Fluorescence visualization of the distribution of microfilaments in gonads and early embryos of the nematode Caenorhabditis elegans

Several intracellular motility events in the Caenorhabditis elegans zygote (pseudocleavage, the asymmetric meeting of the pronuclei, the segregation of germ line-specific granules, and the generation of an asymmetric spindle) appear to depend on microfilaments (MFs). To investigate how MFs participate in these manifestations of zygotic asymmetry, the distribution of MFs in oocytes and early embryos was examined, using both antibodies to actin and the F-actin-specific probe rhodamine-phalloidin. In early-stage zygotes, MFs are found in a uniform cortical meshwork of fine fibers and dots or foci. In later zygotes, concomitant with the intracellular movements that are thought to be MF mediated, MFs also become asymmetrically rearranged; as the zygote undergoes pseudocleavage and as the germ line granules become localized in the posterior half of the cell, the foci of actin become progressively more concentrated in the anterior hemisphere. The foci remain anterior as the spindle becomes asymmetric and the zygote undergoes its first mitosis, at which time fibers align circumferentially around the zygote where the cleavage furrow will form. A model for how the anterior foci of actin may participate in zygotic motility events is discussed. Phalloidin and anti-actin antibodies have also been used to visualize MFs in the somatic tissues of the adult gonad. The myoepithelial cells that surround maturing oocytes are visibly contractile and contain an unusual array of MF bundles; the MFs run roughly longitudinally from the loop of the gonad to the spermatheca. Myosin thick filaments are distributed along the MFs in a periodic manner suggestive of a sarcomere-like configuration. It is proposed that these actin and myosin filaments interact to cause sheath cell contraction and the movement of oocytes through the gonad.     

An analysis of the role of microfilaments in the establishment and maintenance of asymmetry in Caenorhabditis elegans zygotes

Microfilaments are needed to generate asymmetry during the first cell cycle in Caenorhabditis elegans zygotes. To investigate when and how microfilaments participate in this process, we have "pulsed" zygotes with the microfilament inhibitor cytochalasin D (CD) at different times during the cell cycle. We have shown that microfilaments are only required during a narrow time interval approximately three-quarters of the way through the first cell cycle for the manifestations of asymmetry that occur during and subsequent to this interval. When CD treatment spans this critical time interval, pseudocleavage, pronuclear migration, germ-granule segregation (all of which occur during the interval), and movement of the mitotic spindle to an asymmetric position (which occurs later in the cell cycle) are perturbed. In contrast, embryos briefly treated with CD before or after the critical time interval manifest normal asymmetry. Our results suggest that in C. elegans microfilaments participate in the generation of zygotic asymmetry by providing spatial cues and/or serving as a part of the necessary machinery only during a brief period in the first cell cycle, and are not required to maintain asymmetries that have already been established.     

MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.     

Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germ-cell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.     

Arp2/3-dependent pseudocleavage [correction of psuedocleavage] furrow assembly in syncytial Drosophila embryos

BACKGROUND: In syncytial blastoderm Drosophila embryos, actin caps assemble during telophase. As the cell cycle progresses through interphase, these small caps expand and fuse to form pseudocleavage furrows that are structurally related to the cleavage furrows that assemble during somatic cell division. The molecular mechanism driving cell cycle coordinated actin reorganization from the caps to the furrows is not understood. RESULTS: We show that Drosophila embryos contain a typical Arp2/3 complex and that components of this complex localize to the margins of the expanding caps, to mature pseudocleavage furrows, and to somatic cell cleavage furrows during the postcellularization embryonic divisions. A mutation that disrupts the arpc1 subunit of Arp2/3 leads to spindle fusions that are characteristic of pseudocleavage furrow disruption. By contrast, this mutation does not significantly affect nuclear positioning during interphase, which is dependent on actin cap function. In vivo analysis of actin reorganization demonstrates that the arpc1 mutation does not prevent assembly of small actin caps but blocks cap expansion and furrow assembly as the cell cycle progresses through interphase. The scrambled gene is also required for cap expansion and furrow assembly, and Scrambled is required for Arp2/3 localization to the cap margins. CONCLUSIONS: The Drosophila Arp2/3 complex and Scrambled protein are required for actin cap expansion and pseudocleavage furrow formation during the syncytial blastoderm divisions. We propose that Scrambled-dependent localization of Arp2/3 to the margins of the expanding caps triggers local actin polymerization that drives cap expansion and pseudocleavage furrow assembly.     

Brief cytochalasin-induced disruption of microfilaments during a critical interval in 1-cell C. elegans embryos alters the partitioning of developmental instructions to the 2-cell embryo

We are investigating the involvement of the microfilament cytoskeleton in the development of early Caenorhabditis elegans embryos. We previously reported that several cytoplasmic movements in the zygote require that the microfilament cytoskeleton remain intact during a narrow time interval approximately three-quarters of the way through the first cell cycle. In this study, we analyze the developmental consequences of brief, cytochalasin D-induced microfilament disruption during the 1-cell stage. Our results indicate that during the first cell cycle microfilaments are important only during the critical time interval for the 2-cell embryo to undergo the correct pattern of subsequent divisions and to initiate the differentiation of at least 4 tissue types. Disruption of microfilaments during the critical interval results in aberrant division and P-granule segregation patterns, generating some embryos that we classify as 'reverse polarity', 'anterior duplication', and 'posterior duplication' embryos. These altered patterns suggest that microfilament disruption during the critical interval leads to the incorrect distribution of developmental instructions responsible for early pattern formation. The strict correlation between unequal division, unequal germ-granule partitioning, and the generation of daughter cells with different cell cycle periods observed in these embryos suggests that the three processes are coupled. We hypothesize that (1) an 'asymmetry determinant', normally located at the posterior end of the zygote, governs asymmetric cell division, germ-granule segregation, and the segregation of cell cycle timing elements during the first cell cycle, and (2) the integrity or placement of this asymmetry determinant is sensitive to microfilament disruption during the critical time interval.     

P granules extend the nuclear pore complex environment in the C. elegans germ line

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.     

RMD-1, a novel microtubule-associated protein, functions in chromosome segregation in Caenorhabditis elegans

For proper chromosome segregation, the sister kinetochores must attach to microtubules extending from the opposite spindle poles. Any errors in microtubule attachment can induce aneuploidy. In this study, we identify a novel conserved Caenorhabditis elegans microtubule-associated protein, regulator of microtubule dynamics 1 (RMD-1), that localizes to spindle microtubules and spindle poles. Depletion of RMD-1 induces severe defects in chromosome segregation, probably through merotelic attachments between microtubules and chromosomes. Although rmd-1 embryos also have a mild defect in microtubule growth, we find that mutants of the microtubule growth regulator XMAP215/ZYG-9 show much weaker segregation defects. This suggests that the microtubule growth defect in rmd-1 embryos does not cause abnormal chromosome segregation. We also see that RMD-1 interacts with aurora B in vitro. Our results suggest that RMD-1 functions in chromosome segregation in C. elegans embryos, possibly through the aurora B–mediated pathway. Human homologues of RMD-1 could also bind microtubules, which would suggest a function for these proteins in chromosome segregation during mitosis in other organisms as well.     

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.     

Anillin-mediated targeting of peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.     

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.     

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.     

Studies on Differentiation Without Cleavage in Chaetopterus variopedatus

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis on differentiation without cleavage and on normal development have been studied in Chaetopterus . Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.     

Cell wall assembly in Fucus zygotes. II. Cellulose synthesis and deposition is controlled at the post-translational level

An alkali-insoluble cell wall component of Fucus distichus embryos is shown to be cellulose. Cellulose is not found in eggs, but within 20 min following fertilization is present in cell walls of zygotes. Incorporation of [³H]glucose or NaH¹⁴CO₃ confirms that this cellulose in zygotes of F. distichus and F. vesiculosus is newly synthesized, with the highest rate of synthesis occurring within the first 60 min. Use of specific inhibitors indicates that the increase in cellulose does not require protein or RNA synthesis. The evidence is consistent with the hypothesis that the initiation of cellulose synthesis triggered by fertilization is controlled at the post-translational level.     

Differential requirements for DNA replication in the activation of mitotic checkpoints in Saccharomyces cerevisiae.

Checkpoints prevent inaccurate chromosome segregation by inhibiting cell division when errors in mitotic processes are encountered. We used a temperature-sensitive mutation, dbf4, to examine the requirement for DNA replication in establishing mitotic checkpoint arrest. We used gamma-irradiation to induce DNA damage and hydroxyurea to limit deoxyribonucleotides in cells deprived of DBF4 function to investigate the requirement for DNA replication in DNA-responsive checkpoints. In the absence of DNA replication, mitosis was not inhibited by these treatments, which normally activate the DNA damage and DNA replication checkpoints. Our results support a model that indicates that the assembly of replication structures is critical for cells to respond to defects in DNA metabolism. We show that activating the spindle checkpoint with nocodazole does not require prior progression through S phase but does require a stable kinetochore.     

Diploid,but not haploid,human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.     

Distinct roles for two C. elegans anillins in the gonad and early embryo

Anillins are conserved proteins that are important for stabilizing and remodeling the actin cytoskeleton. Anillins have been implicated in cytokinesis in several systems and in cellularization of the syncytial Drosophila embryo. Here, we examine the functions of three C. elegans proteins with homology to anillin (ANI-1, ANI-2 and ANI-3). We show that ANI-1 and ANI-2 contribute to embryonic viability by performing distinct functions in the early embryo and gonad, respectively. By contrast, ANI-3 appears to be dispensable for embryonic development. ANI-1 is essential for cortical ruffling and pseudocleavage, contractile events that occur in embryos prior to mitosis. ANI-1 is also required for the highly asymmetric cytokinetic events that extrude the two polar bodies during oocyte meiosis, but is dispensable for cytokinesis following mitotic chromosome segregation. During both meiosis and mitosis, ANI-1 targets the septins, but not myosin II, to the contractile ring and does not require either for its own targeting. In contrast to ANI-1, ANI-2 functions during oogenesis to maintain the structure of the rachis, the central core of cytoplasm that connects the developing oocytes in the syncytial gonad. In ANI-2-depleted worms, oocytes disconnect prematurely from the defective rachis, generating embryos of varying sizes. Our results highlight specialization of divergent anillin family proteins in the C. elegans life cycle and reveal conserved roles for this protein family in organizing syncytial structures and cortical contractility.     

Wnt-dependent spindle polarization in the early C. elegans embryo

Correct orientation of the mitotic spindle is crucial for the proper segregation of localized determinants and the correct spatial organization of cells in early embryos. The cues dividing cells use to orient their mitotic spindles are currently the subject of intensive investigation in a number of model systems. One of the cues that cells use during spindle orientation is provided by components of the Wnt signaling pathway. Because of its stereotypical cleavage divisions, the availability of Wnt pathway mutants and the ability to perform RNAi, and because cell-cell interactions can be studied in vitro, the C. elegans embryo continues to be a useful system for identifying specific cell-cell interactions in which Wnt-dependent signals polarize the mitotic spindle. This review discusses the evidence for involvement of Wnt signaling during spindle orientation in several contexts in the early C. elegans embryo, a process that involves upstream Wnt effectors but does not involve downstream nuclear effectors of Wnt signaling, and places this Wnt spindle orientation pathway in the larger context of other known modulators of spindle orientation in animal embryos.