Worm Breeder's Gazette 15(3): 33 (June 1, 1998)

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.

gad-1: evidence for a role in pharynx as well as gut specification and interaction with pop-1

Jennifer Knight, Bill Wood

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

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
        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