Live imaging has revolutionized how many biologists track the process of development in organisms today. This ability to follow natural events that occur at the tissue and cellular levels allows biologists to have a deeper understanding of developmental processes in key model organisms. With that being said, Drosophila imaginal discs are of particular interest because of their important role in the organism’s development. In fact, Drosophila limbs (including legs, wings, antennae, mouth parts, etc.) are derived from these imaginal discs. However, because of their small size and location within the larva and pupa, several studies have neglected the application of live imaging techniques.
One of the main problems with imaginal disc morphogenesis is understanding how a disc of approximately 5-40 epithelium cells generates a complex three dimensional structure of 30,000-50,000 cells (1). To address this concern, the method used to examine imaginal disc development via live imaging has become a growing topic of interest. For instance, Aldaz et al. (2010) demonstrated in her study that the use of viscous culture medium allows high resolution imaging of imaginal disc development. To validate this, the transformation that occurs during the process of metamorphosis of the wing imaginal disc into the mature wing was observed; this examination reported several previously unobserved stages of this model of organogenesis. While this technique is helpful in studying the dynamic changes that occur during morphogenesis, live imaging can also play a crucial role in understanding several other developmental processes.
What exactly are Drosophila Imaginal Discs? And why do we need live imaging to follow their development?
Imaginal discs start as small clusters of cells that are set aside during embryogenesis. These cells then begin to proliferate during larval development, in which they form into folded, single layer, epithelial sacs. It is important to note here that each imaginal disc forms a separate structure. As depicted in Figure 1, there is a separate imaginal disc that forms wings while another imaginal disc forms legs. Furthermore, the cells in the disc stop dividing before differentiation, which begins at the time of pupation. However, once the discs begin differentiating, they ultimately fuse to form a continuous adult head and thoracic cuticle.
Figure 1: (a) A third-instar larva illustrates the position of all imaginal discs. Images of wing, haltere and leg discs corresponding to their imaginal discs are also shown. (b) The body of the adult Drosophila is depicted, indicating the trunk and the different appendages that exist (2). Click on image to enlarge.
Time-lapse microscopy allows biologists to follow living tissue in hopes to examine events that occur during cell division, migration, and morphogenesis. Drosophila embryo live imaging was used in the Aldaz et al. study to track dorsal closure, germ band extension, wound healing, myoblast fusion, and cell death. The image displayed below in Figure 2 illustrates a typical live imaging microscope apparatus.
Figure 2: Live cell Imaging Microscope (3). Click on image to enlarge.
During Drosophila metamorphosis, extensive remodeling of larval tissues generates the adult. For instance, the adult abdomen forms from the histoblasts, while all other exoskeleton structures derive from imaginal discs. Imaginal discs are typically formed by two contigous epithelia, a columnar layer, squamous layer, the disc proper (DP), and the peripodial epithelium (PE).
Imaginal discs are remodeled in an evagination process made of two discrete stages: elongation and eversion. During elongation, the columnar epithelium lengthens and changes in shape, while during eversion, contraction of the PE is assumed to drive the appendages through the larval epidermis. While fluorescence tomography(imaging by sections through the use of penetrating waves) has been used to capture evagination in vivo, limited resolution is known to occur.
Aldaz et al. developed in her study an ex vivo culture system that allowed direct confocal imaging of imaginal discs, lasting up to about 24 hours. The method was in fact a continuation of the organ studies developed by Milner in the 1970s that provided culture conditions for high resolution confocal microscopy of disc development under real time conditions (4). Milner’s technique was altered in that Aldaz et al. cultured discs attached or unattached to the pupal epidermis. To validate this technique, wing disc eversion was described. Various processes were observed during the study, some of which include: the formation of sensory organs, pupal cell divisions at the dorsal/ventral boundary, patterned apoptosis, and wound healing (4). By comparing live processes ex vivo with fixed tissue dissected at different stages, Aldaz et al. concluded with confidence that their technique produced normal events.
What experiments were conducted to illustrate these findings?
Image Imaginal Disc Development ex vivo
Aldaz et al. modified Milner’s technique to enhance the explanted discs’ viscosity to a point where the tissue was held in a precise position; therefore allowing long-term imaging without drifting while also leaving it unconstrained for normal tissue remodeling not to be obstructed. It was determined that 2.5 % methyl cellulose was the best viscosity agent. The technique permitted imaging with either an inverted or upright microscope, which in turn made it easy to employ within any laboratory that had access to a confocal microscope.
Figure 3: Wing imaginal disc and preparation of samples. (A) A late third instar imaginal disc. (Upper) A frontal view with the squamous peripodial epithelium (PE) in green. (Lower) An axial section showing a more detailed view of the three types of cells in the imaginal disc. (B) Custom observation chamber for use with an upright microscope. (C) Anterior half of a prepupa (3–4 h APF) cut along the axial plane. (D) Same pupa bisected along the sagittal plane. (E) One of the half-pupae showing a wing imaginal disc (arrow) close to the inner side of the pupal case. (F) Culture dish containing the sample embedded in agarose and immersed in liquid medium. (4)
Aldaz et al. provided another modification to earlier ex vivo approaches. Since imaginal discs are normally attached to the larval and pupal epidermis, they wanted to be able to image development in similar tissue environments. Because of this, they dissected the pupa in culture medium, but left the discs attached as normal to the pupal epidermal wall. This was achieved with two specific cuts: first, they removed the posterior of the pupa; second, they bisected the remaining anterior portion along the line of the L-R symmetry. The two halves then contained imaginal discs that were sufficiently exposed to image.
The development of the wing imaginal discs during metamorphosis was used to validate the live imaging approaches. The medium’s viscosity allowed the disc to be situated so that images could be recorded in the plane of the disc epithelium or perpendicular to it. The technique was ultimately compatible with any fluorescent labeling method. In the case described above, Aldaz et al. followed the developmental processes via fluorescent reporter proteins. A transgene with the protein Armadillio (Arm) linked to GFP-labeled apical cell contours of both the columnar and squamous epithelial layers. GFP-tagged regulatory light chain of nonmuscle myosin II, Spaghetti squash (Sqh), had a stronger signal in the PE and even in the disc proper, the cytoplasmic signal was sufficient to see the whole cell.
Imaging Evagination of the Wing Imaginal Disc
The process of evagination of the imaginal wing disc required approximately 12 hours for this study. Having combined the information acquired from over 40 movies, they were able to divide evagination into two separate processes: folding and retraction.
Figure 4: Ex vivo Imaginal disc eversion. (a) Prepupal imaginal disc during the eversion process. Disc is labeled with the Sqh-GFP transgene (green) and grunge-Gal4 driving UAS-mRFP (red) (b) Prepupal imaginal disc mounted on its side showing the distribution of Sqh-GFP (green) and His2A-RFP (red). (4)
Are there any future applications that we can take from these ex vivo culture and imaging techniques?
Although wing disc eversion was used to validate disc culturing and imaging, the application of viscous medium is also applicable for other developmental studies. To illustrate the versatility of such approaches, Aldaz et al. also imaged other processes while performing her study on eversion. For instance, it was observed that the reorganization of intracellular myosin-II rich fibers changes while the cell shapes. Ex vivo culture is also advantageous because it allows the application of exogenous markers, compounds, and drugs (4).
Figure 5: Further applications of ex Vivo culture. (A) Frames that illustrate a wing disc labeled with Sqh-GFP. The red arrows point to the nuclei of two peripodial cells. Using a 100x objective, intracellular structures like myosin II fibers can be followed. (B) Prepupal imaginal disc labeled with Sqh-GFP (green) and annexin V-Cy3 (red), which was added to the medium after dissection, as a reporter of apoptosis during retraction of the PE. (C) Numerous dividing cells in a wing disc labeled with Arm-GFP (D) Wing disc labeled with His2A-mRFP (red) and Sqh-GFP (green). (E) Initial (upper) and final (lower) frames that illustrate the healing of a wound (red arrow) in the notum of a wing disc labeled with Sqh-GFP. (F) Wing disc labeled with Arm-GFP and the development of two wing vein sensilae in the wing pouch (4).
What new information has Aldaz et al. provided us with?
Drosophila imaginal discs provide further investigation into the organism’s developmental mechanisms. Their growth and differentiation ultimately represents a model of many general processes, some of which include: proliferation, growth, differentiation, morphogenesis, and programmed cell death.
Aldaz et al. introduced in her study live imaging methods that further the scope of imaginal discs as developmental models. In hopes of validating the use of live imaging by analyzing morphogenesis in viscous medium, Aldaz et al. focused on metamorphosis as a model of epithelium and organogenesis. Previous work had relied heavily on observing dissected, fixed tissue-this method made it difficult to interpret complex remodeling and consequently did not allow high resolution of the sequence of events. However, in this study, their description of disc eversion as well as their complementary data from attached and unattached discs provided much confidence that the culture system was faithfully reproducing in vivo development.
The particular strengths of the culture method included the ability to follow disc development over long periods (at least 24 hours), viscous medium that supported discs in virtually any orientation, and the application of confocal imaging that reconstructed tissue development in 3D (4). The technique was appealing in that offered simplicity, for it did not require the use of any atypical equipment or reagents. In addition, the viscous culture media could easily be transformed for live imaging of other organ systems in Drosophila and other species. However, because other studies have not yet been published in this specific realm of study, it was particularly difficult to make experimental comparisons.
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(1) Rossomando, Edward F., and Stephen Alexander, 1992. Morphogenesis: an Analysis of the Development of Biological Form. New York: Marcel Dekker: 154-155.
(2)Morata, G, 2001.How Drosophila appendages develop, Nature Reviews Molecular Cell Biology 2: 89-97.
(4) Aldaz, S., 2010. Live imaging of Drosophila imaginal disc development, PNAS 107:14217-14222.
(5) Milner, MJ. 1997. The eversion and differentiation of Drosophila melanogaster leg and wing imaginal discs cultured in vitro with an optimal concentration of beta-ecdysone, J Embryol Exp Morphol 37: 105-117.
(6) Milner, MJ. 1987. The cell biology of Drosophila wing metamorphosis in vitro. Rouxs Arch Dev Biol 196: 191-201.