Cortical cytasters: a highly conserved developmental trait of Bilateria with similarities to Ctenophora
© Salinas-Saavedra and Vargas; licensee BioMed Central Ltd. 2011
Received: 12 August 2011
Accepted: 1 December 2011
Published: 1 December 2011
Cytasters (cytoplasmic asters) are centriole-based nucleation centers of microtubule polymerization that are observable in large numbers in the cortical cytoplasm of the egg and zygote of bilaterian organisms. In both protostome and deuterostome taxa, cytasters have been described to develop during oogenesis from vesicles of nuclear membrane that move to the cortical cytoplasm. They become associated with several cytoplasmic components, and participate in the reorganization of cortical cytoplasm after fertilization, patterning the antero-posterior and dorso-ventral body axes.
Presentation of the hypothesis
The specific resemblances in the development of cytasters in both protostome and deuterostome taxa suggest that an independent evolutionary origin is unlikely. An assessment of published data confirms that cytasters are present in several protostome and deuterostome phyla, but are absent in the non-bilaterian phyla Cnidaria and Ctenophora. We hypothesize that cytasters evolved in the lineage leading to Bilateria and were already present in the most recent common ancestor shared by protostomes and deuterostomes. Thus, cytasters would be an ancient and highly conserved trait that is homologous across the different bilaterian phyla. The alternative possibility is homoplasy, that is cytasters have evolved independently in different lineages of Bilateria.
Testing the hypothesis
So far, available published information shows that appropriate observations have been made in eight different bilaterian phyla. All of them present cytasters. This is consistent with the hypothesis of homology and conservation. However, there are several important groups for which there are no currently available data. The hypothesis of homology predicts that cytasters should be present in these groups. Increasing the taxonomic sample using modern techniques uniformly will test for evolutionary patterns supporting homology, homoplasy, or secondary loss of cytasters.
Implications of the hypothesis
If cytasters are homologous and highly conserved across bilateria, their potential developmental and evolutionary relevance has been underestimated. The deep evolutionary origin of cytasters also becomes a legitimate topic of research. In Ctenophora, polyspermic fertilization occurs, with numerous sperm entering the egg. The centrosomes of sperm pronuclei associate with cytoplasmic components of the egg and reorganize the cortical cytoplasm, defining the oral-aboral axis. These resemblances lead us to suggest the possibility of a polyspermic ancestor in the lineage leading to Bilateria.
Keywordscytasters cytoplasmic asters parthenogenesis polyspermic fertilization bilaterian ctenophores microtubule network centrioles.
Despite repeated descriptions of cytasters in animal eggs from different bilaterian phyla, there has been no formal review of their potential developmental and evolutionary relevance. Indeed, cortical cytasters appear to play a crucial role in early development: after fertilization, cortical cytoplasm undergoes reorganization, with movement of cytoplasmic components that are independent from the movements of endoplasmic reorganization ( and references therein). The movements of cortical cytoplasm are crucial to the patterning of the antero-posterior and dorso-ventral body axis ([4, 9, 26] and references therein). A well-known example is cortical rotation and grey crescent formation after fertilization in early amphibian development. The cytoskeleton is required for these cytoplasmic movements, and cytasters are presumed to play a crucial role [27–29]. Cytasters conceivably can act in cytoplasmic reorganization much like the centrosome does during cell division, positioning and transporting various cytoplasmic components [30–33].
Presentation of the hypothesis
We propose that cytasters are an ancient and highly conserved trait of Bilateria that was already present in the most recent common ancestor of Protostomia and Deuterostomia (the "Urbilateria"). Thus, we propose homology and conservation of bilaterian cytasters, in contrast with the alternative possibility of homoplasy: that cytasters evolved independently in different bilaterian groups.
Detailed descriptions of cytasters in oogenesis and early development are published for several 'model system' Bilateria, particularly so sea urchins, amphibians, and holometabolous insects such as Diptera and Hymenoptera. The available information allows us to compare the extent of similarity in the formation, structural dynamics and function of cytasters observed in both deuterostome and protostome taxa. Specific similarities would suggest that an independent evolutionary origin of cytasters is unlikely, providing a first argument for the antiquity and homology of cytasters in Bilateria. In contrast, if the processes of cytaster formation were found to be essentially different for different phyla, this could be consistent with the possibility of independent origins (although it would not prove homoplasy per se). Different mechanisms of cytaster formation would suggest that cytasters could form easily under different biological conditions, supporting the argument for homoplasy.
Developmental similarities in protostomes and deuterostomes
The fact that all the specific developmental processes above have been described in both protostome and deuterostome taxa suggests that cytasters did not originate independently in each lineage but are homologous, having already been present in their most recent common ancestor (the Urbilateria). The specific developmental pathways also suggest that cytasters are not easily formed under different biological processes and conditions. A clear prediction of the hypothesis of homology is that most basic groups of Bilateria should also conserve these specific similarities. In contrast, if taxa with cytasters are nested within groups that otherwise lack cytasters, this would provide evidence of homoplasy, with cytasters appearing independently in different lineages. The absence of cytasters, or their modification, in turn, can be proven to be a secondarily derived condition, if it occurs in taxa that are nested within groups that otherwise exhibit the presence and general developmental pattern of cytasters. By examining published evidence from non-bilaterian outgroups, such as Ctenophora and Cnidaria, we can also test whether cytasters originated exclusively in the lineage leading to Bilateria, or in the ancestors of a larger group of Eumetazoa. To this purpose, we reviewed published data reporting the presence or absence of cytasters in different animal species. Although some of the older descriptions are rudimentary, modern observations with plenty of structural detail are available for several taxa. The techniques used and structures observed are summarized in Additional file 1. This bibliographical review allows identification of the more important gaps in information and future directions of research that will allow further testing of the hypothesis of homology.
Cytasters in protostomes
Within protostomes, it is well accepted that two main lineages exist, the Lophotrochozoa and the Ecdysozoa. The Lophotrochozoa includes well-studied phyla such as Mollusca and Annelida. In zygotes of the leech (an annelid) the cortical cytoplasm is populated by numerous interconnected cytasters that together constitute the whole cortex microtubule network. This ectoplasmic cytoskeletal domain is observable in the egg before fertilization, from the meiotic phase onwards, when no monoaster has yet been formed, which confirms its formation is independent of the monoaster . The reorganization and translocation of the ectoplasmic cytoskeleton is linked to the dynamics of cytasters (see ). No studies have yet confirmed (or discarded) the initial formation of cytasters from nuclear vesicles (See Additional file 1). In the mollusk Crepidula plana, as in the aforementioned groups, cytasters are in the egg cortex, surrounded by cytoplasmic components, closely associated with vesicles (presumably of nuclear origin), and their formation is not related to the centrosome  (See Additional file 1). Another mollusk in which cytasters have been reported is Spisula solidissima. These observations are important since the lineage leading to annelids and mollusks (the Lophotrochozoa) diverged from that leading to insects (the Ecdysozoa) towards the origins of Protostomia. Other Lophotrochozoa in which cytasters have been observed (although in less detail) are the Cestoda Baerietta diana and Distoichometra kozloffi, and the Nemertean Cerebratulus lacteus.
Within the Ecdysozoa, information about cytasters is available in great detail for holometabolous insects like Hymenoptera (as discussed above) and Diptera. They have also been described in Collembolla, indicating that cytasters were already present at the origin of the Hexapoda [17, 23–25, 53, 54]. In other arthropods, cytasters have been described in the eggs of the Vericrustacean Artemia salina. The description of cytasters in the nematode Caenorhabditis elegans is important, because Nematodes diverged early from all other Ecdysozoan lineages . We conclude that the available evidence is consistent with the homology and conservation of egg cytasters in Protostomes, according to a good sample of lineages spanning both Ecdysozoa and Lophotrochozoa.
Cytasters in deuterostomes
The deuterostomes are split into two main groups, the Ambulacraria and the Chordata [56, 57]. Within the Ambulacraria, cytasters are especially well-studied in sea urchins, as discussed above ([15, 16, 18, 21, 58, 59] and references therein). In sea urchins, interconnected cytasters  take part in cytoplasmic rotation (cortical reorganization over 16°C) when the egg is fertilized . Cytasters are also present in other echinoderms such as starfish , sea cucumber , and sand-dollar [35, 60]. Within the chordates, available information is almost entirely restricted to amphibians and mammals. Otherwise, one of us has confirmed the presence of cytasters in the egg cortex of the teleost Danio rerio, the zebrafish (MS personal observation). In the amphibian Cynops (a newt), the cytasters are distributed around the whole unfertilized egg cortex, and form a coarse microtubule network in parallel array, except around the meiotic spindle at the animal pole (cytoplasm is restricted to the animal pole, because of the high yolk content at the vegetal pole; ). This parallel array correlates with the direction of cortical rotation. Cytoplasmic asters also have been described in the oogenesis of the salamander Triturus viridescens and several anurans (see Additional file 1), related to cortical and germ plasm movements [11, 12, 27, 62–65].
In mammals, as expected, the cytasters surround the cortex of the unfertilized egg [22, 47, 66–69] and are independent of the sperm derived-centrosome. Cytasters have been described in the oocytes of human , pig [43, 71], rabbit , opossum  and marsupial rat . Observations in marsupials are especially interesting, since this lineage diverged early within mammals. The Glires (mouse and rabbit) deserve special attention because they are derived: their cytasters do not contain mature centrioles. Rather, the nuclear vesicles (multivesicular aggregates) have been observed to contain CPBs surrounded by pericentriolar matrix [40–42]. The absence of a mature centriole has led to the description of these cytasters as acentriolar [19, 74], but it must be kept in mind that CPB can also organize microtubule polymerization (even in absence of pericentriolar matrix ). Navara  reported that no cytasters are present in the cow. If this is not an artifact, and the cow is really lacking cytoskeletal organizers in the cortical cytoplasm, this would represent a secondary loss of cytasters in evolution: Phylogenetically, the cow is firmly nested within mammals that do have cytasters.
Cytasters in absence of the centrosome: a common role throughout Bilateria
In the early development of Bilateria, the centriole of the sperm provides the centrosome, which drives the migration, encounter, and fusion of the sperm and egg pronuclei as well as the early cycles of cell division. However, in some cases where the sperm-derived centrosome is absent, these functions are carried out by cytasters. As we have described above, the Glires differ from other mammals because their cytasters do not have mature centrioles (see above). Glires are also derived in that the oocyte has no centriole , and there is no sperm-derived centrosome in the fertilized egg. In the absence of a sperm-derived centriole to form the centrosome, the only comparable organizers are cytasters, which take over its function in guiding the migration of the pronuclei towards the center of the egg . Cytasters in Glires also organize and form the mitotic spindle, allowing the cell cycles of early mouse development [19, 22, 67–69, 74, 76] in the absence of a centrosome. Subsequently, at the blastocyst stage, in each cell a cytaster develops into a mature centriole and becomes a centrosome ([19, 40, 42] and references therein). This demonstrates that despite being described as acentriolar, the cytasters of Glires can be functional as cytoplasmic organizers and have the potential to become the centrosome. The functional takeover of centrosomal functions by cytasters has also been repeatedly observed in several bilaterian phyla in cases in which the sperm-derived centrosome may be absent yet development proceeds. This is the case for many Hexapoda that present natural parthenogenesis [17, 23–25, 53, 54] and is also true for experimentally induced parthenogenesis in eggs of the Vericrustacean Artemia salina. In sea urchin and sand dollar, when eggs are artificially activated (without fertilization), the cytasters form a structure resembling a mitotic spindle (bipolar but without asters; ) and development proceeds parthenogenetically [35, 37]. Cytasters also take over the function of early cell divisions when parthenogenesis is artificially induced in the pig and rabbit [42, 43]. In the salamander Triturus, in experiments on androgenic development, eggs are fertilized in the absence of the sperm-derived centrosome, so cytasters must organize the early cell divisions . In all the cases mentioned above, cortical reorganization and cell divisions proceed normally, in the absence of a sperm-derived centrosome. This demonstrates that cytasters can organize and move cytoplasmic components much like a centrosome, which supports the strong inference that cytasters are crucial for the reorganization of cortical cytoplasm and axis establishment. A potential for replacing centrosomal functions in early development, and a capacity to eventually become centrosomes, is another specific trait of cytasters that is ubiquitous across distantly related bilaterian phyla (see Additional file 1).
Development without cytasters: Cnidaria and Ctenophora
A review of published evidence from basic groups of Eumetazoa supports the notion that cytasters are an exclusively bilaterian trait. Outside of Bilateria, in Cnidaria and Ctenophora, no cytasters are formed during the process of oogenesis, as confirmed by immunofluorescence and microtubule polymerization techniques [14, 77] (Figure 4). In cnidarians, animal-vegetal (a-v) polarity of the oocyte is generated during oogenesis and is present in the oocyte before fertilization, with the animal pole at the site of emission of the polar bodies [77, 78]. Maternal determinants that specify the germ line (for example, Nvvas1, and Nvnos2 RNA ) and oocyte polarity (CheFz1 RNA ) are transported throughout the entire cytoplasm involving microtubules [77, 79], and not in the ectoplasm alone as in Bilateria [3, 8, 80–86]. There is no independent ectoplasmic network of microtubules, nor did we find any description suggesting the differentiation of cortical cytoplasm in cnidarians. In this case, the polarization of the egg is directed by the centrosome associated with the meiotic spindle. Transportation and reorganization of maternal determinants in cnidarians begins during oogenesis and is completed after fertilization, mediated by a single microtubule network that is required for their transport (1c in Figure 3) . This microtubule network has no obvious polarity, other than the position at the animal pole of the maternal centrosome of the meiotic spindle during oogenesis. At the moment of fertilization, the maternal centrosome is absent and the sperm-derived centrosome localizes at the animal pole (2c in Figure 3) . At the end of each meiotic and mitotic cycle, when the centrosome ceases its activity, the entire microtubule network is depolymerized , in contrast with bilaterian eggs, in which the cortical network remains. The position of the centrosome of the meiotic/mitotic spindle (both before and after fertilization) is associated with the direction of transportation of maternal determinants during ooplasmic segregation. Under normal conditions, all maternal determinants necessary for normal development are transported towards the animal pole (1c in Figure 3) , where the nucleus and centrosome of the meiotic/mitotic spindle are found . When centrifuged, the centrosome can be moved to a position that is offset from the nucleus. In this case, maternal determinants are now transported towards the new position of the centrosome (rather than the nucleus). The presence of a single microtubule network nucleated by a single centrosome in the egg  confirms that cnidarians do not have an independent ectoplasmic network of microtubules comparable to that formed in bilaterians and ctenophores. Thus, the process of redistribution of maternal determinants in cnidarian eggs involves only the meiotic/mitotic spindle in cytoplasmic reorganization, rather than multiple cytasters. In this sense, we may compare cnidarian development to the endoplasmic domain of Bilateria, which is also reorganized upon fertilization by the microtubule network associated with the sperm-derived centrosome. In contrast, in both Ctenophora and Bilateria, reorganization of cortical cytoplasm is independent of the centrosome.
Cortical reorganization without cytasters: The role of polyspermy in Ctenophora
Cytasters are absent in the eggs of ctenophores (Figure 4) as confirmed by immunofluorescence and microtubule polymerization techniques . However, cortical cytoplasmic movements after fertilization occur that closely resemble those of bilaterian ectoplasm (1 to 3b in Figure 3). The ctenophore Beroe ovata has physiological polyspermic fertilization [87, 88] taking place immediately before or during formation of the first polar body (1 to 2b in Figure 3) . After several spermatozoa enter the egg, cytoplasmic components become associated with the supernumerary sperm cells, each forming a spherical zone called Sperm Pronuclear Zone (SPZ; 2b in Figure 3), that consists of cortical granules, mitochondria, endoplasmic reticulum, and other cytoplasmic components, including the nuclear envelope of the sperm pronucleus. Each SPZ is organized by the centriole associated with each male pronucleus . The formation of this new cortical cytoplasmic configuration is carried out by microtubule-mediated waves and is of utmost importance in establishing the oral-aboral axis , which takes place after the fusion of pronuclei and first cleavage cycle [14, 89]. In the polyspermic Ctenophora, it is the female pronucleus of Beroe that migrates to join a stationary male pronucleus, choosing one (2b in Figure 3). At this site, the zygote nucleus forms, first mitosis occurs and the first cleavage furrow starts [14, 89]. As development and cell division advances, the nuclear envelopes of the supernumerary male pronucleus break down, their DNA is degraded, and the giant asters nucleated by their associated centrosomes shrink and are no longer visible, presumably becoming integrated with the microtubule network (3b in Figure 3) . The centrioles of zygote centrosomes retain their mature form.
Testing the hypothesis
Implications of the hypothesis
Cortical cytoplasmic modules are an ancient and highly conserved Bilaterian trait
Based on the observations in several different bilaterian animals, cytasters are much more than star-like centers of microtubule polymerization. They have a specific ontogeny, developing from precursor bodies (possibly centrin buds) found in fragments of nuclear membrane (often described as accessory nuclei or vesicles) that move to the egg cortex and become associated to several cytoplasmic components, conforming complex cortical modules (Figure 2, Additional file 1). Thus, to go beyond the notion contained in the descriptive term cytaster, we wish to forward the notion and acronym of Cortical Cytoplasmatic Modules (CCM) for the entire assemblage of centriole (or, as in mouse and rabbit, centriolar precursor) associated with the membranous element, aster and associated cytoplasmic components: mitochondria, endoplasmic reticulum, granular material, ribosomes, proteins, maternal mRNA, membranous elements, and others.
Are CCMs inherited in cytoplasm?
The development of CCMs brings up interesting questions about their inheritance. An intriguing possibility suggested by Kallenbach and Mazia  is that the oocyte cytasters upon disappearing may produce themselves the CPBs (seeds) that become allocated to the nuclear surface. Thus, cytaster reduction may be an important source of seeds that become centrioles of new CCMs during oogenesisb. In Bilateria, during the process of cell division, the nuclear membrane (from which vesicles of CCM's are formed) disintegrates into fragments, which thereafter allocate to the cytoplasm of both resulting cells [30, 32, 33, 90]. Thus, it is easy to conceive how CPBs could be inherited through cytoplasm containing these fragments. CPBs may always be present in the nuclear membrane, but only become cytasters during the process of oogenesis. If so, artificial induction of cytaster formation is conceivable. This seems to be the case in cultured mammalian cells, where formation of cytasters similar to that in oogenesis is induced upon the experimental ablation of the centrosome  and when cells are arrested in S-phase . In these cell cultures, the centrioles of cytasters develop from seeds in vesicles of the nuclear membrane that move towards the cortical cytoplasm. These seeds in the nuclear membrane have been shown to be centrin buds containing alpha/gamma-tubulin and centrin 2. Like centrioles, these seeds are capable of self-replication , and are probably the same as the CPB seeds observed in oogenesis. Further research is required to establish if new centrin buds can only be formed by replication, or can also be assembled from isolated centrin proteins [44, 91]. If new CPBs only form by replication, they can only be inherited through cytoplasm.
A polyspermic ancestor?
The early development of Bilateria can be compared to other Eumetazoan outgroups in order to make inferences about the origin of CCMs. In this regard, the capacity of the sperm-derived centrosome to re-organize the egg cytoplasm (as observed in Cnidaria and the endoplasm of Bilateria) is linked in the case of the polyspermic Ctenophora to the establishment of a well-differentiated ectoplasmic domain, which is required for the establishment of the oral-aboral axis. Compelling resemblances exist between the CCMs of bilaterian eggs and the aforementioned SPZ of Ctenophora. The SPZ and CCM both possess a microtubule-nucleating centriole (and aster) initially associated with the nuclear membrane (sperm pronucleus of SPZ, and vesicles of nuclear membrane or accessory nuclei in CCM), which becomes surrounded by associated cytoplasmic components of the egg. Both are required for the early developmental processes of ectoplasmic reorganization and axes establishment, and both have a similar developmental fate, reducing the aster and centrioles, which become incorporated in the microtubule network (see above; compare illustration 2-3b and 2-3a in Figure 3). As discussed above, in the absence of the sperm-derived centrosome, CCMs are capable of taking over its role in the migration, encounter, and fusion of the sperm and egg pronuclei . In the ctenophore Beroe ovata, when the unfertilized egg is artificially activated (SPZs are absent) the female pronucleus migrates randomly in the cytoplasm throughout the egg [14, 93]. When the egg of Beroe is fertilized, SPZs drive the migration, encounter, and fusion of pronuclei . As discussed above, when bilaterian oocytes are artificially activated (no sperm enter the egg), they undergo the normal process of segregation and redistribution of cytoplasm, without reactivation of meiosis ([30, 33] M. Salinas-Saavedra, personal observation in zebrafish and sea urchin) and development often continues parthenogenetically [17, 24, 35, 48, 53, 54, 73, 94, 95]. In contrast, cytoplasmic reorganization and cell division is disrupted in artificially activated eggs of ctenophores, demonstrating that SPZs are required for the same processes carried out by CCMs in Bilateria.
The remarkable similarities to ctenophore SPZs listed above suggest that the development and function of CCMs are derived from early developmental processes similar to those in the polyspermic Ctenophora. More specifically, we propose that the origin of CCMs involved the acquisition by the female germ line of the capacity to produce numerous centriole-based modules of ectoplasm, a process that previously was required for the exogenous contribution of supernumerary sperm. Because CPBs can be inherited through cytoplasm, it is possible that the female germline acquired numerous CPBs directly from the reduction of sperm centrioles. An interesting point to observe in future studies is the distribution of germline determinants (like vasa and nanos) during the development of polyspermic Ctenophores. According to our hypothesis, we expect a cortical localization of these markers associated with the microtubules of SPZs, similar to the distribution observed in bilaterian eggs, associated to microtubules of the egg cortex.
The phylogenetic relationships of Ctenophora to other metazoa are currently controversial [56, 96–102]. However, Ctenophora has often been suggested to be a sister group to Bilateria [96, 99–102], especially on the basis of the morphological evidence. If so, this would support the notion that polyspermy could have been present in the most recent common ancestor of Ctenophora and Bilateria, and the lack of a differentiated cortical cytoplasm, as in cnidarians, may represent a primitive condition for the egg (with a single microtubule network).
The complex egg of Bilateria and its reorganization are crucial in the selective distribution of cytoplasmic domains during cleavage, leading to body axis patterning. Despite the well-acknowledged importance of egg cytoplasmic domains and their movements [1, 4], their evolutionary origin is seldom discussed and thus seems largely mysterious. In this sense, a new explicative framework emerges when we consider the possibility that these cytoplasmic movements may derive from a complex, polyspermic fertilization, like that of Ctenophora, leading to the differentiation of an ectoplasmic domain and the cytoplasmic movements of body axis patterning. More detailed research in Ctenophora and Cnidaria is bound to be informative about the early evolutionary history of cytoplasmic reorganization.
aPreviously, during the formation of the egg, the endoplasm is organized by the germinal vesicle-derived centrosome. In the literature, authors discussing the fate of the oocyte-derived centrosome state that it is missing at the end of meiosis [30, 53].
bThis notion is supported by the experimental injection of centrioles (isolated from adult tissues) into the egg of Xenopus. These centrioles become reduced and cease to be observable, but upon fertilization, an increased number of CCMs shows up at the site of injection ( and references therein). This suggests the injected centrioles provided an increased number of centriole precursor bodies at the injection site.
List of abbreviations
cortical cytoplasmatic module
centriolar precursor body
sperm pronuclear zone.
This work was supported by FONDECYT grant 11080258 to AOV. The authors thank Jorge Mpodozis, Luis Ossa-Fuentes, Juan Salazar, Sebastian Jaramillo, Jõao F. Botelho, Uziel Carrera, and members of 'Rayo' lab for helpful discussions.
- Cantillana V, Urrutia M, Ubilla A, Fernández J: The complex dynamic network of microtubule and microfilament cytasters of the leech zygote. Dev Biol. 2000, 228: 136-149. 10.1006/dbio.2000.9931.PubMedGoogle Scholar
- Gard DL, Cha BJ, Schroeder MM: Confocal immunofluorescence microscopy of microtubules, microtubule-associated proteins, and microtubule-organizing centers during amphibian oogenesis and early development. Curr Top Dev Biol. 1996, 31: 383-431.Google Scholar
- Theusch EV, Brown KJ, Pelegri F: Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm. Dev Biol. 2006, 292: 129-141. 10.1016/j.ydbio.2005.12.045.PubMedGoogle Scholar
- Sardet C, Paix A, Prodon F, Dru P, Chenevert J: From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo. Dev Dyn. 2007, 236: 1716-1731. 10.1002/dvdy.21136.PubMedGoogle Scholar
- Gross F: Memoirs: cleavage of blastomeres in the absence of nuclei. J Cell Sci. 1936, S2-79: 57-72.Google Scholar
- Fernández J, Olea N, Téllez V: Formation of the male pronucleus, organization of the first interphase monaster, and establishment of a perinuclear plasm domain in the egg of the glossiphoniid leech theromyzon rude. Dev Biol. 1994, 164: 111-122. 10.1006/dbio.1994.1184.PubMedGoogle Scholar
- Fuentes R, Fernández J: Ooplasmic segregation in the zebrafish zygote and early embryo: Pattern of ooplasmic movements and transport pathways. Dev Dyn. 2010, 239: 2172-2189. 10.1002/dvdy.22349.PubMedGoogle Scholar
- Fernández J, Valladares M, Fuentes R, Ubilla A: Reorganization of cytoplasm in the zebrafish oocyte and egg during early steps of ooplasmic segregation. Dev Dyn. 2006, 235: 656-671. 10.1002/dvdy.20682.PubMedGoogle Scholar
- Newman SA: Animal egg as evolutionary innovation: a solution to the 'embryonic hourglass' puzzle. J Exp Zool B Mol Dev Evol. 2011Google Scholar
- Hornick JE, Bader JR, Tribble EK, Trimble K, Breunig JS, Halpin ES, Vaughan KT, Hinchcliffe EH: Live-cell analysis of mitotic spindle formation in taxol-treated cells. Cell Motil Cytoskeleton. 2008, 65: 595-613. 10.1002/cm.20283.PubMed CentralPubMedGoogle Scholar
- Van Assel S, Brachet J: Métabolisme des acides nuclféiques et des protéines et formation de cytasters dans les oeufs d'amphibiens sous léaction de l'eau lourde. J Embryol Exp Morphol. 1968, 19: 261-272.PubMedGoogle Scholar
- Van Assel S, Brachet J: Formation de cytasters dans les œufs de Batraciens sous l'action de l'eau lourde. J Embryol Exp Morphol. 1966, 15: 143-151.PubMedGoogle Scholar
- Verde F, Berrez JM, Antony C, Karsenti E: Taxol-induced microtubule asters in mitotic extracts of Xenopus eggs: requirement for phosphorylated factors and cytoplasmic dynein. J Cell Biol. 1991, 112: 1177-1187. 10.1083/jcb.112.6.1177.PubMedGoogle Scholar
- Houliston E, Carré D, Johnston JA, Sardet C: Axis establishment and microtubule-mediated waves prior to first cleavage in Beroe ovata. Development. 1993, 117: 75-87.PubMedGoogle Scholar
- Kallenbach RJ: Ultrastructural analysis of the initiation and development of cytasters in sea-urchin eggs. J Cell Sci. 1985, 73: 261-278.PubMedGoogle Scholar
- Kuriyama R, Borisy GG: Cytasters induced within unfertilized sea-urchin eggs. J Cell Sci. 1983, 61: 175-189.PubMedGoogle Scholar
- Riparbelli MG, Tagu D, Bonhomme J, Callaini G: Aster self-organization at meiosis: a conserved mechanism in insect parthenogenesis?. Dev Biol. 2005, 278: 220-230. 10.1016/j.ydbio.2004.11.009.PubMedGoogle Scholar
- Harris PJ, Clason EL, Prier KR: Tubulin polymerization in unfertilized sea-urchin eggs induced by elevated temperature. J Cell Sci. 1989, 93: 9-17.PubMedGoogle Scholar
- Schuh M, Ellenberg J: Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell. 2007, 130: 484-498. 10.1016/j.cell.2007.06.025.PubMedGoogle Scholar
- Fernández J, Cantillana V, Ubilla A: Reorganization and translocation of the ectoplasmic cytoskeleton in the leech zygote by condensation of cytasters and interactions of dynamic microtubules and actin filaments. Cell Motil Cytoskeleton. 2002, 53: 214-230. 10.1002/cm.10069.PubMedGoogle Scholar
- Harris PJ, Clason EL: Conditions for assembly of tubulin-based structures in unfertilized sea urchin eggs. Spirals, monasters and cytasters. J Cell Sci. 1992, 102: 557-567.PubMedGoogle Scholar
- Tang C-JC, Hu H-M, Tang TK: NuMA expression and function in mouse oocytes and early embryos. J Biomed Sci. 2004, 11: 370-376. 10.1007/BF02254442.PubMedGoogle Scholar
- Tram U, Sullivan W: Reciprocal inheritance of centrosomes in the parthenogenetic Hymenopteran Nasonia vitripennis. Curr Biol. 2000, 10: 1413-1419. 10.1016/S0960-9822(00)00795-8.PubMedGoogle Scholar
- Riparbelli MG, Callaini G: Drosophila parthenogenesis: a model for de novo centrosome assembly. Dev Biol. 2003, 260: 298-313. 10.1016/S0012-1606(03)00243-4.PubMedGoogle Scholar
- Riparbelli MG, Callaini G: Drosophila parthenogenesis: a tool to decipher centrosomal vs acentrosomal spindle assembly pathways. Exp Cell Res. 2008, 314: 1617-1625. 10.1016/j.yexcr.2008.01.030.PubMedGoogle Scholar
- Jesuthasan S, Stähle U: Dynamic microtubules and specification of the zebrafish embryonic axis. Curr Biol. 1997, 7: 31-42. 10.1016/S0960-9822(06)00025-X.PubMedGoogle Scholar
- Dettlaff TA: Action of actinomycin and puromycin upon frog oocyte maturation. J Embryol Exp Morphol. 1966, 16: 183-195.PubMedGoogle Scholar
- Ressom R, Dixon K: Relocation and reorganization of germ plasm in Xenopus embryos after fertilization. Development. 1988, 103: 507-518.PubMedGoogle Scholar
- Houliston E, Elinson RP: Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs. Development. 1991, 112: 107-117.PubMedGoogle Scholar
- Schatten G: The Centrosome and Its Mode of Inheritance: The Reduction of the Centrosome during Gametogenesis and Its Restoration during Fertilization. Dev Biol. 1994, 165: 299-335. 10.1006/dbio.1994.1256.PubMedGoogle Scholar
- Palazzo RE, Vogel JM, Schnackenberg BJ, Hull DR, Wu X: Centrosome maturation. Curr Top Dev Biol. 1999, 49: 449-470.Google Scholar
- Bornens M: Organelle positioning and cell polarity. Nat Rev Mol Cell Biol. 2008, 9: 874-886. 10.1038/nrm2524.PubMedGoogle Scholar
- Wilson P: Centriole inheritance. Prion. 2008, 2: 9-16. 10.4161/pri.2.1.5064.PubMed CentralPubMedGoogle Scholar
- Ferree PM, McDonald K, Fasulo B, Sullivan W: The origin of centrosomes in parthenogenetic hymenopteran insects. Curr Biol. 2006, 16: 801-807. 10.1016/j.cub.2006.03.066.PubMedGoogle Scholar
- Chambers R: The formation of the aster in artificial parthenogenesis. J Gen Physiol. 1921, 4: 33-39. 10.1085/jgp.4.1.33.PubMed CentralPubMedGoogle Scholar
- Kallenbach RJ: 'De novo' centrioles originate at sites associated with annulate lamellae in sea-urchin eggs. Biosci Rep. 1982, 2: 959-966. 10.1007/BF01114903.PubMedGoogle Scholar
- Kallenbach RJ, Mazia D: Origin and maturation of centrioles in association with the nuclear envelope in hypertonic-stressed sea urchin eggs. Eur J Cell Biol. 1982, 28: 68-76.PubMedGoogle Scholar
- Miyazaki A, Kato K, Nemoto S: Role of microtubules and centrosomes in the eccentric relocation of the germinal vesicle upon meiosis reinitiation in sea-cucumber oocytes. Dev Biol. 2005, 280: 237-247. 10.1016/j.ydbio.2005.01.026.PubMedGoogle Scholar
- Shirai H, Hosoya N, Sawada T, Nagahama Y, Mohri H: Dynamics of mitotic apparatus formation and tubulin content during oocyte maturation in starfish. Dev Growth Differ. 1990, 32: 521-529. 10.1111/j.1440-169X.1990.00521.x.Google Scholar
- Calarco P: Centrosome precursors in the acentriolar mouse oocyte. Microsc Res Tech. 2000, 49: 428-434. 10.1002/(SICI)1097-0029(20000601)49:5<428::AID-JEMT4>3.0.CO;2-K.PubMedGoogle Scholar
- Szollosi D, Calarco P, Donahue R: Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci. 1972, 11: 521-541.PubMedGoogle Scholar
- Szöllosi D, Ozil J-P: De novo formation of centrioles in parthenogenetically activated, diploidized rabbit embryos. Biol Cell. 1991, 72: 61-66. 10.1016/0248-4900(91)90079-3.PubMedGoogle Scholar
- Kim NH, Simerly C, Funahashi H, Schatten G, Day BN: Microtubule organization in porcine oocytes during fertilization and parthenogenesis. Biol Reprod. 1996, 54: 1397-1404. 10.1095/biolreprod54.6.1397.PubMedGoogle Scholar
- La Terra S, English C, Hergert P, McEwen B, Sluder G, Khodjakov A: The de novo centriole assembly pathway in HeLa cells. J Cell Biol. 2005, 168: 713-722. 10.1083/jcb.200411126.PubMed CentralPubMedGoogle Scholar
- Collins E, Hornick J, Durcan T, Collins N, Archer W, Karanjeet K, Vaughan K, Hinchcliffe E: Centrosome biogenesis continues in the absence of microtubules during prolonged S-phase arrest. J Cell Physiol. 2010, 225: 454-465. 10.1002/jcp.22222.PubMed CentralPubMedGoogle Scholar
- Gard D, Hafezi S, Zhang T, Doxsey S: Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. J Cell Biol. 1990, 110: 2033-2042. 10.1083/jcb.110.6.2033.PubMedGoogle Scholar
- Schatten G, Simerly C, Asai DJ, Szöke E, Cooke P, Schatten H: Acetylated alpha-tubulin in microtubules during mouse fertilization and early development. Dev Biol. 1988, 130: 74-86. 10.1016/0012-1606(88)90415-0.PubMedGoogle Scholar
- Iwao Y, Yasumitsu K, Narihira M, Jiang J, Nagahama Y: Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs. Mol Reprod Dev. 1997, 47: 210-221. 10.1002/(SICI)1098-2795(199706)47:2<210::AID-MRD13>3.0.CO;2-3.PubMedGoogle Scholar
- Conklin E: Mitosis and amitosis. Biol Bull. 1917, 33: 396-436. 10.2307/1536267.Google Scholar
- Kuriyama R: Effect of taxol on first and second meiotic spindle formation in oocytes of the surf clam, Spisula solidissima. J Cell Sci. 1986, 84: 153-164.PubMedGoogle Scholar
- Douglas L: The development of organ systems in nematotaeniid cestodes. III. Gametogenesis and embryonic development in Baerietta diana and Distoichometra kozloffi. J Parasitol. 1963, 49: 530-558. 10.2307/3275758.PubMedGoogle Scholar
- Yatsu N: Experiments on the development of egg fragments in Cerebratulus. Biol Bull. 1904, 6: 123-136. 10.2307/1535574.Google Scholar
- Karr TL: Centrosome inheritance: a central 'in-egg-ma' solved?. Curr Biol. 2001, 11: R21-24. 10.1016/S0960-9822(00)00037-3.PubMedGoogle Scholar
- Riparbelli M, Giordano R, Callaini G: Centrosome inheritance in the parthenogenetic egg of the collembolan Folsomia candida. Cell Tissue Res. 2006, 326: 861-872. 10.1007/s00441-006-0253-x.PubMedGoogle Scholar
- Hyman AA, White JG: Determination of cell division axes in the early embryogenesis of Caenorhabditis elegans. J Cell Biol. 1987, 105: 2123-2135. 10.1083/jcb.105.5.2123.PubMedGoogle Scholar
- Hejnol A, Obst M, Stamatakis A, Ott M, Rouse GW, Edgecombe GD, Martinez P, Baguna J, Bailly X, Jondelius U, Wiens M, Müller WE, Seaver E, Wheeler WC, Martindale MQ, Giribet G, Dunn CW: Assessing the root of bilaterian animals with scalable phylogenomic methods. Proc Biol Sci. 2009, 276: 4261-4270. 10.1098/rspb.2009.0896.PubMed CentralPubMedGoogle Scholar
- Stach T: Chordate phylogeny and evolution: a not so simple three-taxon problem. J Zool. 2008, 276: 117-141. 10.1111/j.1469-7998.2008.00497.x.Google Scholar
- Schroeder T, Battaglia D: "Spiral asters" and cytoplasmic rotation in sea urchin eggs: induction in Strongylocentrotus purpuratus eggs by elevated temperature. J Cell Biol. 1985, 100: 1056-1062. 10.1083/jcb.100.4.1056.PubMedGoogle Scholar
- Henson JH, Fried CA, McClellan MK, Ader J, Davis JE, Oldenbourg R, Simerly CR: Bipolar, anastral spindle development in artificially activated sea urchin eggs. Dev Dyn. 2008, 237: 1348-1358. 10.1002/dvdy.21533.PubMed CentralPubMedGoogle Scholar
- Fry H: Conditions determining the origin and behavior of central bodies in cytasters of Echinarachnius eggs. Biol Bull. 1928, 54: 363-395. 10.2307/1536884.Google Scholar
- Kaylor C: Studies on experimental haploidy in salamander larvae: II. cytological studies on androgenetic eggs of Triturus viridescens. Biol Bull. 1941, 81: 402-419. 10.2307/1537914.Google Scholar
- Keppel D, Dawson A: Effects of colchicine on the cleavage of the frog's egg (Rana pipiens). Biol Bull. 1939, 76: 153-161. 10.2307/1537855.Google Scholar
- Kubota T: A regional change in the rigidity of the cortex of the egg of Rana nigromaculata following extrusion of the second polar body. Development. 1967, 17: 331-340.Google Scholar
- Skoblina MN: Role of karyoplasm in the emergence of capacity of egg cytoplasm to induce DNA synthesis in transplanted sperm nuclei. J Embryol Exp Morphol. 1976, 36: 67-72.PubMedGoogle Scholar
- Manes ME, Barbieri FD: On the possibility of sperm aster involvement in dorso-ventral polarization and pronuclear migration in the amphibian egg. J Embryol Exp Morphol. 1977, 40: 187-197.PubMedGoogle Scholar
- Calarco-Gillam PD, Siebert MC, Hubble R, Mitchison T, Kirschner M: Centrosome development in early mouse embryos as defined by an autoantibody against pericentriolar material. Cell. 1983, 35: 621-629. 10.1016/0092-8674(83)90094-6.PubMedGoogle Scholar
- Maro B, Howlett S, Webb M: Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. J Cell Biol. 1985, 101: 1665-16672. 10.1083/jcb.101.5.1665.PubMedGoogle Scholar
- Schatten H, Schatten G: Motility and centrosomal organization during sea urchin and mouse fertilization. Cell Motil Cytoskeleton. 1986, 6: 163-175. 10.1002/cm.970060215.PubMedGoogle Scholar
- Palacios MJ, Joshi HC, Simerly C, Schatten G: Gamma-tubulin reorganization during mouse fertilization and early development. J Cell Sci. 1993, 104: 383-389.PubMedGoogle Scholar
- Terada Y, Hasegawa H, Ugajin T, Murakami T, Yaegashi N, Okamura K: Microtubule organization during human parthenogenesis. Fertil Steril. 2009, 91: 1271-1272. 10.1016/j.fertnstert.2008.05.051.PubMedGoogle Scholar
- Katayama M, Zhong Z, Lai L, Sutovsky P, Prather RS, Schatten H: Mitochondrial distribution and microtubule organization in fertilized and cloned porcine embryos: implications for developmental potential. Dev Biol. 2006, 299: 206-220. 10.1016/j.ydbio.2006.07.022.PubMed CentralPubMedGoogle Scholar
- Breed WG, Simerly C, Navara CS, VandeBerg JL, Schatten G: Microtubule configurations in oocytes, zygotes, and early embryos of a marsupial, Monodelphis domestica. Dev Biol. 1994, 164: 230-240. 10.1006/dbio.1994.1194.PubMedGoogle Scholar
- Merry NE, Johnson MH, Gehring CA, Selwood L: Cytoskeletal organization in the oocyte, zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura). Mol Reprod Dev. 1995, 41: 212-224. 10.1002/mrd.1080410212.PubMedGoogle Scholar
- Schatten H, Sun Q-Y: Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation. Molecular Reproduction and Development. 2011Google Scholar
- Navara C: Microtubule organization in the cow during fertilization, polyspermy, parthenogenesis, and nuclear transfer: The role of the sperm aster. Dev Biol. 1994, 162: 29-40. 10.1006/dbio.1994.1064.PubMedGoogle Scholar
- Schatten G, Simerly C, Schatten H: Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc Natl Acad Sci USA. 1985, 82: 4152-4156. 10.1073/pnas.82.12.4152.PubMed CentralPubMedGoogle Scholar
- Amiel A, Houliston E: Three distinct RNA localization mechanisms contribute to oocyte polarity establishment in the cnidarian Clytia hemisphaerica. Dev Biol. 2009, 327: 191-203. 10.1016/j.ydbio.2008.12.007.PubMedGoogle Scholar
- Fritzenwanker JH, Genikhovich G, Kraus Y, Technau U: Early development and axis specification in the sea anemone Nematostella vectensis. Dev Biol. 2007, 310: 264-279. 10.1016/j.ydbio.2007.07.029.PubMedGoogle Scholar
- Extavour CG, Pang K, Matus DQ, Martindale MQ: vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms. Evol Dev. 2005, 7: 201-215. 10.1111/j.1525-142X.2005.05023.x.PubMedGoogle Scholar
- Wu H-R, Chen Y-T, Su Y-H, Luo Y-J, Holland LZ, Yu J-K: Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev Biol. 2011, 353: 147-159. 10.1016/j.ydbio.2011.02.014.PubMedGoogle Scholar
- Yabe T, Ge X, Pelegri F: The zebrafish maternal-effect gene cellular atoll encodes the centriolar component sas-6 and defects in its paternal function promote whole genome duplication. Dev Biol. 2007, 312: 44-60. 10.1016/j.ydbio.2007.08.054.PubMed CentralPubMedGoogle Scholar
- Pelegri F: Maternal factors in zebrafish development. Dev Dyn. 2003, 228: 535-554. 10.1002/dvdy.10390.PubMedGoogle Scholar
- Howley C, Ho RK: mRNA localization patterns in zebrafish oocytes. Mech Dev. 2000, 92: 305-309. 10.1016/S0925-4773(00)00247-1.PubMedGoogle Scholar
- Carré D, Djediat C, Sardet C: Formation of a large Vasa-positive germ granule and its inheritance by germ cells in the enigmatic Chaetognaths. Development. 2002, 129: 661-670.PubMedGoogle Scholar
- Sardet C, Prodon F, Dumollard R, Chang P, Chênevert J: Structure and function of the egg cortex from oogenesis through fertilization. Dev Biol. 2002, 241: 1-23. 10.1006/dbio.2001.0474.PubMedGoogle Scholar
- Sardet C, Dru P, Prodon F: Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos. Biol Cell. 2005, 97: 35-49. 10.1042/BC20040126.PubMedGoogle Scholar
- Gould MC, Stephano JL: Polyspermy prevention in marine invertebrates. Microsc Res Tech. 2003, 61: 379-388. 10.1002/jemt.10351.PubMedGoogle Scholar
- Carré D, Rouvière C, Sardet C: In vitro fertilization in ctenophores: sperm entry, mitosis, and the establishment of bilateral symmetry in Beroe ovata. Dev Biol. 1991, 147: 381-391. 10.1016/0012-1606(91)90296-F.PubMedGoogle Scholar
- Carré D, Sardet C: Fertilization and early development in Beroe ovata. Dev Biol. 1984, 105: 188-195. 10.1016/0012-1606(84)90274-4.PubMedGoogle Scholar
- Bornens M: Centrosome composition and microtubule anchoring mechanisms. Curr Opin Cell Biol. 2002, 14: 25-34. 10.1016/S0955-0674(01)00290-3.PubMedGoogle Scholar
- Kilmartin J: Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication. J Cell Biol. 2003, 162: 1211-1221. 10.1083/jcb.200307064.PubMed CentralPubMedGoogle Scholar
- Lindeman R, Pelegri F: Vertebrate maternal-effect genes: Insights into fertilization, early cleavage divisions, and germ cell determinant localization from studies in the zebrafish. Mol Reprod Dev. 2010, 77: 299-313.PubMed CentralPubMedGoogle Scholar
- Houliston E, Carré D, Chang P, Sardet C: Cytoskeleton and ctenophore development. Curr Top Dev Biol. 1995, 31: 41-63.PubMedGoogle Scholar
- Kaufman M: Early Mammalian Development: Parthenogenetic Studies. 1983, Cambridge University PressGoogle Scholar
- Riparbelli MG, Stouthamer R, Dallai R, Callaini G: Microtubule organization during the early development of the parthenogenetic egg of the hymenopteran Muscidifurax uniraptor. Dev Biol. 1998, 195: 89-99. 10.1006/dbio.1997.8841.PubMedGoogle Scholar
- Wallberg A, Thollesson M, Farris J, Jondelius U: The phylogenetic position of the comb jellies (Ctenophora) and the importance of taxonomic sampling. Cladistics. 2004, 20: 558-578. 10.1111/j.1096-0031.2004.00041.x.Google Scholar
- Philippe H, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Renard E, Houliston E, Quéinnec E: Phylogenomics revives traditional views on deep animal relationships. Curr Biol. 2009, 19: 706-712. 10.1016/j.cub.2009.02.052.PubMedGoogle Scholar
- Ryan J, Pang K: The homeodomain complement of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa. EvoDevo. 2010, 1: 9-10.1186/2041-9139-1-9.PubMed CentralPubMedGoogle Scholar
- Nielsen C, Eibye-Jacobsen D: Cladistic analyses of the animal kingdom. Biol J Linnean Soc. 1996, 57: 385-410. 10.1111/j.1095-8312.1996.tb01857.x.Google Scholar
- Zrzavý J, Mihulka S, Kepka P, Bezděk A, Tietz D: Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics. 1998, 14: 249-285.Google Scholar
- Peterson K, Eernisse D: Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences. Evol Dev. 2001, 3: 170-205. 10.1046/j.1525-142x.2001.003003170.x.PubMedGoogle Scholar
- Nielsen C: Six major steps in animal evolution: are we derived sponge larvae?. Evol Dev. 2008, 10: 241-257. 10.1111/j.1525-142X.2008.00231.x.PubMedGoogle Scholar
- Nadezhdina ES, Skoblina MN, Fais D, Chentsov YS: Exclusively juvenile centrioles in Xenopus laevis oocytes injected with preparations of mature centrioles. Microsc Res Tech. 1999, 44: 430-434. 10.1002/(SICI)1097-0029(19990315)44:6<430::AID-JEMT4>3.0.CO;2-0.PubMedGoogle Scholar
- Regier J, Shultz J, Zwick A, Hussey A, Ball B, Wetzer R, Martin J, Cunningham C: Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature. 2010, 463: 1079-1083. 10.1038/nature08742.PubMedGoogle Scholar
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