We have previously reported that the maternal-effect gene gad-1, encoding a protein with WD repeats, is required for gastrulation initiation (1). In gad-1(ct226ts) mutants at 25C, the E daughter cells divide early, with the timing and division plane of MS daughters, and fail to migrate into the embryo. However, in most mutant embryos as well as gad-1(RNAi) embryos, the E cell progeny correctly express gut differentiation markers, and we have shown by laser ablation that only E progeny can do so. In about 10% of mutant embryos (n=333) and 8% of gad-1(RNAi) defective embryos (n=198), no gut is produced. To ask whether E adopts another fate in these embryos, we recorded several mutant embryos through the 6th cell cycle. We observed no differences between embryos that do and do not make gut; however, many embryos appeared to have a pharyngeal deficit. In further analyses using the pharyngeal muscle antibody 9.2.1 (n=157) we found that 41% of mutant embryos made no pharynx, while 54% made a pharynx of reduced size, with only 1-8 cells stained. The remaining embryos appeared to make a normal sized pharynx, and none showed an overproduction of pharynx, even when they did not make gut. We obtained similar results with gad-1(RNAi) defective embryos and also in experiments using the earlier 3NB12 pharyngeal marker. In order to determine which blastomeres are capable of making pharynx in gad-1 mutants, we did laser ablations, either isolating MS at the 7-cell stage, or ablating the two MS daughters at the 15-cell stage, and assaying for pharyngeal differentiation by 9.2.1 staining. By these criteria, MS gives rise to pharynx in 66% of the embryos, while ABa-derived pharynx is present in only 30%. Because pop-1 also affects MS and E fates, we asked how gad-1 and pop-1 might interact. In pop-1 embryos, MS takes on the fate of the E cell, resulting in an overproduction of gut, but it nevertheless induces ABa descendants to make pharynx in 100% of the embryos. The same experiments in a pop-1;gad-1 double mutant showed that MS never makes pharynx (as in pop-1 alone), but 30% of the embryos still make ABa-derived pharynx (as in gad-1 alone). These results indicate that in gad-1 mutants, the ability of MS (or its E-like counterpart in a pop-1 mutant) to induce pharyngeal fates in ABa progeny is compromised. There also appears to be an interaction between gad-1 and pop-1 with respect to gut formation. In pop-1;gad-1 embryos, 64 % of the animals make no gut (compared to 10% for gad-1 embryos). This result suggests that gad-1 affects the ability of both the E-like MS cell and the true E cell to make gut. In support of this view, laser ablations of the true E cell in pop-1;gad-1embryos showed that in 11%, the E-like MS cell was capable of making gut; ablations of the E-like MS cell showed that in 36%, the true E cell was capable of making gut. Interestingly, whereas the above interactions between gad-1 and pop-1 appear to be complex, the cell lineage of pop-1;gad-1 embryos looks indistinguishable from that of gad-1 embryos. Whether or not gut is made in pop-1;gad-1 embryos, the E cells divide early and on the surface of the embryo, with the timing characteristic of the MS lineage (6/6 embryos, analyzed through the first 6 cell cycles). All other lineages are indistinguishable from wild type. In summary, we have found that: 1) gad-1 affects MS as well as E cell fates; 2) gad-1 and pop-1 interact in a complex manner in determining E cell fates; but 3) gad-1 appears completely epistatic to pop-1with regard to effects on the cell lineage, suggesting that cell behavior and subsequent cell fates in the E lineage can be separated. 1) Knight, J. K. and W. B. Wood (1998), Devel. Biol., in press

Affiliation:
- Department of MCD Biology, University of Colorado Boulder, CO 80309-0347