Worm Breeder's Gazette 5(2): 46
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.
To test whether cytoplasmic determinants are prelocalized in the egg, we have extruded cell fragments, containing presumptive cytoplasm of somatic and germ line precursor cells, from uncleaved eggs or early blastomeres through laser-induced holes in the eggshells. Eggs were mounted in an extrusion medium containing 120 mM NaCl, 10 mM Hepes at pH 7.2, 100 mg/ml streptomycin sulfate, 100 units/ml penicillin, 0.045 mg/ml each of 18 of the 20 standard amino acids ( omitting asparagine and glutamine), and 0.025 mg/ml of the vital dye trypan blue. The eggshell absorbs the blue dye and is thereby sensitized to a Rhodamine 6 G dye laser microbeam (590 nm) that is used to puncture the shell. The cell adjacent to the hole in the eggshell is then partially extruded by pushing gently on the coverslip. Electronmicrographs show that the cell fragment is surrounded by a plasma membrane. The extruded cytoplasm looks very similar to that in the corresponding region of an intact egg containing, for example, ribosomes, mitochondria and yolk granules. From uncleaved eggs, posterior extrusion is the critical experiment because the posterior 15% of egg cytoplasm is normally segregated into the P2 cell , which will give rise to the somatic stem cells C and D and the germ line precursor P4. About 60% of the fertilized, uncleaved eggs from which posterior cell fragments were removed at or after fusion of the pronuclei showed no developmental defects and gave rise to normal larvae. All those transferred to growth plates formed fertile adults that laid a normal number of eggs (over 250). We estimate that from some of these embryos most or all of the presumptive C, D, and P4 cytoplasm and a large area of egg membrane been removed without affecting normal development or fertility. It is more difficult to remove fragments from later-stage blastomeres. Nevertheless, 12 of 28 embryos surviving the laser shot hatched after removal of large portions of the P2 or P3 blastomere. In the P2 extrusions, embryos developed into fertile adults following removal of all presumptive P4 cytoplasm, or all P4 and D cytoplasm. In one embryo 60 % of the P3 cell was extruded including all of the presumptive P4 cytoplasm. The P3 cell divided, but the resultant P4 cell was very small, and did not divide again during embryogenesis as it normally would (to produce two germ line precursor cells at hatching). Nonetheless, P4 proliferated after hatching. The animal formed apparently normal gonads and laid an approximately normal number of eggs (209). Our results suggest that cytoplasmic prelocalization in the early embryo is not responsible for the determinate development of Caenorhabditis e prepatterning seems also to be absent, since large amounts of membrane were extruded in our experiments.