Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.
Before you start
Fluorescent Staining
Mounting for Adult Hearts
Mounting for Larval Hearts
Representative Results
When executed correctly, all components and associated tissues of the dorsal vessel should remain intact and be readily visualized. The background fluorescence should be minimal. For adults, the ventral longitudinal muscle layer stains very well and produces a substantial signal (Figure 1 and Figure 2). The underlying circular cardiomyocytes however, tend not to produce as intense a signal as that of the overlying ventral layer. The myocytes of the anterior “conical chamber” of the adult heart contain a substantial amount of contractile material and, consequently, this region appears as the most robust relative to the remainder of the cardiac tube. The larval heart shows a spiraling myofibrillar arrangement similar to that of the adult contractile cardiomyocytes (Figure 3).
Figure 1: Top. A fluorescent micrograph showing the entire cardiac tube of an adult Drosophila melanogaster that expresses myosin-GFP. The image was taken with a Zeiss Imager Z1 fluorescent microscope equipped with an Apotome sliding module. Myosin-GFP is shown in green, actin is stained with AlexaFluor® 594 phalloidin (red) and α-actinin is labeled with anti-α-actinin antibody (blue). Note the preparative procedure permits recovery of heart samples with well-preserved structures. CC = conical chamber; AM = alary muscle; V = internal valve; VLL = ventral longitudinal muscle layer. Bottom. A region of the adult cardiac tube from the CC through the 3rd abdominal segment of the heart just beneath the ventral longitudinal layer showing the spiraling myofibril arrangement of the contractile cardiomyocytes. Please click here for a larger version of Figure 1a, and here for a larger version of figure 1b
Figure 2: Representative confocal stacks of an anterior portion of the adult heart stained with AlexaFluor® 594 phalloidin (red) and an anti-Pericardin (collagen type IV) (green) antibody. Pericardin is associated with the heart along the ventral surface, likely originating from the longitudinally oriented myofibrils of the ventral muscle layer. Please click here for a larger version of figure 2.
Figure 3: A fluorescent micrograph of segment A7 of the heart proper of a stage three Drosophila larva. The image was taken with a Zeiss Imager Z1 fluorescent microscope equipped with an Apotome sliding module. Actin is labeled with AlexaFluor® 594 phalloidin (green) and α-actinin is stained with anti-α-actinin antibody (red). Please click here for a larger version of figure 3.
Here we present a protocol useful for preparing and staining the Drosophila melanogaster dorsal vessel and associated tissues for imaging via fluorescent or confocal microscopy. We provide a concise account of the steps refined by and commonly employed in our lab for effective staining that permits well-resolved in situ imaging of larval and adult Drosophila heart tubes. Others have described similar methods in abbreviated form2, 3, 4.
Important additional details require consideration when implementing the staining procedure. For example, as found with other biological materials, the fairly compliant Drosophila cardiac muscle fibers become quite brittle following fixation. Of particular note are the multiple pairs of supportive alary fibers attached to the cardiac tube, which are considerably delicate 5, 6, 7. Thus, limiting unnecessary manipulation decreases the physical stress and direct damage to the fixed tissue and it increases the likelihood of visualizing well-preserved and completely intact structures. Further, handling the adult samples only at the extreme edges of the cuticle minimizes the chances for tissue perturbation.
It is also essential to note that the steps of this protocol should be considered as simply a starting point for successful immunostaining of larval and adult Drosophila hearts. The methodology is reduced to the minimum number of steps required for the reagents commonly used in our laboratory. As with any immunohistochemistry protocol, certain antibodies might require different conditions (blocking, variable amounts of detergent, prolonged washing steps, etc.). This protocol can therefore be empirically optimized for such antibodies to meet the unique staining needs of potentially any Drosophila cardiac structure.
Studying the heart using the techniques described here is particularly powerful since Drosophila melanogaster is a highly tractable model system that benefits from a wide variety of well-developed genetic tools. These tools permit unparalleled investigative power to study development, structure and function of cardiac muscle and its components. For example, mutational analysis of cytoarchitectural proteins in the heart has helped identify a variety of cardiomyopathy models in Drosophila 8, 9. Fluorescent labeling of the sarcomeric components of these models has revealed that cardiac dilation in flies is accompanied by myofibrillar disorganization and a loss of contractile material. Additionally, multiple GFP-insertion lines are currently available10. The above protocol can be used in combination with such lines to gather detailed morphological information regarding the localization and the potential molecular interactions made by GFP-tagged proteins. Furthermore, genome-wide RNAi collections of fly lines exist11 that allow selective knockdown of protein constituents within the heart12. Our staining protocol, followed by suitable microscopy, in conjunction with the cardiac-specific knockdown of genes of interest, enable careful analysis of a protein’s contribution to heart structure and function. Together, these techniques make a powerful screening method to help visualize and decipher the relevance of a host of known, or previously uncharacterized, protein components necessary for proper cardiac morphology and physiology.
The authors thank S.I. Bernstein (San Diego State University) for critical reading and helpful suggestions regarding the preparation of this manuscript. This work was supported by NIH grants to S.I. Bernstein, SDSU, and to R. Bodmer, BIMR; and by a post-doctoral fellowship from the Western States Affiliate of the American Heart Association to G. Vogler and to A. Cammarato.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
Ethylene glycol-bis (2-amino-ethylether) -N,N,N’,N’-tetra-acetic acid (EGTA) | Reagent | Sigma-Aldrich® | E4378 | |
Formaldehyde, 10%, methanol free, Ultra Pure | Reagent | Polysciences Inc. | 50-00-0 | |
Triton-X-100 | Reagent | Sigma-Aldrich® | 9002-93-1 | |
Alexa Fluor® 594 phalloidin | Reagent | Invitrogen™ | A12381 | |
Vectashield® Mounting Medium for Fluorescence with DAPI | Reagent | Vector Laboratories, Inc. | H-1200 | |
Tungsten pins | Reagent | Fine Science Tools | 26002-10 | |
Pin holder | Reagent | Fine Science Tools | 26018-17 |