Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.     

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.     

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.     

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.     

Roles of Hroth, the ascidian otx gene, in the differentiation of the brain (sensory vesicle) and anterior trunk epidermis in the larval development of Halocynthia roretzi

Otx genes are expressed in the anterior neural tube and endoderm in all of the chordates so far examined. In mouse embryos, important roles of otx genes in the brain development have been well documented. However, roles of otx genes in other chordate species have been less characterized. To advance our understanding about roles of otx genes in chordates, we have studied Hroth, otx of the ascidian, Halocynthia roretzi. Hroth is expressed in the anterior part of the neural tube (the sensory vesicle), the endoderm and anterior epidermis in the development. In this study, we investigated roles of Hroth in the larval development through an antisense morpholino oligonucleotides (MOs) approach. Embryos injected with Hroth-targeting MO (Hroth knockdown embryos) developed into larvae without the adhesive organ, sensory pigment cells and cavity of the sensory vesicle. The tissues, in which defects were observed, are derived from anterior-animal cells of the embryo in early cleavage stages. During cleavage stages, Hroth is also expressed in the endoderm precursors of the vegetal hemisphere. However, Hroth expression in the anterior endoderm precursors do not seem to be essential for the above defects, since MO injection into the anterior-animal but not anterior-vegetal pair cells at the 8-cell stage gave the defects. Analysis of marker gene expression demonstrated that the fate choice of the sensory vesicle precursors and the specification of the sensory vesicle territory occurred normally, but the subsequent differentiation of the sensory vesicle was severely affected in Hroth knockdown embryos. The anterior trunk epidermis including the adhesive organ-forming region was also affected, indicating that anterior epidermal patterning requires Hroth function. Based on these findings, similarities and differences in the roles of otx genes between ascidians and mice are discussed.     

Nuclear plasticity and timing mechanisms of the initiation of alkaline phosphatase expression in cytoplasm-transferred blastomeres of ascidians

Egg cytoplasm containing endoderm determinants was transferred to presumptive-muscle or presumptive-epidermis blastomeres isolated from cleavage-stage embryos of the ascidian Halocynthia roretzi. We investigated three aspects of the expression of endoderm-specific alkaline phosphatase (ALP) activity. First, we examined whether ectopic ALP expression, an indication of ectopic endoderm formation, was promoted in cytoplasm-transferred blastomeres isolated at late-cleavage stage. The results showed that the cell fate was converted by the introduced cytoplasm, even in recipient blastomeres in which the cell fate was already restricted to muscle or epidermis, and in those where expression of the muscle- or epidermis-specific genes was already initiated. Next, we examined the formation of endoderm and other tissue in embryos by double staining for ALP and muscle- or epidermis-specific marker. Regions positive for ALP and positive for muscle or epidermis marker were mutually exclusive. These results suggested that muscle- or epidermis-specific genes that were already expressed in the recipient blastomeres were down-regulated in ectopically forming endoderm cells. This is evidence for nuclear plasticity during ascidian embryogenesis. In the last series of experiments, we investigated the timing of the appearance of ALP activity in cytoplasm-transferred embryos. In the partial embryos that were derived from various combination of recipient blastomeres and donor cytoplasm obtained from various staged eggs and embryos, the timing seemed to coincide with the time that starts when cell fusion for cytoplasmic transfer was done. Therefore, the clock that determines the timing of the initiation of ALP expression is likely to start at the moment of cell fusion. Several possible hypotheses for the timing mechanism are discussed.     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features in isolated muscle progenitor cells

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a muscle-specific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and muscle-specific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells. The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermal cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.     

Developmental autonomy of muscle fine structure in muscle lineage cells of ascidian embryos

We have observed ultrastructural features of muscle differentiation in the muscle lineage cells of cleavage-arrested whole embryos and partial embryos of ascidians. Whole embryos of Ciona intestinalis and Ascidia ceratodes were cleavage-arrested with cytochalasin B at the 8-cell stage and reared to an age equivalent to several hours after hatching; these embryos formed extensive myofilaments which were often further organized into myofibrils of different sizes and densities in the peripheral cytoplasm of the two muscle lineage blastomeres (B4.1 pair). Developing myofibrils in cleavage-arrested embryos resembled the muscle elements observed in normal hatched larvae, but were less uniformly organized. A similar development of myofilaments and myofibrils occurred in the muscle lineage cells of multicellular partial embryos reared to "hatching" age. These partial embryos resulted from the isolated muscle lineage pair (B4.1) of blastomeres of the 8-cell stage (Ciona and Ascidia), and from a muscle lineage blastomere pair (B5.2) isolated at the 16-cell stage (Ascidia). Muscle lineage cells in the partial embryos were readily identified by the dense aggregates of mitochondria in their cytoplasm. Taken together, these results from the two kinds of partial embryo effectively eliminate inductive interactions with embryonic tissues other than mesodermal as a necessary factor in the onset of self-differentiation in muscle lineage cells. The relative complexity of muscle phenotype expressed in cleavage-arrested and partial embryos attests to an unusually strong developmental autonomy in the ascidian muscle lineages. This autonomy lends further support to the theory that a localized and segregated egg cytoplasmic determinant is responsible for larval muscle development in 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.     

A novel member of the Xenopus Zic family, Zic5, mediates neural crest development

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.     

Notch signaling is involved in nervous system formation in ascidian embryos

Notch signaling plays crucial roles during embryogenesis in various metazoans. HrNotch, a Notch homologue in the ascidian Halocynthia roretzi, has been previously cloned, and its expression pattern suggests that HrNotch signaling is involved in nervous system formation. To determine the function of HrNotch signaling, in the present study we examined the effects of the constitutively activated forms of HrNotch. Overexpression resulted in larvae with defects in neural tube closure and brain vesicle formation. In embryos expressing the activated HrNotch, the expression of a neural marker gene, HrETR-1, was enhanced and expanded in the central nervous system, although ectopic expression decreased during the tailbud stage. The activated HrNotch also suppressed the formation of the adhesive organ (palps) and the peripheral nervous system, which consists of ciliary mechanosensory neurons, whereas it promoted epidermal differentiation. The suppression and promotion of the formation of these respective cell types were confirmed by examination of the expression of relevant tissue-specific markers. We also cloned Hrdelta, an ascidian homologue of DSL family genes, which encode ligands for which Notch acts as a receptor. The expression of Hrdelta was observed in the precursors of palps and peripheral neurons in addition to the CNS. These results suggest that Notch signaling is important for ascidian nervous system formation and that it affects the fate choice between palps and epidermis and between peripheral neurons and epidermis within the neurogenic regions of the surface ectoderm by suppressing the formations of palps and peripheral neurons and promoting epidermal differentiation.     

Muscle lineage cytoplasm can change the developmental expression in epidermal lineage cells of ascidian embryos

Cytoplasm from muscle lineage blastomeres of an ascidian embryo can cause cells of a nonmuscle lineage to produce larval tail muscle acetylcholinesterase. Muscle cytoplasm was partitioned microsurgically into epidermal lineage blastomeres at the eight-cell stage. Posterior half-embryos (the two B3 cells) of Ascidia nigra were obtained first by separating the anterior and posterior blastomere pairs at the four-cell stage. At third cleavage, the two B3 cells divide into an ectodermal cell pair that gives rise solely to epidermal tissues, and a mesodermal-endodermal blastomere pair from which the tail muscle cells are derived. When the ectodermal and mesendodermal blastomere pairs were isolated from one another by microsurgery and reared as partial embryos, only cells originating from the mesendodermal blastomeres produced a histochemical acetylcholinesterase reaction. Immediately after cleavage of the isolated B3 cells into ectodermal and mesendodermal cell pairs, the cleavage furrows could be made to disappear by pressing firmly on the mesendodermal cells with a microneedle. Repeated up and down pressure with the microneedle at a new position across the mesendodermal cells caused furrows to reestablish in the new position, thereby incorporating mesodermal cytoplasm and increasing the size of the ectodermal cells. The cytoplasmically altered ectodermal blastomere pairs, which became detached from the mesendodermal cells by this microsurgical procedure, continued to divide and were reared to “larval” stages. One-third of these epidermal partial larvae produced patches of cells containing acetylcholinesterase. These results lend further support to the theory that choice of particular differentiation pathways (embryonic determination) in ascidian embryos is mediated by segregation of specific egg cytoplasmic determinants.