Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians

BACKGROUND: Orientation and positioning of the cell division plane are essential for generation of invariant cleavage patterns and for unequal cell divisions during development. Precise control of the division plane is important for appropriate partitioning of localized factors, spatial arrangement of cells for proper intercellular interactions, and size control of daughter cells. Ascidian embryos show complex but invariant cleavage patterns mainly due to three rounds of unequal cleavage at the posterior pole. RESULTS: The ascidian embryo is an emerging model for studies of developmental and cellular processes. The maternal Posterior End Mark (PEM) mRNA is localized within the egg and embryo to the posterior region. PEM is a novel protein that has no known domain. Immunostaining showed that the protein is also present in the posterior cortex and the in centrosome-attracting body (CAB) and that the localization is extraction-resistant. Here we show that PEM of Halocynthia roretzi is required for correct orientation of early-cleavage planes and subsequent unequal cell divisions because it repeatedly pulls a centrosome toward the posterior cortex and the CAB, respectively, where PEM mRNA and protein are localized. When PEM activity is suppressed, formation of the microtubule bundle linking the centrosome and the posterior cortex did not occur. PEM possibly plays a role in anchoring microtubule ends to the cortex. In our model of orientation of the early-cleavage planes, we also amend the allocation of the conventional animal-vegetal axis in ascidian embryos, and discuss how the newly proposed A-V axis provides the rationale for various developmental events and the fate map of this animal. CONCLUSIONS: The complex cleavage pattern in ascidian embryos can be explained by a simple rule of centrosome attraction mediated by localized PEM activity. PEM is the first gene identified in ascidians that is required for multiple spindle-positioning events.     

The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo

Many kinds of animal embryos exhibit stereotyped cleavage patterns during early embryogenesis. In the ascidian Halocynthia roretzi, cleavage patterns are invariant but they are complicated by successive unequal cleavages that occur in the posterior region. Here we report the essential roles of a novel structure, called the centrosome-attracting body (CAB), which exists in the posterior pole cortex of cleaving embryos, in generating unequal cleavages. By removing and transplanting posterior egg cytoplasm and by treatment with sodium dodecyl sulfate, we demonstrated that loss of the CAB resulted in abolishment of unequal cleavage, while ectopic formation of the CAB caused ectopic unequal cleavages to occur. Experiments with a microtubule inhibitor demonstrated that the centrosome and nucleus were attracted toward the posterior cortex, where the CAB is located, by shortening of microtubule bundles formed between the centrosome and the CAB. Consequently, the mitotic apparatus was positioned asymmetrically, resulting in unequal cleavage. Immunohistochemistry provided evidence that a microtubule motor protein, a kinesin or kinesin-like molecule, may be associated with the CAB. Formation of the CAB during the early cleavage stage was resistant to treatment with the microtubule inhibitor. In contrast, the integrity of the CAB was lost upon treatment with a microfilament inhibitor. We propose that the CAB plays key roles in the orientation and positioning of cleavage planes during unequal cell division.     

Ultrastructural studies on the centrosome-attracting body: Electron-dense matrix and its role in unequal cleavages in ascidian embryos

In ascidian embryos, three successive unequal cleavages occur at the posterior pole, generating a specific cleavage pattern. A recently reported novel structure designated the centrosome-attracting body (CAB) has been suggested to play essential roles in the unequal cleavages attracting centrosomes and the nucleus towards the posterior pole. To examine the morphological features of the CAB, the ultrastructure of the CAB of two ascidian species, Halocynthia roretzi and Ciona intestinalis was observed by transmission electron microscopy. Detailed observations clarified that the electron-dense matrix (EDM) was a CAB-specific component that was commonly observed in the CAB of both species but was not found in other areas of the embryo. Further observations of the CAB in various staged embryos revealed that the ultrastructure was quite stable, with no difference between points of a cell cycle or between each stage from the 8- to 64-cell stage when unequal cleavage occurred. Observations of extracted embryos implied that the EDM was the extraction-resistant component of the CAB and was tightly anchored to the plasma membrane. It has been proposed that the EDM functions as a physical attachment site at the cell cortex for microtubules emanating from centrosomes and provides a scaffold for the centrosome-attracting machinery. Interestingly, the ultrastructure of the CAB resembled germ plasm reported in other animals, raising the possibility that the CAB-containing posterior-most blastomeres are germline precursors.     

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.     

Maternal determinants and mRNAs in the cortex of ascidian oocytes, zygotes and embryos

The peripheral region of ascidian oocytes and zygotes contains five determinants for morphogenesis and differentiation of the embryo. The determinant for the 24 primary muscle cells of the tadpole, macho1, is one of several cortical mRNAs localized in a gradient along the animal-vegetal axis in the oocyte. After fertilization these mRNAs, together with cortical endoplasmic reticulum (cER) and a subcortical mitochondria-rich domain (myoplasm), relocate in two major reorganization phases forming the posterior plasm (postplasm) of the zygote. At the 8-cell stage cortical mRNAs concentrate in a macroscopic cortical structure called the centrosome-attracting body (CAB), forming a characteristic posterior end mark (PEM) in the two posterior vegetal blastomeres. We propose to call the numerous mRNAs showing this particular cortical localization in the posterior region of the embryo postplasmic/PEM RNAs and suggest a nomemclature. We do not know how postplasmic/PEM RNAs reach their polarized distribution in the oocyte cortex but at least PEM1 and macho1 (and probably others) bind to the network of cER retained in isolated cortical fragments. We propose that after fertilization, these postplasmic/PEM mRNAs move in the zygote cortex together with the cER network (cER/mRNA domain) via microfilament- and microtubule-driven translocations. The cER/mRNA domain is localized posteriorly at the time of first cleavage and distributed equally between the first two blastomeres. After the third cleavage, the cER/mRNA domain and dense particles compact to form the CAB in posterior vegetal blastomeres of the 8-cell stage. We discuss the identity of postplasmic/PEM RNAs, how they localize, anchor, relocate and may be translated. We also examine their roles in unequal cleavage and as a source of posterior morphogenetic and differentiation factors.     

Centrosome-attracting body: A novel structure closely related to unequal cleavages in the ascidian embryo

The mechanism of unequal cleavage is one of the most intriguing subjects in cell biology. Previous studies of unequal cleavage have focused on a limited number of organisms such as yeasts, nematodes, sea urchins and annelids. The cleavage pattern of the ascidian embryo is invariant. In the ascidian embryo, the posterior-most blastomeres divide unequally in three successive cleavages. In the present study, it was shown that the ascidian embryo provides another good experimental system with which to analyze the mechanism of unequal cleavage. A novel structure, designated as CAB (centrosome-attracting body), which was found specifically in the unequally cleaving blastomeres was described. In the course of unequal cleavages, first, a thick microtubule bundle appeared between CAB and one of the centrosomes. Then with the shortening of the microtubule bundle, the nucleus with the centrosome was drawn toward CAB, situated at the posterior cortex of the blastomere. Finally, a cleavage furrow formed in the middle of the asymmetrically located mitotic apparatus and produced two blastomeres of different size, generating a smaller cell that inherits CAB. The CAB seemed to play an essential role in the unequal cleavages in the ascidian embryo.     

Polo-like kinase controls vertebrate spindle elongation and cytokinesis

During cell division, chromosome segregation must be coordinated with cell cleavage so that cytokinesis occurs after chromosomes have been safely distributed to each spindle pole. Polo-like kinase 1 (Plk1) is an essential kinase that regulates spindle assembly, mitotic entry and chromosome segregation, but because of its many mitotic roles it has been difficult to specifically study its post-anaphase functions. Here we use small molecule inhibitors to block Plk1 activity at anaphase onset, and demonstrate that Plk1 controls both spindle elongation and cytokinesis. Plk1 inhibition did not affect anaphase A chromosome to pole movement, but blocked anaphase B spindle elongation. Plk1-inhibited cells failed to assemble a contractile ring and contract the cleavage furrow due to a defect in Rho and Rho-GEF localization to the division site. Our results demonstrate that Plk1 coordinates chromosome segregation with cytokinesis through its dual control of anaphase B and contractile ring assembly.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

Localization and expression pattern of type I postplasmic mRNAs in embryos of the ascidian Halocynthia roretzi

The posterior-vegetal cytoplasm (PVC) of fertilized ascidian eggs plays important roles in embryo development. It has been reported that some maternal RNAs are localized to the PVC. We identified four novel type I postplasmic mRNAs that are localized to the PVC through the use of data from a cDNA project of maternal mRNAs in the eggs of Halocynthia roretzi (MAGEST database). The mRNAs are HrGLUT, HrPEN-1, and HrPEM-3, which show similarity to a glucose transporter, a g1-related protein, and Ciona pem-3, respectively; and HrPEN-2, with no similarity. Maternal mRNAs of all four genes were identically localized to the PVC after ooplasmic segregation. During cleavage, they were concentrated in the centrosome-attracting body (CAB) and were then segregated into the small blastomeres located at the posterior pole. This localization pattern is common to all known type I postplasmic mRNAs found so far. HrGLUT, HrPEN-1, and HrPEM-3 were expressed zygotically in various tissues later in embryogenesis: HrGLUT and HrPEM-3 in the mesenchyme and nervous system, and HrPEN-1 in the ectodermal cells.     

The fat facets gene is required for Drosophila eye and embryo development.

In a screen for mutations affecting Drosophila eye development, we have identified a gene called fat facets (faf) which is required for cell interactions that prevent particular cells in the developing eye from becoming photoreceptors. Analysis of eyes mosaic for faf+ and faf- cells shows that faf is required in cells near to, but outside, normal developing photoreceptors and also outside of the ectopic photoreceptors in mutant facets. faf is also essential during oogenesis, and we show that a faf-lacZ hybrid protein is localized via the first 392 amino acids of faf to the posterior pole of oocytes. Posterior localization of faf-lacZ depends on oskar. oskar encodes a key organizer of the pole plasm, a specialized cytoplasm at the posterior pole of embryos. The pole plasm is required for germ cell formation and contains the determinant of posterior polarity, encoded by nanos. Although other pole plasm components are required for localization of nanos RNA or for nanos protein function, faf is not. We have cloned the faf gene, and have shown that it encodes two similar large (approximately 300 x 10(3) M(r)) proteins that are unique with respect to other known proteins.     

Polo-like kinase 1 may regulate G2/M transition of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2

Polo-like kinase 1(Plk1) has been reported to be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during mammalian early embryonic mitosis. In the present study, we examined the expression of Plk1 at protein and mRNA level in mouse fertilized eggs by Western blot and RT-PCR. We also examined the kinase activity of Plk1. At various developmental phases of mouse one-cell stage embryos, both the protein and the mRNA of Plk1 were uniformly distributed; but the kinase activity of Plk1 increased at G2/M phase and decreased at the end of M phase. At the meantime, the phosphorylation of Tyr 15 of Cdc2 was inhibited at M phase. To investigate its function in mammalian fertilized eggs further, we used specific short hairpin RNAs (shRNA) and scytonemin, the putative inhibitor of Plk1 to suppress the activity of Plk1 in mouse fertilized eggs. Upon blockage of the activation of with Plk1 shRNA and scytonemin in mouse one-cell stage embryos, the cleavage rate decreased and the phosphorylation level of Tyr 15 of Cdc2 increased. These results imply that the Plk1 may regulate cell cycle progression of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2.     

Cleavage pattern,gastrulation, and neurulation in the appendicularian, <Emphasis Type="Italic">Oikopleura dioica</Emphasis>

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short—approximately 5 days—and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20°C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left–right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2 × 4 array along the anterior–posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the localized posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.     

PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos

The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In Caenorhabditis elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles, respectively, and the lowest levels of these proteins correlate with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization, and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.     

Mitochondrial small ribosomal RNA is present on polar granules in early cleavage embryos of Drosophila melanogaster

In Drosophila, formation of the germline progenitors, the pole cells, is induced by polar plasm localized in the posterior pole region of early embryos. The polar plasm contains polar granules, which act as a repository for the factors required for pole cell formation. It has been postulated that the factors are stored as mRNA and are later translated on polysomes attached to the surface of polar granules. Here, the identification of mitochondrial small ribosomal RNA (mtsrRNA) as a new component of polar granules is described. The mtsrRNA was enriched in the polar plasm of the embryos immediately after oviposition and remained in the polar plasm throughout the cleavage stage until pole cell formation. In situ hybridization at an ultrastructural level revealed that mtsrRNA was enriched on the surface of polar granules in cleavage embryos. Furthermore, the localization of mtsrRNA in the polar plasm depended on the normal function of oskar, vasa and tudor genes, which are all required for pole cell formation. The temporal and spatial distribution of mtsrRNA is essentially identical to that of mitochondrial large ribosomal RNA (mtlrRNA), which has been shown to be required for pole cell formation. Taken together, it is speculated that mtsrRNA and mtlrRNA are part of the translation machinery localized to polar granules, which is essential for pole cell formation.     

Murine polo like kinase 1 gene is expressed in meiotic testicular germ cells and oocytes

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding region shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythroleukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the PlK1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage. © 1995 Wiley-Liss, Inc.     

Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.     

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.     

Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex

Localization of maternal mRNAs in the egg cortex is an essential feature of polarity in embryos of Drosophila, Xenopus and ascidians. In ascidians, maternal mRNAs such as macho 1, a determinant of primary muscle-cell fate, belong to a class of postplasmic RNAs that are located along the animal-vegetal gradient in the egg cortex. Between fertilization and cleavage, these postplasmic RNAs relocate in two main phases. They further concentrate and segregate in small posterior blastomeres into a cortical structure, the centrosome-attracting body (CAB), which is responsible for unequal cleavages. By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 mum of the surface of the unfertilized egg, and within 8 mum of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip 'Polarity inside the egg cortex' tells the story and can be downloaded at www.bioclips.com/bioclip.html