Commonly used reagents for visualizing Drosophila embryo structures.

INTRODUCTIONA variety of probes and stains are available for labeling specific structures in Drosophila embryos. This article lists some commercially available reagents used for visualizing specific structures.

Propidium iodide staining of Drosophila embryos.

INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. Propidium iodide (PI), a stain commonly used to visualize nucleic acids, is added directly to the mounting medium. Before staining with PI, the embryos must be treated with RNase to remove the RNA. This protocol presents methods for staining Drosophila embryos with PI, as well as for the subsequent mounting and storage of samples.

DAPI Staining of Drosophila Embryos.

INTRODUCTIONDrosophila embryos can be stained with specific fluorescent probes or antibodies through either direct or indirect immunofluorescence. In particular, several effective probes exist for visualizing DNA. 4',6-diamidino-2-phenylindole (DAPI) is a commonly used DNA-binding dye. Because it is specific for double-stranded DNA, no prior RNase treatment is required. While the embryo staining method described here uses DAPI, other fluorescent DNA probes can be processed similarly.

Drosophila embryo collection.

INTRODUCTIONA number of factors make the early Drosophila embryo particularly amenable to cellular analysis. First, large numbers of specifically staged embryos are easily collected from normal and mutant stocks. Also, the morphological and cellular events of embryogenesis have been characterized extensively. For example, directly after fertilization, the embryo proceeds through a series of rapid nuclear divisions that rely on the highly coordinated dynamics of the microtubules, microfilaments, and other cytoskeletal components. During this time, critical events that establish the axis and patterning in the embryo occur. These events have been thoroughly described and provide an excellent resource in which to analyze the primary cellular defect in newly isolated mutations. Analysis of fixed samples allows many more embryos to be examined in a single session on the microscope, and the fixed preparations are stable for long periods. If fluorescent probes are used, anti-quenching reagents in the mounting media allow extensive documentation of the samples without signal deterioration. Double- and triple-labeling for colocalization studies are easily performed. Finally, images can be recorded from fixed samples over multiple planes for three-dimensional reconstructions. This protocol describes the most common and generally applicable procedure for collecting Drosophila embryos for fixed cellular analysis.

Drosophila embryo dechorionation.

INTRODUCTIONEarly Drosophila embryos are particularly amenable to cellular analysis. However, they are protected by an outer chorion layer, as well as an impermeable and opaque vitelline membrane. Consequently, preparation of Drosophila embryos for fixed-sample analysis must include procedures for chorion removal, vitelline membrane permeabilization, fixation, and finally, vitelline membrane removal. This protocol describes a method for removing the chorion layer.

Fixation of Drosophila embryos.

INTRODUCTIONA number of techniques are available for fixation of Drosophila embryos, each with their own advantages and limitations. The technique appropriate for a specific study will depend on a number of variables, including the preservation qualities of the cellular components to be examined, the position of the components within the embryos, and the probe used. Formaldehyde-based techniques are useful for preserving deep and/or cytoskeletal elements. Fixation with methanol or boiling is harsher, but both result in nearly total devitellinization of the embryos. Fixation without methanol requires devitellinization of the embryos by hand. While the most tedious, this latter method often produces the highest-quality results. It may be necessary to try all of these techniques to determine which one is the most appropriate for each new antibody or reagent.

Rehydration of Drosophila embryos.

INTRODUCTIONFixed Drosophila embryos stored in methanol must be rehydrated before staining. Rehydration methods differ slightly for embryos fixed in methanol versus embryos fixed in formaldehyde; techniques for both are presented. An alternative, gentler rehydration method is also presented for studies where a new probe or antibody is being used.

Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components Nuclear-fallout and Rab11.

Cytokinesis requires a dramatic remodeling of the cortical cytoskeleton as well as membrane addition. The Drosophila pericentrosomal protein, Nuclear-fallout (Nuf), provides a link between these two processes. In nuf-derived embryos, actin remodeling and membrane recruitment during the initial stages of metaphase and cellular furrow formation are disrupted. Nuf is a homologue of arfophilin-2, an ADP ribosylation factor effector that binds Rab11 and influences recycling endosome (RE) organization. Here, we show that Nuf is an important component of the RE, and that these phenotypes are a consequence of Nuf activities at the RE. Nuf exhibits extensive colocalization with Rab11, a key RE component. GST pull-downs and the presence of a conserved Rab11-binding domain in Nuf demonstrate that Nuf and Rab11 physically associate. In addition, Nuf and Rab11 are mutually required for their localization to the RE. Embryos with reduced levels of Rab11 produce membrane recruitment and actin remodeling defects strikingly similar to nuf-derived embryos. These analyses support a common role for Nuf and Rab11 at the RE in membrane trafficking and actin remodeling during the initial stages of furrow formation.