Posterior End Mark Research Articles

Control of Pem protein level by localized maternal factors for transcriptional regulation in the germline of the ascidian, Halocynthia roretzi.

Localized maternal mRNAs play important roles in embryogenesis, e.g. the establishment of embryonic axes and the developmental cell fate specification, in various animal species. In ascidians, a group of maternal mRNAs, called postplasmic/PEM RNAs, is localized to a subcellular structure, called the Centrosome-Attracting Body (CAB), which contains the ascidian germ plasm, and is inherited by the germline cells during embryogenesis. Posterior end mark (Pem), a postplasmic/PEM RNAs member, represses somatic gene expression in the germline during cleavage stages by inhibition of RNA polymerase II activity. However, the functions of other postplasmic/ PEM RNAs members in germline formation are largely unknown. In this study, we analyzed the functions of two postplasmic/PEM RNAs, Popk-1 and Zf-1, in transcriptional regulation in the germline cells. We show that Popk-1 contributes to transcriptional quiescence by controlling the size of the CAB and amount of Pem protein translated at the CAB. Our studies also indicated that zygotic expression of a germline gene starts around the onset of gastrulation and that the decrease of Pem protein is necessary and sufficient for the zygotic germline gene expression. Finally, further studies showed that the decrease of the Pem protein level is facilitated by Zf-1. Taken together, we propose that postplasmic/PEM RNAs such as Popk-1 and Zf-1 control the protein level of the transcriptional repressor Pem and regulate its transcriptional state in the ascidian germline.

Evidence for a centrosome-attracting body like structure in germ-soma segregation during early development, in the urochordate Oikopleura dioica

BackgroundGerm cell formation has been investigated in sessile forms of tunicates. This process involves the release of a subset of maternal transcripts from the centrosome-attracting body (CAB) in the progenitor cells of the germ line. When germ-soma segregation is completed, CAB structures are missing from the newly formed primordial germ cells (PGCs). In free-swimming tunicates, knowledge about germ cell formation is lacking. In this investigation, comparative gene expression and electron microscopy studies were used to address germ cell formation in Oikopleura dioica (O. dioica).ResultsWe found that the RNA localization pattern of pumilio (pum1) is similar to the pattern described for a subset of maternal transcripts marking the posterior end of ascidian embryos. Transcripts marking the posterior end are called postplasmic or posterior-end mark (PEM) transcripts. We found no localization of vasa (vas) transcripts to any sub-region within the germ-line precursor cells. Expression of vas4 was detected in the newly formed PGCs. Electron microscopy studies confirmed the presence of structures with similar morphology to CAB. In the same cytoplasmic compartment, we also identified pum1 transcripts and an epitope recognized by an antibody to histone H3 phosphorylated on serine 28.ConclusionsOur findings support that a CAB-like structure participates in the segregation of maternal pum1 transcripts during germ-soma separation in O. dioica.

A Maternal Factor Unique to Ascidians Silences the Germline via Binding to P-TEFb and RNAP II Regulation

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1-3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3-5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

Polo-like kinase 1 is required for localization of Posterior End Mark protein to the centrosome-attracting body and unequal cleavages in ascidian embryos

In ascidian embryos, the posterior-localized maternal factor Posterior End Mark (PEM) is responsible for patterning embryos along the anterior-posterior axis with regard to both cleavage pattern involving unequal cell divisions and gene expression. Although PEM plays important roles in embryogenesis, its mechanism of action is still unclear because PEM has no known functional domain. In the present study, we explored the candidate of PEM partner proteins in Halocynthia roretzi using yeast two-hybrid screening. We isolated a homologue of Polo-like kinase 1 (Plk1), a key regulator of cell division and highly conserved in eukaryotes, as the first potential binding partner of PEM. We biochemically confirmed that interaction occurred between the Plk1 and PEM proteins. Immunostaining showed that Plk1 protein concentrates in the centrosome-attracting body (CAB) at the posterior pole, where PEM protein is also localized. The CAB is a subcellular structure that plays an important role in generating the posterior cleavage pattern. Plk1 localization to the CAB was dependent on the cell cycle phases during unequal cleavage. Inhibition of Plk1 with specific drugs resulted in failure of the nucleus to migrate towards the posterior pole and formation of a microtubule bundle between the CAB and a centrosome, similarly to inhibition of PEM function, suggesting that both proteins are involved in the same process of unequal cleavages. This interrupted nuclear migration was rescued by overexpression of PEM. In Plk1-inhibited embryos, the localization of PEM protein to the CAB was impaired, indicating that Plk1 is required for appropriate localization of PEM.

Localized PEM mRNA and Protein Are Involved in Cleavage-Plane Orientation and Unequal Cell Divisions in Ascidians

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. 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. 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.

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.

Posterior end mark 3 (pem-3), an Ascidian Maternally Expressed Gene with Localized mRNA Encodes a Protein with Caenorhabditis elegans MEX-3-like KH Domains

Maternal factors localized in the posterior-vegetal cytoplasm of an ascidian egg are essential for cell specification and pattern formation of the embryo. The molecular identification of the localized factors and the elucidation of the machinery associated with the localization are therefore key research subjects. I report here the isolation and characterization of a novel maternally expressed gene, posterior end mark 3 (pem-3). The pem-3 cDNA was obtained from a cDNA library of fertilized egg mRNAs subtracted with gastrula mRNAs of Ciona savignyi. As in the case of pem (Yoshida et al., 1996, Development 122, 2005–2012), the pem-3 maternal transcript was gradually concentrated after fertilization in the posterior-vegetal cytoplasm of the egg, and it later marked the posterior end of developing embryos. The PEM-3 protein was also detected in the posterior end of early embryos. The nucleotide sequence predicted that pem-3 encodes a probable RNA-binding protein with two KH domains that have an extensive similarity with those of Caenorhabditis elegans MEX-3. MEX-3 is also localized in nematode embryos (Draper et al., 1996, Cell 87, 205–216), suggesting that PEM-3 is a candidate homologue of MEX-3. In addition to maternal expression, a zygotic transcript of pem-3 and its gene product were detected in cells of the neural plate, mesenchyme, and epidermis of embryos after the neural-plate stage. Inhibition of zygotic expression using an antisense oligonucleotide resulted in the development of abnormal larvae without sensory pigment cells, suggesting that the zygotic PEM-3 plays a role in the differentiation of the brain of the ascidian larva.

Regulation of the trunk-tail patterning in the ascidian embryo: a possible interaction of cascades between lithium/beta-catenin and localized maternal factor pem.

Embryonic cell specification and pattern formation in the ascidian embryo are controlled by prelocalized egg cytoplasmic determinants. In previous studies, we showed that overexpression of a maternal gene, posterior end mark (pem), whose transcript localizes to posterior-vegetal cytoplasm of the fertilized egg, causes a loss of the anterior and dorsal structures of the larva (Yoshida et al., Development 122, 2005-2012, 1996). In the present study, first we observed that lithium treatment resulted in reduction of the larval tail. Lineage tracing analyses revealed that descendants of the A4.1 blastomere of the 8-cell-stage embryo (which forms the greater part of notochord and nerve cord) were missing from the tail region, that they were translocated anteriorly into the trunk region, and that the fate of the A4.1-line notochord cells had changed to endoderm. These results suggest that lithium treatment affects the trunk-tail patterning during embryogenesis by changing the cell fate of specific cell lineages. Second, we showed that lithium treatment could rescue the anterior and dorsal structures in pem-overexpressed larvae. This result suggests that pem plays a role in the patterning of the ascidian embryo via a signaling cascade that is affected by lithium. Third, we isolated an ascidian beta-catenin gene and found that overexpression of beta-catenin in the A4.1 blastomere had effects very similar to lithium treatment, such as reduction of the tail and anterior translocation of A4.1 descendants. These results suggest that the target of lithium is, at least in part, the Wnt-signaling cascade and that pem may also function via this cascade.

Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 ( Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark ( pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex.

Posterior end mark 2 (pem-2),pem-4,pem-5, andpem-6: Maternal Genes with Localized mRNA in the Ascidian Embryo

The posterior–vegetal cytoplasm of an ascidian egg contains maternal factors required for pattern formation and cell specification of the embryo. We report here the isolation and characterization of cDNA clones for novel maternal genes,posterior end mark 2(pem-2),pem-4,pem-5, andpem-6. We obtained these clones from a cDNA library ofCiona savignyifertilized egg mRNAs subtracted with gastrula mRNAs by examining the localization of the corresponding mRNAs of randomly selected clones by whole-mountin situhybridization. As in the case ofpem, all of these mRNAs were localized in the posterior–vegetal cytoplasm of the egg, and they later marked the posterior end of early embryos. The predicted amino acid sequence suggested that PEM-2 contains a signal for nuclear localization, an src homology 3 (SH3) domain, and a consensus sequence of the CDC24 family guanine nucleotide dissociation stimulators (GDSs). PEM-4 has a signal for nuclear localization and three C2H2-type zinc finger motifs, while PEM-5 and PEM-6 show no similarity to known proteins. These results provide further evidence that the ascidian egg contains maternal messages that are localized in the posterior–vegetal cytoplasm.

116 Facilitation of Ca 2+ action potential frequency by a small molecular weight G protein in rat pituitary GH 3 cells

Suppression of zygotic transcription in early embryonic germline cells is tightly linked to their separation from the somatic lineage. Many invertebrate embryos utilize localized maternal factors that are successively inherited by the germline cells for silencing the germline. Germline quiescence has also been associated with the underphosphorylation of Ser2 of the C-terminal domain (CTD-Ser2) of RNA polymerase II [1, 2, 3]. Here, using the ascidian Halocynthia roretzi, we identified a first deuterostome example of a maternally localized factor, posterior end mark (PEM), which globally represses germline transcription. PEM knockdown resulted in ectopic transcription and ectopic phosphorylation of CTD-Ser2 in the germline. Overexpression of PEM abolished all transcription and led to the underphosphorylation of CTD-Ser2 in the somatic cells. PEM protein was reiteratively detected in the nucleus of the germline cells and coimmunoprecipitated with CDK9, a component of posterior transcription elongation factor b (P-TEFb). These results suggest that nonhomologous proteins, PEM and Pgc of Drosophila [3, 4, 5] and PIE-1 of C. elegans [1, 6, 7], repress germline gene expression through analogous functions: by keeping CTD-Ser2 underphosphorylated through binding to the P-TEFb complex. The present study is an interesting example of evolutionary constraint on how a mechanism of germline silencing can evolve in diverse animals.

Posterior end mark, a novel maternal gene encoding a localized factor in the ascidian embryo.

Ascidian embryogenesis is regarded as a typical 'mosaic' type. Recent studies have provided convincing evidence that components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for establishment of the anteroposterior axis of the embryo. We report here isolation and characterization of a novel maternal gene, posterior end mark (pem). After fertilization, the pem transcript is concentrated in the posterior-vegetal cytoplasm of the egg and later marks the posterior end of developing ascidian embryos. Despite its conspicuous localization pattern, the predicted PEM protein shows no significant homology to known proteins. Overexpression of this gene by microinjection of synthesized pem mRNA into fertilized eggs results in development of tadpole larvae with deficiency of the anteriormost adhesive organ, dorsal brain and sensory pigment-cells. Lineage tracing analysis revealed that the anterior epidermis and dorsal neuronal cells were translocated posteriorly into the tail region, suggesting that this gene plays a role in establishment of anterior and dorsal patterning of the embryo. The ascidian tadpole is regarded as a prototype of vertebrates, implying a similar function of pem in vertebrate embryogenesis.