The C. elegans genome project has identified two genes homologous to
p21CIP/p27KIP family of cyclin-dependent kinase inhibitors (CKI). These genes,
cki-1 and
cki-2, are on chromosome II, and covered by cosmid T05A6. Of the two,
cki-1 has the greater similarity to
p27KIP and so we focused our attention on its expression, regulation and its potential role in mediating developmental signals to the cell cycle apparatus.
cki-1 expression pattern was first examined using a GFP reporter construct carrying 8kb of
cki-1 upstream sequence and the
cki-1 3'UTR.
cki-1::GFP shows a temporal and spatial pattern of expression that precisely coincides with temporal patterns of cell cycle progression during postembryonic development. Resting blast cells, including V, Z1/Z4, VPC, and SM, show strong expression of
cki-1::GFP before cell division, but greatly reduced or undetectable expression while dividing. When their progeny cells withdraw from the cell cycle to terminally differentiate,
cki-1::GFP expression increases again dramatically.
cki-1::GFP also shows high expression concomitant with cell cycle arrest in dauer larvae and starvation-arrested L1s. These results suggest that
cki-1 may play a role in maintaining cells in G1 stage of the cell cycle during devlopmentally-regulated cell cycle arrest and terminal differentiation. Preliminary promoter analysis indicates the presence of multiple temporal and cell-type specific cis-acting elements such as VPC-, SM- and lateral hypodermal-specific enhancers throughout the 8kb promoter sequence of
cki-1 (Rosalind Lee, unpublished). Detailed promoter analysis is underway to delineate such elements. To examine the role of
cki-1 as a mediator of developmental cell cycle control, we focused on the vulva cell lineage, wherein the length of G1 phase of the vulva precursor cell (VPC) cell cycle is regulated by the heterochronic gene pathway includinglin-14 . The VPC G1 phase is shortened in
lin-14(lf) and consequently VPCs divide and generate vulval cells precociously in the L2 instead of the L3. Conversely,
lin-14(gf) mutants can severely delay VPC division. In the wild type,
cki-1::GFP expression is seen weakly in VPCs starting from the L1 molt or early L2 and peaks at the L2 molt, shortly before VPCs enter the S-phase of the first round of division. Removing
lin-14 activity almost completely abolishes
cki-1::GFP expression in VPCs in this period, whereas with elevated
lin-14 activity, VPCs that are blocked from division show prolonged GFP expression. These results suggest that
lin-14 activates
cki-1 expression in the VPCs. To further explore the possibility that
cki-1 might be a downstream effector of
lin-14 in VPC cell cycle control, we tested whether expression of CKI-1 is sufficient to arrest a VPC in G1. CKI-1 or CKI-1::GFP fusion proteins were expressed specifically in P6.p using the
egl-17 promoter (a generous gift from R. Burdine and M. Stern), and were found to efficiently block P6.p from dividing while P5.p and P7.p (which did not express
egl-17::CKI-1::GFP) were unaffected. Using a S-phase reporter, rnr::GFP, we confirmed that
egl-17::CKI-1 blocks P6.p prior to S-phase, probably in G1. Second, we used RNAi to test for whether
cki-1 is required to maintain VPCs in G1 during wild type development.
cki-1(RNAi) animals display pleiotropic abnormalities, and among these are precocious VPC cell divisions during the L2, one stage earlier than in the wild type. Although the precocious VPC divisions in
cki-1(RNAi) animals are reminiscent of precocious vulva development in
lin-14(lf) (consistent with
lin-14 controlling VPC cell cycle by temporally regulating
cki-1 expression), there are important differences:
cki-1(RNAi) VPCs divide precociously, but unlike
lin-14(lf) VPCs, they do not generate differentiated vulval progeny. Rather,
cki-1(RNAi)-induced precocious VPC divisions generate duplicated, fully competent VPCs.
cki-1(RNAi);
lin-12(gf) animals show a "hypermuv" phenotype with as many as twelve psuedovulva as a result of these extra competent VPCs. Therefore, in VPCs
cki-1(RNAi) does not appear to alter cell fates but rather permits resting cells to divide precociously without premature commitment to further development. This suggests that there may be multiple regulatory controls on VPC cell cycle progression. By regulating
cki-1 activity,
lin-14 prevents VPCs from dividing in the L2, but
lin-14 also acts independently of
cki-1 to control when VPCs divide in L3 and when they can respond to signals which commit them to the production of differentiated vulval cells.