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Live Imaging of Caenorhabditis elegans: Examples

Adapted from "Live Imaging of Caenorhabditis elegans," Chapter 20, in Live Cell Imaging: A Laboratory Manual (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

First Divisions in Early Embryos and Chromatin Dynamics in Lamin Mutants

To image early cleavages and chromatin dynamics, it is convenient to use histone H2B fused to GFP or lamin::GFP. Time-lapse movies can be obtained using conventional confocal microscope systems and their included software. Early embryos dissected from transgenic hermaphrodites are placed with egg salts on agar pads (e.g., Live Imaging of Caenorhabditis elegans: Preparation of Samples). Chromatin dynamics can be followed easily, and wild-type embryonic cells can be compared with mutants or RNAi-treated embryos [for normal lamin staining, see Fig. 1 and for chromosomal defects in lamin(RNAi) embryos, see Movie 1 ].

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Figure 1. A stereo pair showing three-dimensional views of transgenic L1 larva expressing Ce-lamin fused to GFP (Liu et al. 2000). The animal was viewed with a Bio-Rad MRC-1024 confocal scanhead coupled to a Zeiss Axiovert 135M inverted microscope. The VoxBlast 3-Dimensional Measurement and Volume Visualization Software (VayTek) were used for the three-dimensional reconstruction.

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Movie 1. Arrested embryos injected with lmn-1 dsRNA. The expression of the lmn-1 gene (Liu et al. 2000) was down-regulated in C. elegans hermaphrodites expressing histone H2B-GFP (AZ212 line) (Praitis et al. 2001). The F1 progeny of hermaphrodites injected with double-stranded RNA of lmn-1 were mounted on an agar pad with egg salts solution, sealed with vacuum grease, and viewed with a Zeiss Axioplan II microscope equipped with an epifluorescence illuminator. An Axiocam CCD camera and the AxioVision Image Analysis package were used to collect the time-lapse data every 30 seconds. The video shows that most nuclei have disorganized chromatin. One nucleus shows the typical anaphase bridges during mitosis.

Simultaneous Nomarski and GFP Imaging of Elongating Epidermal Cells in Embryos

To image embryos using time-lapse movies in different focal planes, it is convenient to use the four-dimensional imaging system developed by John White and collaborators (Eliceiri et al. 2002). Suitable commercial software is also available with different confocal or deconvolution microscopy systems (see Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection). Embryos are placed on poly-L-lysine-coated coverslips in a small drop of egg salts solution. Using a 1-ml syringe, a thin layer of high-vacuum grease (or petroleum jelly) is applied around the edge of the coverslip, and a second coverslip or a slide with an agar pad is placed on top. The embryos attach to the poly-L-lysine-coated coverslip, and the vacuum grease or petroleum jelly seals the chamber and protects the embryos from being squashed. This closed chamber allows extended observation of embryos expressing multiple fluorescence signals (GFP) and DIC (Rabin and Podbilewicz 2000) (see Fig. 2 , Movie 2 , and Movie 3 ).

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Figure 2. Projections of embryo expressing AJM-1::GFP in the zonulae adherens (ZA) and eff-1p::GFP in the cytoplasm of epidermal cells during skin syncytiogenesis (Rabin and Podbilewicz 2000; Shemer and Podbilewicz 2002). The animal was viewed with a Bio-Rad MRC-1024 confocal scanhead coupled to a Nikon E800 microscope. The Lasersharp software was used for the three-dimensional reconstruction of the three z-series images shown that represent three time points in minutes. Arrows point to the ZA between two dorsal epithelial cells before (0 min) and after (25 and 35 min) cell fusion. Strong cytoplasmic and nuclear staining is from the transcriptional reporter gene eff-1promoter::GFP. Cytoplasmic mixing confirms cell fusion, and ZA disappearance shows plasma membrane fusion.

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Movie 2. Simultaneous imaging of Nomarski (blue) and eff-1promoter::GFP (green) in developing embryos reveals timing of cell fusion. Confocal microscopy was performed as in Figure 2 (Rabin and Podbilewicz 2000). The initial expression of the reporter gene was observed in the precursors of the epidermal cells. Cytoplasmic mixing of the GFP signal in the dorsal and ventral hypodermis occurs during elongation (morphogenesis) of the embryo, reflecting cell fusion between epidermal cells.

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Movie 3. Expression of AJM-1::GFP in epithelial cells during embryonic morphogenesis. Expression of AJM-1::GFP in epithelial cells during embryonic morphogenesis. Animation of confocal projections collected every 5 minutes shows changes in epidermal cell morphology and cell fusion during elongation.

Analysis of Individual Larvae or Adults Using Confocal Microscopy and Three-Dimensional Reconstructions of Vulval Rings

To analyze organogenesis of the vulva, including the stages of cell generation, cell migration, ring formation, and intraring cell fusion, a laser-scanning confocal microscope is used to follow different GFP reporters expressed in the vulval cells (Sharma-Kishore et al. 1999; Shemer et al. 2000). It is possible to follow individual worms throughout development, using successive recovery of the worms as described in Live Imaging of Caenorhabditis elegans: Preparation of Samples, even after exposure to laser confocal microscopy. We anesthetize larvae with 0.01% levamisole to completely block movement in an agar pad. After rapid analysis using the 488-nm line of the 100-mW argon laser at 0.3% intensity (Rabin and Podbilewicz 2000), the coverslip is slowly removed, and the worms are gently transferred from the slide into a fresh plate and recovered with a drop of M9 buffer at 20°C. After 2 hours or more, the worms are returned to a new agar pad with anesthesia, imaged, and recovered again (Shemer and Podbilewicz 2002) (Movie 4 and Movie 5 ).

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Movie 4. z-series of two vulval toroids expressing egl-17/FGFpromoter::GFP (Burdine et al. 1998). In the L4 stage, the vulva is composed of a stack of seven rings or toroids. Only two vulval syncytial cells express fibroblast growth factor (FGF/egl-17). The vulD ring on top (dorsal) contains two nuclei, and the vulC toroid contains four nuclei. Two additional rings above vulD and three ventral rings do not express this reporter gene at this stage (Burdine et al. 1998).

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Movie 5. Projection of the z-series and rotation of two vulval rings expressing fibroblast grown factor (egl-17) from Movie 4. Using software from Lasersharp (Bio-Rad), it is possible to animate the vulval rings. The center (hole) allows the passage of eggs to the outside and sperm in the opposite direction during mating.

REFERENCES

Burdine R.D., Branda C.S., Stern M.J. 1998. Egl-17(fgf) expression coordinates the attraction of the migrating sex myoblasts with vulval induction in C. elegans. Development 125: 1083–1093.[Abstract]

Eliceiri K.W., Rueden C., Mohler W.A., Hibbard W.L., White J.G. 2002. Analysis of multidimensional biological image data. BioTechniques 33: 1268–1273.[Medline]

Liu J., Rolef Ben-Shahar T., Riemer D., Treinin M., Spann P., Weber K., Fire A., Gruenbaum Y. 2000. The Caenorhabditis elegans lamin gene is essential and is required for nuclear organization, mitotic progression, chromosome segregation and spatial organization of nuclear pore complexes. Mol. Biol. Cell 11: 3937–3947.[Abstract/Free Full Text]

Praitis V., Casey E., Collar D., Austin J. 2001. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157: 1217–1226.[Abstract/Free Full Text]

Rabin Y. and Podbilewicz B. 2000. Temperature-controlled microscopy for imaging living cells: Apparatus, thermal analysis, and temperature dependency of embryonic elongation in C. elegans. J Microsc 199: 214–223.[Medline]

Sharma-Kishore R., White J.G., Southgate E., Podbilewicz B. 1999. Formation of the vulva in C. elegans: A paradigm for organogenesis. Development 126: 691–699.[Abstract]

Shemer G. and Podbilewicz B. 2002. LIN-39/Hox triggers cell division and represses EFF-1/Fusogen-dependent vulval cell fusion. Genes & Dev. 16: 3136–3141.[Abstract/Free Full Text]

Shemer G., Kishore R., Podbilewicz B. 2000. Ring formation drives invagination of the vulva in C. elegans: Ras, cell fusion and cell migration determine structural fates. Dev. Biol. 221: 233–248.[Medline]