We modified earlier Drosophila primary cell culture protocols (see Materials and Methods) to examine muscle fiber formation and differentiation, with a focus on myoblast fusion and muscle identity. Briefly, we dissociate late stage 8/stage 9 embryos (3.25–4.25 hours), remove debris, plate and grow for 8 hours. We begin analysis 8 hours after plating, since it takes this amount of time for the cells to settle and adhere to the culture dish. To confirm that our protocol could be used for studies of muscle differentiation, we first determined that myotubes formed in culture as evidenced by the presence of multinucleate cells grown for 24 hours expressing myosin heavy-chain (MHC) (). We next visualized the actin cytoskeleton with phalloidin and found that actin becomes striated by day 12, indicating that these myotubes undergo terminal differentiation to form myofibers with sarcomeres (). To establish the ideal time for assessing myoblast fusion in culture, we performed a time course analysis of fusion by counting the number of multinucleate myotubes in a given 20x image field. We found that myoblast fusion commences in the initial 8 hours after plating and increases rapidly between 12–30 hours post-plating, reaching a plateau around 48 hours after plating (). The myotubes in culture adopt a variety of morphologies including long, finger-like cells as well as branched and polygon-shaped myotubes ( and
and data not shown). The number of nuclei in the myotubes ranges from two to greater than seven, with a mean of 3.7 nuclei per myotube (). We quantified the number of nuclei per myotube as a function of time () and found that, although the total number of myotubes increases over time, the distribution of myotubes containing 2, 3, 4, 5, 6 or 7+ nuclei remains consistent. These results are consistent with previous cell culture experiments11
and delineated\ a discrete period of time, between 12–48 hours after plating, as optimal for studying myoblast fusion.
Figure 1 Primary embryonic Drosophila cells fuse and form muscles in culture. (A) Representative image from a field of myotubes in a culture of primary cells grown for 24 h from twipromoter-actin-GFP, apME-NLS::GFP embryos. The culture was immunostained for GFP (more ...)
Figure 3 Muscle identity is preserved in myotubes in primary cell culture. (A–D) Confocal maximum intensity projections of primary myotubes formed from cells isolated from apME-NLS::dsRed embryos at 24 h after plating. Myotube identity was determined by (more ...)
Despite previous work demonstrating that multinucleate myotubes form in primary cell cultures;7–12
how the process of myoblast fusion proceeds in culture and compares to what is observed in embryos has not been studied. We therefore sought to determine whether embryonic myoblast fusion proteins were similarly expressed and localized in our primary culture system. We focused on two well-characterized proteins that are required for fusion in the embryo: the intracellular adaptor protein Rolling pebbles/Antisocial (Rols/Ants) and the fusion protein Blown-fuse (Blow).13–20
In the embryo, Rols is found at the fusion site in FCs and myotubes, while Blow is found initially in FCMs and subsequently in myotubes after fusion.4
Blow and Rols/Ants are expressed in primary cells and localize to the sites of attachment between myoblasts and nascent myotubes as has been shown in embryos ( and B
). Additionally, staining with phalloidin revealed an F-actin structure at the myoblast attachment site ( and B
and white arrowheads). This accumulation of F-actin has been shown through time lapse microscopy in embryos to mark the site of fusion19
and is designated the actin focus ( and B″
We measured the size of the actin foci that formed in culture. These foci ranged in size from 1.2 µm2
to 3.4 µm2
with a mean area of 2.3 µm2
(SD ± 0.7 µm). This size range is within the previously observed range for normal foci of 0.7 µm2
to 4.5 µm2
and the mean size we observed is not significantly different from the reported mean of 1.9 µm2
(SD ± 0.7 µm) observed in embryos (p = 0.18). The localization of these fusion proteins and the actin focus at the site of fusion is similar to what our lab and others have observed in Drosophila embryos.13–21
Therefore, based on the localization of fusion proteins and the size of the actin focus, fusion between myoblasts and multinucleate myotubes in primary culture appears to proceed via mechanisms similar to fusion in embryos.
Figure 2 Proteins important for myoblast fusion are properly expressed and localized in primary myoblasts in culture. (A and B) Confocal maximum intensity projections of representative myotubes generated from apME-NLS::dsRed embryos after 24 h in culture immunostained (more ...)
Having established that the early events of myoblast fusion in primary culture are analogous to those observed in embryos, we set out to perturb myotube formation and myoblast fusion by treating primary cultures with drugs. Recognizing that the actin cytoskeleton and actin regulators are integral components of the fusion process, we grew primary cells in the presence of the actin polymerization inhibitor latrunculin B.15,19,21–26
We performed a time course and quantified the number of myotubes in primary cultures that were treated with two different concentrations of latrunculin B 8 hours after plating (). We found that fusion was severely decreased in cultures treated with the drug and that fusion was reduced to a greater extent in the cultures treated with a higher concentration of drug.
To further characterize this reduction in fusion, we counted the number of nuclei per myotube at time points from 8 to 48 hours after plating (). The mean number of nuclei/myotube was 3.7 in cultures treated with 250 nM of latrunculin B and 3.5 in cultures treated with 50 nM latrunculin B (p = 0.25), numbers which are not significantly different from the 3.7 nuclei/myotube observed in untreated controls (). The total number of myotubes in the drug-treated culture increases only slightly, if at all, between 8 hours and 48 hours after plating; we do not observe any significant change to the average number of nuclei/myotube during this time period. These data indicated that drug treatment beginning 8 hours after plating prevented the formation of new myotubes, as well as additional rounds of myoblast fusion to existing myotubes. Moreover, any myotubes that formed prior to the drug treatment at 8 hours post-plating remained intact, and disruption of the actin cytoskeleton with these drug concentrations had no effect on the viability of these myotubes, their morphology, or their attachment to the coverslip ( and data not shown). This disruption of fusion in primary cells was consistent with the known role for actin during fusion in embryos. In addition, these data indicated that the primary cell culture system is suitable for drug treatment experiments aimed at understanding the mechanisms of myoblast fusion.
Prior work in mammalian C2C12 cells has implicated the microtubule-binding proteins EB1 and EB3 in myoblast fusion;27,28
we therefore tested whether microtubules were essential for myoblast fusion in Drosophila cells. The role of microtubules in fusion has not been examined in Drosophila because embryos completely lacking tubulin do not survive long enough to make muscle. Therefore the ability to transiently and reversibly interfere with microtubules in primary culture is an ideal system to determine whether microtubules have a function during fusion. Eight hours after plating, we treated cells with the microtubule depolymerization drug nocodazole. By performing a time course analysis of myotube formation, we observed that treatment with nocodazole resulted in a reduction of fusion and myotube formation (), and that an increased concentration of drug in the media further decreased fusion. We counted the average number of GFP-positive nuclei per myotube in cultures treated with 100 nM nocodazole, and found that the mean number of nuclei per myotube was 4.0 (sd ± 0.3), which was not significantly different (p = 0.44) from untreated cells (3.7, sd ± 0.4) (). Similar to treatment with latrunculin B, the average number of nuclei per myotube did not change over time. While our data, thus far, do not pinpoint which step(s) in the fusion process require microtubules, these results provide the first evidence that microtubule integrity is important for myoblast fusion in Drosophila.
An additional aspect of muscle differentiation in Drosophila embryos is the adoption of specific muscle identities, the result of the expression of particular factors known as FC identity genes. These identity genes are transcriptional regulators that are expressed in incompletely overlapping subsets of FCs and muscles, and are thought to determine final muscle properties such as size, shape and orientation.1–3
The earliest identity gene expression can be detected in stage 10 embryos (4.5–5.5 hours), and expression of some identity genes continues in the developing myotube and final muscle. To determine whether specific muscle identities are established and maintained in primary cell culture, we examined expression of five such identity genes: Krüppel (Kr), apterous (ap), slouch (slou), Muscle segment homeobox Msh) and even-skipped (eve)
( and data not shown).29–33
We detected myotubes expressing Kr, Slou, Msh or Eve proteins, as well as myotubes expressing the ap promoter-driven transgene, apME-NLS::dsRed
. Since identity genes are expressed in incompletely overlapping patterns within the 30 muscles per hemisegment in the embryo, we expected to detect expression of single identity genes in myotubes as well as overlapping expression.1–3,34
As predicted, we observe myotubes that co-express Msh and apME-NLS::dsRed
in myotubes (), as well as co-expression of Kr and apME-NLS::dsRed
(data not shown). We therefore conclude that aspects of muscle identity are established and maintained in our primary culture system.