An ascidian homologue of the gonadotropin-releasing hormone receptor is a retinoic acid target gene

Transdifferentiation of the multipotent atrial epithelium is a key event during budding of the ascidian Polyandrocarpa misakiensis. The transdifferentiation is induced by mesenchyme cells that were stimulated by retinoic acid. The fluorescent differential display identified a few cDNA fragments for retinoic acid-inducible genes. One of the cDNA clones, named Pm-GnRHR, encoded a seven-pass transmembrane receptor similar to gonadotropin-releasing hormone receptors. Putative amino acid sequence showed high similarity to Ciona intestinalis GnRHRs and formed a cluster with other GnRHR proteins in a phylogenetic tree. The level of expression of the Pm-GnRHR mRNA increased during the early stage of bud development, suggesting that the Pm-GnRHR function is involved in some aspects of bud development.     

Ascidian Budding as a Transdifferentiation-Like System: Multipotent Epithelium is not Undifferentiated

During bud development of the ascidian Polyandrocarpa misakiensis , most of the new tissues are formed from foldings of atrial epithelium. Although the atrial epithelium has been believed to be undifferentiated, we found that this epithelium of P. misakiensis strongly expressed a tissue-restricted antigen, named Pae 1. Cross-reactivity of the antibody was found only in a few differentiated tissues such as branchial epithelium and phagocyte-like cells. In developing buds, the antigen disappeared selectively from the regions where the atrial epithelium forms organ rudiments. These regions corresponded with that of mitotic activity, thickening of the epithelium, swelling of nuclei, the appearance of nucleoli and accumulation of a large amount of RNA. From these observations, we assume that the change in antigen expression indicates a change in the state of differentiation of the atrial epithelium. Although Pae 1 antigen was never detected in functional gut, it was detected in the invaginating gut epithelium. This result indicates that gut cells were derived from the cells which had expressed the antigen. We therefore conclude that the conversion of the atrial epithelium into gut can be regarded as a transdifferentiation-like process.     

Development of irradiated tunicate buds: Is cell division cycle required for morphallaxis?

In the tunicate, Polyandrocarpa misakiensis , transdifferentiation occurs in the multipotent atrial epithelium during morphallactic bud development. Irradiation (10–80 Gy) or aphidicolin (10 μg/mL) blocked this process severely, although the atrial epithelium could form organ placodes. The placodes consisted of cuboidal cells with a high nucleus : cytoplasm ratio and were lacking the alkaline phosphatase antigen from the cell surface, suggesting that the atrial epithelium might undergo dedifferentiation without initiating cell cycling. Irradiated buds could resume organogenesis in temporal accordance with the restoration of mitotic activity. Bud pieces irradiated at 40 Gy were juxtaposed with unirradiated counterparts. In the operated buds, irradiated, non-dividing cells participated in organogenesis at the site of juxtaposition in cooperation with the unirradiated, dividing cells. These results have shown that in P. misakiensis the cell division cycle, probably DNA replication, is indispensable for transdifferentiation of the atrial epithelium, although every cell in the organ rudiment need not enter cell cycling. We suggest that homoiogenetic induction occurs between dividing cells and non-dividing cells.     

Budding-specific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates.

We have examined immunocytochemically the expression, localization and in vivo function of a calcium-dependent and galactose-binding 14 x 10(3) Mr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner epithelium of a double-walled bud vesicle. Soon after the bud entered the developmental phase, the granules were secreted into the mesenchymal space, where the lectin-positive extracellular matrix (ECM) developed. The lectin was also produced and secreted by granular leucocytes during budding. Hemoblasts, pluripotent stem cells in the blood, were often found in association with the ECM and they aggregated with epithelial cells to form organ rudiments. The lectin showed a high binding affinity for hemoblast precursors. The blockage of epithelial transformation of stem cells by galactose in in vivo bioassy was ineffective in the presence of the lectin. Polyclonal anti-lectin antibody prevented the hemoblasts spreading on the ECM and moving toward the epithelium, but it did not block the cell-cell adhesion of hemoblasts. By three days of bud development, lectin granules and ECM have almost disappeared from the developing bud together with a cessation of hemoblast aggregation. These results show that Polyandrocarpa lectin is a component of the ECM induced specifically in budding and suggest strongly that it plays a role in bud morphogenesis by directing the migration of pluripotent stem cells to the epithelium.     

Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates

The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.     

Cellular and molecular characterization of transdifferentiation in the process of morphallaxis of budding tunicates

In the budding tunicate,Polyandrocarpa misakiensis, a bud consists of two epithelial sheets, of which the inner, atrial epithelium shows developmental multipotency. It contains pigment granules in the cytoplasm and expresses a few differentiation markers on the cell surface. During bud development, these features are lost and new differentiation markers appear in organ rudiments that arise from the atrial epithelium. This transdifferentiation of the multipotent epithelium requires at least one cycle of cell division. It may be triggered by endogenous retinoids, probably retinoic acid (RA). RA acts on mesenchymal cells, which then secrete proteases that would serve as an actual transdifferentiation factor of the atrial epithelium.     

Retinoic Acid can Induce a Secondary Axis in Developing Buds of a Colonial Ascidian, Polyandrocarpa misakiensis

We have examined the effect of retinoic acid (RA) on axial pattern formation during bud development of the ascidian, Polyandrocarpa misakiensis . A bead containing various concentrations of RA was implanted into the distal portion of a bud at a site where morphogenic events do not normally occur. Control buds were implanted with beads containing dimethyl sulfoxide (DMSO), the solvent of RA. No apparent effect was observed in these buds containing beads treated with DMSO. In contrast, beads containing 100 μg/ml of RA could induce ectopic structures in the distal portion of buds in about 30% of the cases. The resulting animals had completely duplicated antero-posterior axes. Histological studies showed that, within two days of RA treatment, atrial epithelial cells situated just beneath the implanted bead became thickened and formed a gut rudiment that resembled the posterior structure of the animal. The effect of RA treatment was dose-dependent. The minimum concentration of RA required to induce a secondary axis was 100 ng/ml. Beads containing 1 mg/ml of RA had a lethal effect on the cells that surrounded the beads. These results are discussed in relation to the role of RA in axis formation and the mechanism by which positional values are specified during normal and aberrant bud development in ascidians.     

The postbranchial digestive tract of the ascidian, Polyandrocarpa misakiensis (Tunicata: Ascidiacea). 1. Oesophagus

The organization of the oesophagus in the budding styelid ascidian, Polyandrocarpa misakiensis, is described. The oesophagus consists of external and internal epithelium, and there are loose connective tissue, blood sinuses, and a muscular layer between them. The internal epithelium is simple columnar, except for the bottom of three folds. The external epithelium is simple squamous. The internal epithelium contains four cell types, i.e., ciliated mucous cells, band cells, endocrine cells, and undifferentiated cells. The ciliated mucous cells have apical cilia and microvilli, and two types of mucous vesicle. The band cells also have apical cilia and electron-dense granules in the apical cytoplasm. The endocrine cells are bottle-shaped, and have electron-dense granules both above and below the nucleus. The undifferentiated cells form pseudostratified epithelium at the bottom of each fold, and they have nuclei with prominent nucleoli. One type of coelomic cell, which has retractile cytoplasm, often migrates in the internal epithelium. Near the stomach, there are many darkly stained round cells clustered around the posterior end of the oesophagus. These two types of coelomic cells may be involved in the defense mechanism against the invasion of foreign organisms. The basic organization of the oesophagus of P. misakiensis is similar to those of other ascidians. However, the presence of three folds is a characteristic of a solitary species, rather than of a colonial species. Although ascidians are chordate invertebrates, the organization of their oesophagus is not very complex, which might reflect their life style.     

The dorsal strand of Polyandrocarpa misakiensis (Protochordata: Ascidiacea): a light and electron microscopic study

The dorsal strand of a budding ascidian, Polyandrocarpa misakiensis , was studied by light and electron microscopy. The length and morphology of this organ vary even among zooids in the same colony. After curving between the paired posterior nerve trunks, the strand turns left and terminates in a dead end. The dorsal strand of this species is one of the simplest types among ascidians, since there is no branching, no extensive nerve plexus, and no neuronal or non-neuronal endocrine cells surrounding it. The strand mainly consists of a simple cuboidal epithelium, but has several variations, such as multilayered regions, and protrusions of a cellular chain. The strand cells usually have mitochondria, free ribosomes and a Golgi complex of dictyosomal type. There are many mitotic cells in the strand epithelium. The presence, between the cerebral ganglion and dorsal strand epithelium, of cells with morphology intermediate between the strand and neuronal cells suggests that the dorsal strand might supply cellular components, such as neurones, to the cerebral ganglion in adult zooids. At the caudal end, the strand cells show ultrastructural features suggesting active protein synthesis and secretion. These cells appear to be liberated from the epithelium to release the content of their granules.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

Molecular collaborations between serpins and trefoil factor promote endodermal cell growth and gastrointestinal differentiation in budding tunicates

We present evidence supporting novel collaborations between the serine protease inhibitor (serpin) and the trefoil factor during the budding stage of the tunicate Polyandrocarpa misakiensis. Using a maltose-binding protein/P-serpin fusion protein, two polypeptides of 40 kDa and 45 kDa were pulled down from Polyandrocarpa homogenates. Based on their partial amino acid sequence data, a single cDNA (928 bp) was cloned. It encodes a polypeptide that has five tandem repeats of a trefoil consensus motif. Thus, we termed the cDNA P-trefoil. Both P-trefoil and P-serpin were expressed exclusively by coelomic cells during budding. P-Trefoil was expressed mainly by coelomic cells throughout the asexual life cycle of Polyandrocarpa, while P-Serpin was localized particularly in coelomic cells and in the extracellular matrix in developing buds. The native P-Trefoil protein showed aminopeptidase activity. It induced cell growth in cultured Polyandrocarpa cells at a concentration of 8 microg/mL. P-Serpin reinforced this activity of P-Trefoil. Further, a mixture of P-Trefoil and P-Serpin exhibited the in vitro induction of a gut-specific alkaline phosphatase. These results show for the first time that a serpin can interact with a trefoil factor to play a role in the cellular growth and differentiation of the gastric epithelium.     

Mitosis and body patterning during morphallactic development of palleal buds in ascidians

Spatiotemporal distribution of mitosis and anteroposterior body patterning during morphallactic development of palleal buds in the ascidian, Polyandrocarpa misakiensis, have been studied histologically in the presence or absence of 1.5 mM colchicine. Local cell division became evident at the proximal end of the inner, atrial epithelium of 1.5-day intact buds. This and other histological evidence showed that the primary cell activation took place at that region. In 2-day intact buds, mitotic activity spread out from the proximal end toward the lateral epithelial wall that had the lower (more anterior) positional information, referred to as the secondary cell activation. These primary and secondary activation sites were the presumptive domains of the gut and pharyngeal rudiments which specified the anteroposterior body pattern of a bud. Surgical manipulations to induce the reversal of bud polarity caused the conversion of the secondary activation site and of the pharyngeal domain, but had no effect on the primary cell activation. Thus, positional information in ascidians contributes to the formation of the pharynx by specifying the secodary cell activation site. On the other hand, a large discontinuity in positional information enhanced the primary cell activity. When two positional information gaps were constructed in a single bud, the primary cell activation occurred at two sites, resulting in an additional gut rudiment. The results of this study are discussed in the context of the possible basic mechanism that the budding in ascidians shares with epimorphic fields.     

A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.     

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.     

Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells

The surface distribution of the envelope glycoproteins of influenza, Sendai and Vesicular Stomatitis viruses was studied by immunofluorescence and immunoelectromicroscopy in infected epithelial cell monolayers, from which these viruses bud in a polarized fashion. It was found that before the onset of viral budding, the envelope proteins are exclusively localized into the same plasma membrane domains of the epithelial cells from which the virions ultimately bud: the glycoproteins of influenza and Sendai were detected at the apical surface, while the G protein of Vesicular Stomatitis virus was concentrated at the basolateral region. On the other hand, Sendai virus nucleocapsids, which can be easily identified in the cytoplasm before viral assembly, could be observed throughout the cell, not showing any preferential localization near the surface that the virions utilize for budding. These results are consistent with a model in which the asymmetric distribution of viral envelope proteins, rather than a polarized delivery of nucleocapsids, directs the polarity of viral budding. Furthermore, the asymmetric surface localization of viral glycoproteins suggests that these proteins share with intrinsic surface proteins of epithelial cells common biogenetic mechanisms and informational features or "sorting out" signals that determine their compartmentalization in the plasma membrane.     

Developmental pattern of colonies in the polystyelid ascidian,Polyandrocarpa misakiensis

Summary

The growth pattern of zooids formed asexually by budding was studied in the colonial ascidian, Polyandrocarpa misakiensis. Each colony started from a blas- tozooid (the first generation) on the glass plate in two series of experiments. To evaluate the growth of colonies, lineage of all the zooids of three successive generations was traced on photographs which were taken once a week. The zooids of the first generation produced many buds from any basal margin of the zooidal body, and those of the second generation produced a small number of buds mainly from anterior parts of the zooidal body. The zooids of the second generation produced by early budding of mother zooids were clearly more prolific than those produced by late budding. Circular colonies which developed around a zooid of the first generation consisted of stratified zones of successive generations. Each zone was composed of two subzones; the outer one mainly containing early-produced zooids, and the inner one mainly containing late-produced zooids. The zooids in the marginal area of colony are early-produced ones from generation to generation. The seawater temperature may influence the growth of zooids and/or the frequency of budding.