[
Worm Breeder's Gazette,
1994]
LOW TEMPERATURE AFFECTS EMBRYONIC HANDEDNESS REVERSAL Bill Wood, Arny Florance and Dominique Bergmann Department of MCD Biology, University of Colorado, Boulder C. elegans embryos become left-right asymmetric at the 6-cell stage witlf a handedness which is essentially invariant among N2 animals reared at 20, and which persists into adulthood, determining the asymmetric placement of the gonad, intestine, the coelomocytes, and several other cells. Reversal of embryonic handedness at the 6-cell stage by micromanipulation results in development of healthy adults with all left-right asymmetries reversed. l Spontaneous reversal occurs at a frequency of a few percent among animals developing embryos treated with chitinase at the 2-cell stage to remove the egg shell.2 We have now found that when N2 are reared at 10, the frequency of animals with reversed gonad handedness as adults increases to above 0.5%. Those examined by Nomarski microscopy exhibited handedness reversal of the ventral nerve cord (cell bodies on the left) and reversed placement of the coelomocytes as well as the intestine and gonad, suggesting that these animals developed from reversed embryos. To determine the cold-sensitive period for handedness reversal, young gravid adults reared at 20 were placed at 10 for 2 hours, then transferred to new 20 plates and subsequently transferred to fresh 20 plates at 2-hr intervals. Eggs laid during the cold pulse (interval l; exposed to 10 after fertilization), the first 2 hr after return to 20 (interval 2; exposed to 10 either just before or just after fertilization), and each of the four subsequent 2-hr intervals at 20 (3-6, exposed to 10 before fertilization) were allowed to hatch and develop at 20 into adults, which were then scored for gonad handedness. Among animals developing from embryos exposed to 10 after fertilization (interval 1), no reversals were' observed (N=1,956), whereas among animals developing from oocytes and sperm exposed to 10 before fertilization (intervals 3-6), the frequency of reversal was 0.21% + 0.09% (N=9,223). Surprisingly, although left-right asymmetry first appears at the 6-cell stage, and the embryo's left and right are highly unlikely to be specified until the 4-cell stage, I the cold-sensitive period for reversal is before fertilization. This result suggests that the effect is on the gametes. We can imagine two possible explanations: 1) Production of some eggshell precursor component, synthesized during oogenesis, could be cold sensitive, such that embryos with "soft" eggshells are produced at 10, leading to a low frequency of reversal as seen in chitinased embryos.2 2) Some handed structure in a gamete, such as the sperm centriole for example, may normally dictate the skewing of the ABa and ABp spindles that leads to handed asymmetry in the embryo;l when this structure is exposed to 10 its subsequent ability to dictate handedness could be impaired, leading to a low frequency of reversal. We are currently carrying out experiments with chilled males to determine whether egg or sperm is the cold-sensitive gamete, as well as examining effects of other treatments on handedness reversal. Finally, we are continuing small scale screens for maternal-effect mutations that result in a high frequency of embryonic reversal. None have been found, but so far we have screened only about 1000 EMS-mutagenized genomes. I Wood, W. B. (1991) Nature 349: 536-538. 2 Wood, W. B. and Kershaw, D. (1991) In Biological Asymmetry and Handedness, CIBA Foundation Symposium 162: 143-164.