Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis

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Publisher Summary

The early embryo of Caenorhabditis elegans is one of the most powerful model systems to study cell division. This chapter develops a correlative light and electron microscopic approach to stage early C. elegans embryos prior to high-pressure freezing and electron tomography. Cellulose microcapillaries are used to contain the embryos in short transparent tubes. These permit the viewing of early mitotic events under a light microscope. Next step is to stage selected embryos by light microscopy (LM), to immobilize these staged embryos by high-pressure freezing, and to fix them by freeze-substitution. The chapter describes the thin-layer embedding and 3D reconstruction of spindle components by electron tomography. Using the newly developed rapid transfer system (RTS) of the Leica EMPACT2 high-pressure freezer reduces the time between light microscopic observation and cryoimmobilization to about 5 sec. The correlative approach allows systematic structure-function studies in staged early wild-type embryos and those treated with RNA-mediated interference (RNAi) to reduce the level of specific gene products.

Introduction

The Caenorhabditis elegans early embryo is one of the most powerful model systems in which to study various subprocesses of mitosis, such as nuclear envelope assembly/disassembly, centrosome dynamics, centriole assembly, formation of the mitotic spindle, kinetochore assembly, chromosome segregation, and cytokinesis (for a review see Oegema and Hyman, 2005). For analysis of these processes by correlative microscopy, C. elegans offers several advantages. (1) The stereotypic early development of the embryo has been studied and characterized in detail (for reviews see Cowan 2004, Pelletier 2004a, Schneider 2003). (2) The appearance, migration, rotation, and disappearance of the pronuclei around prometaphase can be easily recognized by noninvasive microscopy, allowing precise staging of embryonic development. (3) Mitotic spindle components, such as microtubules, kinetochores, or centrosomes, can be visualized using GFP‐tagged strains. Moreover, strains are available which express multiple‐tagged proteins (Oegema et al., 2001). (4) RNA‐mediated interference (RNAi) is used routinely to generate oocytes whose cytoplasm is reproducibly depleted of targeted essential gene products. Following fertilization, these oocytes are then analyzed as they attempt to go through the first round of mitosis (reviewed in Oegema and Hyman, 2005). Application of RNAi allows the comparison of wild‐type and mutant embryos. (5) The reliability of RNAi technology has it made possible to perform genome‐wide screens to identify all the components involved in the early mitoses of C. elegans (Sönnichsen et al., 2005). Taken together, these features allow one to study the dynamics and ultrastructure of cell division in a systematic manner that was not possible until recently.

Correlative approaches in microscopy have been used to obtain complementary information about a given system (Kapoor 2006, Rieder 1999). For example, light microscopy (LM) in combination with GFP‐tagging, on the one hand, offers the advantage of studying centrosome dynamics in living systems, but with obvious limitations in the obtained resolution. Electron microscopy (EM), on the other hand, delivers high‐resolution “snapshot” images of centrosomes, either purified (Chrétien et al., 1997) in vitro (Schnackenberg et al., 1998) or in vivo (O'Toole et al., 2003b), but the yield of information about the dynamics of the process under study is low. On the basis of only “snapshot” images, it is not always possible to unambiguously reconstruct a complex process, like the assembly of a cellular structure. For analysis of an organelle like the centrosome in the early C. elegans embryo, one would like to be able to observe spindle dynamics by live‐cell imaging and stop the process immediately at a specific stage by rapid cryofixation; this would permit subsequent, high‐resolution analysis in situ, preferably by electron tomography. The prerequisites for such a correlative approach are: (1) to optimize the time resolution of the technique and (2) to use a method that gives optimal preservation of the ultrastructure.

One approach to achieving the first goal (i.e., to stop the developing C. elegans embryo at specific stages of early mitosis) has been laser‐assisted chemical fixation (Dammermann 2004, Kirkham 2003, Pelletier 2004b, Priess 1986). Taking advantage of the fact that early embryos are protected by coverings that allow them to continue to divide in the presence of fixative, selected embryos were attached to an Aclar plastic surface and “bathed” in M‐9 buffer containing 2% glutaraldehyde. A laser beam was used to perforate the embryo's eggshell, allowing the fixative to diffuse into the embryo through a small (1.5 μm) hole and to stop development. Since this method achieved a rapid cessation of the embryonic development and permitted a staging of the embryo prior to conventional EM, it has been applied to understand the role of specific genes in centriole duplication (Dammermann 2004, Kirkham 2003, Pelletier 2004b). The major disadvantage of laser‐assisted fixation, however, is its poor rate of fixation and the resulting insufficient structural preservation of the early embryo (for a general discussion on the disadvantages of chemical fixation on cellular fine structure, the reader is referred to Murk et al., 2003). In order to achieve the kind of resolution necessary for electron tomographic studies, we have previously used high‐pressure freezing as the means of primary fixation (O'Toole et al., 2003b). Whole worms were high‐pressure frozen and embedded in a thin layer of resin, so individual worms could be remounted and sectioned longitudinally. Embryos at the desired stages can be identified in serial sections of these longitudinally oriented worms. Using electron tomography of such high‐pressure frozen material, we have described the three‐dimensional (3D) structure of the mitotic C. elegans centrosome and identified both open and closed morphologies of centrosome‐associated microtubule ends (O'Toole et al., 2003b). However, a disadvantage of this procedure is that the whole worms contain populations of embryos at many phases of development, so the stage of mitosis had to be determined after sectioning. Here, we describe a method that combines the advantages of: (1) a correlative LM approach that allows us to stage development and fixation times with high time resolution, (2) high‐pressure freezing for the best possible preservation of ultrastructure, and (3) electron tomography for visualization and analysis of high‐resolution structure in 3D.

Section snippets

Rationale

Rationale of the described technique is to combine the observation of live mitotic embryos with high‐pressure freezing of clearly defined stages, allowing us to obtain tomographic reconstructions of mitotic spindle components in well‐preserved embryos of C. elegans as they develop. The methods presented here include procedures to transfer the isolated embryos into capillary tubes, to stage selected embryos by LM, to immobilize these staged embryos by high‐pressure freezing, and to fix them by

Staging of Isolated Embryos by LM

This section describes the preparation of isolated C. elegans embryos for live‐cell imaging under conditions suitable for subsequent high‐pressure freezing. The idea is to make use of capillary tubing to contain the embryos in short transparent tubes so that one can observe early developmental events under the light microscope, prior to time‐resolved high‐pressure freezing (McDonald et al., 2006).

As a first step, we prepare a “loading device” to collect isolated early embryos into capillary

Staging of Early Embryos

Instrumentation: Stereo dissecting microscope with light source, light microscope equipped with either DIC or epifluorescence (for example, a Zeiss Axioplan 2 with a 20 × 0.5 NA Plan‐Neofluar Apochromat dry objective, Hamamatsu Orca 12 bit digital camera controlled by MetaView Software, time‐lapse intervals of 10–15 sec).

Materials: C. elegans strain XA3501 expressing GFP::β‐tubulin and GFP::histone to visualize microtubules and chromatin, strain TH31 expressing GFP::γ‐tubulin and GFP::histone to

Discussion

We are interested in the 3D analysis of spindle components in early C. elegans embryos that have a known developmental history. Our method for correlative LM/EM has enabled us for the first time to select and stage mitotic C. elegans embryos prior to high‐pressure freezing and subsequent electron tomography. In the next sections, we discuss various methodological aspects of this technique and, briefly, its potential for the analysis of other systems.

Acknowledgments

The authors would like to thank Paul Verkade for sharing expertise on the newly developed EMPACT2+RTS and Jana Mäntler and Susanne Kretschmar for excellent technical assistance.

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