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. 2009:559:357-70.
doi: 10.1007/978-1-60327-017-5_25.

Detecting apoptotic cells and monitoring their clearance in the nematode Caenorhabditis elegans

Nan Lu  1 , Xiaomeng Yu, Xiangwei He, Zheng Zhou
Affiliations

Detecting apoptotic cells and monitoring their clearance in the nematode Caenorhabditis elegans

Nan Lu et al. Methods Mol Biol. 2009.

Abstract

Apoptosis is a genetically controlled process of cell suicide that plays an important role in animal development and in maintaining homeostasis. The nematode Caenorhabditis elegans has proven to be an excellent model organism for studying the mechanisms controlling apoptosis and the subsequent clearance of apoptotic cells, aided with cell-biological and genetic tools. In particular, the transparent nature of worm bodies and eggshells makes C. elegans particularly amiable for live cell microscopy. Here we describe a few methods for identifying apoptotic cells in living C. elegans embryos and adults and for monitoring their clearance during embryonic development. These methods are based on Differential Interference Contrast microscopy and on fluorescence microscopy using GFP-based reporters.

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Figures

Fig. 1
Fig. 1
A diagram describes the fate of an apoptotic cell in metazoans.
Fig 2
Fig 2
Apoptotic cells display the same distinct morphology detectable by DIC microscopy in different embryonic development stages. (A) DIC images of a wild-type embryo at different developmental stages. Time (labeled in min) represents the time point the embryo enters the corresponding stage after the first cleavage. (e) and (f) were captured at mid-3-fold (~550 min) and late 4-fold (~770 min) stages, respectively. Dorsal is up and anterior is to the left. Scale bars: 20 μm in (a–f) and 50 μm in (g). Arrows indicate cell corpses. (B) The number of cell corpses in wild-type embryos and newly hatched L1 larvae scored under the DIC microscope by two different individuals in independent experiments. Data are presented as mean ± SD. n, number of animals scored. aData published in (15). bData published in (16). cScored in the head of L1 larvae hatched within 1 h.
Fig. 3
Fig. 3
CED-1 and 2xFYVE as markers for apoptotic cells. (A) Diagrams of the reporter constructs. Pced-1ced-1 promoter, ICD Intracellular domain, SS Signal sequence, TM transmembrane domain. (B) DIC and fluorescence images illustrating that CED-1:GFP and 2×FYVE:GFP are enriched on the engulfing cell membrane surrounding cell corpses. (a–d) ~330 min-stage wild-type embryos. Anterior is to the top. Ventral faces readers. Scale bars: 10 μm. Arrows indicate phagosomes containing cell corpses. Arrowheads label the three ventral hypodermal cells as engulfing cells for C1, C2, and C3. (e–h) Part of the gonad in wild-type adult hermaphrodites. Mid-body is to the left. Scale bars: 20 μm. Arrows indicate phagosome scontaining germ cell corpses.
Fig. 4
Fig. 4
Time-lapse recording of the engulfment and degradation of apoptotic cells in C. elegans embryos. (A) DIC image of a ~310 min-stage embryo at which stage the time-lapse recording should begin. Anterior is to the top. Ventral faces readers. Arrowheads indicate the three ventral hypodermal cells that will engulf C1, C2, and C3. C1, C2, and C3 have not displayed the distinct DIC morphology of cell corpses at this time point. (B) Time lapse images of the co-expressed CED-1:GFP (a–g) and 2xFYVE:mRFP (h–u) around cell corpse C3 in a wild-type embryo. 0 min: the time point when the engulfing cell extends pseudopods halfway around C3. The scale bar: 5 μm. (C) The relative PI3P signal intensity on the surface of the phagosome containing C3 measured from images in (B) (h–u) plotted over time. (D) The volume of the phagosome containing C3 measured from images in (B) (h–u) plotted over time.
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