Worm Breeder's Gazette 15(1): 31 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Zoological Institute University of Koeln, 50923 Koeln, Germany
Gut differentiation in C. elegans is easily detectable due to the autofluorescence and birefringence of rhabditin granules (Babu, 1974; Laufer et al., 1980). However, in experimental manipulated embryos both markers are sometimes hardly detectable. In other nematodes we found these markers to be completely absent. Alternative markers for gut differentiation such as visualization of gut esterase activity (L. Edgar, 1995; see below) requires experimental experience and antibody staining detects gut in late embryogenesis, only. Moreover, both methods are time consuming and only fixed material can be processed. Here, we present an easy and fast method to reliably detect gut differentiation of early and living embryos in a variety of nematode species. The method relies on the endocytotic activity of the gut primordium which can be visualized by the uptake of fluorescently-labelled transferrin (Bossinger et al., 1996). Embryos of appropriate stages are mounted in a drop of distilled water on a polylysine-coated slide. The water is replaced by culture medium supplemented with 0.1 mg/ml Texas Red-coupled transferrin (Molecular Probes, T- 2871). We advise either to use the Leibovitz L- 15 medium based recipe or EGM (both developed and described by L. Edgar: Blastomere culture and analysis in: Methods in Cell Biology 48: 303- 321,1995) or the medium used by C.A. Shelton and B. Bowerman (Development 122: 2043-2050, 1996). Embryos are covered with a coverslip sealed with vaseline on the edges to prevent desiccation. In order to allow the access of transferrin to the blastomeres we perforate the eggshell and the underlying vitelline membrane by short laser pulses. Depending on the laser used it will be necessary to stain eggshells to allow absorption of laser energy. We add 8mg/ml trypan blue (Sigma) to the culture medium and use a nitrogen-pumped dye- laser with Rhodamin 6G as laser dye. Medium containing trypan blue should be replaced by medium with transferrin after perforation of the eggshell, because trypan blue inhibits the uptake of this endocytotic marker. However, instead of laser perforation it is sufficient to squeeze embyros by applying pressure on the coverslip in order to disrupt the vitelline layer. Alternatively, embryos may be devitellinized as described by L. Edgar (1995; see above). Embryos are incubated for 5-30 min at room temperature. Finally, the excess of free transferrin-conjugate is removed in order to reduce background fluorescence by replacing it with transferrin-free medium. Endocytosis of transferrin is analyzed with epifluorescence at 515- 565 nm. Applying this method in the soil nematode Acrobeloides nanus we found that endocytotic activity is already detectable at the 2-E cell stage which is considerably earlier than in C. elegans, where uptake of transferrin starts with 16-E cells present. Nevertheless, in both nematodes the endocytosis is among the first markers of tissue- specific differentiation. In all nematodes species we tested so far, transferrin is specifically accumulated in the gut primordium. This method may also be usefull to identify genes involved in endocytosis.