Worm Breeder's Gazette 15(2): 35 (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.

Developmental Regulation of a Cyclin-Dependent Kinase Inhibitor Controls Postembryonic Cell Cycle Progression in C. elegans

Yang Hong, Richard Roy, Victor Ambros

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755

        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
        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promoter sequence of cki-1 (Rosalind Lee, unpublished).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.