Department of Developmental and Molecular Biology, Albert Einstein College of Medicine
Jenny, A. Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis. J. Vis. Exp. (54), e2959, doi:10.3791/2959 (2011).
Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.
1. Fly head dissection
2. Fixation and embedding (use gloves for all steps!)
3. Embedding and arrangement in molds
4. Trimming and sectioning
5. Staining and microscopic analysis
6. Representative results:
Most frequently, eyes are embedded in plastic for a detailed analysis of external rough eye phenotypes detected as part of a genetic screen or in the process of testing genetic interactions with genes known to affect either general eye structure or polarity. Typical results of eye sections are shown in Figure 3. Wild-type ommatidia (Fig. 3A) show the full photoreceptor complement surrounded by a lattice of pigment cells. In contrast, in a strabismus (stbm, a.k.a. van-gogh7) mutant, the ommatidial polarity is lost and, even though the full photoreceptor complement is present, rotation and chirality are randomized (Fig. 3B). Furthermore, since the stbm allele sectioned was in a w- background, the pigment granules are missing in Fig. 3B. Figure 3C shows an example of a clonal analysis. Drosophila Rho kinase (drok) is required for ommatidial rotation as well as for the structural integrity of the eye8. Homozygous drok2 clones can be identified by the absence of pigment in the pigment cells and the rhabdomeres (typically, clones are induced using eyeless-FLP and a P-element with a strongly expressed white marker gene complementing a background w- mutation in the wild-type and heterozygous tissue). It is thus possible to identify mutant areas by their lack of pigment. Due to the cell-autonomy of the pigmentation in the R-cells mentioned earlier, genotypes of individual R-cells can be determined (see arrowheads in Fig. 3C).
Figure 1. The Drosophila eye is an excellent model system for biologists since, in contrast to the smooth surface of a wild-type eye (A), the surface of a mutant eye is externally frequently rough (B), indicative of underlying eye phenotypes. In all figures, anterior is to the left and dorsal is up. Images Courtesy of Dr. Jennifer Curtiss, NMSU, Las Cruces, NM, USA.
Figure 2. Light microscopic images of single ommatidia sectioned using the described method. Only seven R-cells are visible at a time per ommatidium, since R7 lies on top of R8. (A, A') On the R7 level, the cell body of R7 is detected between R1 and R6 (yellow arrow in A'). In contrast, at the R8 level (B), the cell body of R8 is detected between R1 and R2 (yellow arrow in B'). Blue arrowheads mark the pigment granules that can be used as a cell-autonomous pigment marker.
Figure 3. Tangential sections through wild-type (A), stbm (B) and drok (C) mutant adult Drosophila eyes. Schematics below the sections indicate the polarity of ommatidia (see (A) for arrows). Circles represent ommatidia with defects in the photoreceptor complement. Yellow lines represent the dorsal/ventral line of symmetry (equator). In contrast to the well-oriented ommatidia of wild-type (A), the planar organization is lost in the stbm mutant (B). Note that the section of the stbm mutant shown lacks pigment due to its w- mutant background. (C) Because drok mutations are lethal, they have to be analyzed in clones. Thus, pigmented cells are wild-type or heterozygous (filled blue arrowheads), while R-cells lacking pigment (open blue arrowheads) are homozygous mutant. In addition to rotation defects, drok mutants also show structural defects including missing or excess numbers of R-cells. Note that for clonal analysis, the sections are not stained to avoid obscuring the pigment granules.
Using Drosophila as model organism, genetic screens led to the identification of many of the founding members of gene families essential for most of the highly conserved signaling pathways in higher eukaryotes including humans. Since, under laboratory conditions, a functional eye is dispensable for survival, the eye is a particularly well suited tissue for the discovery of novel gene functions and the assessment of genetic networks. Ultrastructural analysis of the fly eye using the described method thus led to fundamental discoveries relevant for development and disease. Initially, single cell clonal analysis was performed using X-ray induced clones combined with known closely associated cell-autonomous recessive markers. More recently, the availability of the FLP/FRT system to generate clones has greatly facilitated the phenotypic analysis of lethal mutations in eye sections6,9.
Analysis of eye sections is not limited to tangential sections described here. If desired, the heads can be aligned in any orientation in the molds and transverse sections can be obtained to study deeper layers in the head such as the lamina and medulla. The protocol described here is thus a versatile method for analyses of eye and head structures Drosophila adults.
No conflicts of interest declared.
I would like to thank Dr. Jennifer Curtiss for the pictures in Fig. 1 and Jeremy Fagan and Dr. Florence Marlow for critically reading the manuscript. Our work is supported by NIH grant 1R01GM088202.
Glutaraldehyde and OsO4 are highly toxic and should be handled with extreme care in a well-ventilated hood. Use filter tips when handling OsO4 to prevent blackening of your pipette.
Dehydration: Make 30%, 50%, 70%, (80%), 90%, and 100% ethanol solutions. Preferably cool 30%, 50%, 70% ethanol on ice before use.
1% Toluidine-blue in 1% Borax.
Table 1. Resin preparation
To prepare the resin, carefully, but thoroughly mix all ingredients in a plastic beaker using a magnetic stirrer with a large stir bar. Avoid bubbles. After mixing, aliquot in standard scintillation vials or similar containers and freeze at -20°C. Use soft resin for all applications unless your EM facility asks for the harder formulation. Use gloves to handle resin (carcinogenic in its unpolymerized form). To polymerize resin on equipment and waste, bake at 70°C overnight. Waste may then be safely discarded and equipment cleaned and reused. Spilled unpolymerized resin can be cleaned with isopropanol.
Table 2. Materials