The BMP signaling pathway is required together with the FGF pathway for notochord induction in the ascidian embryo

The 40 notochord cells of the ascidian tadpole invariably arise from two different lineages: the primary (A-line) and the secondary (B-line) lineages. It has been shown that the primary notochord cells are induced by presumptive endoderm blastomeres between the 24-cell and the 64-cell stage. Signaling through the fibroblast growth factor (FGF) pathway is required for this induction. We have investigated the role of the bone morphogenetic protein (BMP) pathway in ascidian notochord formation. HrBMPb (the ascidian BMP2/4 homologue) is expressed in the anterior endoderm at the 44-cell stage before the completion of notochord induction. The BMP antagonist Hrchordin is expressed in a complementary manner in all surrounding blastomeres and appears to be a positive target of the BMP pathway. Unexpectedly, chordin overexpression reduced formation of both primary and secondary notochord. Conversely, primary notochord precursors isolated prior to induction formed notochord in presence of BMP-4 protein. While bFGF protein had a similar activity, notochord precursors showed a different time window of competence to respond to BMP-4 and bFGF. Our data are consistent with bFGF acting from the 24-cell stage, while BMP-4 acts during the 44-cell stage. However, active FGF signaling was also required for induction by BMP-4. In the secondary lineage, notochord specification also required two inducing signals: an FGF signal from anterior and posterior endoderm from the 24-cell stage and a BMP signal from anterior endoderm during the 44-cell stage.     

An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo

The major mesodermal tissues of ascidian larvae are muscle, notochord and mesenchyme. They are derived from the marginal zone surrounding the endoderm area in the vegetal hemisphere. Muscle fate is specified by localized ooplasmic determinants, whereas specification of notochord and mesenchyme requires inducing signals from endoderm at the 32-cell stage. In the present study, we demonstrated that all endoderm precursors were able to induce formation of notochord and mesenchyme cells in presumptive notochord and mesenchyme blastomeres, respectively, indicating that the type of tissue induced depends on differences in the responsiveness of the signal-receiving blastomeres. Basic fibroblast growth factor (bFGF), but not activin A, induced formation of mesenchyme cells as well as notochord cells. Treatment of mesenchyme-muscle precursors isolated from early 32-cell embryos with bFGF promoted mesenchyme fate and suppressed muscle fate, which is a default fate assigned by the posterior-vegetal cytoplasm (PVC) of the eggs. The sensitivity of the mesenchyme precursors to bFGF reached a maximum at the 32-cell stage, and the time required for effective induction of mesenchyme cells was only 10 minutes, features similar to those of notochord induction. These results support the idea that the distinct tissue types, notochord and mesenchyme, are induced by the same signaling molecule originating from endoderm precursors. We also demonstrated that the PVC causes the difference in the responsiveness of notochord and mesenchyme precursor blastomeres. Removal of the PVC resulted in loss of mesenchyme and in ectopic notochord formation. In contrast, transplantation of the PVC led to ectopic formation of mesenchyme cells and loss of notochord. Thus, in normal development, notochord is induced by an FGF-like signal in the anterior margin of the vegetal hemisphere, where PVC is absent, and mesenchyme is induced by an FGF-like signal in the posterior margin, where PVC is present. The whole picture of mesodermal patterning in ascidian embryos is now known. We also discuss the importance of FGF induced asymmetric divisions, of notochord and mesenchyme precursor blastomeres at the 64-cell stage.     

Role of the FGF and MEK signaling pathway in the ascidian embryo

In the ascidian embryo, a fibroblast growth factor (FGF)-like signal from presumptive endoderm blastomeres between the 32-cell and early 64-cell stages induces the formation of notochord and mesenchyme cells. However, it has not been known whether endogenous FGF signaling is involved in the process. Here it is shown that 64-cell embryos exhibit a marked increase in endogenous extracellular signal-regulated kinase (ERK/MAPK) activity. The increase in ERK activity was reduced by treatment with an FGF receptor 1 inhibitor, SU5402, and a MEK (ERK kinase/MAPKK) inhibitor, U0126. Both drugs blocked the formation of notochord and mesenchyme when embryos were treated at the 32-cell stage, but not at the 2- or 110-cell stages. The dominant-negative form of Ras also suppressed notochord and mesenchyme formation. Both inhibitors suppressed induction by exogenous basic FGF. These results suggest that the FGF signaling cascade is indeed necessary for the formation of notochord and mesenchyme cells during ascidian embryogenesis. It is also shown that FGF signaling is required for formation of the secondary notochord, secondary muscle and neural tissues, and at least ERK activity is necessary for the formation of trunk lateral cells and posterior endoderm. Therefore, FGF and MEK signaling are required for the formation of various tissues in the ascidian embryo.     

Binary specification of nerve cord and notochord cell fates in ascidian embryos

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.     

Conservation of the Developmental Role ofBrachyuryin Notochord Formation in a Urochordate, the AscidianHalocynthia roretzi

The notochord is one of the characteristic features of the phylum Chordata. The vertebrateBrachyurygene is known to be essential for the terminal differentiation of chordamesoderm into notochord. In the ascidian, which belongs to the subphylum Urochordata, differentiation of notochord cells is induced at the late phase of the 32-cell stage through cellular interaction with adjacent endoderm cells as well as neighboring notochord cells. The ascidianBrachyurygene (As-T) is expressed exclusively in the notochord-lineage blastomeres, and the timing of gene expression at the 64-cell stage precisely coincides with that of the developmental fate restriction of the blastomeres. In addition, experimental studies have demonstrated a close relationship between the inductive events andAs-Texpression. In the present study, we show that overexpression ofAs-Tby microinjection of the synthesizedAs-TRNA results in the occurrence, without the induction, of notochord-specific features in the A-line presumptive notochord blastomeres. We also show that overexpression ofAs-TRNA leads to ectopic expression of notochord-specific features in non-notochord lineages, including those of spinal cord and endoderm. These results strongly suggest that the developmental role of theBrachyuryis conserved throughout chordates in notochord formation.     

Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme : II. The 16- and 32-cell stages

Cell lineages during development of ascidian embryos were analyzed by injecting horseradish peroxidase as a tracer enzyme into identified cells of the 16-cell and 32-cell stage embryos of Halocynthia roretzi. Most of the blastomeres of these embryos developed more kinds of tissues than have hitherto been reported, and therefore, the developmental fates of each blastomere are more complex. It has been thought that every blastomere of the 64-cell stage ascidian embryo gives rise to only one kind of tissues, but the finding that the several blastomeres at the 32-cell stage developed into at least three different kinds of tissues, clearly indicates that the stage at which the fates of every blastomere are determined to one tissue is later than the 64-cell stage. The results also clearly demonstrate that muscle cells are derived not only from B-line cells (B5.1, B5.2, B6.3, and B6.4) but also from A-line cells (A5.2 and A6.4) and b-line cells (b5.3 and b6.5). Based on the present analysis as well as other studies, complete cell lineages of muscle cells up to their terminal differentiation have been proposed. In addition, lineages of nervous system, notochord, and epidermis are also discussed.     

Synergistic action of HNF-3 and Brachyury in the notochord differentiation of ascidian embryos.

In vertebrate embryos, the class I subtype forkhead domain gene HNF-3 is essential for the formation of the endoderm, notochord and overlying ventral neural tube. In ascidian embryos, Brachyury is involved in the formation of the notochord. Although the results of previous studies imply a role of HNF-3 in notochord differentiation in ascidian embryos, no experiments have been carried out to address this issue directly. Therefore the present study examined the developmental role of HNF-3 in ascidian notochord differentiation. When embryos were injected with a low dose of HNF-3 mRNA, their tails were shortened and when embryos were injected with a high dose of HNF-3 mRNA, which was enough to inhibit differentiation of epidermis and muscle, no obvious ectopic differentiation of endoderm or notochord cells was observed. However, co-injection of HNF-3 mRNA along with Brachyury mRNA resulted in ectopic differentiation of notochord cells in the animal hemisphere, suggesting that HNF-3 acts synergistically with Brachyury in ascidian notochord differentiation. Notochord differentiation of the A-line precursor cells depends on inducing signal(s) from endodermal cells, which can be mimicked by bFGF treatment. Treatment of notochord precursor cells isolated from the 32-cell stage embryoswith bFGF resulted in upregulation of both the HNF-3 and Brachyury genes.     

Brain induction in ascidian embryos is dependent on juxtaposition of FGF9/16/20-producing and -receiving cells

Coordinated regulation of inductive events, both spatially and temporally, during animal development ensures that tissues are induced at their specific positions within the embryo. The ascidian brain is induced in cells at the anterior edge of the animal hemisphere by fibroblast growth factor (FGF) signals secreted from vegetal cells. To clarify how this process is spatially regulated, we first identified the sources of the FGF signal by examining the expression of brain markers Hr-Otx and Hr-ETR-1 in embryos in which FGF signaling is locally inhibited by injecting individual blastomeres with morpholino oligonucleotide against Hr-FGF9/16/20, which encodes an endogenous brain inducer. The blastomeres identified as the inducing sources are A5.1 and A5.2 at the 16-cell stage and A6.2 and A6.4 at the 24-cell stage, which are juxtaposed with brain precursors at the anterior periphery of the embryo at the respective stages. We also showed that all the cells of the animal hemisphere are capable of expressing Hr-Otx in response to the FGF signal. These results suggest that the position of inducers, rather than competence, plays an important role in determining which animal cells are induced to become brain tissues during ascidian embryogenesis. This situation in brain induction contrasts with that in mesoderm induction, where the positions at which the notochord and mesenchyme are induced are determined mainly by intrinsic competence factors that are inherited by signal-receiving cells.     

Early embryonic expression of a LIM-homeobox gene Cs-lhx3 is downstream of beta-catenin and responsible for the endoderm differentiation in Ciona savignyi embryos

In early Ciona embryos, nuclear accumulation of beta-catenin is most probably the first step of endodermal cell specification. If beta-catenin is mis- and/or overexpressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if beta-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied with the differentiation of extra epidermal cells ( Imai, K., Takada, N., Satoh, N. and Satou, Y. (2000) Development 127, 3009-3020). Subtractive hybridization screens of mRNAs between beta-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential beta-catenin target genes that are responsible for endoderm differentiation in Ciona savignyi embryos. We found that a LIM-homeobox gene (Cs-lhx3), an otx homolog (Cs-otx) and an NK-2 class gene (Cs-ttf1) were among beta-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 were evident only in the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 was expressed again in a set of neuronal cells in the tailbud embryo, while Cs-otx was expressed in the anterior nervous system of the embryo. Expression of all three genes was upregulated in beta-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx did not affect the embryonic endoderm differentiation, although the formation of the central nervous system was suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also failed to suppress the endoderm differentiation, although injection of its synthetic mRNAs resulted in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppressed the endodermal cell differentiation and this suppression was rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs resulted in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescued the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the beta-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation.     

Developmental potential for tissue differentiation of fully dissociated cells of the ascidian embryo

Summary Initially, each tissue-progenitor blastomere of embryos of the ascidian Halocynthia was identified and isolated manually at the 110-cell (late-blastula) stage, the time at which most of the blastomeres have assumed a particular fate, such that each gives rise to a single type of tissue. The isolates were allowed to develop as partial embryos, then tissue differentiation was examined by monitoring the expression of specific molecular markers for differentiation of epidermis, endoderm, muscle and notochord. Essentially, all of the precursor blastomeres of these four kinds of tissue expressed the appropriate features of tissue differentiation in isolation, indicating that determination is already complete in most of the blastomeres by the 110-cell stage. Next, in order to evaluate the absolute capacity of cells for autonomous development, embryos were maintained continuously in a dissociated state from the first cleavage to the 110-cell stage, then the cells were allowed to develop into partial embryos. Tissue differentiation in the partial embryos was examined. The results showed the striking autonomy of the processes of segregation of developmental potential, as well as the autonomy of the processes of expression of differentiated phenotypes, namely those of epidermis and endoderm. Autonomous muscle differentiation was also observed; however, excess formation of muscle partial embryos occurred. The hypothesis that fate determination is mediated by localized maternal information in the egg cytoplasm is supported by the evidence of development of these tissues. By contrast, no evidence of notochord differentiation was observed in the partial embryos.     

Developmental autonomy of presumptive notochord cells in partial embryos of an ascidian

Summary

Ultrastructural features of larval notochord cell differentiation, sheath (membrane leaflets and filaments) and vacuoles of intracellular colloid, were found in some cells of certain partial embryos of the ascidian, Ciona intestinalis. As expected from established lineage fate maps, mature quarter-embryos developing from microsurgically isolated anterior-vegetal blastomeres (A4.1 pair) at the 8-cell stage had some cells with the notochord features. Such cells, however, also occurred in quarter-embryos resulting from the posterior-vegetal blastomere pair (B4.1) and in partial embryos derived from the B5.1 cell pair isolated at the next cleavage of the B4.1 blastomeres. These findings confirm a prediction of additional notochord cell fates from a recent revision of the ascidian lineage map based on cell marking with microinjected horseradish peroxidase. Partial embryos obtained from other lineages of the 8- and 16-cell stages did not develop notochord cells.     

Induction of ascidian peripheral neuron by vegetal blastomeres.

Ascidian tadpole larvae have a similar dorsal tubular nervous system as vertebrates. The induction of brain formation from a4.2-derived (a-line) cells requires signals from the A4.1-derived (A-line) cells. However, little is known about the mechanism underlying the development of the larval peripheral nervous system due to the lack of a suitable molecular marker. Gelsolin, an actin-binding protein, is specifically expressed in epidermal sensory neurons (ESNs) that mainly constitute the entire peripheral nervous system of the ascidian young tadpoles. Here, we address the role of cell interactions in the specification of ESNs using immunostaining with an anti-gelsolin antibody. Animal half (a4.2- and b4.2-derived) embryos did not give rise to any gelsolin-positive neurons, indicating that differentiation of ESNs requires signals from vegetal cells. Cell isolation experiments showed that A4.1 blastomeres induce gelsolin-positive neurons from a-line cells but not from b4.2-derived (b-line) cells. On the other hand, B4.1 blastomeres induce gelsolin-positive neurons both from b-line cells and a-line cells. This is in sharp contrast to the specification of brain cells which is not affected by the ablation of B4.1-derived (B-line) cells. Furthermore, basic fibroblast growth factor (bFGF) induced ESNs from the a-line cells and b-line cells in the absence of vegetal cells. Their competence to form ESNs was lost between the 110-cell stage and the neurula stage. Our results suggested that the specification of the a-line cells and b-line cells into ESNs is controlled by distinct inducing signals from the anterior and posterior vegetal blastomeres. ESNs in the trunk appear to be derived from the a8.26 blastomeres aligning on the edge of presumptive neural region where ascidian homologue of Pax3 is expressed. These findings highlight the close similarity of ascidian ESNs development with that of vertebrate placode and neural crest.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Fates and Roles of the Presumptive Organizer Region in the 32-cell Embryo in Normal Development of Xenopus laevis

Using 32-cell Xenopus embryos series of extirpation experiments were performed in order to clarify whether or not the dorsal equatorial blastomeres were committed to differentiate to the axial mesodermal structures. First, these blastomeres designated as B1, B1', C1 and C1' and C1' were labeled using the technique of HRP injection or vital staining. They produce descendants which become localized in the organizer region of the early gastrula. These cells form the prechordal plate, notochord, somites, pharyngeal endoderm and neural tube at early neurula stage. The results of extirpation of the medial two or four of these blastomeres show that the entire head lacks or the tissues and organs of the head greatly reduce. This indicates that already at the 32-cell stage they have been committed to autonomously differentiate to form the axial mesodermal tissues of the head and that their roles in the head formation can neither be replaced nor complemented by any other blastomeres surrounding them. It is also shown that the vegetal yolk cells do not seem to play essential roles for development of the axial organs of the head. On the basis of the present results a view of establishment of the organizer of Xenopus eggs is proposed.