Jimmy Ouellet and Richard Roy. . The intestinal lineage of C. elegans provides an excellent model to examine how developmental cues control the cell cycle due to the invariant, developmentally-regulated cell cycle transitions characteristic of these cells. We have isolated 5 mutants that show irregularities in the control of intestinal nuclear division timing during post-embryonic development. We classified these mutants based on whether they possessed more intestinal nuclei (
rr33 and
rr45) or less intestinal nuclei (
rr42,
rr43 and
rr44) than wild-type. Since all these mutants hatch with the wild-type complement of intestinal cells they all affect the cell cycle transition from mitosis to karyokinesis and finally the onset of the endocycles that normally occurs at the first larval stage. By performing lineage analysis on
rr33 mutants we found that the intestinal nuclear divisions occur normally at the L1 stage, but they do not make the appropriate transition to endocycle that usually occurs at the L1 moult. Consequently they undergo an extra round of nuclear division early in the L2 stage and become tetranucleate. We mapped this
rr33 mutant close to
dpy-5 on LGI and we performed systemic RNAi with all the predicted genes in this interval to determine if any of these candidates could phenocopy
rr33. Only C32F10.2 (RNAi) phenocopied the
rr33 phenotype and this gene corresponds to
lin-35: the C. elegans orthologue of the Retinoblastoma(Rb) gene.
lin-35/Rb is part of the synMuv B complex, which is required for the proper regulation of vulva cell fates by inhibiting the ras signaling pathway. We found that all synMuv B components tested so far enhance the intestinal phenotype of
lin-35(
rr33), suggesting that the same complex that is required for vulva specification is also required for the appropriate regulation of intestinal nuclear divisions. The reiteration of a L1-specific division during the L2 stage (heterochronic phenotype) prompted us to look at some of the components of the miRNA as well as PTGS/RNAi pathway. We found that the
lin-35(
rr33); al! g-1
(gk214) double mutant is synthe! tically lethal, whereas neither of the single mutants show this phenotype suggesting that these two genes might function in a similar essential pathway or process in addition to their roles in regulating nuclear division. We also found that components of the endoRNAi pathway (
rrf-3 and
eri-3) suppress the intestinal phenotype of
lin-35(
rr33) suggesting that DCR-1 protein play a role in this transition and is limiting. When the endoRNAi pathway is abrogated DCR-1 becomes more available for the microRNA or exoRNAi pathway to restore the wild-type complement of intestinal division. We therefore conclude that LIN-35 and probably other components of the synMUV B are required for the timely cell cycle transition of the intestinal cells at the L1 stage and that
lin-35 cooperates with components of the PTGS and/or microRNA pathway to appropriately regulate this cell cycle transition. Distinct and Redundant Functions for C. elegans HP1 Proteins in Post-Embryonic Development. Palladino Francesca, Sonia Schott, Thomas Simonet, Vincent Coustham, Cecile Bedet, Marianthi Karali Laboratory of Molecular and Cellular Biology, UMR5161, Ecole Normale Superieure de Lyon, 69007 Lyon, France The highly conserved HP1 family proteins play an important role in the dynamic organization of nuclear architecture and in the epigenetic control of gene expression. Although most species contain more than one HP1 family member which differ in their chromosomal distribution, it is not known to what extent their function is redundant or specific in a developmental context. C. elegans has two HP1 homologues, HPL-1 and HPL-2. While
hpl-2 is required for the maintenance of a functional germline and for vulval development by acting in the Rb related synMuvB pathway, no obvious function has so far been attributed to HPL-1. We report the characterization of an
hpl-1 null allele. We show that while the absence of
hpl-1 alone results in no obvious phenotype,
hpl-1;
hpl-2 double mutants show synthetic, temperature sensitive phenotypes including larval lethality and severe defects in the development of the somatic gonad. Furthermore, we find that
hpl-1 has an unexpected role in vulval development by acting redundantly with
hpl-2, but not other genes previously implicated in vulval development. Localization studies show that like HPL-2, HPL-1 is a ubiquitously expressed nuclear protein. However, HPL-1 and HPL-2 show clear differences in their localization. This is most clearly evident in embryos, where HPL-1 and HPL-2 are found concentrated in non-overlapping nuclear foci. We find that the LIN-13 protein, which forms a complex with HPL-2, is absolutely required for recruitment of HPL-2, but not HPL-1, in these foci. Our results show that HPL-1 and HPL-2 play both unique and redundant functions in post-embryonic development. Altogether, these studies contribute to an understanding of the function of HP1 proteins in gene regulation throughout development, and in a more general way on how a general chromatin interacting protein may play specific roles in given developmental pathways.