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.     

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.     

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

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.     

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.     

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.     

A signalling relay involving Nodal and Delta ligands acts during secondary notochord induction in Ciona embryos

The notochord is one of the defining features of chordates. The ascidian notochord is a rod like structure consisting of a single row of 40 cells. The anterior 32 ;primary' notochord cells arise from the A-line (anterior vegetal) blastomeres of the eight-cell stage embryo, whereas the posterior 8 ;secondary' notochord cells arise from the B-line (posterior vegetal) blastomeres of the eight-cell stage embryo. Specification of notochord precursors within these two lineages occurs in a spatially and temporally distinct manner. We show that specification of the secondary but not the primary notochord in Ciona intestinalis requires a relay mechanism involving two signalling pathways. First, we show evidence that acquisition of secondary notochord fate is dependent upon lateral Nodal signalling sources, situated in the adjacent b-line animal cells. Expression of the notochord specific gene Ci-Brachyury in the secondary notochord precursor was downregulated following selective inhibition of Nodal signal reception in B-line derivatives and also, strikingly, following selective inhibition of Nodal signal reception in A-line cell derivatives. Within the A-line, Nodal signals are required for localised expression of Delta2, which encodes a divergent form of Delta ligand. Using four distinct reagents to inhibit Delta2/Notch signals, we showed that Delta2 signalling from A-line cells, which activates the Notch/Su(H) pathway in adjacent B-line cells, is required for specification of the secondary notochord precursor. We propose a model whereby laterally produced Nodal acts to specify the secondary notochord precursor both directly in the B-line cells and via Delta2 induction in adjacent A-line 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.     

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.     

Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.