The Interactive Fly

Zygotically transcribed genes

Cell Cycle Genes

How does the cell cycle function?

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

Identification of pathways regulating cell size and cell-cycle progression by RNAi

Developmental control of late replication and S phase length

Female meiosis


Genes involved in cell cycle

The Cyclins

Cyclin dependent kinases

Other genes

How does the cell cycle function?

Cell Cycle in Drosophila

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).

These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).

The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).

Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).

In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).

The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions andincreases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).

Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).

The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).

Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells

Double-stranded RNA-mediated interference (RNAi) was used to study Drosophila cytokinesis. Double-stranded RNAs for anillin, RacGAP50C, pavarotti, rho1, pebble, spaghetti squash, syntaxin1A, and twinstar all disrupt cytokinesis in S2 tissue culture cells, causing gene-specific phenotypes. The phenotypic analyses identify genes required for different aspects of cytokinesis, such as central spindle formation, actin accumulation at the cell equator, contractile ring assembly or disassembly, and membrane behavior. Moreover, the cytological phenotypes elicited by RNAi reveal simultaneous disruption of multiple aspects of cytokinesis. These phenotypes suggest interactions between central spindle microtubules, the actin-based contractile ring, and the plasma membrane, and led to a proposal that the central spindle and the contractile ring are interdependent structures. Finally, these results indicate that RNAi in S2 cells is a highly efficient method to detect cytokinetic genes, and predict that genome-wide studies using this method will permit identification of the majority of genes involved in Drosophila mitotic cytokinesis (Somma, 2002).

The finding that chicadee, four wheel drive (fwd), and Kinesin-like protein at 3A (klp3A) are not required for cytokinesis in S2 cells is not surprising, because previous studies pointed toward a specific involvement of these genes in meiotic cytokinesis of males. Null mutations in klp3A, a gene encoding a kinesin-like protein expressed both in testes and somatic tissues, disrupt meiotic cytokinesis but have no effect on larval neuroblast division. Similarly, flies homozygous for null mutations in fwd, which encodes a phosphatidyl-inositol kinase, are viable but male sterile, and are specifically defective in male meiotic cytokinesis. In contrast with fwd and klp3A that are not required for viability, chic is an essential gene that specifies a Drosophila homolog of profilin. However, both male sterile chic mutants and heteroallelic chic combinations resulting in lethality, display severe disruptions in meiotic cytokinesis but have no defects in neuroblast cytokinesis (Somma, 2002).

It was initially surprising to find that RNAi depletion of the Pnut protein, which shares homology with the yeast septins, does not markedly affect cytokinesis in S2 cells. This protein concentrates in the cleavage furrow of several Drosophila cell types; null pnut mutants die at the larval/pupal boundary and exhibit polyploid cells in their brains, consistent with a defect in cytokinesis. It is possible that the lack of an effect in pnut (RNAi) cells reflects a small amount of residual Pnut protein in these cells. However, it is instead believed that Pnut's role in cytokinesis is not fundamental to the process. The larval brains of null pnut mutants were reexamined and the presence of polyploid cells was confirmed. However, polyploid cells represent only 10.5% of the mitotic figures, indicating that most neuroblasts can undergo cytokinesis even in the absence of Pnut. In addition, Pnut is not required for cytokinesis during either male meiosis or the cystoblast divisions in the female germline. Taken together, these findings indicate that the Pnut function is either partially or totally dispensable for cytokinesis in Drosophila (Somma, 2002).

The phenotypical analyses of RNAi-induced mutants in the RacGAP50C, rho1, and sqh genes provide the first description of the cytological defects that lead to cytokinesis failures when the function of these genes is ablated. Previous studies have shown that mutations in rho1 and sqh disrupt mitotic cytokinesis but have not defined the cytological phenotypes elicited by these mutations. In addition, pav and pbl (RNAi) cells have been characterized; the phenotypes of these (RNAi) cells are consistent with those previously observed in animals homozygous for mutations in these genes (Somma, 2002 and references therein).

Cells in which the RacGAP50C, pav, pbl, rho1, and sqh genes are ablated by RNAi normally undergo anaphase A, but they then fail to elongate and to undergo anaphase B. After anaphase A, mutant cells proceed toward telophase and decondense their chromosomes, forming typical telophase nuclei. However, these cells fail to develop a central spindle, to assemble an actomyosin contractile ring and to concentrate anillin in the cleavage furrow. This results in the formation of short, aberrant telophases that are unable to undergo cytokinesis and will thus give rise to binucleated cells (Somma, 2002).

The functional ablation of genes influencing either the actin or the microtubule cytoskeleton have similar effects on cytokinesis. The genes pbl, rho1, and sqh likely play primary roles in controlling the actin cytoskeleton. The sqh gene encodes a regulatory light chain of myosin II. Rho1 is a member of the Rho family GTPases that cycle from an inactive GDP-bound state to an active GTP-bound state under the regulation of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs enhance the exchange of bound GDP for GTP, whereas GAPs increase the GTPase activity of Rho. Rho proteins and Rho GEFs, such as Drosophila Pbl and human ECT2, localize to the cleavage furrow and are required for contractile ring assembly. In contrast, the activities of RacGAP50C and pav are likely to primarily influence the function of the central spindle. The Pav kinesin-like protein, a homolog of the C. elegans ZEN-4, is localized in the central spindle, and is thought to mediate microtubule cross-linking at the central spindle midzone. The RacGAP50C gene encodes a Rho GAP, and it is orthologous to the cyk-4 gene of C. elegans. CYK-4 interacts with ZEN-4, and the two proteins are mutually dependent for their localization to the central spindle. The complete absence of Pav immunostaining in RacGAP50C (RNAi) telophases suggests a similar interaction between RacGAP50C and Pav, pointing to a role of RacGAP50C in central spindle assembly. In summary, the cytological phenotypes of pbl, rho1, and sqh (RNAi) cells indicate that a primary defect in acto-myosin ring formation results in a secondary defect in central spindle assembly. The phenotypes of RacGAP50C- and Pav-depleted cells suggest the converse: that a primary defect in the central spindle can secondarily disrupt contractile ring formation. Thus, taken together, these data indicate that the central spindle and the actomyosin ring are interrelated structures. Although the molecular mechanisms underlying the cross talk between these structures is not conpletely understood, two possibilities can be envisioned. The formation and maintenance of both the central spindle and the actomyosin ring could be mediated by physical interactions between interzonal microtubules and components of the contractile ring. Alternatively, the central spindle and the contractile ring could be coupled by a checkpoint-like regulatory mechanism, which would inhibit the formation of either of these structures when the other is not properly assembled (Somma, 2002).

Although RacGAP50C, pav, pbl, rho1, and sqh (RNAi) cells display similar terminal phenotypes, the aberrant telophases observed in these cultures differ in both actin and anillin distribution. In rho1 telophases these proteins are excluded from the cell equator, in pbl they are uniformly distributed, and in RacGAP50C, pav, and sqh they concentrate in a wide equatorial band. This suggests that rho1 and pbl are required for actin and anillin accumulation in the equatorial region of the dividing cell. In contrast RacGAP50C, pav, and sqh seem to be required for the assembly of the contractile machinery from proteins already concentrated at the cell equator. In sqh (RNAi) cells the failure to assemble an actomyosin ring is likely to be a direct consequence of the depletion of an essential component of the ring. In RacGAP50C and pav cells this failure is instead likely to be a secondary effect of problems in central spindle assembly (Somma, 2002).

An interplay between the central spindle and the contractile ring has been suggested by studies on Drosophila male meiosis. Mutant spermatocytes in the chic, and dia loci, which encode products thought to be involved in contractile ring formation, and mutants in the kinesin-encoding gene klp3A, all display severe defects in both structures. Although all the extant results on Drosophila cells strongly suggest an interdependence of the central spindle and the contractile ring, it is currently unclear whether this is true in all animal cells. Studies on mammalian cells have shown that central spindle plays an essential role during cytokinesis. However, these experiments have provided limited information on whether perturbations in the actomyosin ring assembly disrupt the central spindle. The best evidence of an interplay between the central spindle and the contractile ring has been in rat kidney cells. By puncturing these cells with a blunt needle a physical barrier is created between the central spindle and the equatorial cortex. This barrier not only abrogates actomyosin ring assembly on the side of perforation facing the cortex, but also disrupts the organization of central spindle microtubules on the opposite side (Somma, 2002 and references therein).

In contrast, studies on C. elegans embryos indicate that, at least in the early stages of cytokinesis, the actomyosin ring and the central spindle can assemble independently. Why do Drosophila, and possibly mammalian cells, differ from C. elegans in the interactions between the central spindle and the contractile ring? It is believed that the answer to this question reflects differences in the distance between the central spindle and the equatorial cortex. In Drosophila and mammalian cells during central spindle assembly the equatorial cortex is very close to the interzonal microtubules. In contrast, in C. elegans embryos the central spindle assembles in the center of the cell when the cleavage furrow has just began to ingress, so that during their assembly the actomyosin ring and the central spindle lie a considerable distance apart. Only later in cell division, after substantial furrow ingression, can the actomyosin ring and the central spindle come into contact. It is thus hypothesized that in embryonic cells of C. elegans the cytokinetic process consists of two steps: an early step, where the central spindle and the contractile ring assemble independently in distant cellular regions, and a late step that begins when the central spindle and the contractile ring have come into contact. The early stage might be mediated by interactions between astral (rather than central spindle) microtubules and the contractile ring. The late step of C. elegans cytokinesis may then require that the contractile ring and the central spindle interact cooperatively to complete cytokinesis successfully. This two-step hypothesis also applies to other large cells, such as echinoderm eggs, where the central spindle and the cortex are separated by large masses of cytoplasmic material and seem to assemble independently (Somma, 2002 and references therein).

The syx1A gene, which encodes a t-SNARE, plays an essential role in embryonic cellularization, but its direct role in cytokinesis has not been demonstrated. In syx1A (RNAi) cells approximately half of the telophases are shorter that those of control cells and display severe defects in both the central spindle and the contractile ring. These findings are rather surprising, because there is abundant evidence that syntaxins are specifically involved in membrane fusion processes. Thus, the observations on syx1A (RNAi) cells raise the question of how a defect in membrane formation can affect both the central spindle and contractile ring assembly. Studies of C. elegans embryos depleted of the cytokinesis-specific Syntaxin-4 protein by RNAi have shown that in some of these embryos there is a complete failure of cleavage furrow ingression, suggesting an underlying defect in the contractile ring machinery. It has been thus proposed that formation of new membrane may positively regulate contractile ring assembly. In agreement with this hypothesis, it is suggested that RNAi-induced Syx1A depletion in S2 cells disrupts membrane formation at the site of cleavage furrow, causing a secondary defect in contractile ring formation and thus also in central spindle assembly (Somma, 2002).

Identification of pathways regulating cell size and cell-cycle progression by RNAi

Many high-throughput loss-of-function analyses of the eukaryotic cell cycle have relied on the unicellular yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. In multicellular organisms, however, additional control mechanisms regulate the cell cycle to specify the size of the organism and its constituent organs. To identify such genes, this study analysed the effect of the loss of function of 70% of Drosophila genes (including 90% of genes conserved in human) on cell-cycle progression of S2 cells using flow cytometry. To address redundancy, genes involved in protein phosphorylation were also targeted simultaneously with their homologues. Genes that control cell size, cytokinesis, cell death and/or apoptosis, and the G1 and G2/M phases of the cell cycle were identified. Classification of the genes into pathways by unsupervised hierarchical clustering on the basis of these phenotypes shows that, in addition to classical regulatory mechanisms such as Myc/Max, Cyclin/Cdk and E2F, cell-cycle progression in S2 cells is controlled by vesicular and nuclear transport proteins, COP9 signalosome activity and four extracellular-signal-regulated pathways (Wnt, p38βMAPK, FRAP/TOR and JAK/STAT). In addition, by simultaneously analysing several phenotypes, a translational regulator, eIF-3p66, was identified that specifically affects the Cyclin/Cdk pathway activity (Bjorklund, 2006).

The cell cycle can be divided into distinct phases including a synthesis (S) phase, where DNA is replicated, and a mitosis (M) phase, where cell division occurs. In animal cells, growth and the synthesis of components required for these phases are regulated by extracellular growth factors and occur mainly in two gap phases, G1 (between M and S) and G2 (between S and M). The ordered progression of the cell-cycle phases is orchestrated by cyclin-dependent kinases (Cdks), whose activity is controlled by phosphorylation and by association with specific regulatory subunits, the cyclins (Bjorklund, 2006).

The cell cycle is a robust system in which compensatory mechanisms control its overall length. Therefore the effect of RNA-mediated interference (RNAi)-induced loss of function of Drosophila genes was analyzed in S2 cell-cycle distribution using flow cytometry, which allows direct determination of the fraction of cells in different phases of the cell cycle (Bjorklund, 2006).

Consistent with the high efficiency of RNAi in Drosophila, clear phenotypes including arrest in G1, G2/M and S, cytokinesis and DNA replication defects, and apoptosis and/or cell death were observed when targeting known regulators of these processes. A pilot screen was carried out targeting Drosophila kinases and phosphatases individually, and in pools of homologues to address redundancy. Unexpectedly, the analysis of redundancy did not reveal any additional cell-cycle regulators. These results suggest that a considerably lower fraction of Drosophila kinases regulate the cell cycle (19%) than has been reported previously (35%). Despite this, the screen identified also kinases for which a role in the S2 cell cycle has not been appreciated (for example, AKT1, CG7177 and MEKK1/MEKK4) (Bjorklund, 2006).

It was reasoned that, by screening most Drosophila genes, it should be possible to identify the pathways regulating the S2 cell cycle. Screening of 11,971 double-stranded RNAs (dsRNAs) generated from the Drosophila Gene Collection (DGC) releases 1 and 2 showed that loss of 270 and 169 genes resulted in significant changes in G1 and G2 populations, respectively. A strong correlation between cell size at the G1 and G2 phases was observed in individual samples, suggesting that Drosophila cells do not have a 'strong' cell-size checkpoint that forces cells to a particular size at a defined cell-cycle phase. Components acting on the same direction in a particular process, such as ribosomal proteins or Dp, E2f and Cyclin E, appeared in discrete areas of a plot describing G1 cell size as a function of fraction of cells in G1. The strongest phenotypes, characterized by specifically decreased cell size, were observed with dsRNAs targeting Rbf, a negative regulator of E2f pathway, and cropped (crp), the Drosophila AP4 transcription factor (Bjorklund, 2006).

To identify genes involved in cell death and cytokinesis, changes were analyzed in the populations of cells with less than 2N DNA or more than 4N DNA, respectively. The increase in the <2N population in all cases consisted of apoptotic or dead cells; the genes whose loss caused this phenotype included previously unknown effectors, the known inhibitor of apoptosis Thread and several mitosis regulators, consistent with induction of apoptosis by mitotic catastrophe. The increase in the >4N population was due to cells that had undergone two rounds of DNA replication without cytokinesis (8N DNA); dsRNAs causing this phenotype targeted several previously undescribed genes and known cytokinesis regulators (Bjorklund, 2006).

There have been several large-scale RNAi screens, and in many there is very little overlap between the gene sets identified even when the same phenotypes are analysed. This is apparently due to a very large number of false positives. To reduce the number of false positives, all phenotypes were analysed by using criteria that excluded all 564 control samples. Roughly 4% of genes had a cell-size, cell-cycle or cell-death phenotype. From an analysis of 19 different pathways and/or protein complexes, it was estimated that the screen identified ~80% of strong non-redundant cell-cycle regulators. Despite the general effectiveness of the RNAi, however, some genes with clearly defined functions in the cell cycle were not identified. In different cases, the weak RNAi phenotype could be explained by high protein levels or stability, redundant function and/or the fact that defects in mitotic or DNA replication fidelity often do not appreciably alter overall cell-cycle phase distribution (Bjorklund, 2006).

Of the 78 known interactions between the genes identified, 70 were consistent with the observed phenotypes. A high proportion of the diseases linked to the human orthologues of the identified genes are related to cancer, consistent with the known relationship between cell-cycle regulation and cancer. As expected, the Drosophila protein-protein interaction map indicated a much larger number of interactions between proteins than were identified than between a random set of proteins, and most interactions were consistent with the observed phenotypes (Bjorklund, 2006).

Of the 488 genes that identified, 319 have not been identified in previous Drosophila and mammalian RNAi screens. A principal difference between this screen and previous studies is that, by using flow cytometry, six distinct phenotypes can be simultaneously identified, allowing unbiased classification of the identified genes into pathways using hierarchical clustering. Positive components of a given pathway should segregate into the same cluster, because their loss is expected to cause similar phenotypes. This was indeed observed, for example 43 of 56 dsRNAs in one 'translation' cluster targeted ribosomal subunits. Another 'G1 cell-cycle' cluster contained the classical positive G1 regulators of the Cdk/E2f pathway, including E2f, Dp, Cyclin E, Cdk2 and Cdk4. Because of an opposite phenotype, Rbf, a known negative regulator of this pathway, was not included in this cluster (Bjorklund, 2006).

The other genes in the G1 cell-cycle cluster included two cullins (Cul-1/lin19 and Cul-4), involved in ubiquitin-mediated protein degradation, and five components of the COP9 signalosome, a protein complex linked to regulation of cullin activity. In mammals Cul-4 has been linked to DNA damage response, but the results indicate that Cul-4 is also involved in normal cell-cycle regulation in animals. Consistent with the similar phenotypes of the cullins, COP9 subunits (CSNs) and several Cdk/E2f pathway components, the increase in G1 induced by dsRNAs targeting CSNs could be reversed by simultaneously targeting the Cdk inhibitor Dacapo. The results are thus consistent with a loss of COP9 leading to G1 arrest owing to failure of cullin-mediated degradation of Dacapo. The positive role of COP9 in the cell cycle is in agreement with mouse knockout studies. In Drosophila ovaries, however, COP9 negatively regulates the cell cycle, suggesting that its effects may be tissue-specific (Bjorklund, 2006).

Notably, a translational initiation factor, eIF-3p66, clustered with the G1 cell-cycle regulators. Expression of eIF-3p66 also correlates well with that of Rbf, E2f and Cyclin E during Drosophila development. dsRNAs targeting eIF-3p66 resulted in an increased cell size, indicating that, unlike loss of other translational regulators, loss of eIF-3p66 does not generally inhibit translation. Furthermore, the phenotype caused by dsRNA targeting eIF-3p66 was reversed by dsRNA targeting dacapo, indicating that eIF-3p66 specifically affects the Cyclin/Cdk pathway (Bjorklund, 2006).

The cluster of genes whose loss resulted in an increase in G1 cell size with no G1 DNA content phenotype contained several genes involved in transcription and messenger RNA processing. The lack of G1 phenotype is probably due to a uniform effect of transcriptional inhibition on all cell-cycle phases, and continued growth of the cells is possible owing to translation of stable mRNAs. The distinct phenotype of transcriptional and translational regulators suggests that cell size may be controlled at the RNA level, either directly by regulatory RNAs or through differential stability of mRNAs of proteins controlling cell division and growth (Bjorklund, 2006).

A number of genes whose loss resulted in increased G1 content were classified into a defined 'signalling' cluster, which contained two regulators of translation (eIF-4G and Ef1α48D), a ubiquitin E3-ligase (URE-B1/CG8184), and components of several extracellular ligand-regulated signalling pathways. These genes differed from those in the G1 cell-cycle cluster in that the dsRNAs targeting them resulted in decreased cell size. Thus, the signalling pathways either affect cell division through regulation of growth, or affect both cell growth and cell division in parallel (Bjorklund, 2006).

By manually comparing the genes identified with literature, four signalling pathways were identified that regulate the cell cycle: the FRAP/TOR, JAK/STAT, Wnt and p38βMAPK pathways. The FRAP/TOR pathway is known to regulate cell proliferation and, consistently, eight known components of this pathway were identifed as G1 regulators. In S2 cells, another known cell-cycle regulatory pathway, the JAK/STAT pathway, had a relatively weak effect, as indicated by the mild phenotypes observed by dsRNAs targeting several of its components. The identified genes known to regulate the Wnt pathway indicated that this pathway is not active in S2 cells, but that treatments that activate it inhibit S2 cell proliferation (Bjorklund, 2006).

Several genes linked to the p38βMAPK pathway were also identified, and the function of MEKK1/MEKK4 and MEK3 in this pathway was varified by analysing p38βMAPK phosphorylation in response to dsRNA treatments. Although regulation of the cell cycle by the p38βMAPK pathway in the absence of stress or DNA damage has not previously been appreciated, the data are consistent with the identification of MAPk-Ak2 (Manke, 2005) as a checkpoint kinase (Bjorklund, 2006).

Loss of several kinases that negatively regulate Cdk1 activity led to an increase in G1 content, probably owing to an accelerated G2/M phase. It was also observed that Chk1 and MAPk-Ak2, two kinases that have been linked to DNA damage checkpoints, operate in normal cell-cycle regulation of cultured S2 cells. Together with the finding that in human cells Chk1 shields Cdk1 from premature activation, the results suggest that during normal cell cycle these kinases have basal activity that slows down G2 progression. The basal activity could represent partial activation that occurs locally in response to damage during normal replication, possibly effecting localized changes in DNA replication and repair (Bjorklund, 2006).

In summary, several pathways and processes are involved in cell-cycle and cell-size regulation, including basal transcription, vesicular trafficking, nuclear export, ubiquitin-mediated protein degradation, and the Cdk/E2f, JAK/STAT, FRAP/TOR, p38βMAPK and Wnt signalling pathways. The genes identified are likely to include previously unknown components of these pathways, ubiquitin ligase and kinase substrates, and targets of transcription factors such as E2f and Myc/Max that are relevant for cell-cycle progression. Thus, these results will also serve as a solid foundation for a systems biology analysis of the metazoan cell cycle (Bjorklund, 2006).

Developmental control of late replication and S phase length

Fast, early embryonic cell cycles have correspondingly fast S phases. In early Drosophila embryos, forks starting from closely spaced origins replicate the whole genome in 3.4 min, ten times faster than in embryonic cycle 14 and a hundred times faster than in a wing disc. It is not known how S phase duration is regulated. This study examined prolongation of embryonic S phases, its coupling to development, and its relationship to the appearance of heterochromatin. Imaging of fluorescent nucleotide incorporation and GFP-PCNA gave exquisite time resolution of S phase events. In the early S phases, satellite sequences replicated rapidly despite a compact chromatin structure. In S phases 11-13, a delay in satellite replication emerged in sync with modest and progressive prolongation of S phase. In S phase 14, major and distinct delays ordered the replication of satellites into a sequence that occupied much of S phase. This onset of late replication required transcription. Satellites only accumulated abundant heterochromatin protein 1 (HP1) after replicating in S phase 14. By cycle 15, satellites clustered in a compact HP1-positive mass, but replication occurred at decondensed foci at the surface of this mass. It is concluded that the slowing of S phase is an active process, not a titration of maternal replication machinery. Most sequences continue to replicate rapidly in successive cycles, but increasing delays in the replication of satellite sequences extend S phase. Although called constitutively heterochromatic, satellites acquire the distinctive features of heterochromatin, compaction, late replication, HP1 binding, and aggregation at the chromocenter, in successive steps coordinated with developmental progress (Shermoen, 2010).

This work shows that heterochromatin, long recognized as a key factor in the developmental programming of gene expression, also plays an integral role in the timing of the early embryonic cell cycles. Satellite sequences successively acquire features of heterochromatin, becoming late replicating by cycle 14, which prolongs S phase. This prolongation of S phase slows the early cell cycles and allows the progression to MBT (Shermoen, 2010).

This description of the successive introduction of the features of heterochromatin reveals a lack of interdependency of these features. For example, because it occurs earlier, the compaction of the satellite sequences is independent of late replication and of HP1 binding. It was also possible to visualize the events of late replication with unprecedented spatial and temporal resolution that gives insights into the replication of compacted HP1-bound chromatin (Shermoen, 2010).

Origin spacing could contribute to S phase length. However, electron microscopy studies show that origin spacing changes only slightly, from 7.9 to 10.6 kb, from preblastoderm embryos to cycle 14. Because forks are thought to converge at a rate of 3 kb/min, the additional separation would extend S phase by about 1 min, a minor contribution to the change from a 3.4 to a 50 min S phase (Shermoen, 2010).

S phase duration would also increase if all of the replicons did not replicate at the same time. However, asynchrony in replicon firing can occur in two ways, organized and unorganized. Unorganized asynchrony means that origins fire at different times without regard to their position in the genome. In this case, early- and late-firing origins can be juxtaposed. When replication from an early-firing origin reaches an adjacent later-firing origin just before it fires, one fork, rather than two, replicates the interorigin distance, doubling replication time. Greater unorganized asynchrony will result in passive replication of later origins and reduce the number of origins. Thus, given the known origin spacing, unorganized asynchrony is unlikely to make a very major contribution to the more than 10-fold increase in S phase length between preblastoderm cycles and cycle 14 (Shermoen, 2010).

If replication asynchrony is organized so that large regions of the genome (replication units) have many similarly behaving replicons, early-initiated forks invading a late region from the outside will not have time to replicate a significant portion of the large domain. The insulation resulting from distance can greatly magnify the impact of asynchrony on S phase duration. Organization of genomes into large replication units is widespread but poorly understood. This study shows that the satellite sequences are replication units and that embryonic changes in S phase duration result from change in the schedules of their replication. In preblastoderm cycles, satellite sequences replicate early, finishing in synch with general replication. Subsequently, satellite replication is increasingly delayed in parallel to S phase prolongation. Importantly, when satellite replication is late, it is deferred, not slow. For example, a 3.4 Mb Y-chromosomal repeat of the simple sequence AATAC begins to replicate 18 min into S phase 14. Each type of satellite sequence exhibits distinct replication delays. The 11 Mb X-chromosomal repeat of 359 bases, termed the 359 sequence has almost no delay in S phase 14, whereas AATAT and AATAACATAG finish replicating after 359 but before AATAC. The stereotyped schedules suggest that each replication unit has a characteristic 'lateness' parameter. This lateness parameter appears to be continuously variable in that there are many replication units, each with its own schedule of replication (Shermoen, 2010).

Though its replication is delayed, a unit such as AATAC replicates quickly once initiated (10 min). Although the 359 satellite is more slowly replicating (~15 min), it is suggested that it may be composed of separately and asynchronously replicating subdomains that were sometimes resolved. It is concluded that the dynamics of replication within a replication unit change only modestly during the early cycles (from 3.4 to roughly 10 min) (Shermoen, 2010).

In summary, the results argue that by S phase 14, the genome is replicated as a series of units, each of which replicates relatively quickly, but that a temporal program of sequential replication of these units creates a long S phase. This replication program resembles a consensus view of replication in slowly replicating cells of mammals and plants. It is concluded that progression from coincident replication of all of the replication units in a rapid S phase to sequential replication in a prolonged S phase 14 underlies prolongation of early embryonic S phases in Drosophila (Shermoen, 2010).

In widely divergent species and biological settings, heterochromatin has common characteristics including compaction, transcriptional quiescence, late replication, 'repressive' histone modifications, and association of specific heterochromatin proteins. This intimate association suggests mechanistic coupling of these features. If this were so, the various heterochromatin characteristics would emerge coordinately at the same moment during development. Instead, the current observations show temporal uncoupling during early Drosophila embryogenesis (Shermoen, 2010).

Although it was suggested that heterochromatin appears at cycle 14, both the cytological and biochemical manifestations of heterochromatin develop progressively. Foci of compacted chromatin that align with satellite sequences appeared in preblastoderm embryos prior to, and hence independently of, late replication and HP1 recruitment. Furthermore, HP1 binding to satellite sequences occurred late in cycle 14, after the onset of late replication. Because HP1 would have to decorate the satellite sequences at the onset of cycle 14 if it were required to suppress early replication and promote late replication, it is concluded that the late replication of satellite sequences is specified independently of the HP1 binding. Finally, satellite sequences reorganize; the previously independent foci of satellites aggregate into a large coherent HP1-positive region at the very beginning of interphase 15. This intimate association of satellites, which makes the chromocenter more coherent, is downstream of cycle 14 events and the MBT (Shermoen, 2010).

Together, these findings show that satellite sequences acquire the features of heterochromatin progressively. Compaction is present early, late replication is introduced subsequently, and recruitment of HP1 and then chromocenter maturation follow. Onset of position-effect variegation suggests that heterochromatic suppression of transcription begins in G2 of cycle 14 and mounts subsequently. Thus, heterochromatin does not form in a single step, and it acquires increasing influence during critical developmental events surrounding the MBT and gastrulation (Shermoen, 2010).

If compaction of chromatin prevents replication, decompaction might accompany or provoke replication. Real-time observations of PCNA and HP1 in cycle 15 show replication adjacent to, but not overlapping, HP1-bright foci of compacted chromatin. A more diffuse HP1 region appears adjacent to bright HP1 foci; PCNA overlies these fainter partner foci. Each partner focus appears and disappears as the PCNA signal rises and declines. It is concluded from this that replication does not occur in the compacted domain and that the sequences in the compacted HP1-bright focus unfurl during replication (Shermoen, 2010).

The persistence of in situ foci for 359 and AATAC shows that the satellites are not fully decondensed during replication. The size of partner HP1 foci also argues for limited decompaction. If an entire focus of compacted HP1-bright chromatin were to disperse, it would expand in volume, but the partner focus is about the same size as the brighter parent focus. Thus, it is suggested that a partner focus represents decompaction of a portion of the sequences harbored in the adjacent HP1-bright focus (Shermoen, 2010).

Following replication, heterochromatic sequences rapidly recompact. After an initial expansion, the partner HP1 focus does not grow throughout replication, and it shrinks and disappears as replication declines. When pulsed with fluorescent nucleotides for less than the replication time of the satellite, fluorescence overlies the compacted satellite sequence. Thus, it is suggested that DNA is “spooled” out of compacted foci, replicated, and returned to compacted foci shortly after replication. It the duration of replication-associated decompaction in embryonic cycle 15 is roughly estimated as 1 min. The dynamics, which are not easily consistent with decompaction of large topological domains, suggest that active replication forks drive local unfolding of chromatin structure, but the possibility cannot be excluded that transient decompaction might promote replication (Shermoen, 2010).

Mechanisms that couple the changing cell-cycle behavior with development are of great interest. Previous work suggested that the gradual prolongation of early cycles is secondary to gradual prolongation of S phase. A model in which the exponentially increasing amounts of DNA titrate replication components to prolong S phase is attractive but not presently supported (Shermoen, 2010).

The current results show that if a titration mechanism governs S phase duration, it is indirect. Injection of α-amanitin in cycle 13 prevented onset of late replication, accelerated S phase 14, and caused an early synchronous mitosis. Thus, activity of at least one of the DNA-dependent RNA polymerases is required to slow S phase, and the replication “hardware” needed for a rapid S phase is not limiting. Accordingly, if a titration mechanism were involved, the titrated component would regulate an upstream process. For example, transcription is restricted prior to cycle 14, and titration of a repressor might derepress transcription in late cycle 13, indirectly triggering onset of late replication (Shermoen, 2010).

Three findings suggest an abrupt switch to late replication at the beginning of cycle 14: the dramatic increase in S phase length, the accompanying switch of satellite sequences to delayed replication, and the requirement for transcription in cycle 13 for this transition. However, the late replication program of cycle 14 is anticipated by slight delays in replication of satellite sequences in cycles 12 and 13. These early changes suggest a more progressive process. It is proposed that early slight changes in replication timing and transcription initiate a positive feedback process that precipitates an abrupt change at the MBT. Rapid cell cycles suppress transcription and limit the time available to modify newly replicated chromatin, but, once the cycle begins to slow, transcription and heterochromatin modifications would accelerate to create conditions permissive for late replication, which would further slow the cycle (Shermoen, 2010).

list of genes active in cell cycle


References

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date revised: 15 October 2011

Zygotically transcribed genes

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