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.