Worm Breeder's Gazette 3(2): 20
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.
In 1973 Whittaker showed that when early ascidian blastulae are treated with a cleavage inhibitor and subsequently examined for appearance of tissue specific enzymes or pigments, these differentiation markers appear on schedule, and only in the precursor cells of the appropriate tissues. The pattern changes at each of the first few cleavages, apparently reflecting segregation of developmental potential. In order to carry out similar experiments with C. elegans, the permeability barrier imposed by the eggshell must be circumvented to allow application of cleavage inhibitors. This can be accomplished using a permeable eggshell mutant (Laufer, this issue), or by gently bursting wild-type eggs between a slide and a coverslip. When this procedure is carried out in a suitable culture medium (ibid.) many of the blastomeres of early cleavage stage embryos survive and continue development to twitching tissue masses. Most of our experiments have been done with embryos from burst eggs, which generally appear healthier and give better results than the permeable shelI embryos. As a differentiation marker we have used the 'rhabditin' granules that appear in gut cells late in embryonic development; they can be seen in the light microscope as fluorescent bodies when viewed with epifluorescence or as bright spots on a dark background when viewed with crossed polarizers (Ward, personal communication). The precursors of the gut cell lineage are the P1, EMSt, and E blastomeres at the 2-, 4-, and 8- cell stages, respectively (Deppe et al., 1978). Cleavage of burst egg preparations was stopped by addition of 20 g/ml cytochalasin B and 50 g/ml colchicine to the medium. Embryos then were incubated overnight at 16 C and scored for appearance of the marker. In arrested 2-cell embryos, granules were observed in 17 out of 21 P1 cells and in 0 out of 24 AB cells; in some of these embryos granules were observed in the P1 cell even though the AB cell had been destroyed when the egg was burst. In arrested 4-cell embryos, the granules were seen in 6 out of 20 EMSt cells and in none of the AB or P2 cells. Although the biochemical nature of this marker is poorly defined, the results appear to show segregation of potential for a tissue-specific activity that can be expressed independently of cell division, in agreement with the findings of Whittaker. We are in the process of examining other tissue-specific markers, and screening egg- lethal mutants for anomalous segregation of developmental potential.