Live imaging of Drosophila myoblast fusion

Abstract

Myoblast fusion requires a number of cellular behaviors, including cell migration, recognition, and adhesion, as well as a series of subcellular behaviors, such as cytoskeletal rearrangements, vesicle trafficking, and membrane dynamics, leading to two cells becoming one. With the discovery of fluorescent proteins that can be introduced and studied within living cells, the possibility of monitoring these complex processes within the living embryo is now a reality. Live imaging, unlike imaging techniques for fixed embryos, allows the opportunity to visualize and measure the dynamics of these processes in vivo. This chapter describes the development and use of live imaging techniques to study myoblast fusion in Drosophila.

Figure 1. FC and FCM arrangements and fusion profile of individual muscles

(A–C) Stage 12 (A), 13 (B) and 14 (C) rp298-lacZ embryos were stained with antibodies against ß-gal to label FC/myotube nuclei (green) and Lmd to label FCMs (blue). Three-dimensional renderings of single mesodermal hemisegments at stage 12 (A, 1 grid unit = 5.7 µm), 13 (B, 1 grid unit = 10.9 µm) and 14 (C, 1 grid unit = 14.1 µm) are shown. Each panel shows an external view (left) and a side view rotated 90° clockwise (right). Red arrows point to dorsal, green arrows point to anterior and blue arrows point to external. SM stands for somatic mesoderm and VM stands for visceral mesoderm. (A) At stage 12 the somatic mesoderm folds into two layers so that the FCs (green) are concurrently the most external and internal cells (yellow arrows, A), with the FCMs (blue) in between. (B) At stage 13, the internal FCs and FCMs have moved externally to underlay the overlaying epidermis (not labeled). The FCs (green) appear to rest on top of the FCMs (blue) at this stage and the cells are tightly packed together. (C) By stage 14, the number of rp298-lacZ expressing nuclei (green) have increased due to fusion. The FCMs (blue) have separated from one another. (D) Wild type stage 12–15 embryos were stained with antibodies against Eve (DA1), Runt (DO2) or Slouch (DT1, VT1 and VA2) in combination with phalloidin to assist accurate staging. The number of nuclei for each muscle and stage were counted in 50 hemisegments (A2-4). Graph showing the percentage of fusion events that occur during each stage for each muscle during the course of fusion (7.5–13 hours AEL). The mean number of nuclei observed for each muscle at stage 15 is 100% and a single nucleus is 0%. (E) Schematic showing which muscles were analyzed for wild type fusion profiles (blue). Adapted from (7).

Figure 2. All of Drosophila myogenesis can be visualized using live imaging

Lateral view of live twi-GAL4; Dmef2-GAL4 × UAS-GFP::actin embryo. These GAL4 lines strongly express throughout myogenesis. Each image represents a single frame from a time lapse sequence over the course of 5 hrs, covering stages 12–15. During this time, most of myoblast fusion is completed. Images are single optical slices. (arrow points to mesodermal hemisegment. * denotes autofluorescence of amnioserosa)

Figure 3. Dynamic actin-bases behaviors of myoblasts

Lateral views of live twi promoter-GFP::actin embryos. Actin labeling is concentrated at the cell cortices and in cellular extensions such as lamellipodia and filopodia. Such behaviors appear critical for myoblast fusion. The nucleus is evident as a non-labeled structure in the middle of the cells. Each panel represents a single time point from a time lapse sequence. Images are single optical slices. (A) FC for a segment border muscle prior to fusion extends lamellipodia (arrowhead) and filopoida (arrow). (B) FCM extends lamellipodia (arrowhead) and a filopodium (arrow) prior to fusion.

Figure 4. Single myoblast fusion event

Lateral view of live twist promoter-GFP::actin, apME580-NLS::dsRed.T4 embryo. Each row of panels represents a time point from a time-lapse sequence. (A) Single optical slice. In this sequence, an actin focus (white arrow) forms at the site of adhesion between an FCM and an apterous-labeled myotube. This focus resolves, followed by fusion and addition of an additional labeled nucleus (yellow arrowhead) to the myotube. (B) Same sequence as in A, but as an optical projection displaying 9 µm of the Z-axis.