slouch is expressed in several regions: in small subsets of cells in the somatic mesoderm, in specific muscle founder cells, in the CNS, and in a small region of the endoderm. slouch is first expressed at midstage 11, during the third mesodermal mitosis. It is restricted to muscle precursors. At stage 15, after the yolk sac has been surrounded by the fusion of anterior and posterior primordia of the midgut, slouch is expressed in a narrow band of endodermal cells. Expression in the CNS starts during early stage 11, in very specific neural precursors (Dohrmann, 1990).

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that act as founders for larval muscles or form precursors of adult muscles. The formation of individual muscle fibers is seeded by a special class of founder myoblasts that fuse with neighboring mesodermal cells to form the syncytial precursors of particular muscle. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. During normal development (embryonic stage 11), two ventrally located progenitor cells divide once to produce three muscle founders and the precursor of an adult muscle (known as a persistent Twist cell because of its continued expression of twist). The more dorsal of the two progenitors divides, first giving rise to the founders of muscles VA1 and VA2, followed by the more ventral progenitors which produce the VA3 founder and the ventral adult persistent Twist precursor (VaP). As the progenitors divide, Numb is included in one of the two dorsal progenitors and in one of the two ventral progenitors. Thus the division of a muscle progenitor produces an unequal distribution of Numb between the founders: one contains Numb, the other does not. In numb mutants, some muscles are lost and others are transformed. For example VA1 and VaP are duplicated and VA2 and VA3 are lost. Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. Kruppel, S59 and even skipped expression mark a subset of the developing muscles. In numb mutants the expression of Kruppel, S59 and even skipped is initiated normally but is lost from both founder cells after they are formed. Thus in numb mutants there are no muscles that express Kr, eve or S59. In contrast, when numb is ectopically expressed throughout the mesoderm, Kr, S59 and eve expression are maintained in both founders and in the muscle precursors to which they give rise. In these embryos, Kr, S59 and eve-expressing muscles are duplicated (Gomez, 1997).

Effects of Mutation or Deletion

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).

The results from the injection of nautilus dsRNA point to a more general approach for the analysis of gene function during Drosophila development and suggest that the RNA interference method essentially would mimic a gene knock-out in the injected generation of Drosophila embryos. To test this idea a variety of cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null mutant. Daughterless mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region). In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern for embryos injected with S59 dsRNA, although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).

Downstream of identity genes: muscle-type-specific regulation of the fusion process

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. To understand how cell fate information residing within founders is translated during differentiation, this study focused on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Muscle protein 20 (Mp20), Paxillin (Pax), and M-spondin (mspo), three genes that regulate actin dynamics and cell adhesion and, as is shown in this study, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation (Bataillé, 2010).

The myoblast fusion is asymmetric and takes place between founder cells (FCs) and fusion competent myoblasts (FCMs). Previous reports originated the idea that FCMs are not 'naive' myoblasts and contribute to the modulation of fusion process. In contrast, the current results support a view that FCs rather than FCMs carry the instructive information and lead to the conclusion that FCMs do not play an active role in setting the number of fusion events. However, because the spatial distribution of FCMs seems to be nonuniform, it is conceivable that the local distribution of FCMs is coordinated with the requirements of FCs to facilitate fusion process (Bataillé, 2010).

The identity genes lb, slou, and eve are required to specify FCs at the origin of five muscles the DA1, DT1, SBM, VA2, and VT1. This study provides evidence that these identity genes are also required for setting the muscle-specific number of fusions and demonstrates how this identity information is executed. After specification step, FCs fuse, between the embryonic stage 12 and 15, with a determined number of FCMs to generate muscles with a specific number of nuclei. During this time period eve, lb, and slou continue to be expressed in subsets of developing muscles and the data show that they are sufficient to establish the muscle-specific fusion programs in DA1, SBM, and VT1 (11, 7, and 4 nuclei, respectively). Furthermore, slou in combination with other factors contributes to two other programs that end up with seven to eight fusion events in muscles DT1 and VA2. To regulate number of fusion events eve, lb, and slou act by modulating expression of genes involved in dynamics of actin cytoskeleton or cell adhesion. Starting from stage 13, they establish a muscle-specific combinatorial code of expression levels of three targets: Mp20, Pax, and mspo. The combination of expression of the targets leads to the muscle-specific control of the number of fusion events. This notion is supported by the fact that each of identity genes is able to impose at ectopic locations the combinatorial realisator code of Mp20, Pax, and mspo expression, and thus, ectopically execute its fusion program. Given that the code of Mp20, Pax, and mspo is not sufficient to explain fusion programs in all muscles, it is hypothesized that other identity gene targets exist that modulate fusion counting (Bataillé, 2010).

The data support a two-step model of myoblast fusion according to which a muscle precursor is formed between stage 12 and 13 by an initial fusion, and then, between stage 13 and 15, fuses with additional myoblasts until the muscle reaches its final size. The fact that Mp20, Pax, and mspo are expressed from stage 13 suggests that the transition point between the two steps depends not only on the timing of FCM migration but also on the activation of limiting factors such as the identity gene targets which modulate the number of additional fusions. Since no nuclear divisions were observed in FCs or in growing myotubes in any of the genetic contexts analyzed, it can confidently be said that the number of nuclei present in each muscle is determined only by the number of fusion events (Bataillé, 2010).

Specification of FCs requires combinatorial code of activities of identity genes. This study shows that the same identity genes play instructive roles in subsequent muscle-type-specific differentiation process. Importantly, the data enlighten the fact that the identity genes are not equivalent and have distinct, context-dependent mode of action. eve, lb, and slou are sufficient to set the fusion programs in DA1, SBM, and VT1 muscles; however, in VA2 and DT1 slou functions in a different way and seems not to have a decisive role in this process. Because the specification of the VA2 and DT1 FCs also involves functions of Poxm, Kr, and ap, it is hypothesized that they act together with slou in setting fusion programs of VA2 and DT1. This raises an important question about hierarchy of identity genes during execution of muscle identity programs and their roles in acquisition of specific properties of muscles such as number of nuclei, attachment points, and innervation (Bataillé, 2010).

The data presented in this study demonstrate that the number of fusion events in developing muscles is regulated by a muscle-specific combinatorial realisator code of identity gene targets. In contrast to the previously identified fusion genes acting in all muscles, the identified identity targets, Mp20, Pax, and mspo, display muscle-type specific expression and modulate fusion in a muscle-type-specific manner proportionally to the level of their expression. The loss and gain of function of each of them lead to subtle fusion phenotypes indicating that the range of fusion events controlled by these three candidates is limited. Indeed, the loss of function of Mp20 results in loss of two nuclei in a subset of muscles, whereas its overexpression induces the recruitment of maximum two FCMs. A similar range of defects in number of fusion events is observed in Pax and mspo mutant embryos indicating that they influence fusion process at the same level (Bataillé, 2010).

Mp20 encodes a cytoskeletal protein displaying restricted expression in adult muscles and sharing sequence homology with the lineage-restricted mouse proteins SM22alpha, SM22beta, and NP25. These proteins contain calponin-like repeats, and, in mammals, interact with F-actin and participate in the organization of the actin cytoskeleton. In Drosophila S2R cells, the RNAi knockdown of Mp20 induces a phenotype of round and nonadherent cells supporting its role in regulation of fusion process (Bataillé, 2010 and references therein).

The second candidate, Pax (DPxn37), is a scaffold protein that recruits structural and signaling molecules to the sites of focal adhesion. Pax has been shown to be involved in the actin cytoskeleton organization, cell adhesion, cell migration, and cell survival. In the developing Drosophila muscles, Pax protein localizes at muscle-tendon junctions suggesting that it may play a role in muscle attachment. The current analyses of Pax mutant embryos do not reveal muscle-tendon adhesion defects but show discrete myoblast fusion phenotypes, which correlate with differential muscle-specific expression of Pax. The role of Pax in modulating fusion is consistent with previously described implications of Pax interacting proteins, including ARF6 in myoblast fusion in both Drosophila and vertebrates, and FAK in vertebrates (Bataillé, 2010 and references therein).

Finally, mspo belongs to the F-Spondins, a conserved family of ECM proteins, which maintain cell-matrix adhesion in multiple tissues. In vertebrates, F-Spondins have context-dependent effects on axon outgrowth and cell migration. As Mp20, Pax, and Mspo are expressed in FC cells and growing myotubes, one possibility is that they modify the spreading and/or motility of FC protrusions required to attract FCMs. Alternatively, by modulating actin cytoskeleton, Mp20, Pax, and Mspo may also influence the stability of adhesion between the growing muscle and the FCM creating permissive conditions or blocking the progression of fusion process (Bataillé, 2010).

The muscle-type-specific regulation of fusion programs by the identity genes and their targets raises an intriguing question of how this regulation is executed from the mechanistic point of view. Because different levels of expression of Mp20, Pax, and mspo correlate with different fusion programs in both wild-type and genetically manipulated embryos, it was thought that by following kinetics of fusion in small and big muscles insights would be gained into how the fusion programs are modulated. It turns out that the rate of fusion is proportional to the size of muscle, meaning the number of fusion events, thus revealing that the identity genes acting via their targets set up the frequency of fusion events. Accordingly, loss and gain of function of identity genes and their targets identified here results in modulations of fusion programs by accelerating or slowing down the fusion rate. This finding provides insights into mechanistic understanding of muscle-type-specific regulation of fusion process and raises an important question about whether this mechanism is broadly conserved (Bataillé, 2010).


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NK1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 April 2013

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