The embryonic expression pattern of the sns transcript was examined using a digoxigenin labeled cDNA fragment. The earliest expression of sns is seen during stage 11 in the visceral mesoderm, and in the somatic mesoderm prior to the onset of myoblast fusion. Although expression in the visceral mesoderm diminishes as germ band retraction proceeds, expression persists in the somatic mesoderm at high levels until stage 14, during which time myoblast fusion is taking place. No expression was evident in the visceral musculature or in the dorsal vessel at this time. By stage 15, transcript levels have also declined in the somatic musculature, and by stage 17 only faint expression could be detected. During stage 17, weak expression is also seen in the muscle attachment sites (Bour, 2000).
Antisera generated against the carboxy-terminal portion of the Sns protein confirmed that the pattern of protein expression is similar to that of the transcript. Confocal microscopy confirmed the co-expression of Sns and FASIII in the visceral musculature. As anticipated for a putative cell adhesion molecule, both colorimetric and immunofluorescent confocal staining revealed the enrichment of the Sns protein at the cell membrane. In addition, Sns appears to become localized to discrete sites in the membrane as fusion progresses . This membrane localization of Sns clusters has been confirmed by examination of serial confocal sections through the embryo. Like its transcript, Sns also appears to decline significantly as fusion progresses, such that little expression is observed in multinucleate syncitia (Bour, 2000).
Three independent experimental approaches have been used to address whether Sns is expressed in both the founder cells and putative fusion competent cells of the somatic mesoderm. In the first approach, wild-type embryos were double-labeled with antisera against Sns and various founder cell markers, and examined by confocal microscopy. Markers included the enhancer trap rP298-lacZ and even-skipped. In brief, Sns expression is not detected in isolated cells that express rP298-lacZ. These isolated cells were observed in many confocal sections, in embryos in several orientations. Because most of these are not near the ventral nerve cord, it is inferred that these are unfused founder cells rather than rP298-lacZ expressing glial cells. A similar analysis addressed co-expression of the Eve founder cell marker and Sns. At the earliest appearance of the Eve-expressing founder cell, no Sns expressing cells are observed in its vicinity, consistent with the pattern of expression observed with rP298-lacZ. After a brief period of time, Sns-expressing cells are observed in close proximity, and begin to fuse almost immediately. These data suggest that Sns marks the fusion-competent cells shortly before fusion, and is not expressed in the founder cells (Bour, 2000).
Two additional analyses addressed whether Sns expression in the somatic musculature might be exclusive to the fusion competent cells. Both a non-null allele of sns and a null allele of mbc were used to determine whether Sns is expressed in morphologically distinct founder cells. Elongated cells were observed in both snsXS5 and mbcD11.2 mutant embryos immunostained with MHC but not with Sns, consistent with the interpretation that Sns is not expressed in the founder cells. A second analysis relied on the hypothesis that Notch mediates a cell-fate decision between muscle progenitors (from which the founder cells arise) and the putative fusion competent cells. Notch NXK11 mutant embryos were double-labeled with vestigial (Vg) another founder cell marker, and Sns. Wild-type embryos exhibit normal distribution patterns of both markers. By comparison, one observes a dramatic expansion in the number of Vg-expressing founder cells in NXK11 mutant embryos, in agreement with studies using other founder cell markers. Of most significance is a dramatic reduction in the number of Sns-expressing cells in these mutant embryos. This observation supports the hypothesis that Notch mediates a cell-fate decision between muscle progenitors and fusion-competent cells, that Notch is necessary for the selection of fusion-competent myoblasts, and that Sns specifically marks this population of myoblasts (Bour, 2000).
The sns locus, which is essential for myoblast fusion, was uncovered during an F2 lethal screen for EMS-induced point mutations in cytological region 95A on the third chromosome. In this screen, the original mutagenized fly was later found to have contained two recessive lethal mutations, one in the region of interest on the third chromosome and one on the second chromosome. Genetic mapping revealed that the muscle defect segregated with the second chromosome, and the recovered mutant locus was named sticks and stones (sns). Examination of the developing body wall muscles in snsA3.24 mutant embryos revealed an almost complete block in myoblast fusion (Bour, 2000).
The muscle phenotype of embryos homozygous for the original snsA3.24 allele includes a large number of unfused myosin-expressing cells and a corresponding absence of differentiated muscle fibers. Embryos transheterozygous for this sns allele and Df(2R)BB1, which deletes the entire sns region, exhibit the same mutant phenotype. Thus snsA3.24 behaves as a null allele by genetic criteria. The presence of founder cells was then assessed using an antibody directed against Nautilus. Nau-expressing cells are detected in their correct positions in sns mutant embryos, but do not appear to fuse. These cells are capable of producing myosin heavy chain (MHC) protein, and appear to be analogous to the muscle founders described in mbc mutant embryos. The entire myoblast population was also examined using a polyclonal antibody directed against Mef2, an early marker for most if not all cells of the somatic musculature. Mef2 expression is detected in the myoblasts of sns mutant embryos, in numbers approximately equivalent to those observed in wild-type embryos. These results imply that the precursors of the somatic musculature begin their differentiation program in sns mutant embryos, but become blocked at the point of myoblast fusion. In contrast to the severe defects in the somatic musculature, only subtle defects were observed in constrictions of the visceral musculature (Bour, 2000).
The rost locus, located at cytological position 30, encodes another protein that was reported to be essential for myoblast fusion (Paululat, 1995; Paululat, 1997). However, the original rostP20-containing chromosome was recently shown to contain two mutations that affect muscle development, a P-element insertion in the rost locus and a second mutation that maps to genetic position 43-49 (Paululat, 1999). Because the doubly-mutant chromosome was used in an EMS mutagenesis screen to isolate noncomplementing mutations (Paululat, 1995), the resulting alleles could be in either locus. Due to the localization of this second mutation to cytological region 43-49, it was of interest to determine whether any of the EMS alleles obtained in this screen were, in fact, allelic to sns. Indeed, all sns mutant alleles fail to complement the 43-49 mutation. Recombinants of rost2023 that separated cytological regions 30 and 44 were also isolated and crossed to sns6.1. Recombinants that retained region 44 did not complement sns6.1, whereas recombinants that retained region 30 complemented sns6.1. These data strongly suggest that the rost2023 mutation is allelic to sns. In addition, the sns sequence was examined in several putative rost alleles by NIRCA. Through this analysis, rost202 was found to contain a C to T transition that results in a nonsense mutation at amino acid residue 367 of the sns gene (Bour, 2000).
Although the cytoplasmic and transmembrane domains of neither Duf/Kirre nor Sns play a significant role in the ability of these proteins to direct interaction of cultured S2 cells or become localized to points of cell-cell contact between the associated cells, the cytoplasmic domains of other members of the IgSF have been implicated in critical cell signaling events. It was therefore of interest to determine whether the cytoplasmic or transmembrane domain of Sns is required in the embryo to direct events more complex than those observed in cultured cells. To determine if the cytoplasmic/transmembrane domains of Sns are essential for myoblast fusion, the full length sns cDNA and sns-GPI were placed under UAS control, and transformed into flies. These transgenes were then introduced into an sns mutant background and their expression directed by the mesodermal expression of GAL4. Interestingly, mesodermal expression of the full length Sns is sufficient to rescue myoblast fusion in an sns mutant background, and these embryos grow into viable adults. However, the GPI-anchored form of Sns, which lacks both the transmembrane and cytoplasmic domains, is unable to rescue muscle formation in sns mutant embryos even when expressed from multiple copies of the transgene. The UAS-sns and UAS-sns-GPI transgenes have been sequenced in their entirety, express similar levels of protein in membrane preparations from embryos, as detected by Western blot, and do not exhibit dominant effects when expressed in wild-type embryos. Moreover, the fusion competent myoblasts did not appear to migrate toward the founder cells in these embryos. These data suggest that, in contrast to the ability to direct aggregation of S2 cells, the presence of the transmembrane and/or cytoplasmic domain of Sns is essential for Sns to direct myoblast fusion in the embryo (Galletta, 2004).
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. 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 (duf or kirre) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganized while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. These results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes (Klapper, 2002).
The gene mbc, a homolog of human DOCK180, is expressed in all somatic myoblasts during the fusion processes. In embryos mutant for mbc, syncytia within somatic muscles are almost absent, presumably due to defects in the rearrangement of the cytoskeleton during preceding myoblast fusion. Mbc is also expressed in the visceral mesoderm from stage 12 onward. Embryos mutant for mbc not only exhibit defects in the formation of midgut constrictions but also show severe abnormalities in the formation of visceral muscles. At stage 12 the visceral band is randomly interrupted and the elongated FAS III-expressing cells seem to be disoriented. During further development, parts of the visceral band either stretch out in the dorsoventral direction, as in the wild type, or form disarranged patches. The status of the founder cells in an mbc mutant background was analyzed by expressing rP298-LacZ in the strain mbcC1. At stage 14 the number of rP298-LacZ-expressing cells within the remaining population of myoblasts appears to be reduced and large gaps within the visceral band are visible that are not present in the wild type. Many globular cells are still observed at the margins of the visceral band that appear to be unfused myoblasts. Antibody staining reveals a prolonged expression of SNS within a corresponding subpopulation of myoblasts. As described for duf and sns mutant embryos, no myoblast fusions were detectable. While the circular musculature shows severe defects and is apparently reduced, the longitudinal musculature is completely absent at the end of embryogenesis (Klapper, 2002).
By using the GAL4/UAS transplantation system for cell lineage analyses of the mesoderm anlage, syncytia were detected not only within somatic muscles but also in the visceral musculature. Since the visceral muscles have classically been described as mononuclear, this surprising observation led an examination of syncytia formation within this tissue. Evidence is provided that the circular visceral muscles of the midgut are likewise organized as syncytia. The first signs of GFP expression (driven by daGAL4) within these muscles were observed in embryos at stage 15. Since there is a considerable delay of about 2-4 h between the activation of the UAS-GFP construct and the formation of the fluorescent product, it is assumed that the formation of syncytia begins at stage 12. This is consistent with the observation of the first fusion processes within the visceral band. Using GFP expression as an in vivo marker, individual syncytia could be followed throughout development. In contrast to longitudinal muscle fibers, which have been found to contain up to six nuclei, the circular muscles of hindgut as well as midgut always comprise two nuclei each. Thus, fusion processes within this tissue stop after the formation of binucleate minimal syncytia that each cover one-half of the gut tube. This is a curious finding since in other muscle types of Drosophila many nuclei share a common cytoplasm to generate a large structural and functional unit (Klapper, 2002).
Following labelled syncytia through all stages of development, it has been shown that the visceral musculature is not replaced by a newly formed imaginal tissue but persists through metamorphosis. Since the visceral musculature plays a crucial role for the proper formation of the midgut during embryonic development, the persisting visceral musculature might again serve as a template for remodelling of the gut tube during metamorphosis (Klapper, 2002).
Since myoblast fusions were discovered within the visceral musculature, whether these syncytia are also formed by Duf-expressing founder and Sns-expressing fusion-competent cells was examined. duf can be detected in muscle precursors as long as they incorporate further fusion-competent cells into the syncytia. The expression of Duf and Sns within the visceral mesoderm suggests a function of specifying founder and fusion-competent cells similar to that observed for the somatic musculature. To clarify whether these genes indeed play a functional role in visceral myoblast fusion, the phenotypes of sns and duf mutant embryos was examined with respect to syncytia formation within the developing visceral musculature. In wild type embryos the ventrally located cells of the visceral band exhibit all characteristics of muscle founder cells, in that they express Duf, become elongated and fuse specifically with Sns-expressing myoblasts. Without duf function no signs of visceral muscle fusion are detectable. It is therefore proposed that duf is the key component in the visceral muscle founder cells. This observation is in agreement with the postulated role of Duf as an attractant for fusion-competent cells in the somatic musculature (Klapper, 2002).
These results indicate that loss of duf function leads to a complete loss of fusion even though the two populations of founders and fusion-competent myoblasts are already aligned within the visceral band at stage 11. Therefore, in the visceral mesoderm, Duf expression seems not only to attract the fusion-competent cells but also to play a crucial role in the fusion process itself. Probably the gaps within the visceral band of duf mutant embryos may indicate an early Duf adhesion function necessary for a proper anterior-posterior alignment of the visceral myoblasts. Furthermore, despite initial cell alignment at stage 11, this contact between founder cells and fusion-competent myoblasts is lost during further development, so that additional gaps appear between these cell layers. In sns mutant embryos, again, no myoblast fusions were detectable. The phenotype closely resembles that of duf mutant embryos, since the two populations of founders and fusion-competent cells seem to reject one another. However, the large gaps within the visceral band of duf embryos were never observed in sns mutants. Whereas in the anterior part of the midgut severe defects of the visceral musculature are obvious at later stages of embryogenesis, the posterior part looks quite normal. This regional difference is somewhat difficult to explain, since at earlier stages no such regional distinctions were observed. It is thought that either in the posterior visceral band sns function can be mimicked or bypassed later on by another spatially restricted gene product, or that even unfused myoblasts can form a seemingly normal musculature in the posterior part. The stronger phenotype at later stages in duf mutant embryos may indicate that the founder cells play a more crucial role in visceral myogenesis than the fusion-competent cells. In somatic myogenesis, founder and fusion-competent cells play specific roles in the recognition process, while in mbc mutants the fundamental capability to fuse is lost in both cell types. Because here intracellular components of the fusion apparatus are affected, fusion-competent cells still aggregate around founders, but the tight membrane junctions are not formed and fusion does not occur. As is consistent with the somatic phenotype, in visceral myogenesis, fusion is totally blocked although founder and fusion-competent cells are in direct contact and still express Sns and Duf (Klapper, 2002).
Taken together, these results provide evidence that the founder-cell hypothesis also applies to visceral myogenesis employing the same genetic components as used in the somatic myoblast fusion processes. Thus, the specification of myoblasts as either founder or fusion-competent cells might be a fundamental step preceding syncytia formation (Klapper, 2002).
Previous analyses have provided evidence that the primordium for the longitudinal midgut musculature is located at the posteriormost tip of the mesoderm anlage. In byn mutant embryos the hindgut as well as the longitudinal musculature are absent. The longitudinal musculature can be rescued, if byn is ectopically expressed exclusively within the posterior tip of the mesoderm anlage, leading to the conclusion that the entire anlage of the longitudinal musculature is located within this posterior region. However, after transplantation within the central region of the trunk, mesoderm labelled longitudinal muscles were frequently obtained. Taking into account that the founder-cell hypothesis is also valid for this tissue, this seeming contradiction can now be solved. The founder cells of the longitudinal musculature that comprise the genetic information for the specific tissue identity arise from the posterior tip of the mesoderm anlage, while the fusion-competent cells originate from the entire trunk region as indicated by confocal analysis. During the fusion processes it is possible to distinguish between two different events of syncytia formation. (1) At stage 12 all the ventrally located founder cells simultaneously become extended dorsally. Each of them fuse with a single SNS-expressing fusion-competent cell, eventually differentiating into a binucleate circular muscle. (2) The remaining SNS-expressing cells migrate to both margins of the visceral band and successively fuse with the longitudinal founder cells that invade from the posterior tip of the mesoderm. It is thus reasonable that in byn mutant embryos the entire longitudinal musculature is missing, since the fusion-competent cells alone are not capable of differentiating longitudinal muscles (Klapper, 2002).
The body wall muscle of a Drosophila larva is generated by fusion between founder cells and fusion-competent myoblasts (FCMs). Initially, a founder cell recognizes and fuses with one or two FCMs to form a muscle precursor, then the developing syncitia fuses with additional FCMs to form a muscle fiber. These interactions require members of the immunoglobulin superfamily (IgSF), with Kin-of-IrreC (Kirre) and Roughest (Rst) functioning redundantly in the founder cell and Sticks-and-stones (Sns) serving as their ligand in the FCMs. Previous studies have not resolved the role of Hibris (Hbs), a paralog of Sns, suggesting that it functions as a positive regulator of myoblast fusion and as a negative regulator that antagonizes the activity of Sns. The results reported in this study resolve this issue, demonstrating that sns and hbs function redundantly in the formation of several muscle precursors, and that loss of one copy of sns enhances the myoblast fusion phenotype of hbs mutants. It was further shown that excess Hbs rescues some fusion in sns mutant embryos beyond precursor formation, consistent with its ability to drive myoblast fusion, but show using chimeric molecules that Hbs functions less efficiently than Sns. In conjunction with a physical association between Hbs and SNS in cis, these data account for the previously observed UAS-hbs overexpression phenotypes. Lastly, it was demonstrated that either an Hbs or Sns cytodomain is essential for muscle precursor formation, and signaling from IgSF members found exclusively in the founder cells is not sufficient to direct precursor formation (Shelton, 2009).
Sns and Hbs function redundantly in the initial fusion event between founder cells and FCMs. As observed in other mutants, precursor formation in sns mutant embryos is delayed over that occurring in wild-type embryos, but is readily observed in stage 13 embryos in at least some segments. By contrast, no fusion was observed by late stage 15 in sns, hbs double mutant embryos. Although the possibility of a temporal delay of fusion in sns, hbs double mutants cannot be eliminated because reporter expression declines after this stage, a model is favored in which a crucial first step is not occurring in the absence of both Sns and Hbs. Using new FCM reporters that facilitate quantitation of unfused myoblasts, re-examination of the hbs loss-of-function phenotype reveals that the loss of one copy of sns actually worsens the hbs mutant phenotype, as expected if these proteins have some functional redundancy. Finally, both snsGal4 and mef2Gal4 directed Hbs can drive a significant amount of fusion in sns mutants, arguing that Hbs is capable of directing fusion beyond precursor formation (Shelton, 2009).
Although Hbs can rescue the sns mutant phenotype beyond precursor formation, replacing any domain of Hbs with the comparable domain of Sns improves the ability of the chimeric protein to rescue fusion over that achieved by Hbs alone. The activity of the Hbs cytodomain is most dramatically different from that of Sns, providing an explanation for the observation that intact Hbs or a membrane-anchored Hbs cytoplasmic domain both interfere with myoblast fusion in wild-type embryos. Rather than acting as an antagonist of Sns, these high levels of Hbs probably interfere competitively with endogenous Sns. First, an excess of Hbs may drive its interaction with a limiting component that is normally used more efficiently by Sns. Alternatively, given their ability to form hetero- and homodimers in vivo, excess Hbs may sequester Sns in a less functional form. Although the data do not fully resolve this issue, the co-localization of Hbs and Sns is consistent with the latter model. Of note, dimer formation between the related IgSF proteins Boc and Cdo can be directed by sequences in both the extracellular and intracellular domains (Kang, 2002), and both the extracellular and intracellular domains of Sns are capable of mediating its interaction with Hbs, raising the possibility that either full-length Hbs or a membrane-anchored cytodomain may sequester Sns under conditions of overexpression (Shelton, 2009).
The finding that Hbs functions positively but much less efficiently than Sns in directing later rounds of myoblast fusion provides an explanation for the previously observed behavior of Hbs in overexpression assays (Artero, 2001; Dworak, 2001). Additionally, the data appear to be inconsistent with a model in which excess Sns is deleterious, as inferred if a decrease in sns copy number compensates for the loss of hbs (Artero, 2001). The possibility that Sns activity is negatively regulated cannot be excluded. Possible mechanisms could include limitations in the machinery for tyrosine phosphorylation, such that unphosphorylated Sns even in excessive amounts would be unable to transduce a signal to downstream events. Downstream targets of Sns may also be limiting, such that no further activation of the pathway can be accomplished by Sns. It is also noted that Sns protein is transient, appearing just before fusion and being eliminated shortly thereafter. Despite the issue of whether Sns activity is regulated in some fashion, the data are not consistent with a model in which its activity is negatively regulated by endogenous Hbs (Shelton, 2009).
Current models for myoblast fusion suggest that it occurs in two steps that differ genetically and/or temporally. Consistent with the two genetically distinct steps, fusion does not occur in embryos mutant for genes encoding the guanine nucleotide exchange factors Schizo, Mbc or Duf and Rst. By contrast, precursor formation is observed in embryos lacking the Hem-2/Nap1 homolog Kette, the Kirre-associated protein Rols, the Arp14D/66B regulators WASp and Vrp1 or Sns. These data support a model in which the molecular requirements for precursor formation differ from those for subsequent myotube formation. An alternative model, using three dimensional analyses and quantitating fusing myoblasts over time, revealed that fusion occurs in two temporal phases, comprising an initial phase of limited fusion between cells that are in close proximity and a second phase when most myoblast fusion occurs. Moreover, precursor formation is temporally delayed in embryos lacking molecules such as Rols and Kette, suggesting that these molecules do influence the first step in fusion (Shelton, 2009).
The present study does not address whether the genetic requirements for precursor formation differ from those for subsequent rounds of fusion, or whether these steps utilize the same set of proteins. The data do not eliminate the possibility of two distinct genetic steps, with Sns and Hbs acting redundantly in precursor formation but not in later events. Hbs is capable of directing precursor formation in the absence of Sns. However, the ability of Hbs to drive fusion beyond precursor formation when in excess, and the observation that removal of one copy of sns enhances fusion defects in hbs mutants, suggests that Hbs can assist in later rounds of myoblast fusion. These data are consistent with models in which molecular interactions in precursor formation and subsequent fusion differ kinetically but not genetically. One possibility, independent of the process of fusion itself, is that Sns and Hbs differ in their ability to drive FCM cell migration. Although the role of cell migratory behavior in myoblast fusion is unclear, the ability to migrate may contribute to the rate of fusion. While these questions remain to be addressed, the present study advances the understanding of fusion by resolving the interaction of two proteins that function early in the process, thereby providing additional perspectives for sorting out the different mechanisms of myoblast fusion (Shelton, 2009).
Reference names in red indicate recommended papers.
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date revised: 10 February 2010
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