We are interested in how the heterochronic genes affect the cell division cycle of P3.p- P8.p, the vulva precursor cells (VPCs). In precocious
lin-14(lf) and
lin-28(lf) mutants the VPCs divide approximately one stage earlier (mid-L2) than in wild-type (mid-L3), although the VPCs themselves are born at the normal time. Thus, the length of the VPC cell cycle is shortened in
lin-14(lf) and
lin-28(lf) mutants (approximately 16 hours) compared to the wild-type ( approximately 24 hours). We have observed no alterations in the timing of the subsequent vulval cell divisions in
lin-14 and
lin-28 mutants. Mutations in the heterochronic genes
lin-4,
lin-29) affect the length of the VPC cell cycle. Epistasis analysis indicates that
lin-14 and
lin-28 are epistatic to
lin-4 and that
lin-29 plays no role in the VPCs. Thus, among these genes,
lin-14 and
lin-28 are the most direct regulators of the timing of VPC divisions. The epistatic relationship between
lin-14 and
lin-28 has not yet been established for the VPC defect. To determine when
lin-14 activity is required to inhibit early VPC divisions, we performed temperature shift experiments with
lin-14(
n179ts) animals.
lin-14 (
n179ts) animals were shifted at various times during development and then scored at the L2 molt for whether VPC divisions had occurred. In the figure below, '% mutant' refers to the portion of animals shifted at the indicated time that exhibited one or more VPC that had divided by the L2 molt. The number of animals shifted at each time is indicated).
n179ts animals maintained at 15 C showed no mutant phenotype and approximately 70% of animals maintained at 25 C were mutant. These preliminary results suggest that
lin-14 can act as early as approximately 2 hours after hatching and as late as the L1 molt. Although more animals need to be analyzed, these results suggest that
lin-14 is not a direct regulator of the VPC cell cycle since it can act prior to VPC birth (P cell division). Perhaps
lin-14 acts by activating a more direct regulator of the cell cycle. Genes downstream of
lin-14 would be candidates for such VPC cell cycle regulators. To examine the length of time required for lin- 14 activity to inhibit early VPC divisions, pulse shift experiments are planned. We also plan to isolate
lin-28ts alleles in order to determine the time of
lin-28 action. In order to determine which phase or phases of the cell cycle are altered in
lin-14 and
lin-28 mutants we are using direct quantitation of VPC DNA to locate the time of VPC S phase in the wild type and mutants. The earliest time point at which a doubling of DNA is observed in the VPCs relative to neurons in the ventral cord will delineate the S/G2 border. We quantitate the DNA of VPC nuclei using propidium iodide staining and confocal microscopy (White et al., 1987). This technique is similar to that used previously (e.g., by Hedgecock and White, 1985) to determine cell ploidy. However, in previous studies, conventional microscopy was used and VPCs were not analyzed. Our preliminary results indicate that wild type VPC cells do not enter G2 before the L2 molt. [See Figure 1]