Susan M. Abmayr

SNS: adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts

The body wall muscles in the Drosophila larva arise from interactions between Duf/Kirre and Irregular chiasm C-roughest (IrreC-rst)-expressing founder myoblasts and sticks-and-stones (SNS)-expressing fusion competent myoblasts in the embryo. Herein, we demonstrate that SNS 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.

Cell and molecular biology of myoblast fusion

In organisms from Drosophila to mammals, the musculature is comprised of an elaborate array of distinct fibers that are generated by the fusion of committed myoblasts. These muscle fibers differ from each other in features that include location, pattern of innervation, site of attachment, and size. The sizes of the newly formed muscles of an embryo are controlled in large part by the number of cells that form the syncitial fiber. Over the past few decades, an extensive body of literature has described the process of myoblast fusion in vertebrates, relying primarily on the strengths of tissue culture model systems. More recently, genetic studies in Drosophila embryos have provided new insights into the process. Together, these studies define the steps necessary for myoblast differentiation, the acquisition of fusion competence, the recognition and adhesion between myoblasts, and the fusion of two lipid bilayers into one. In this review, we have attempted to combine insights from both Drosophila and vertebrate studies to trace the processes and molecules involved in myoblast fusion. Implicit in this approach is the assumption that fundamental aspects of myoblast fusion will be similar, independent of the organism in which it is occurring.

Embryonic development of the larval body wall musculature of Drosophila melanogaster

The somatic, or body wall, muscles of the larva of Drosophila melanogaster are composed of an elaborate pattern of segmentally repeating fibers that form during embryogenesis. The primordia of these muscles progress from morphologically indistinct mesodermal cells to multinucleate syncytia with unique characteristics that include shape, size, location and attachment to the epidermis. Although relatively little is known about the development of the musculature and the mechanisms by which this elaborate pattern is achieved, recent progress has begun to reveal key players in this process.

Identification of a Drosophila homologue to vertebrate Crk by interaction with MBC

The vertebrate adapter protein termed Crk was initially identified from the chicken CT10 retrovirus on the basis of its transforming activity (Mayer et al., 1988. Nature 332, 272-275). We have identified a Drosophila protein with homology to vertebrate Crk, termed dCRK, by interaction with the protein encoded by the Drosophila myoblast city (mbc) gene. The dCRK protein has extensive homology to the both the Crk-II form of vertebrate Crk and the Crk-related protein CRKL, and includes one SH2 domain followed by two SH3 domains. A single protein of approx. 37kDa is detected in extracts from embryos, and Northern analysis revealed a single transcript of 1.3kb. The dCrk mRNA is abundant throughout embryogenesis, declines during the larval stages, and reappears during pupation. In addition, maternally-provided transcripts have been detected. During embryogenesis, the spatial distribution of this transcript is relatively broad and appears to include all germ layers. Finally, dCrk is located on the fourth chromosome, approximately at cytological position 101F-102A.

2 Drosophila Myogenesis and insights into the Role of nautilus

Several aspects of muscle development appear to be conserved between Drosophila and vertebrate organisms. Among these is the conservation of genes that are critical to the myogenic process, including transcription factors such as nautilus. From a simplistic point of view, Drosophila therefore seems to be a useful organism for the identification of molecules that are essential for myogenesis in both Drosophila and in other species. nautilus, the focal point of this review, appears to be involved in the specification and/or differentiation of a specific subset of muscle founder cells. As with several of its vertebrate and invertebrate counterparts, it is capable of inducing a myogenic program of differentiation reminiscent of that of somatic muscle precursors when expressed in other cell types. We therefore favor the model that nautilus implements the specific differentiation program of these founder cells, rather than their specification. Further analyses are necessary to establish the validity of this working hypothesis. Studies have revealed a critical role for Pax-3 in specifying a particular subset of myogenic cells, the progenitors of the limb muscles. These myogenic cells migrate from the somite into the periphery of the organism, where they differentiate. These myoblasts do not express MyoD or myf5 until they have arrived at their destination and begin the morphologic process of myogenesis (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). They then begin to express these genes, possibly to put the myogenic plan into action. Thus, as with nautilus, MyoD and myf5 may be necessary for the manifestation of a muscle-specific commitment that has already occurred. By comparison with vertebrates, it was anticipated that the single Drosophila gene would serve the purpose of all four vertebrate genes. However, its restricted pattern of expression and apparent loss-of-function phenotype are inconsistent with this expectation. It remains to be determined whether nautilus functions in a manner similar to just one of the vertebrate genes. Since the myf5- and MyoD-expressing myoblasts are proliferative, the loss of one cell type appears to be compensated by proliferation of the remaining cell type. This apparent plasticity may obscure differences in mutant phenotype resulting from the loss of particular cells that express each of these genes. In Drosophila, by comparison, nautilus-expressing cells committed to the myogenic program undergo few, if any, additional cell divisions, and thus no other cells are available to compensate for the loss of nautilus. Therefore, the apparent differences between the Drosophila nautilus gene and its vertebrate counterparts may reflect, at least in part, differences in the developmental systems rather than differences in the function of the genes themselves.