Worm Breeder's Gazette 15(2): 36 (February 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.
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755
In C. elegans, the fates of the six multipotent vulva precursor
cells (VPCs) are determined by extracellular signals in the late
L2/early L3, toward the end of the developmentally-regulated VPC cell
cycle (see also Hong et al, this Gazette). Wild-type VPC nuclei complete
S-phase in the early L3, approximately 1-2 hours after the L2 molt. A
regulatory pathway of heterochronic genes controls the timing of VPC
S-phase and also controls the timing of their competence to respond to
extracellular signals that determine their fates. Since VPC S-phase and
VPC fate determination both coincide with the beginning of the L3, and
are both controlled by the heterochronic gene pathway, the question
arises of whether completion of S-phase in VPCs may be mechanistically
linked to cell fate determination.
To test whether steps in the determination of vulval cell fates
are associated with steps in the VPC cell cycle, I examined the cell
cycle expression of two reporters for the primary fate, egl-17::GFP (a
gift from R. Burdine and M. Stern) and lin-12::GFP (a gift from D.
Levitan and I. Greenwald). In normal development, egl-17::GFP is
up-regulated in presumptive primary VPCs, and lin-12::GFP is
down-regulated in association with the primary fate. Hydroxyurea was
used to arrest the VPC cell cycle in G1, and the G1-arrested VPCs were
examined for the up-regulation of egl-17::GFP or the down-regulation of
lin-12::GFP (in separate transgenic lines). egl-17::GFP expression was
easily detectable specifically in P6.p of essentially all animals with
G1-arrested VPCs. Similarly, lin-12::GFP was distinctly reduced or
absent specifically from P6.p of animals with G1-arrested VPCs. These
results indicate that induction of the primary fate is initiated prior
to S-phase.
The patterns of expression of egl-17::GFP in G1-arrested VPCs of
wild type, lin-15(lf) or lin-12(gf) animals suggest that, in G1, VPCs
signal laterally via the Lin-12 pathway to inhibit adjacent primary
fates. To determine when in the cell cycle VPCs commit to secondary
fates, temperature-shift experiments were performed using
temperature-sensitive lin-12(gf) and lin-12(intra) mutations (gifts from
I. Greenwald) in conjunction with transient G1-arrest via reversible
hydroxyurea treatment. Surprisingly, these experiments indicated that
secondary fates are not irreversibly determined by Lin-12 until after
S-phase, despite the fact that as described above, lateral signalling
seems to be evident before S-phase. These results suggest that S-phase
of the VPC cell cycle is a pivotal step in vulval determination. Before
S-phase, VPCs begin the process of selecting primary and secondary fates
in response to extracellular signals, but the final pattern
(particularly with respect to secondary fates) is not irreversibly
determined until after completion of S-phase.
Now, VPC S-phase can occur precociously without resulting in
precocious cell fate determination, for example in cki-1(RNAi) animals
where the heterochronic gene pathway is still active (see Hong et al,
this Gazette). This indicates that completion of S-phase in VPCs is not
sufficient for cell fate determination, and underscores the parallel
roles of cell cycle progress and the heterochronic gene pathway in
gating VPC competence.