The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.

Centrosome duplication is a critical step in assembly of the bipolar mitotic spindle, but the molecular mechanisms regulating this process during the cell cycle and during animal development are poorly understood. Here, we report that the zyg-1 gene of Caenorhabditis elegans is an essential regulator of centrosome duplication. ZYG-1 is a protein kinase specifically required for daughter centriole formation that localizes transiently to centrosomes and acts at least one cell cycle prior to each spindle assembly event. In the embryo, ZYG-1 participates in a unique regulatory scheme whereby paternal ZYG-1 regulates duplication and bipolar spindle assembly during the first cell cycle, and maternal ZYG-1 regulates these processes thereafter. ZYG-1 is therefore a key molecular component of the centrosome/centriole duplication process.

The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry.

During the first cell cycle of Caenorhabditis elegans embryogenesis, asymmetries are established that are essential for determining the subsequent developmental fates of the daughter cells. The maternally expressed par genes are required for establishing this polarity. The products of several of the par genes have been found to be themselves asymmetrically distributed in the first cell cycle. We have identified the par-4 gene of C. elegans, and find that it encodes a putative serine-threonine kinase with similarity to a human kinase associated with Peutz-Jeghers Syndrome, LKB1 (STK11), and a Xenopus egg and embryo kinase, XEEK1. Several strong par-4 mutant alleles are missense mutations that alter conserved residues within the kinase domain, suggesting that kinase activity is essential for PAR-4 function. We find that the PAR-4 protein is present in the gonads, oocytes and early embryos of C. elegans, and is both cytoplasmically and cortically distributed. The cortical distribution begins at the late 1-cell stage, is more pronounced at the 2- and 4-cell stages and is reduced at late stages of embryonic development. We find no asymmetry in the distribution of PAR-4 protein in C. elegans embryos. The distribution of PAR-4 protein in early embryos is unaffected by mutations in the other par genes.

RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans.

As a step towards comprehensive functional analysis of genomes, systematic gene knockout projects have been initiated in several organisms [1]. In metazoans like C. elegans, however, maternal contribution can mask the effects of gene knockouts on embryogenesis. RNA interference (RNAi) provides an alternative rapid approach to obtain loss-of-function information that can also reveal embryonic roles for the genes targeted [2,3]. We have used RNAi to analyze a random set of ovarian transcripts and have identified 81 genes with essential roles in embryogenesis. Surprisingly, none of them maps on the X chromosome. Of these 81 genes, 68 showed defects before the eight-cell stage and could be grouped into ten phenotypic classes. To archive and distribute these data we have developed a database system directly linked to the C. elegans database (Wormbase). We conclude that screening cDNA libraries by RNAi is an efficient way of obtaining in vivo function for a large group of genes. Furthermore, this approach is directly applicable to other organisms sensitive to RNAi and whose genomes have not yet been sequenced.

Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast.

Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30-50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.

PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditis elegans embryos.

The par genes are required to establish polarity in the Caenorhabditis elegans embryo. Mutations in two of these genes, par-3 and par-6, exhibit similar phenotypes. A third gene, pkc-3, gives a similar phenotype when the protein is depleted by RNA interference. PAR-3 and PKC-3 protein are colocalized to the anterior periphery of asymmetrically dividing cells of the germline lineage and the peripheral localizations of both proteins depends upon the activity of par-6. Here we report the molecular cloning of par-6 and the immunolocalization of PAR-6 protein. We found that par-6 encodes a PDZ-domain-containing protein and has homologues in mammals and flies. Moreover, we discovered that PAR-6 colocalizes with PAR-3 and that par-3 and pkc-3 activity are required for the peripheral localization of PAR-6. The localization of both PAR-3 and PAR-6 proteins is affected identically by mutations in the par-2, par-4 and par-5 genes. The co-dependence of PAR-3, PAR-6 and PKC-3 for peripheral localization and the overlap in their distributions lead us to propose that they act in a protein complex.

An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3.

Cell polarity is fundamental to differentiation and function of most cells. Studies in mammalian epithelial cells have revealed that the establishment and maintenance of cell polarity depends upon cell adhesion, signaling networks, the cytoskeleton, and protein transport. Atypical protein kinase C (PKC) isotypes PKCzeta and PKClambda have been implicated in signaling through lipid metabolites including phosphatidylinositol 3-phosphates, but their physiological role remains elusive. In the present study we report the identification of a protein, ASIP (atypical PKC isotype-specific interacting protein), that binds to aPKCs, and show that it colocalizes with PKClambda to the cell junctional complex in cultured epithelial MDCKII cells and rat intestinal epithelia. In addition, immunoelectron microscopy revealed that ASIP localizes to tight junctions in intestinal epithelial cells. Furthermore, ASIP shows significant sequence similarity to Caenorhabditis elegans PAR-3. PAR-3 protein is localized to the anterior periphery of the one-cell embryo, and is required for the establishment of cell polarity in early embryos. ASIP and PAR-3 share three PDZ domains, and can both bind to aPKCs. Taken together, our results suggest a role for a protein complex containing ASIP and aPKC in the establishment and/or maintenance of epithelial cell polarity. The evolutionary conservation of the protein complex and its asymmetric distribution in polarized cells from worm embryo to mammalian-differentiated cells may mean that the complex functions generally in the organization of cellular asymmetry.

Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans.

Asymmetric cell divisions, critically important to specify cell types in the development of multicellular organisms, require polarized distribution of cytoplasmic components and the proper alignment of the mitotic apparatus. In Caenorhabditis elegans, the maternally expressed protein, PAR-3, is localized to one pole of asymmetrically dividing blastomeres and is required for these asymmetric divisions. In this paper, we report that an atypical protein kinase C (PKC-3) is essential for proper asymmetric cell divisions and co-localizes with PAR-3. Embryos depleted of PKC-3 by RNA interference die showing Par-like phenotypes including defects in early asymmetric divisions and mislocalized germline-specific granules (P granules). The defective phenotypes of PKC-3-depleted embryos are similar to those exhibited by mutants for par-3 and another par gene, par-6. Direct interaction of PKC-3 with PAR-3 is shown by in vitro binding analysis. This result is reinforced by the observation that PKC-3 and PAR-3 co-localize in vivo. Furthermore, PKC-3 and PAR-3 show mutual dependence on each other and on three of the other par genes for their localization. We conclude that PKC-3 plays an indispensable role in establishing embryonic polarity through interaction with PAR-3.

Early patterning of the C. elegans embryo.

Studies of about 20 maternally expressed genes are providing an understanding of mechanisms of patterning and cell-fate determination in the early Caenorhabditis elegans embryo. The analyses have revealed that fates of the early blastomeres are specified by a combination of intrinsically asymmetric cell divisions and two types of cell-cell interactions: inductions and polarizing interactions. In this review we summarize the current level of understanding of the molecular mechanisms underlying these processes in the specification of cell fates in the pregastrulation embryo.

PAR-2 is asymmetrically distributed and promotes association of P granules and PAR-1 with the cortex in C. elegans embryos.

The par genes participate in the process of establishing cellular asymmetries during the first cell cycle of Caenorhabditis elegans development. The par-2 gene is required for the unequal first cleavage and for asymmetries in cell cycle length and spindle orientation in the two resulting daughter cells. We have found that the PAR-2 protein is present in adult gonads and early embryos. In gonads, the protein is uniformly distributed at the cell cortex, and this subcellular localization depends on microfilaments. In the one-cell embryo, PAR-2 is localized to the posterior cortex and is partitioned into the posterior daughter, P1, at the first cleavage. PAR-2 exhibits a similar asymmetric cortical localization in P1, P2, and P3, the asymmetrically dividing blastomeres of germ line lineage. This distribution in embryos is very similar to that of PAR-1 protein. By analyzing the distribution of the PAR-2 protein in various par mutant backgrounds we found that proper asymmetric distribution of PAR-2 depends upon par-3 activity but not upon par-1 or par-4. par-2 activity is required for proper cortical localization of PAR-1 and this effect requires wild-type par-3 gene activity. We also find that, although par-2 activity is not required for posterior localization of P granules at the one-cell stage, it is required for proper cortical association of P granules in P1.

par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3.

The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a partitioning defective phenotype (par) have identified several genes involved in this process. We have identified a new gene, par-6, which acts in conjunction with other par genes to properly localize cytoplasmic components in the early embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity is revealed by our genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the PAR-3 protein at the cell periphery. In addition, we find that loss-of-function par-6 mutations act as dominant bypass suppressors of loss-of-function mutations in par-2.

Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo.

Asymmetric cleavage plays an important role in Caenorhabditis elegans embryogenesis. In addition to generating cellular diversity, several early asymmetric cleavages contribute to the spatial organization of the embryo. Genetic and molecular analyses of several genes, including six par genes and the mex-1 and mes-1 genes, together with experimental embryological studies, have provided insights into mechanisms controlling polarity and spindle orientations during these cleavages. In particular, these studies focus attention on microfilament-based motility and changing protein distributions at the cell cortex.

A non-muscle myosin required for embryonic polarity in Caenorhabditis elegans.

Daughter cells with distinct fates can arise through intrinsically asymmetrical divisions. Before such divisions, factors crucial for determining cell fates become asymmetrically localized in the mother cell. In Caenorhabditis elegans, PAR proteins are required for the early asymmetrical divisions that establish embryonic polarity, and are asymmetrically localized in early blastomeres, although the mechanism of their distribution is not known. Here we report the identification in C. elegans of nonmuscle myosin II heavy chain (designated NMY-2) by means of its interaction with the PAR-1 protein, a putative Ser/Thr protein kinase. Furthermore, injections of nmy-2 antisense RNA into ovaries of adult worms cause embryonic partitioning defects and lead to mislocalization of PAR proteins. We therefore conclude the NMY-2 is required for establishing cellular polarity in C. elegans embryos.

Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos.

The par-3 gene is required for establishing polarity in early C. elegans embryos. Embryos from par-3 homozygous mothers show defects in segregation of cytoplasmic determinants and in positioning of the early cleavage spindles. We report here that the PAR-3 protein is asymmetrically distributed at the periphery of the zygote and asymmetrically dividing blastomeres of the germline lineage. The PAR-3 distribution is roughly the reciprocal of PAR-1, another protein required for establishing embryonic polarity in C. elegans. Analysis of the distribution of PAR-3 and PAR-1 in other par mutants reveals that par-2 activity is required for proper localization of PAR-3 and that PAR-3 is required for proper localization of PAR-1. In addition, the distribution of the PAR-3 protein correlates with differences in cleavage spindle orientation and suggests a mechanism by which PAR-3 contributes to control of cleavage pattern.

par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed.

The first cleavage of C. elegans is asymmetric, generating daughter cells with different sizes, cytoplasmic components, and fates. Mutations in the par-1 gene disrupt this asymmetry. We report here that par-1 encodes a putative Ser/Thr kinase with similarity to kinases from yeasts and mammals. Two strong alleles have mutations in the kinase domain, suggesting that kinase activity is essential for par-1 function. PAR-1 protein is localized to the posterior periphery of the zygote and is distributed in a polar fashion preceding the asymmetric divisions of the germline lineage. Because PAR-1 distribution in the germline correlates with the distribution of germline-specific P granules, it is possible that PAR-1 functions in germline development as well as in establishing embryonic polarity.

Pseudocleavage is dispensable for polarity and development in C. elegans embryos.

The first cleavage of the Caenorhabditis elegans embryo is asymmetrical, producing daughters with different cell fates. During the first cell cycle, P granules, cytoplasmic components that are segregated to the germ-line, are localized to the posterior of the embryo. It has been hypothesized that the asymmetrical behavior of the daughters of the first division results from a similar localization of developmental determinants. A process called pseudocleavage also occurs during the first cell cycle: Anterior cortical contractions culminate in a single partial constriction of the embryo called the pseudocleavage furrow. Coincident with pseudocleavage, there is an anteriorly directed flow of cortical cytoplasm and a posteriorly directed flow of internal cytoplasm. Foci of filamentous cortical actin become asymmetrically distributed into an anterior cap. Roles for these various first cell cycle events in cytoplasmic localization and development have been suggested but remain unclear. We have isolated a maternal effect mutation, nop-1(it142), which abolishes the anterior cortical contractions and the pseudocleavage furrow. In addition, cortical actin foci remain uniformly distributed in most embryos. Despite these defects, cytoplasmic and cortical streaming is present and P granules are localized to the posterior of early embryos. In most embryos from mutant mothers, development proceeds normally and the embryos hatch and grow into fertile adults. We conclude that the pseudocleavage contractions and furrow are dispensable for the development of C. elegans.

Control of cleavage spindle orientation in Caenorhabditis elegans: the role of the genes par-2 and par-3.

Polarized asymmetric divisions play important roles in the development of plants and animals. The first two embryonic cleavages of Caenorhabditis elegans provide an opportunity to study the mechanisms controlling polarized asymmetric divisions. The first cleavage is unequal, producing daughters with different sizes and fates. The daughter blastomeres divide with different orientations at the second cleavage; the anterior blastomere divides equally across the long axis of the egg, whereas the posterior blastomere divides unequally along the long axis. We report here the results of our analysis of the genes par-2 and par-3 with respect to their contribution to the polarity of these divisions. Strong loss-of-function mutations in both genes lead to an equal first cleavage and an altered second cleavage. Interestingly, the mutations exhibit striking gene-specific differences at the second cleavage. The par-2 mutations lead to transverse spindle orientations in both blastomeres, whereas par-3 mutations lead to longitudinal spindle orientations in both blastomeres. The spindle orientation defects correlate with defects in centrosome movements during both the first and the second cell cycle. Temperature shift experiments with par-2(it5ts) indicate that the par-2(+) activity is not required after the two-cell stage. Analysis of double mutants shows that par-3 is epistatic to par-2. We propose a model wherein par-2(+) and par-3(+) act in concert during the first cell cycle to affect asymmetric modification of the cytoskeleton. This polar modification leads to different behaviors of centrosomes in the anterior and posterior and leads ultimately to blastomere-specific spindle orientations at the second cleavage.

par-2, a gene required for blastomere asymmetry in Caenorhabditis elegans, encodes zinc-finger and ATP-binding motifs.

The par-2 gene of Caenorhabditis elegans functions in early embryogenesis to ensure an asymmetric first cleavage and the segregation of cytoplasmic factors. Both processes appear to be required to generate daughter blastomeres with distinct developmental potential. We isolated an allele of par-2 by using a screen for maternal-effect lethal mutations in a strain known for its high frequency of transposition events. A transposable element was found to be linked to this allele. Sequences flanking the site of transposon insertion were cloned and found to rescue the par-2 mutant phenotype. DNA in the par-2 region hybridized to a 2.3-kb germ-line-enriched mRNA. The cDNA corresponding to this germ-line-enriched message was cloned, sequenced, and used to identify the molecular lesions associated with three par-2 alleles. Sequence analysis of the par-2 cDNA revealed that the predicted protein contained two distinct motifs found in other known proteins: an ATP-binding site and a cysteine-rich motif which identifies the par-2 gene product as a member of a growing class of putative zinc-binding proteins.

par-4, a gene required for cytoplasmic localization and determination of specific cell types in Caenorhabditis elegans embryogenesis.

Specification of some cell fates in the early Caenorhabditis elegans embryo is mediated by cytoplasmic localization under control of the maternal genome. Using nine newly isolated mutations, and two existing mutations, we have analyzed the role of the maternally expressed gene par-4 in cytoplasmic localization. We recovered seven new par-4 alleles in screens for maternal effect lethal mutations that result in failure to differentiate intestinal cells. Two additional par-4 mutations were identified in noncomplementation screens using strains with a high frequency of transposon mobility. All 11 mutations cause defects early in development of embryos produced by homozygous mutant mothers. Analysis with a deficiency in the region indicates that it33 is a strong loss-of-function mutation. par-4(it33) terminal stage embryos contain many cells, but show no morphogenesis, and are lacking intestinal cells. Temperature shifts with the it57ts allele suggest that the critical period for both intestinal differentiation and embryo viability begins during oogenesis, about 1.5 hr before fertilization, and ends before the four-cell stage. We propose that the primary function of the par-4 gene is to act as part of a maternally encoded system for cytoplasmic localization in the first cell cycle, with par-4 playing a particularly important role in the determination of intestine. Analysis of a par-4; par-2 double mutant suggests that par-4 and par-2 gene products interact in this system.

Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis elegans embryo.

We describe interactions between maternal-effect lethal mutations in four genes of Caenorhabditis elegans whose products appear to be involved in the meiotic and mitotic divisions of the one-cell embryo. Mitosis is disrupted by two dominant temperature-sensitive gain-of-function maternal-effect lethal mutations, mei-1(ct46) and mel-26(ct61), and by recessive loss-of-function maternal-effect lethal mutations of zyg-9. The phenotypic defects resulting from these mutations are similar. Doubly mutant combinations show a strong enhancement of the maternal-effect lethality under semipermissive conditions, suggesting that the mutant gene products interact. We isolated 15 dominant suppressors of the gain-of-function mutation mei-1(ct46). Thirteen of these suppressors are apparently intragenic, but 11 of them suppress in trans as well as cis. Two extragenic suppressors define a new gene, mei-2. The suppressor mutations in these two genes also result in recessive maternal-effect lethality, but with meiotic rather than mitotic defects. Surprisingly, most of these suppressors are also able to suppress mel-26(ct61) in addition to mei-1(ct46). The products of the four genes mei-1, mei-2, zyg-9 and mel-26 could be responsible for some of the specialized features that distinguish the meiotic from the mitotic divisions in the one-cell embryo.