THE ORIENTATION OF FIRST CLEAVAGE IN THE SEA-URCHIN EMBRYO, LYTECHINUS-VARIEGATUS, DOES NOT SPECIFY THE AXES OF BILATERAL SYMMETRY

One blastomere of the two-cell stage sea urchin embryo (Lytechinus var iegatus) was labeled with an intracellular fluorescent lineage tracing stain to determine, from the lineage of that blastomere, the orientat ion of the first cleavage furrow with regard to the axes of bilateral symmetry in the gastrula and pluteus larva. Two methods were used to m ark the blastomere: In the first, the lipophilic carbocyanine dye DiIC (16) was microinjected directly into the blastomere after first cleava ge was completed. In the second, caged (nonfluorescent) fluorescein-de xtran was microinjected into the single-celled zygote and uncaged (mad e fluorescent) in one of the blastomeres at the two-cell stage using t wo-photon excitation microscopy (TPEM). This is the first use of TPEM for embryonic lineage tracing. In both methods the dye proved to be no ntoxic and fluorescence was confined to lineally related cells. The re sults from both methods were similar and showed that the first cleavag e furrow was variable in its orientation. Results were similar using a nimals obtained from different geographic locations. These results dif fer from those of McCain and McClay (Development, 1994, 120, 395-404), who reported that the median orientation was invariant in this specie s. The differences between the two studies are discussed. We conclude that first cleavage does not specify nor is it predictive of the bilat eral axes in this species. The technique of TPEM is proffered as a pow erful new tool that will enable the marking and tracing of embryonic c ell lineages with less injury and more precision than current methods. (C) 1996 Academic Press, Inc.