Characterisation of the role of Vrp1 in cell fusion during the development of visceral muscle of Drosophila melanogaster
© Eriksson et al; licensee BioMed Central Ltd. 2010
Received: 7 December 2009
Accepted: 11 August 2010
Published: 11 August 2010
In Drosophila muscle cell fusion takes place both during the formation of the somatic mesoderm and the visceral mesoderm, giving rise to the skeletal muscles and the gut musculature respectively. The core process of myoblast fusion is believed to be similar for both organs. The actin cytoskeleton regulator Verprolin acts by binding to WASP, which in turn binds to the Arp2/3 complex and thus activates actin polymerization. While Verprolin has been shown to be important for somatic muscle cell fusion, the function of this protein in visceral muscle fusion has not been determined.
Verprolin is specifically expressed in the fusion competent myoblasts of the visceral mesoderm, suggesting a role in visceral mesoderm fusion. We here describe a novel Verprolin mutant allele which displays subtle visceral mesoderm fusion defects in the form of mislocalization of the immunoglobulin superfamily molecule Duf/Kirre, which is required on the myoblast cell surface to facilitate attachment between cells that are about to fuse, indicating a function for Verprolin in visceral mesoderm fusion. We further show that Verprolin mutant cells are capable of both migrating and fusing and that the WASP-binding domain of Verprolin is required for rescue of the Verprolin mutant phenotype.
Verprolin is expressed in the visceral mesoderm and plays a role in visceral muscle fusion as shown by mislocalization of Duf/Kirre in the Verprolin mutant, however it is not absolutely required for myoblast fusion in either the visceral or the somatic mesoderm.
In general there are three major muscle types in vertebrates as well as in insects; visceral muscle, cardiac muscle and skeletal muscle. Drosophila muscle progenitors, i.e. myoblasts, arise during embryogenesis and undergo the central process of myoblast fusion during the development of both the visceral and the somatic muscles. The mechanisms underlying cell fusion are actively studied in musculature of Drosophila melanogaster, with significant focus on the process of fusion within the somatic mesoderm (SM), although the phenomenon of myoblast fusion also occurs during the formation of the visceral muscle. The visceral mesoderm (VM) of the fruitfly consists of an inner layer of circular muscles, formed after one round of myoblast fusion, surrounded by an outer layer of longitudinal muscles [1–3]. Although the process of fusion in the VM is generally considered to be similar to SM fusion, VM fusion has not been as extensively studied and is not entirely understood [4–7]. To date, a number of molecules that are required for SM fusion have been identified, leading to the development of models describing the process of SM fusion . Central to this, two different myoblast subtypes have been identified, founder cells (FCs) and fusion competent myoblasts (FCMs), which differentially express a number of transcription factors and adhesion molecules . The FC is destined to become the first cell of each SM muscle, fusing with FCMs to generate the multinucleated muscle. FCMs continue to fuse with the growing myotube ultimately resulting in a muscle of the appropriate mass [10, 11]. Attraction between the FC and the FCM is mediated, at least in part, by immunoglobulin-domain containing proteins such as protein Dumbfounded/Kin of Irre (Duf/Kirre) and Sticks and Stones (SNS) which are expressed on the cell membrane of the FCs and FCMs respectively [12–15]. The subsequent fusion of the myoblast plasma membrane is to a large extent dependent on signaling pathways regulating the actin cytoskeleton.
The significance of the actin machinery in SM fusion has become evident from studies of mutants of the Scar-Wasp signaling network. Scar (WAVE in mammals) and Wiskott-Aldrich syndrome protein (Wasp) are multidomain proteins which are structurally different at their NH2 -terminal domains, but which both contain a common Verprolin-homology, cofilin-homology, and highly acidic (VCA) - region at the COOH-terminal region, through which they bind to and activate the Arp2/3 complex . The Arp2/3 complex is a well characterized actin nucleator, and thus Scar and Wasp are important regulators of actin polymerization . A number of additional proteins are necessary for the proper function of both Scar and Wasp; Scar acts in a complex with four other proteins, including Kette (NAP125 in mammals), while Wasp functions in a complex with Verprolin (Vrp). Vrp is also known as Wasp interacting protein (WIP) in mammals  and in Drosophila Vrp is known as Verprolin1 (Vrp1) /D-WIP /Solitary /and Solas . Both Scar and Wasp are activated by small GTPases such as Rac and Cdc42 . Rac, in turn, is regulated by the guanine nucleotide exchange factor Myoblast city (Mbc) . Drosophila mutants in Scar, Wasp, Vrp, Arp2, Kette, mbc, Rac1, Rac1-Rac2-mtl and Cdc42 all show SM fusion defects during embryonic stages, although the severity of fusion phenotypes varies extensively between the different mutants, probably due to redundancy as well as maternal contribution in certain cases [20, 21, 25–28]. The fusion defects in these mutants, characterized by unfused SM cells as well as abnormal actin accumulations at the cell-cell attachment sites (in the case of Scar, Wasp, Kette, Rac1-Rac2-mtl), confirm the importance of the actin machinery in SM cell fusion [25, 29].
In this work we have investigated the process of myoblast fusion in the VM. VM cells in Drosophila melanogaster express the ALK (Anaplastic lymphoma kinase) receptor tyrosine kinase (RTK), which activates a signaling cascade resulting in the specification of VM FCs [4–6]. The immunoglobulin-domain containing molecules Duf/Kirre and Sns are expressed in the VM FCs and FCMs respectively, and play a role in VM fusion, mediating adhesion between the FCs and FCMs. We identified the actin regulatory protein Vrp1 as a molecule important in the process of muscle fusion the SM and VM development, based on a deficiency screen for VM fusion mutants carried out in our laboratory. A role for Vrp in the SM fusion process has previously been reported [20–22, 26], however, Vrp is also strongly expressed in the FCMs of the VM suggesting a role in VM fusion. Here we show that Vrp1 mutants display defects in the development of the visceral muscle, although the defects observed in the VM are more subtle than those observed in the SM.
Vrp1 f06715 is an insertion in the Vrp locus which exhibits severe somatic muscle fusion defects
Vrp expression pattern
Vrp is specifically expressed in the FCMs of the VM
The VM of Vrp1 mutant embryos displays a subtle phenotype
We also investigated the development of the longitudinal visceral muscles in vrp1 f06715 mutants, employing UAS-LacZ expressed under the control of 5053-GAL4 as a readout. At stage 12 in both vrp1 f06715 and control embryos (Figure 4H and 4I) the longitudinal muscles surround the circular musculature, and at later stages, both in mutants and controls, the longitudinal muscles form a characteristic longitudinal pattern (Figure 4F and 4G). These results indicate that longitudinal muscle development is not obviously affected by Vrp1 mutation.
Since actin foci have been reported to be formed at the cell-cell attachment sites between fusing FCs and FCMs, and to contain fusion proteins such as Sns, Rols, Loner, Blow and Mbc , we investigated if the Duf/Kirre accumulations in the Vrp1 f06715 mutant could involve such actin structures. For this we employed the twip-GFP-actin fly strain in which a GFP-actin fusion protein is expressed under the control of the twist promoter , and examined actin localization in Vrp1 f06715 mutant animals. Analysis of these mutants revealed that the Duf/Kirre accumulations do not contain elevated levels of actin (Figure 5D arrowheads), suggesting that the Duf/Kirre containing structures we observe are different than the above described actin foci.
Mutations in additional components of the Scar-Wasp signaling network display similar phenotypes as Vrp1 f06715
Vrp1 mutant VM cells are capable of migrating and fusing
In addition to having a role in muscle cell fusion, Vrp1 and other actin regulating proteins have, in other experimental systems, been suggested to have roles in cell motility [36–39]. In order to test the role of Vrp1 in both muscle fusion and cell motility experimentally we analyzed Alk mutant embryos. In Alk mutants, it has previously been shown that FCMs of the VM are able to migrate towards and fuse with the somatic muscle cell population [4–6].
Expression of Vrp1 in the FCM population rescues fusion
In parallel, we examined the effect of the various Vrp1 proteins on the organization of the actin cytoskeleton in porcine aortic endothelial (PAE) cells, reasoning that in this system we would be able to analyze the effect of the various Vrp1 protein domains on the morphology of the actin cytoskeleton. We have previously found that ectopic expression of mammalian Verprolin results in a profound reorganization of filamentous actin . We observe a shift in the balance between monomeric and filamentous actin, seen as the bundling of stress fibers into thick actin filaments and the formation of actin foci (Figure 8B). Here, the full length Vrp1 transgene, but not the truncated forms, induced thick bundles, actin dots and stress fiber loss (Figure 8B and quantification in C), indicating that ectopic expression of Vrp1 regulates the organization of the actin cytoskeleton in PAE cells, in a similar manner to the mammalian Verprolins WIRE and WIP [Additional file2: Supplemental Figure 2E].
Discussion and Conclusions
Df(2R)ED3943 was identified in a deficiency screen designed to identify novel genes with roles in VM development. Subsequent work led to the identification of the Vrp1 WHF06715 mutant allele, present in the Exelixis mutant collection maintained at Harvard , which carries a piggyBac insertion in the Vrp1 gene. Closer examination of both Df(2R)ED3943 and the Vrp1 f06715 mutant, lead to the identification of a subtle VM-phenotype as well as a severe somatic mesoderm (SM) fusion phenotype. At this time the SM fusion phenotype of independent mutants in the Vrp1 locus, which is characterized by a large number of unfused myoblasts, was unpublished. However, several elegant studies have subsequently described the role of Vrp1/D-WIP/Solitary/solas [20–22]. Therefore, we have focused upon investigation of the role of Vrp1 in the development of the visceral musculature.
The VM phenotype observed in Vrp1 f06715 mutants is not as explicit as that in the SM. Both Df(2R)ED3943 and Vrp1 f06715 exhibit defects in gut structure, however, we cannot definitively address how much of this is due to the lack of structural support of a surrounding somatic musculature. More detailed analysis of the developing VM of Vrp1 f06715 mutant embryos was performed, leading to the discovery of a VM phenotype characterized by mislocalization of the adhesion molecule Duf/Kirre (see below for further discussion).
To date, there are few published mutants with strong VM fusion phenotypes, and even mutants with a complete block of fusion between myoblasts in both the SM and the VM, such as sns  and myoblast city [1, 3] mutants, display subtle VM fusion phenotypes which can be difficult to identify. While mutants such as Alk and Jeb, which do not specify founder cells [4–6, 44, 45] display clear fusion phenotypes which are easily identified during embryonic development, many more muscle specific genes which are expressed both in the SM and the VM, have been reported to have weak VM phenotypes when mutated, although they give severe fusion phenotypes in the SM. Examples include mutants in rolling pebbles , antisocial  roughest , blown fuse [48, 49], lame duck [50, 51], loner  and kette . Our work adds Vrp1 the list of mutants belonging to this category.
The Vrp1 protein contains several domains, which are conserved throughout evolution (Figure 2A), [18, 19]. By asking which domains of Vrp1 are required to rescue the Vrp f06715 mutant phenotype we have investigated the importance of the different domains of Vrp1 in Drosophila, and find that only the WASP-binding domain is required for muscle fusion, while the actin binding domains are dispensable. These findings are contradictory to results previously published by Kim et. al 2007, who reported that the WH2 domains were required for rescuing the solitary mutant phenotype . Our results indicate that the Vrp1-WASP interaction is critical in muscle fusion. However, the effects on the organization of the actin cytoskeleton, caused by Vrp1 expression in PAE cells, indicate that all conserved domains have actin cytoskeleton modulating properties, suggesting that the WH2 domains may be of importance in other contexts than myoblast fusion. Two additional proteins - Wasp and Scar - are nucleation promoting factors that act in parallel to activate the Arp2/3 complex, and mutants for the genes that encode these proteins display similar SM fusion phenotypes as the Vrp1 f06715 mutant , indicating that many members of the Scar-Wasp signaling network work together to regulate myoblast fusion. We have analyzed VM fusion in additional single and double mutants for some of the components in this pathway; kette, wasp, and arp3-wasp, and observed that these mutants also develop a gut, suggesting that either VM fusion takes place in these mutants as in Vrp1 f06715 , or that the VM manages to develop normally despite fusion blockage. Interestingly accumulation of Duf/Kirre is observed in all examined mutants of the Scar-Wasp signaling network.
Taken together, we suggest that VM fusion is initiated in mutants of components in the Scar-Wasp signaling network, and that these molecules are involved in an increased efficiency of the fusion process.
In addition to the Arp2/3 complex, other molecular pathways are able to nucleate actin. These include proteins such as formins, Spire and Cordon-bleu. Molecules of these protein families are structurally different to the Arp2/3 complex and produce linear instead of branched actin filaments. (discussed in Campellone and Welch 2010 , and Aspenstöm 2010 ). Spire and several formins, including Diaphanous and Cappuccino, have been identified in Drosophila, were they have been associated with cellular processes such as vesicle transport and actin-microtubule interactions , but not yet with muscle development. Thus, loss of Arp2/3 function does not inhibit all actin polymerization in the cell, although the strong SM phenotypes observed in different Scar-Wasp signaling pathway mutants suggests that the Arp2/3 complex is an important actin nucleator in muscles. Our data suggests that actin polymerization by the Arp2/3 complex pathway is not required for VM fusion. Whether additional modes of actin assembly contribute to VM fusion is an interesting prospect and remains to be further investigated.
Duf/Kirre, together with Sns, is important for myoblast fusion in both the VM and the SM, as these immunoglobulin receptors facilitate attachment between FCs and FCMs, and therefore a mislocalisation of this molecule suggests that the process of fusion does not proceed in the normal fashion. We observe that Duf/Kirre protein is not downregulated in the VM of Vrp1 f06715 mutants, possibly reflecting a stalled or inefficient fusion process. However, a recognizable embryonic gut is developed despite this phenotype, and the longitudinal muscles of Vrp1 f06715 mutants appear morphologically wild type, suggesting that fusion defects do not affect VM development. Interestingly, we also observed a significant accumulation of Duf/Kirre protein in the SM of the analyzed Vrp1 f06715 mutants, strengthening the hypothesis that this particular phenotype is the result of an inability of myoblasts to fuse properly. Accumulation of Duf/Kirre in the SM has previously been reported and suggested to reflect an imbalance in Duf/Rols signaling during fusion , a conclusion that is supported by recent study investigating Duf/Kirre signaling in myoblast fusion efficiency . Our findings in the VM of Vrp1 mutants, together with our and others reports in the SM [20–22, 26] indicate that Vrp1 and components of the Scar-Wasp signaling network are also important for fusion efficiency. Ultrastructural analysis with electron microscopy has shown that SM cell fusion is a process of many steps, including the adherence of the myoblasts to each other, the appearance of vesicles and elongated plaques on both sides of the plasma membranes, the formation of fusion pores which lead to mixing of cell content, and then an anticipated enlargement of the pores as the plasma membranes are broken down, which finally results in complete fusion of the two cells . The Duf/Kirre accumulation in the mutants examined in this study may reflect an inability of fusing cells to proceed through all the above described fusion steps, resulting in an incomplete or stalled fusion event. This would still produce an obvious fusion defective phenotype in the SM, but appears to have little effect in the embryonic VM. Clearly, it remains to be investigated whether loss of Vrp1 results in later developmental defects.
As a result of our experiments investigating Vrp1 function in the VM we conclude that Vrp1 is not absolutely required for muscle cell fusion in vivo. This is evidenced by the fact that Alk10-Vrp1 double mutant FCMs originating from the VM are clearly capable of fusing with FCs of the SM. Naturally, one major difference between the fusion process in the VM and the fusion process in the SM, is that in the VM one FC fuses with only one FCM, whereas in the SM one FC per myotube fuses with up to 25 FCMs to form much larger muscle syncytia. It is possible that the many fusion events that take place in the SM require significantly more efficient actin rearrangement machinery than the few fusion events in the VM, and this would then explain why the fusion phenotypes that are caused by Vrp1, scar, wasp and arp3 disruption are more visible in the SM than in the VM. It follows that evaluation of VM developmental defects will be difficult given current markers, and that study of the VM during larval stages will provide insight. Thus, although the VM of the Vrp1 f0671 mutant displays only minor defects at embryonic stages, the gut may be non-functional as the animal develops further. Unfortunately, at present time we are unable to test the functionality of the mutant larval gut since the Vrp1 mutation causes an embryonic lethal phenotype precluding an investigation of the mutant larval gut. For this, a SM specific tissue rescue would be required, something which is currently not possible. Future development of tools to allow investigation of the function of Vrp1, and indeed other molecules, in the Drosophila visceral muscle at later stages must now be a priority for analyzing the gut muscle specific function of Vrp1 in vivo.
Standard Drosophila husbandry procedures were followed. The following stocks were used: w 1118 , referred to as WT in Figures and text (Bloomington, stock number 5905), Df(2R)ED3943 (Bloomington, stock number 9158), P(Tub-PBac\T)2/wg Sp-1 (Bloomington, stock number 8285), rp298lacZ , Vrp1 f06715 (Exelixis Collection at the Harvard Medical School ), sns20 23 , referred to as sns in Figures and text , twistp-GFP-actin , UAS-LacZ, 5053-GAL4 , kette j4-48 , Arp3 schwächling wasp 3D3-035 , referred to as Arp3-WASP in text , wasp 3D3-035 , Alk10 , Sns-GAL4 . Transgenic fly strains: UAS-Vrp1 full length , UAS-Vrp1ΔWH2, UAS-Vrp1ΔProΔWASP and UAS-Vrp1ΔWASP were generated as described below.
P(Tub-PBac\T)2/wg Sp-1 flies were crossed to Vrp1 f06715 flies to induce expression of piggyBac transposase, in order to remobilize the WHf06715 element. To drive LacZ expression in the longitudinal muscles of Vrp1 f06715 mutant as well as heterozygous controls, flies with the genotype Vrp1-UAS:lacZ/CyOWgLacZ were crossed to flies with the genotype Vrp1/CyOWgLacZ; 5053-GAL4. For studies of migration and fusion of VM cells in the SM, fly strains with the genotype Alk 10 -Vrp1/CyOWgLacZ were generated as well as flies with the genotype rp298lacZ;Alk 10 -Vrp1/CyOWgLacZ . For rescue experiments flies of the genotype Vrp1 f06715 /CyOWgLacZ;UAS-Vrp1 transgene (all four UAS-transgenes, Figure 8A) were crossed with flies of the genotype Vrp1 f06715 -sns-GAL4/CyOWgLacZ, and in the case of rescue of lethality straight winged flies were counted. For studies of actin expression in muscles a twistp-GFP-actin-Vrp1 f06715 fly strain was generated via recombination.
Generation of Vrp1 transgenic constructs
The Vrp1 cDNA clone GH25793 (Drosophila Genomics Resource Center) was used as a PCR template to generate four different myc tagged Vrp1 transgenic constructs; Vrp1 full length (2250 bp), Vrp1 2XΔWH2 (1830 bp), Vrp1 ΔProΔWBD (450 bp) and Vrp1 ΔWBD (2140 bp). The primers added a BamHI restriction site to the 5' end of the PCR product and a XhoI restriction site and a myc sequence to the 3' end. Primers for Vrp1 full length were; 5' primer: GGA TCC GCC ATG GCT ATT CCG CCA CCC CCG GGA, 3' primer: CTC GAG CTA CAG ATC CTC TTC AGA GAT GAG TTT CTG CTC CAT ACC ATT GGT GGC CTT AAA. Primers for Vrp1 ΔWH2 were; 5' primer: GGA TCC GCC GCC ATG ACA ACG AAC TCA TCC GCT CAG, 3' primer: CTC GAG CTA CAG ATC CTC TTC AGA GAT GAG TTT CTG CTC CAT ACC ATT GGT GGC CTT AAA. Primers for Vrp1 ΔProΔWBD were; 5' primer: GGA TCC GCC ATG GCT ATT CCG CCA CCC CCG GGA, 3' primer: CTC GAG CTA CAG ATC CTC TTC AGA GAT GAG TTT CTG CTC TTG GCG CTT CAA CGT CAA GTG. Primers for Vrp1 ΔWBD were; 5' primer: GGA TCC GCC ATG GCT ATT CCG CCA CCC CCG GGA, 3' primer: CTC GAG CTA CAG ATC CTC TTC AGA GAT GAG TTT CTG CTC GGT CTC CAA GTC GTT GAC CAG. Standard PCR programs were used to amplify DNA fragments. PCR products were then digested with BamHI and XhoI and subcloned into the pUAST plasmid  and pcDNA3 (Invitrogen), and the resulting constructs were confirmed by DNA sequencing prior to injection and generation of transgenic fly strains (BestGene Inc).
Embryo Immunostainings and in situ hybridization
Unless otherwise stated, embryos were collected, fixed and immunostained as described previously , prior to dehydration and mounting in methylsalicylate on glass slides for analysis. The following primary antibodies were used: Rabbit anti-β3 Tubulin (1:5000) , guinea pig anti- β3 Tubulin (1:10 000) , rabbit anti-βGal (1:150, Cappel), mouse anti-βGal (1:1000, Promega), mouse anti-Mef2 (1:500, gift from B. Paterson), rabbit anti-Alk (1:1000), guinea pig anti-Alk (1:1000), mouse anti-FasIII (1:50, Developmental Studies Hybridoma Bank), rabbit anti-Duf/Kirre (1:300). Guinea pig anti-Vrp1 was generated by injection of guinea pigs with recombinant HIS-tagged protein corresponding to residues 837-936 of Vrp1 in pETM11 . The resulting guinea pig antiserum (Medprobe) was IgG-purifed on a Protein A column (Pierce) prior to use at 1:1000 for immunostaining. Fluorescent secondary antibodies employed were: goat anti-rabbit Cy3 (1:1000, Amersham), goat anti-mouse Cy3 (1:1000, Jackson), donkey anti-guinea pig Cy3 (1:200, Jackson), goat anti-rabbit Cy2 (1:1000, Amersham), goat anti-mouse Cy2 (1:1000, Amersham), donkey anti-guinea pig Cy2 (1:1000, Jackson), donkey anti-rabbit Cy5 (1:200, Jackson), donkey anti-mouse Cy5 (1:200, Jackson), donkey anti-guinea pig Cy5 (1:400, Jackson). For in situ hybridization a digoxigenin-labelled RNA probe was made using cDNA encoding Vrp1 and a DIG RNA labelling kit (Roche). In situ hybridization of whole-mount wild type Drosophila embryos was carried out as described .
Cell line experiments
Porcine aortic endothelial (PAE ) cells were cultured in Ham's F12 medium, Supplemented with 10% FBS and penicillin/streptomycin at 37°C in an atmosphere of 5% CO2. For immunstaining experiments, the cells were seeded on coverslips and transiently transfected by Lipofectamine (Invitrogen Life Technologies) employing the protocol provided by the manufacturer. Twenty hours post-transfection, the cells were fixed in 3% paraformaldehyde in phosphate buffered saline (PBS) for 20 minutes at 37°C and washed with PBS. The cells were thereafter permeabilized in 0.2% Triton X-100 in PBS for 5 minutes, washed again in PBS and incubated in 5% FBS in PBS for 30 minutes at room temperature. To visualize filamentous actin, cells were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin (Sigma) diluted in 5% FBS in PBS for 30 minutes at room temperature. The coverslips were washed in PBS and mounted on object slides by the use of Fluoromount-G (Southern Biotechnology Associates). Cells were photographed by a Hamamatsu ORCA CCD digital camera employing the QED Imaging System software using a Zeiss Axioplan2 microscope. Thick bundles, actin dots and stress fibers were quantified manually in microscope by calculating the percentage of transfected PAE cells displaying these structures or cells displaying extensive loss of stress fibers (see legends to Figure 8). All samples were analyzed blind.
List of abbreviations
Actin-related protein 2 and 3
Anaplastic lymphoma kinase
Dumbfounded/Kin of Irre
fusion competent myoblasts
porcine aortic endothelial
receptor tyrosine kinase
suppressor of cAMP receptor
Sticks and Stones
Verprolin-homology, cofilin-homology, and highly acidic
Wasp interacting protein
Wiskott-Aldrich syndrome protein
The authors would like to thank members of the RHP laboratory for helpful discussions during the course of this work. RHP is a Swedish Cancer Foundation Research Fellow. The work was supported by grants from the Swedish Research Council (621-2003-3399 to RHP; K2010-67X-15378-06-3 to PA), the Swedish Childhood Cancer Foundation (08/074 to RHP), the Swedish Cancer Foundation (3670-B07-13XCC to PA and 09 0741 to RHP) and the Association for International Cancer Research (AICR 08-0177).
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