Organ-specific cell division abnormalities caused by mutation in a general cell cycle regulator in C. elegans

The precise control of cell division during development is pivotal for morphogenesis and the correct formation of tissues and organs. One important gene family involved in such control is the p21/p27/p57 class of negative cell cycle regulators. Loss of function of the C. elegans p27 homolog, cki-1, causes extra cell divisions in numerous tissues including the hypodermis, the vulva, and the intestine. We have sought to better understand how cell divisions are controlled upstream or in parallel to cki-1 in specific organs during C. elegans development. By taking advantage of the invariant cell lineage of C. elegans, we used an intestinal-specific GFP reporter in a screen to identify mutants that undergo cell division abnormalities in the intestinal lineage. We have isolated a mutant with twice the wild-type complement of intestinal cells, all of which arise during mid-embryogenesis. This mutant, called rr31, is a fully dominant, maternal-effect, gain-of-function mutation in the cdc-25.1 cell cycle phosphatase that sensitizes the intestinal lineage to an extra cell division. We showed that cdc-25.1 acts at the G1/S transition, as ectopic expression of CDC-25.1 caused entry into S phase in intestinal cells. In addition, we showed that the cdc-25.1(gf) requires cyclin E. The extra cell division defect was shown to be restricted to the E lineage and the E fate is necessary and sufficient to sensitize cells to this mutation.     

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.     

CDC-25.1 stability is regulated by distinct domains to restrict cell division during embryogenesis in C. elegans

Cdc25 phosphatases are key positive cell cycle regulators that coordinate cell divisions with growth and morphogenesis in many organisms. Intriguingly in C. elegans, two cdc-25.1(gf) mutations induce tissue-specific and temporally restricted hyperplasia in the embryonic intestinal lineage, despite stabilization of the mutant CDC-25.1 protein in every blastomere. We investigated the molecular basis underlying the CDC-25.1(gf) stabilization and its associated tissue-specific phenotype. We found that both mutations affect a canonical beta-TrCP phosphodegron motif, while the F-box protein LIN-23, the beta-TrCP orthologue, is required for the timely degradation of CDC-25.1. Accordingly, depletion of lin-23 in wild-type embryos stabilizes CDC-25.1 and triggers intestinal hyperplasia, which is, at least in part, cdc-25.1 dependent. lin-23(RNAi) causes embryonic lethality owing to cell fate transformations that convert blastomeres to an intestinal fate, sensitizing them to increased levels of CDC-25.1. Our characterization of a novel destabilizing cdc-25.1(lf) intragenic suppressor that acts independently of lin-23 indicates that additional cues impinge on different motifs of the CDC-25.1 phosphatase during early embryogenesis to control its stability and turnover, in order to ensure the timely divisions of intestinal cells and coordinate them with the formation of the developing gut.     

Control of cell cycle timing during C. elegans embryogenesis

As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P₃ (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.     

Fate Specification and Tissue-specific Cell Cycle Control of the Caenorhabditis elegans Intestine

Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.     

MEL-47, a novel protein required for early cell divisions in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50–80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A mutation of <Emphasis Type="Italic">cdc</Emphasis>-25.1 causes defects in germ cells but not in somatic tissues in <Emphasis Type="Italic">C. elegans</Emphasis>

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.     

Oncogenic potential of a C.elegans cdc25 gene is demonstrated by a gain-of-function allele

In multicellular organisms, developmental programmes must integrate with central cell cycle regulation to co-ordinate developmental decisions with cell proliferation. Hyperplasia caused by deregulated proliferation without significant change to other aspects of developmental behaviour is a probable step towards full oncogenesis in many malignancies. CDC25 phosphatase promotes progression through the eukaryotic cell cycle by dephosphorylation of cyclin-dependent kinase and, in humans, different cdc25 family members have been implicated as potential oncogenes. Demonstrating the direct oncogenic potential of a cdc25 gene, we identify a gain-of-function mutant allele of the Caenorhabditis elegans gene cdc-25.1 that causes a deregulated proliferation of intestinal cells resulting in hyperplasia, while other aspects of intestinal cell function are retained. Using RNA-mediated interference, we demonstrate modulation of the oncogenic behaviour of this mutant, and show that a reduction of the wild-type cdc-25.1 activity can cause a failure of proliferation of intestinal and other cell types. That gain and loss of CDC-25.1 activity has opposite effects on cellular proliferation indicates its critical role in controlling C.elegans cell number.     

Inhibition of GA3-induced antheridiogenesis in Anemia phyllitidis by peptidic extracts from male sex organs of Chara

Low molecular weight peptidic component extracted from maturing male sex organs of Chara tomentosa, capable of inducing increased condensation of chromosomes and profound changes in the cell cycle progression, was applied to gametophytes of Anemia phyllitidis. Morphogenetic effects were studied with regard to cell divisions and GA₃-induced antheridiogenesis. As compared with both the GA ₃ ⁻ and GA ₃ ⁺ control samples, the extract-treated prothallia exhibited considerably lowered number of cells and altered morphology. Antheridial differentiation in prothallia of A. phyllitidis was severely inhibited when peptidic extract was added to medium containing GA₃. Considering endocytotic uptake, evidenced in root meristems, and those effects which have been observed in plant and human cells, the activity of extracts obtained from male sex organs of Chara may be interpreted as inhibitory influence acting via repression or modification of the genetic device of the cells rather, than a direct consequence of the retardation of cell division cycles.     

CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans

In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.     

Plasticity of proximal-distal cell fate in the mammalian limb bud

Maintenance of the shape and diameter of biological tubules is a critical task in the development and physiology of all metazoan organisms. We have cloned the exc-9 gene of Caenorhabditiselegans, which regulates the diameter of the single-cell excretory canal tubules. exc-9 encodes a homologue of the highly expressed mammalian intestinal LIM-domain protein CRIP, whose function has not previously been determined. A second well-conserved CRIP homologue functions in multiple valves of C. elegans. EXC-9 shows genetic interactions with other EXC proteins, including the EXC-5 guanine exchange factor that regulates CDC-42 activity. EXC-9 and its nematode homologue act in polarized epithelial cells that must maintain great flexibility at their apical surface; our results suggest that CRIPs function to maintain cytoskeletal flexibility at the apical surface.     

PAR proteins direct asymmetry of the cell cycle regulators Polo-like kinase and Cdc25

Cell cycle lengths vary widely among different cells within an animal, yet mechanisms of cell cycle length regulation are poorly understood. In the Caenorhabditis elegans embryo, the first cell division produces two cells with different cell cycle lengths, which are dependent on the conserved partitioning-defective (PAR) polarity proteins. We show that two key cell cycle regulators, the Polo-like kinase PLK-1 and the cyclin-dependent kinase phosphatase CDC-25.1, are asymmetrically distributed in early embryos. PLK-1 shows anterior cytoplasmic enrichment and CDC-25.1 shows PLK-1-dependent enrichment in the anterior nucleus. Both proteins are required for normal mitotic progression. Furthermore, these asymmetries are controlled by PAR proteins and the muscle excess (MEX) proteins MEX-5/MEX-6, and the latter is linked to protein degradation. Our results support a model whereby the PAR and MEX-5/MEX-6 proteins asymmetrically control PLK-1 levels, which asymmetrically regulates CDC-25.1 to promote differences in cell cycle lengths. We suggest that control of Plk1 and Cdc25 may be relevant to regulation of cell cycle length in other developmental contexts.     

Vegetative and reproductive morphology of Herpochondria corallinae (Martens) Falkenberg and H. elegans (Okamura) Itono (Ceramiaceae,Rhodophyta) from Korea

The vegetative and reproductive morphology of two epiphytic Herpochondria species, H. corallinae (Martens) Falkenberg (the type species) and H. elegans (Okamura) Itono from Korea were investigated. They are bilaterally compressed and alternate-distichously branched, and lateral branches are produced by oblique divisions of the apical cell. Procarps are restricted to the first periaxial cell of the branches. Spermatangial mother cells produce one or two spermatangia. H. corallinae forms tetrasporangia on six periaxial cells, whereas H. elegans has them on only the two lateral periaxial cells. The prostrate habit, the production of six periaxial cells in opposite pairs, and tetrasporangial stichidia are confirmed as diagnostic characters of the genus Herpochondria.     

Extra divisions and nuclei fusions in microspores from Brassica allohexaploid (AABBCC) × Orychophragmus violaceus hybrids

Abnormal meiosis and microspore development and related defective mutants have often been reported in plants and wide hybrids. Here extra divisions and nuclei fusions were observed to occur in microspore nuclei of partial hybrids between synthetic Brassica hexaploid (2n=54, AABBCC) and another crucifer Orychophragmus violaceus (2n=24). Abnormal spindle were formed and chromosomes were separated into several nuclei of variable sizes after bi-, or multi-polar divisions in the four cells of tetrads. As a consequence, more than eight mini-microspores of different sizes were produced by one tetrad. Genomic in situ hybridization results indicated that no chromosome replication occurred during such divisions. In some tetrads, the four nuclei were fused to form one large cell with increased chromosome number. The extra divisions or fusions appeared only in some flower buds of one plant, some anthers in the same buds, or even in individual cells of tetrads. The possible mechanisms behind these cytological phenomena are discussed.     

The cyclin-dependent kinase inhibitors,cki-1 and cki-2, act in overlapping but distinct pathways to control cell-cycle quiescence during C. elegans development

Cyclin-dependent kinase inhibitors (CKIs) are major contributors to the decision to enter or exit the cell cycle. The Caenorhabditis elegans genome encodes two CKIs belonging to the Cip/Kip family, cki-1 and cki-2. cki-1 has been shown to act as a canonical negative regulator of cell-cycle entry, while the role of cki-2 remains unclear. We identified cki-2 in a genome-wide RNAi screen to reveal genes essential for developmental cell-cycle quiescence. Examination of cki-2 knockout animals revealed extra rounds of cell divisions, verifying a role in establishing or maintaining the temporary cell-cycle arrest. Despite the overlapping defects, the pathways mediated by cki-1 and cki-2 are discrete since the extra cell phenotype conferred by a putative cki-2(null) mutation is enhanced upon additional loss of cki-1 activity. Moreover, the extra cell division defect of cki-2 is not increased with the additional loss of lin-35 Rb, as is seen with cki-1. Thus, both cki-1 and cki-2 mediate cell-cycle quiescence, but our genetic and phenotypic analyses demonstrate that they act within distinct pathways to exert control over the cell-cycle machinery.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

A study of polynucleotide phosphorylase production by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a hollow fibre reactor

A 30-l hollow fibre reactor with continuous fermentation for cell recycling of Escherichia coli AS 1.183 was used to remove the inhibitory effects on cell growth and extend the fast growth phase to increase the yield of polynucleotide phosphorylase (PNPase) in E. coli cells. When the dilution rate was 1.5 h⁻¹, the cell concentration of E. coli reached 235 g/l (wet wt, 70% moisture content), with PNPase activity above 90 u/g (wet wt). With the dilution rate is 1.0 h⁻¹, the fermentor volumetric productivity of PNPase in a hollow fiber reactor can reach 974 (u/h * l) compared to 20 (u/h * l) in a conventional batch culture.     

Metabolic processes sustaining the reviviscence of lichen <Emphasis Type="Italic">Xanthoria elegans</Emphasis> (Link) in high mountain environments

To survive in high mountain environments lichens must adapt themselves to alternating periods of desiccation and hydration. Respiration and photosynthesis of the foliaceous lichen, Xanthoria elegans, in the dehydrated state were below the threshold of CO₂-detection by infrared gas analysis. Following hydration, respiration totally recovered within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to the quick and efficient reactivation of lichen physiological processes, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using ³¹P- and ¹³C-NMR. It appeared that the recovery of respiration was prepared during dehydration by the accumulation of a reserve of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pools, whereas glycolytic and photosynthetic intermediates like glucose 6-P and ribulose 1,5-diphosphate were absent. The large pools of polyols present in both X. elegans photo- and mycobiont are likely to contribute to the protection of cell constituents like nucleotides, proteins, and membrane lipids, and to preserve the integrity of intracellular structures during desiccation. Our data indicate that glcn-6-P accumulated due to activation of the oxidative pentose phosphate pathway, in response to a need for reducing power (NADPH) during the dehydration-triggered down-regulation of cell metabolism. On the contrary, glcn-6-P was metabolised immediately after hydration, supplying respiration with substrates during the replenishment of pools of glycolytic and photosynthetic intermediates. Finally, the high net photosynthetic activity of wet X. elegans thalli at low temperature may help this alpine lichen to take advantage of brief hydration opportunities such as ice melting, thus favouring its growth in harsh high mountain climates.