007).

The lack of significant sequence homology between the cytoplasmic portions of the Duf and SNS proteins, and the comparatively tighter correspondence between D-WIP and SNS localizations, may be indicative of distinct modes of association between D-WIP and the two types of adhesion proteins. It is interesting to note in this context that mammalian Nephrin, which shares structural and sequence similarities with SNS, employs direct binding of its cytoplasmic portion to the adaptor protein Nck, as a means of establishing a functional link to the actin-based cytoskeleton (Massarwa, 2007).

WASp-family proteins are thought to reside in an auto-inhibited conformation, which prevents productive interaction with Arp2/3 and is alleviated only by binding of signaling molecules. Scenarios consistent with a recruiting role for Vrp/WIP proteins have been described, including involvement of WASp in actin-based motility of intracellular pathogens and in cytoskeletal remodeling of the immune synapse. However, Vrp/WIP proteins on their own fail to stimulate, or may even inhibit, WASP-based Arp2/3 activation (Martinez-Quiles, 2001: Ho, 2004), implying a requirement for additional activating elements. The observation that Wsp^Myr, a membrane-tethered form of Wsp, can partially compensate for loss of D-WIP function is consistent with an exclusive recruitment role for D-WIP. However, it should be born in mind that an additional step of Wsp activation may be required after its recruitment. Since the results of phenotypic rescue experiments further imply that established activators of WASp-type proteins such as CDC42 and PIP₂ do not operate in this context, the identity of an independent Wsp activator during myoblast fusion, if one indeed exists, is currently unknown (Massarwa, 2007).

Activation of the Arp2/3 complex promotes the generation of branched networks of polymerizing actin filaments, in close proximity to both the cell surface and to internal cell membranes. The physical force liberated by this energetically favorable process can be harnessed to push against, or otherwise influence, membrane behavior. A key challenge stemming from the experimental observations is to identify the mechanism by which Arp2/3-based force production contributes to the progress of myoblast fusion (Massarwa, 2007).

The detailed TEM-level description of Drosophila myoblast fusion has stipulated a series of events, including formation of pores next to sites of accumulated electron-dense material along the apposed myoblast membranes, vesiculation/fragmentation of the membranes between the pores, and removal of the residual membrane material. Analysis of the D-WIP and Wsp mutant phenotypes demonstrates a requirement for the Arp2/3 system at a relatively late stage of the fusion process, after formation of the initial fusion pores (Massarwa, 2007).

Much of what is known about the mechanisms driving cell-cell (including myoblast) fusion relates to recognition and adhesion between pairs of cells and construction of initial fusion pores, while the more advanced processes of pore enlargement and the eventual establishment of full cytoplasmic continuity between the fusing cells remain mostly unexplored. The demonstration of a requirement for the cellular actin-polymerization machinery at these stages holds the promise of establishing a mechanistic basis for these late events (Massarwa, 2007).

Several possible mechanisms can be proposed for the manner by which polymerization-based forces drive fusion to completion, after initial pore formation. Pore enlargement during membrane fusion poses considerable energy requirements, which Arp2/3-based polymerization seems well suited to satisfy. The 'pushing' forces inherent in this cellular machinery can be applied to the contours of nascent fusion pores, thereby ensuring their continuous expansion. Alternatively, myoblast membranes may be broken down by vesiculation, akin to endocytosis. Detailed genetic and cellular studies have demonstrated essential roles for the Vrp/WIP-WASp-Arp2/3 machinery during endocytosis of clathrin-coated vesicles in budding yeast, and mechanistic interpretations of the forces involved have been put forward. In keeping with previous discussions of these issues, it is tempting to suggest that electron-dense structures, common to the contact sites of myoblasts in both Drosophila and vertebrate species, may provide a structural framework through which polymerization-based forces exert their influence. Finally, a role for the Arp2/3 machinery can be invisioned in an even more advanced step in the fusion process, namely, the final removal of residual, vesiculated membrane material from the disrupted sites of membrane contact to create full cytoplasmic continuity (Massarwa, 2007).

In summary, these observations linking myoblast cell-surface adhesion proteins in Drosophila embryos with the WIP/WASp module suggest a mechanism through which the conserved cellular machinery promoting force production via microfilament nucleation can be harnessed to drive muscle fiber formation to completion. Future studies will determine the finer mechanistic details of the cellular mechanism employed in this instance, and the degree to which this link can be generalized to myogenesis in vertebrate species, as well as other processes of cell fusion (Massarwa, 2007).

The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner

Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner (Schizo). Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. This study analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. This study shows that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. It was also shown that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf (Bulchand, 2010).

This study has shown that in order to ensure successful fusion a large part of the intracellular region of Duf is required for its function. Serial truncations of the intracellular domain reveal that the efficiency of fusion is decreased as larger regions are removed. Also, conserved putative phosphorylation signalling sites function additively resulting in efficient myoblast fusion and the formation of a mature myotube. Several parallels can be drawn from this data and that published by Kocherlakota (2008), on the intracellular domain of the Duf ligand SNS. Similar to what has been found for SNS, the PDZ binding domain is not required for the function of Duf during myoblast fusion. This is contrary to the role of this domain in the function of Rst in the developing eye. While the intracellular domain of SNS is important for its function, the C terminal end of SNS is dispensable similar to that of Duf as shown by Duf ^ΔCT1-flag in the Rols/Loner translocation assay in S2 cells and rescue of the fusion defect in duf, rst embryos. The membrane proximal intracellular regions of SNS and Duf are more important for their functions. While SNS is phosphorylated on tyrosine residues, the ability of Duf ^{4 phos-flag} to only partially rescue the duf, rst mutant, implies that phosphorylation of these sites also contributes to Duf function (Bulchand, 2010).

Membrane anchored forms of Duf irrespective of the sequence of the transmembrane domain, appear to be sufficient for successful fusion. This suggests that the transmembrane domain of Duf does not perform any essential role or contribute to downstream signalling activity and only serves to anchor Duf to the plasma membrane. The PADVI motif, though not essential for myoblast fusion, might have a function in the context of a different tissue type that has not been tested so far. That the functions of Duf cannot be attributed to particular motifs might be a strategy utilised to ensure that normal myotube development occurs in a robust manner and compromising the function of any of these motifs singly, does not drastically affect the overall process. As has been suggested for the downstream functions of SNS, Duf too might transduce signals to cytoskeletal elements via its intracellular domain, to ensure successful myoblast fusion (Bulchand, 2010).

Previous studies proposed that myoblast fusion molecules can be categorised into those that participate in the early versus later phases of fusion. More recently it has been proposed that all fusion molecules are required in all fusion events. Molecules like Rols and Loner have been individually shown to function in the second phase of fusion after the formation of the bi/trinucelate precursor. This study has shown that removal of both rols and loner completely blocks fusion similar to the duf, rst mutant. Analyses of other similar double mutants demonstrate that genes involved in myoblast fusion might interact with each other to affect fusion efficiency. It is possible that what this study has shown with a few myoblast genes is true for other genes that have thus far been characterised for their role in the later stages of fusion. Such interactions have been shown for Kette/Hem/Nap1/GEX-3 and Blow (Bulchand, 2010).

This study has shown that membrane anchored Duf without its intracellular domain and without any interaction with Rols and Loner, is sufficient to initiate fusion. It is possible that even in the absence of robust Duf dependent signal transduction, requirements for the formation of a bi/trinucleate precursor are met. It was also shown that Rols and Loner are required, albeit redundantly, for precursor formation or the initial phase of fusion suggesting that this 'early function' of these molecules appears to be independent of Duf. This fusion defect was observed in late stage 15-early stage 16 embryos to ensure that the observations and interpretation thereof are not due to a delay in fusion. Rols and Loner may perform different roles early versus later on during myoblast fusion. In the later phase of fusion, Rols and Loner appear to sustain fusion by interacting with and translocating Duf to the surface of the myotube. As has been suggested in the case of Rols, Loner too might serve to regulate Duf at the surface of the myotube through as yet unknown mechanisms. It is possible that these supposed distinct early versus late mechanisms are used in mutant conditions in an effort to overcome fusion blocks, thus leading to delayed fusion events (Bulchand, 2010).

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies

Drosophila nephrocytes are functionally homologous to vertebrate kidney podocytes. Both share the presence of slit diaphragms that function as molecular filters during the process of blood and haemolymph ultrafiltration. The protein components of the slit diaphragm are likewise conserved between flies and humans, but the mechanisms that regulate slit diaphragm dynamics in response to injury or nutritional changes are still poorly characterised. This study shows that Dumbfounded/Neph1, a key diaphragm constituent, is a target of the Src kinase Src64B. Loss of Src64B activity leads to a reduction in the number of diaphragms, and this effect is in part mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation. The phosphorylation of Duf by Src64B, in turn, regulates Duf association with the actin regulator Dock. Diaphragm damage induced by administration of the drug puromycin aminonucleoside (PAN model) directly associates with Src64B hyperactivation, suggesting that diaphragm stability is controlled by Src-dependent phosphorylation of diaphragm components. These findings indicate that the balance between diaphragm damage and repair is controlled by Src-dependent phosphorylation of diaphragm components, and point to Src family kinases as novel targets for the development of pharmacological therapies for the treatment of kidney diseases that affect the function of the glomerular filtration barrier (Tutor, 2013).

Many of the vertebrate slit diaphragm proteins are tyrosine phosphorylated in normal glomeruli. Furthermore, tyrosine phosphorylation of nephrin and Neph1 by the SFK Fyn is crucial for the stability of this protein complex and therefore for glomerular filtration function. Thus, upon phosphorylation, the cytoplasmic regions of nephrin and Neph1 recruit the intracellular adaptors Nck and Grb2, among others, that in turn regulate actin cytoskeleton reorganisation. This study found that the post-translational regulation by phosphorylation of Duf, the orthologue of Neph1, is conserved and that Src64B, a member of the non-receptor Src family tyrosine kinases, is responsible for Duf tyrosine phosphorylation in nephrocytes. Furthermore, Src64B function is necessary for the structural integrity of the filtration diaphragm and for normal nephrocyte morphology. In Src64B loss-of-function or knockdown conditions, there is a reduction in the density of filtration diaphragms at the nephrocyte cell membrane. Presumably, this is due in part to their internalisation, as suggested by the accumulation of both Duf and Pyd at protrusions extending inwards from the membrane and the presence in these locations of structures dense to electrons reminiscent of filtration diaphragms. In addition, delocalisation was observed of Duf/Pyd complexes to cell contact membranes that, at the ultrastructural level, are rich in adherens junctions. The presence of adherens junctions in apposed membranes is never found in wild-type mature nephrocytes but is characteristic of embryonic nephrocytes prior to the formation of filtration diaphragms. All these alterations are concomitant with changes in nephrocyte architecture, revealed by their smoother surface and their agglutination. As the loss of filtration diaphragms result in regression of labyrinthine channels, and this is always associated with nephrocyte agglutination, these morphological changes are interpreted as being due to reallocation of Duf/Pyd complexes from SD to adherens junctions. It is suggested that these morphological alterations are the nephrocyte equivalent of podocyte foot process effacement, a feature common to all proteinuric diseases and believed to be initiated by changes in the actin cytoskeleton (Tutor, 2013).

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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, N^ts1. Because of the close proximity of the duf and Notch loci, duf-lacZ, N^ts 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 N^ts 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 N^ts 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).

Coordinated development of muscles and tendons of the Drosophila leg

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 regulate myoblast proliferation and patterning in Drosophila: Expression of the founder-cell marker Dumbfounded/Kirre in imaginal pioneer cells

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).

Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes

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).

Live imaging provides new insights on dynamic F-Actin filopodia and differential endocytosis during myoblast fusion in Drosophila

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 (see Drosophila Arp2/3 component Arpc1) pathways. This study utilized the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. Differential endocytosis of cell surface components was observed at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube (Haralalka, 2014: PubMed).

EFFECTS OF MUTATION

The requirement for duf was analyzed in Df(1)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30 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)w^67k30, attempts were made to rescue the phenotype by supplying duf function in the mesoderm of Df(1)w^67k30 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)w^67k30 embryos is sufficient to rescue the fusion phenotype (Rúiz-Gomez, 2000).

The expression pattern of duf, the failure of myoblast aggregation in Df(1)w^67k30 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).

Function of roughest and its paralog kirre during muscle development

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 mbc^C1 genetic background. In mbc^C1 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)w^67k30 causes embryonic lethality and displays an almost complete lack of myoblast fusion. The genomic interval removed by Df(1)w^67k30 extends from white to kirre. As yet, there is no single embryonic lethal locus known within this region. Hence, the Df(1)w^67k30 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)w^67k30 phenotype. Therefore, removal of kirre is partly responsible for the Df(1)w^67k30 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)w^67k30 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)w^67k30 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 rst^irreC1 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)w^67k30 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 formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc

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 mbc^C1. 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).

REFERENCES

Search PubMed for articles about Drosophila Kirre/Dumbfounded

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date revised: 2 February 2023

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