Gaku Kumano

Ascidian embryonic development: an emerging model system for the study of cell fate specification in chordates.

The ascidian tadpole larva represents the basic body plan of all chordates in a relatively small number of cells and tissue types. Although it had been considered that ascidians develop largely in a determinative way, whereas vertebrates develop in an inductive way, recent studies at the molecular and cellular levels have uncovered several similarities in the way developmental fates are specified. In this review, we describe ascidian embryogenesis and its cell lineages, introduce several characteristics of ascidian embryos, describe recent advances in understanding of the mechanisms of cell fate specification, and discuss them in the context of what is known in vertebrates and other organisms. Developmental Dynamics 236:1748–1757, 2007.

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.

Revisions to the Xenopus gastrula fate map: Implications for mesoderm induction and patterning

A revised fate map of the gastrula Xenopus embryo predicts the existence of patterning mechanisms that operate within the animal/vegetal axis of the mesoderm‐forming marginal zone. We review here molecular and embryologic data that demonstrate that such mechanisms are present and that they operate independently of the Spemann organizer. Evidence suggests that polarized fibroblast growth factor activity in the animal/vegetal axis patterns this axis. We present a model of mesoderm induction and patterning that integrates the new data on Spemann organizer‐independent animal/vegetal patterning with data on other inductive pathways known to act on the gastrula marginal zone.

Segregation of Germ Layer Fates by Nuclear Migration-Dependent Localization of Not mRNA

An important step in early embryonic development is the allocation and segregation of germ layer fates into distinct embryonic regions. However, the mechanism that segregates the mesendoderm into mesoderm and endoderm fates remains largely unknown in most animals. Here, using ascidians, a primitive chordate, we show that these fates are segregated by partitioning of asymmetrically localized Not mRNA from the mesendoderm cell to its mesodermal daughter. Migration of the mesendoderm cell nucleus to the future mesoderm-forming region, release of Not mRNA from the nucleus, Wnt5α-dependent local retention of the mRNA, and subsequent repositioning of the mitotic spindle to the center of the cell are each required for the asymmetric localization and partitioning of Not mRNA. Our results show that nuclear migration plays an unexpected role in asymmetric cell divisions that segregate germ layer fates in chordate embryos.

Cell fate polarization in ascidian mesenchyme/muscle precursors by directed FGF signaling and role for an additional ectodermal FGF antagonizing signal in notochord/nerve cord precursors

Asymmetric cell division plays a fundamental role in generating various types of embryonic cell. In ascidian embryos, asymmetric cell divisions occur in the vegetal hemisphere in a manner similar to those found in Caenorhabditis elegans. Early divisions in embryos of both species involve inductive events on a single mother cell that result in production of daughters with different cell fates. Here we show in the ascidian Halocynthia roretzi that polarity of muscle/mesenchyme mother precursors is determined solely by the direction from which the FGF9/16/20 signal is presented, a role similar to that of Wnt signaling in the EMS and T cell divisions in C. elegans. However, polarity of nerve cord/notochord mother precursors is determined by possible antagonistic action between the FGF signal and a signal from anterior ectoderm, providing a new mechanism underlying asymmetric cell division. The ectoderm signal suppresses MAPK activation and expression of Hr-FoxA, which encodes an intrinsic competence factor for notochord induction, in the nerve cord lineage.

Nuclear accumulation of β-catenin and transcription of downstream genes are regulated by zygotic Wnt5α and maternal Dsh in ascidian embryos

Nuclear β‐catenin plays crucial roles in the establishment of the embryonic axis and formation of mesendoderm tissues in ascidians and other animals. However, the cue responsible for nuclear accumulation of β‐catenin in the vegetal hemisphere is still unknown in ascidians. Here, we investigated the roles of Wnt5α and Dsh in the nuclear accumulation of β‐catenin and activation of its downstream genes in the ascidian Halocynthia roretzi. Wnt5α knockdown embryos lost nuclear accumulation of β‐catenin at the 64‐cell stage but not at the 32‐cell stage, and expression of Hr‐lim, one of the targets of β‐catenin, was impaired in the anterior region of the embryo. Zygotic Wnt5α expression in the anterior‐vegetal blastomeres was primarily responsible for these defects. Dsh knockdown showed no effect on nuclear localization of β‐catenin, but inhibited Hr‐lim expression in the posterior region. These results suggest that maintenance of nuclear Hr‐β‐catenin after the 64‐cell stage is regulated by zygotic Hr‐Wnt5α, and that expression of its target genes is modulated by both Hr‐Wnt5α and Hr‐Dsh. Our results also highlight the importance of nuclear accumulation of β‐catenin up to the 32‐cell stage through a still unclarified mechanism. Developmental Dynamics 236:1570–1582, 2007.

Neurula rotation determines left-right asymmetry in ascidian tadpole larvae

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.

Patterning of an ascidian embryo along the anterior–posterior axis through spatial regulation of competence and induction ability by maternally localized PEM

In ascidian embryos, anterior-posterior (A-P) patterning of the vegetal cells is regulated by posteriorizing activities of a localized egg region known as posterior-vegetal cortex/cytoplasm (PVC). PEM is an essential component of the PVC and is involved in the posterior-specific cell cleavage pattern. Here we report a novel function of PEM independently of its function in cleavage regulation; it controls cell fate by excluding competence to respond to the FGF signal for notochord induction from posterior-vegetal cells. PEM was found to regulate the nuclear accumulation of beta-catenin, an upstream activator of the competence factor. PEM also influences A-P patterning in the animal hemisphere. It was found to regulate FGF signal expression and restrict the occurrence of brain induction only in the anterior region. Our results suggest a model in which PEM patterns the embryo along the A-P axis through regulation of the spatial distribution of competence and induction ability.

MATERNAL AND ZYGOTIC EXPRESSION OF THE ENDODERM-SPECIFIC ALKALINE PHOSPHATASE GENE IN EMBRYOS OF THE ASCIDIAN, HALOCYNTHIA RORETZI

Alkaline phosphatase (AP) activity is expressed by endodermal cells of ascidian larvae. It was reported previously that the expression of AP activity is resistant to treatment with actinomycin D, a transcription inhibitor that inhibits the appearance of several other tissue-specific molecules and morphological markers of tissue formation in developing ascidians. The resistance of AP expression to actinomycin D treatment suggests that endodermal AP activity does not depend on zygotic transcription and that its appearance is mediated by the translational activation of maternal AP mRNA present in ascidian eggs. However, it was also shown that anucleate merogons do not develop AP activity. To directly examine whether maternal AP transcripts are present in the cytoplasm of eggs, we isolated a cDNA of an endoderm-specific AP in Halocynthia roretzi and examined the temporal and spatial expressions of this gene during embryogenesis using Northern blots and in situ hybridization. Maternal AP transcripts were detected in oocytes, cleaving-stage embryos, and in gastrulae, and endoderm-specific AP transcripts dramatically increased about 14 times from the neurula stage to the larval stage in endoderm precursor cells. These results suggest that the differentiation of endoderm is primarily correlated with the activation of zygotic transcription of the AP gene, presumably by egg cytoplasmic factors, similar to how muscle and epidermis are believed to develop.

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi

Background information. In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells.