The cellular anatomy of embryos of the nematode Caenorhabditis elegans

Krieg, C.J.; Cole, T.; Deppe, Uwe; Schierenberg, Einhard; Schmitt, David; Yoder, Bonita L.; Ehrenstein, Gerald

doi:10.1016/0012-1606(78)90190-2

Cited by 61 publications

(13 citation statements)

References 8 publications

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“…The pattern of asymmetric cleavages, cell positions, and cell fates in early C. elegans embryos is invariant (7,8). Cleavage-arrest and cell-ablation experiments have shown that the gut precursor cells in two-cell, four-cell, and eight-cell embryos require neither cell division nor the presence of normal cell neighbors to subsequently express an intestinal differentiation marker (12).…”

Section: Discussionmentioning

confidence: 99%

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Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.

Strome¹,

Wood²

1982

Proc. Natl. Acad. Sci. U.S.A.

352

245

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By using fluorescent antibody staining, we have followed cytoplasmic granules unique to germ-line cells throughout the life cycle of Caenorhabditis elegans. These elements, designated P granules, are segregated exclusively to germ-line precursor cells during early embryogenesis. Prior to mitosis at each ofthe early cleavages that produce a somatic and germ-line daughter cell, the granules become localized in the region of cytoplasm destined for the germ-line daughter. After the 16-cell stage, the granules appear to be associated with the nuclear envelope. P granules persist in the germ cells throughout the larval and adult stages. The P granules are similar in number, size, and distribution to germ-line-specific structures identified as "germinal plasm" by electron microscopy in C. elegans embryos.Asymmetric segregation ofcytoplasmic components is observed during the early cleavages of many invertebrate embryos (1 (4) or by digesting adult worms with 1% NaOCl in 0.5 M NaOH (5). The embryos were transferred to a drop of M9 salt solution on a polylysine-coated slide (6), covered with a silanized coverslip, made permeable by freezing the slide on dry ice and then popping off the coverslip, fixed in absolute methanol at 40C for 20 min, and air dried. Larvae were picked from plates and fixed as described for embryos. Adult gonads and gametes were obtained by cutting open adult worms in M9 salt solution on polylysine-coated slides, frozen as described above, fixed in acetone at -200C for 20 min, and air dried. Fixed preparations were incubated with undiluted nonimmune rabbit serum for 1 hr at 250C and then overnight with F-RAM (1:40) in phosphate-buffered saline (150 mM NaCl/3 mM KCV8 mM Na2HPOJ1.5 mM KH2POJ1 mM MgCl2) at 4°C. The slides were washed for 90 min with three changes of the same buffer at 10°C, treated with diamidinophenylindole (DAPI) hydrochloride (0.5 ,ug/ ml; Boehringer-Mannheim) in phosphate-buffered saline, rinsed with H20, and mounted in Gelutol (Monsanto) mounting fluid.Microscopy. A Zeiss photomicroscope equipped with Nomarski and epifluorescence optics was used for observation and photography. Each embryo preparation was first photographed with 440-to 490-nm epi-illumination to visualize the immunofluorescence and then simultaneously with visible transmitted light and 365-nm epi-illumination to visualize the embryo and the DAPI-stained chromosomes. For larval and gonad preparations, the Nomarski, DAPI, and immunofluorescence images were photographed separately. RESULTSCleavage of the fertilized C. elegans egg (P0) includes four successive asymmetric divisions (Fig. 1), each generating a larger somatic precursor cell and a smaller P cell (7). The resultant P4

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Section: Discussionmentioning

confidence: 99%

“…8; N. Wolf and D. Hirsh, personal communication). These structures, like P granules, are restricted to the P cells during early cleavage stages.…”

Section: Discussionmentioning

confidence: 99%

Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans.

Strome¹,

Wood²

1982

Proc. Natl. Acad. Sci. U.S.A.

352

245

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“…Embryos were transferred to the fixative and incubated for 24 hours at 40°C (Krieg et al, 1978). Similar experiments were done at 4°C and at room temperature.…”

Section: Using Elevated Temperaturementioning

confidence: 99%

“…Agar blocks were incubated either in osmium or reduced osmium as a postfixative. Dehydration was done through two changes of ethanol of increasing concentration (40, 60, 80, 100%) as usual, but 2-minute rinses were preferred over 20-minute rinses in order to avoid shrinkage (Krieg et al, 1978), followed by impregnation in Spurr. Eighty nm-thick sections were made using a Leica Ultracut-S Ultramicrotome.…”

Section: Buffers Usedmentioning

confidence: 99%

“…The three distinct layers that cover the embryo (an inner vitelline layer, a median chitinous layer or chorion, and an outer protein coat; Chitwood and Chitwood, 1974;Bird and Bird, 1991) are almost completely impermeable for fixatives and embedding media under standard electron microscopy procedures. Although methods for TEM of C. elegans embryos have been described previously (Krieg et al, 1978;Wolf et al, 1983;Priess and Hirsh, 1986;Schierenberg et al, 1986), ultrastructural preservation was unsatisfactory because a choice had to be made between visualising either outer cell membranes or internal cellular ultrastructure (Krieg et al, 1978).…”

Section: Introductionmentioning

confidence: 99%

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