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.
In mammals and other vertebrates, developmentally restricted stem cells regulate tissue homeostasis in adult organisms (Hall & Watt 1989). For example, a small number of crypt cells in the digestive tract continue to proliferate and differentiate into functional enterocytes (Potten & Loeffler 1987; Ishizuya-Oka 2007). Consequently, they compensate for cell decay occurring in the small intestine due to cell aging, microbial infection, etc. This type of highly organized stem cell system also operates in some invertebrate organs such as the gonads (Yamashita et al. 2003) and intestine (Ohlstein & Spradling 2007) in Drosophila. A discrete unipotent or multipotent cell system appears to play a role in the overall renewal of cells and tissues, particularly in the process of regeneration. For example, when adult mammalian livers are injured, all the hepatic cells multiply properly to restore the original size and volume of the liver (Hata et al. 2007; Yoshizato 2007). Some sea cucumbers can regenerate the whole digestive tract. The new organ develops from both the coelomic and cloacal epithelium, indicating that transdifferentiation (appearance of new type[s] of cell from differentiated cells) probably occurs in adult organisms (Mashanov et al. 2005). In planarians and annelids, only mesenchymal multipotent cells, i.e. neoblasts, can form a regeneration blastema (Salo 2006). The blastema contributes to tissue renewal directly or indirectly via the formation of the body-organizing center (Agata et al. 2007). However, the cellular and molecular understanding of the cell renewal system in invertebrates remains limited.
Colonial tunicates propagate both sexually and asexually. Asexual reproduction occurs either by outgrowth development (budding) or by self-division (autotomy or strobilation) (Berrill 1950; Brien 1968). Circumstantial evidence suggests that asexual reproduction depends on the regenerative potential of somatic cells since a good correlation can be observed between asexual reproduction and regeneration in tunicates. For example, in the solitary species Ciona intestinalis that only propagates sexually, the regenerative potential is limited to the extremities of the body, such as the pigmented eye cells in the oral and atrial siphons (Loeb's observation, cited by Morgan 1901). In contrast, in the colonial species Clavelina lepadiformis that propagates both sexually and asexually, when the body is divided into anterior and posterior segments, these segments can regenerate the missing body parts (Brien 1968).
In colonial tunicates, several types of multipotent cells and tissues function in renewing and remodeling the body architecture during both regeneration and asexual reproduction (Fig. 1) (Berrill 1950; Nakauchi 1982). Single multipotent cells, referred to as hemoblasts, are present in the coelomic body cavity (hemocoel) (Wright 1981). They play important roles in differentiation and morphogenesis of cardiac muscle cells, body muscle cells, multipotent epithelium, gonadal cells, and germ cells, which has recently been reviewed elsewhere (Kawamura et al. submitted). Other types of multipotent cells are present in epithelial tissues. The epicardium and the septum are known in Aplousobranchiata and Phlebobranchiata of the order Enterogona (Lefevre 1898; Brien 1932; Berrill 1950; Koguchi et al. 1993). The septum is located in the epidermal diverticulum called stolon (Fig. 1A,B). The atrial epithelium is a major formative tissue in Stolidobranchiata of the order Pleurogona (Fig. 1C) (Berrill 1941a; Kawamura & Nakauchi 1986a; Casagrande et al. 1993; Kawamura & Fujiwara 1994; Burighel et al. 1998).
In this paper, we consider the characteristics of multipotent epithelial cells in colonial tunicates. In addition, we summarize several intrinsic molecules that are involved in dedifferentiation, cell growth, and redifferentiation. This is the first review that deals with three major multipotent epithelia in colonial tunicates.
Multipotent epithelial cells (1): Epicardium
The epicardium is generally present in all the organisms belonging to suborders Aplousobranchiata and Phlebobranchiata, except those belonging to the Perophoridae family in which the zooids lack the epicardium. The epicardium extends from the floor of the pharynx toward the pericardium and is variable in its general structure. In organisms belonging to Aplousobranchiata, it is usually a simple tube-like or V-shaped sac that runs parallel to the digestive tract (Figs 1A and 2A), whereas in Phlebobranchiata species such as Ciona intestinalis, it encompasses the entire viscera (Fig. 1B). Many researchers agree with the assumption that the epicardium arises as a pharyngeal evagination and opens into the pharyngeal cavity (Berrill 1948a, 1950; Brien 1968). However, Scott (1952) insists that the epicardium develops from the larval yolk sac. According to her observation, the yolk sac is initially continuous with the pharyngeal epithelium, but later, in young oozooids, it is physically separated from the pharynx and develops into a hollow tube due to consumption of the yolk mass. This hollow tube develops into the epicardium.
In Clavelinidae, when the thorax (pharynx, brain, and siphons) is excised from the abdomen, a new thorax is regenerated exclusively from the epicardium (Brien 1932, 1968). When zooids are cut into segments and implanted into colonies, the segments that contain the epicardium can regenerate with high frequency, while those devoid of the epicardium do not exhibit regenerative potency (Nakauchi 1966). The epicardium also plays an important role in tissue renewal during asexual reproduction in many species belonging to Aplousobranchiata and Phlebobranchiata. These species exhibit self-division (strobilation) that is negatively controlled by the cell population present in the dorsal half of the thorax (Freeman 1971). Therefore, thorax removal or other similar surgical interventions can induce the self-division of the body (Nakauchi 1966; Freeman 1971). The epidermis is responsible for this constriction and the resultant bud formation (Berrill 1948a; Scott 1952; Katow & Watanabe 1981). In contrast, the epicardium is able to renew not only itself but also the pharynx, neural structures, digestive tract, heart, and pericardium in developing buds (Berrill 1948a,b, 1950; Scott 1957; Brien 1968). The gonads and some other tissues are derived from a compact mass of cells associated with the epicardial sac, although it is unclear whether this compact mass is of mesenchymal origin or is an epicardial derivative (Berrill 1948a). According to Scott (1957), morphogenetic events occur in the epicardium even when cell division is arrested with nitrogen mustard.
Recently, the epicardium has been examined ultrastructurally in Aplousobranchiata species (Sugino & Kawamura, in preparation). The epicardial cells are flat (Fig. 2B,D), and various organelles are present in the cytoplasm, such as residual bodies (Fig. 2D arrowhead), rod-like structures (Fig. 2E), and a large aggregate of glycogen granules (Fig. 2F). A large number of coelomic cells are associated with the epicardium (Fig. 2B). They contain fibroblast-like cells (Fig. 2B arrowheads) and undifferentiated cells (Fig. 2C). During strobilation, abundant cytoplasmic debris, including glycogen granules and membrane vesicles of variable sizes, appear in the epicardial lumen (Fig. 2G). In some cases, a portion of cytoplasm seems to be discarded (Fig. 2H). The remaining cell bodies look multilayered (Fig. 2I). Then, the epicardial cells attain a cuboidal shape (Fig. 2J); in most of them, the nucleus becomes swollen and a nucleolus is prominent (Fig. 2K arrowhead), although some organelles such as glycogen granules are still observable in the cytoplasm (Fig. 2L). These observations indicate that the epicardial cells are considerably differentiated, although they are a major source of multipotent cells during regeneration and asexual reproduction in Enterogona. In addition, it is evident that during strobilation, epicardial cells undergo dedifferentiation that would accompany the exocytosis of cytoplasmic components into the epicardial cavity (this issue and Sugino & Kawamura, in preparation).
Multipotent epithelial cells (2): Septum
In some species of Aplousobranchiata and Phlebobranchiata, new buds develop in the epidermal diverticulum (stolon); hence, this type of asexual reproduction is referred to as stolonial budding (Berrill 1950; Nakauchi 1982). The septum is a single-layered, multipotent epithelium located in the stolon. According to Brien (1968), the septum cells are mesodermal in origin. In living colonies, they appear to develop from lymphocytes (hemoblasts) in the blood (Deviney 1934; Freeman 1964).
Pioneering works in the 19th century had already reported that in Perophora cuboidal epithelium derived from the septum plays a key role in bud morphogenesis and tissue renewal (Kowalevsky 1874; Lefevre 1898). The budding zone is usually restricted to an area that lies more than 100 µm proximal to the distal tip of the stolon. It comprises a tube-like hollow vesicle (Fig. 3A) (Deviney 1934; Koguchi et al. 1993). The tube-like vesicle evaginates along with the epidermal cells of the stolon to form a stolonial bud (Fig. 3B) (Lefevre 1898; Deviney 1934). The septum in remaining regions is a simple and squamous epithelium (Fig. 3C). Each cell in the septum exhibits a high level of mitotic activity (MI = approximately 5%) (Fig. 3D–F) (Koguchi et al. 1993).
The ultrastructure of septum cells has been investigated in Perophora japonica (Koguchi et al. 1993). At the distal tip of the stolon, the tube-like septum comprises cuboidal cells that are rich in mitochondria and polysomes, while the ER are poorly developed (Fig. 3G). The basal lamina underlies the basal surface of the cuboidal cells. Cells in the budding zone attain a columnar shape (Fig. 3H). They are characterized by the presence of a large nucleolus and an increased number of large intercellular spaces and mitochondria. The septum cells of the non-budding zone possess an elongated nucleus (Fig. 3I). Rough endoplasmic reticulum (ER) and phagosomes are visible in the cytoplasm, and a basal lamina is present on each side of the cell body. Cells that are morphologically similar to septum cells can be observed in the hemocoel of the stolon (Fig. 3J), suggesting that some part of the multipotent epithelial cells in the stolon may be recruited from the hemocoel (Deviney 1934). In accordance with this notion, the irradiated stolons that have lost both activities of stolonial outgrowth and stolonial budding can restore these activities when unirradiated coelomic cells are injected into the host stolon (Freeman 1964).
As mentioned above, the budding zone in Perophora is usually restricted to the distal region of the stolons; however, this does not necessarily imply that the interzooidal stolons have lost their budding potential. When an interzooidal (non-budding zone) stolon is excised and allowed to develop, one or two buds are normally formed at the cut end or on the new stolon outgrowths, while in exceptional cases, buds regenerate on the original stolon itself (Goldin 1948). In this regard, Fukumoto (1971) has reported that in Perophora thyroid hormones facilitate the elongation of interzooidal stolon, whereas thiourea, an inhibitor of thyroid hormone biosynthesis, induces stolonial budding. Recently, a thyroid hormone receptor gene has been cloned from C. intestinalis that belongs to the same Phlebobranchiata as Perophora (Yagi et al. 2003). These results indicate that in Perophora, the interzooidal septum epithelium can regenerate the bud primordium both under normal and the experimental conditions. Furthermore, when a zooid of Perophora viridis, from which the stolon has been excised in advance, is allowed to regress, the resultant cell mass gives rise to a new zooid without inducing cell proliferation (Barth & Barth 1966). This indicates that in Perophora, formative cells can also develop from adult tissues and/or mesenchymal cells in the blood other than septum cells.
The atrial (peribranchial) epithelium is a specialized tissue that exists in Protochordata, including Urochordata and Cephalochordata, but not in Vertebrata. It underlies the epidermis and encircles the pharynx (Fig. 1C). In some species belonging to Stolidobranchiata, the atrial epithelium evaginates along with the epidermis to form the inner vesicle of a palleal bud (Berrill 1940, 1941a, 1947, 1948c; Abbott 1953; Sabbadin 1955; Izzard 1973; Sugimoto & Nakauchi 1974; Fujimoto & Watanabe 1976). In Botryllus and Symplegma, the bud primordium is a thickened disc of the atrial epithelium. In Botryllus, it comprises approximately 14 cells visible in an optical section and 150 cells in total, and the closed inner vesicle comprises approximately 210 cells in total (Berrill 1941b). In contrast, in polystyelid tunicates such as Metandrocarpa, Polyandrocarpa, and Polyzoa, the bud primordium remains squamous during bud formation. This difference in cell morphology may be related to the time at which dedifferentiation of the multipotent epithelium occurs; in Botryllus and Symplegma, it occurs during bud formation, while in polystyelid tunicates, it occurs during bud development (discussed later).
In Botryllus and Symplegma, the bud primordium is restricted to predictable region(s) of the atrial epithelium (Berrill 1940, 1941a,b; Sabbadin 1955; Sugimoto & Nakauchi 1974), while in polystyelid tunicates including Polyandrocarpa, the atrial epithelium is able to form the bud primordium anywhere along the zooidal body axis (Abbott 1953; Fujimoto & Watanabe 1976; Watanabe & Newberry 1976; Kawamura & Watanabe 1982). Likewise, when Polyandrocarpa zooids are cut into pieces, the atrial epithelium can regenerate the missing parts of zooidal fragments, irrespective of its position of origin (Taneda 1985, unpubl. data, 1992), suggesting that in polystyelid tunicates the atrial epithelium, wherever it is located in the zooid body, should be equivalent concerning morphogenetic potential.
One of the open questions is why and how the atrial epithelium in Stolidobranchiata has acquired the tissue-forming activity, although in Aplousobranchiata and Phlebobranchiata the atrial epithelium never exhibits such an activity. It has long been believed that the atrial epithelium originates from larval ectodermal cells in various species belonging to Enterogona (Scott 1946; Berrill 1948a; Brien 1968). Scott (1946) presented histological evidence that the peribranchial sac appears as a pair of ectodermal invaginations, one on each side of the sensory vesicle. In contrast, tracer experiments using horseradish peroxidase (HRP) have revealed that in Halocynthia roretzi that belongs to Pleurogona (Stolidobranchiata), the atrial epithelium is derived from endodermal blastomeres (Hirano & Nishida 2000). More precisely, the endodermal blastomeres in the 110-cell embryo are A7.1, A7.2, A7.5, B7.1, and B7.2, of which A7.2 and A7.5 contribute to the anterior half of the atrial epithelium and A7.1 and B7.1, to the posterior half. What causes these contradictory results? The ‘endoderm-origin’ theory appears perfect and does not present any practical problems, whereas the ‘ectoderm-origin’ theory does not clarify whether the ectodermal invaginations are in fact identical with the atrial epithelium. If both the ectoderm- and endoderm-origin theories are true, the contradictory results in these experiments may be attributable to the phylogeny of the tunicates, Enterogona or Pleurogona. In this respect, it is interesting that only species belonging to Pleurogona can carry out palleal (peribranchial) budding. The endodermal origin of the atrial epithelium may explain why the atrial epithelium in botryllid and polystyelid tunicates is a formative tissue that enables regeneration and asexual reproduction.
The developmental multipotency of the bud's inner vesicle has been investigated mainly in polystyelid tunicates. In Stolonica, Distomus and Polyzoa, buds are elongated to a large extent. Even when they are subdivided into several pieces under natural or experimental conditions, the inner vesicle of each piece can carry out morphogenesis completely (Berrill 1948c; Fujimoto & Watanabe 1976; Nakauchi et al. 1989). Bud grafting and chimera analyzes using Polyandrocarpa misakiensis have shown that the inner vesicle takes either the anterior fate (including the pharynx) or the posterior fate (including the digestive tract), depending on the parental positional information (Kawamura & Watanabe 1983; Kawamura 1984). It is therefore evident that in P. misakiensis and probably other polystyelid tunicates, the inner vesicle of a bud is developmentally flexible and multipotent.
Although it is multipotent, the inner vesicle of the bud is not undifferentiated in P. misakiensis. Previously, we showed that cells of the vesicle have orange pigment, and they express alkaline phosphatase that is serologically discernible from germline alkaline phosphatase (Kawamura & Fujiwara 1994). The nucleus is ellipsoidal, lacking a prominent nucleolus (Fig. 4A). In the cytoplasm, the mitochondria and ER are poorly developed and fine granules are observable (Fig. 4A arrowheads). Interdigitation is usually visible between neighboring cells. Dedifferentiation and redifferentiation of these cells are similar to transdifferentiation in amphibian retina regeneration to some extent (Araki 2007). In P. misakiensis, cells of organ placodes derived from the inner vesicle undergo extensive morphological and physiological changes during bud development. First, the cell morphology is changed from squamous to cuboidal through a spherical, multilayered shape (Fig. 4A–C) (Kawamura & Fujiwara 1994). Second, the nucleus becomes swollen, having a prominent nucleolus (Fig. 4B,C arrow). The cytoplasm loses gradually fine granules (Fig. 4C arrowheads), and instead, they become enriched with polysomes (Kawamura & Fujiwara 1994). Third, alkaline phosphatase and other antigens disappear gradually, although new differentiation antigens appear soon (Fujiwara & Kawamura 1992; Kawamura & Fujiwara 1994).
In P. misakiensis, the cell proliferation by itself seems insufficient to explain how organ placodes become multilayered temporarily before organogenesis. In the gut rudiment, for example, multilayered cells are observable in 1–1.5 days of bud development earlier than mitotic figures that become abundant from 1.5 to 2.0 days (Kawamura & Nakauchi 1986b). Moreover, the multilayered cells and resultant organ rudiments are sometimes observable even when buds have been γ-irradiated or buds have been treated with aphidicolin, an inhibitor of DNA polymerase α (Kawamura et al. 1995). At least a portion of the multilayered cells may have come from coelomic cells without cell division (Kawamura et al. 1991). Actually, a number of fibroblast-like coelomic cells aggregate around the organ placodes (Fig. 4B). They contain fine granules in the cytoplasm, similar to the atrial epithelium (Fig. 4B arrowheads). A galactose-binding lectin (TC14) plays a role in this aggregation of coelomic cells, because the anti-TC14 polyclonal antibody can block their spreading on the extracellular matrix (ECM) and their movement toward the multipotent epithelium (Kawamura et al. 1991). Taken together, tissue remodeling during budding in P. misakiensis consists of two parts; dedifferentiation of multipotent inner vesicle, and the epithelial transformation of coelomic precursor cells.
Dedifferentiation-regulating factors in colonial tunicates
In this section, we describe concisely the intrinsic factors that regulate dedifferentiation of multipotent cells in colonial tunicates (Fig. 5). Most studies have focused on the atrial epithelium of P. misakiensis and a few on that of Botryllus species.
TC14 (recently redesignated as TC14-1) is a calcium-dependent galactose-binding lectin isolated from P. misakiensis (Suzuki et al. 1990). It is a budding-specific polypeptide expressed by the multipotent epithelium (Kawamura et al. 1991). It can form homodimers by creating hydrophobic bonds with the α-helices of the neighboring polypeptides (Poget et al. 1999). Three additional polypeptides and cDNAs related to TC14-1 have been isolated and termed TC14-2, TC14-3, and TC14-4, respectively (Shimada et al. 1995; Kawamura & Fujiwara 2000; Matsumoto et al. 2001). A recombinant TC14-3 protein inhibits the in vitro proliferation of multipotent cells and induces their aggregation (Fig. 5) (Matsumoto et al. 2001). The aggregates express a homolog of the integrin α-chain (Matsumoto et al. 2001). In developing buds, TC14-3 disappears from the region where dedifferentiation takes place (Fig. 4G–I). Therefore, TC14-3 may act in vivo as a cytostatic factor in order to stabilize the differentiation state of the multipotent epithelium (Fig. 5). Recent studies suggest that in P. misakiensis, cAMP and phosphorylated cAMP-responsive element-binding protein (CREB) are probably downstream effectors of TC14-3 (Kawamura et al. in preparation).
Retinoic acid may act indirectly on multipotent cells because coelomic cells treated with RA become competent to induce dedifferentiation in the atrial epithelium (Hara et al. 1992). The coelomic cells produce a serine protease known as tunicate retinoic acid-inducible modular protease (TRAMP) (Fig. 5) (Ohashi et al. 1999). TRAMP contains several types of protein–protein interaction domains at the N-terminal region and a catalytic domain at the C-terminal region. The catalytic domain of TRAMP expressed in bacteria exhibits trypsin-like protease activity (Ohashi et al. 1999). It may elicit directly a mitogenic response of the atrial epithelium, or otherwise it would act indirectly (Arai et al. 2004). A water-insoluble fraction of P. misakiensis homogenate was treated with trypsin. Mitogenic activity was detected in the water-soluble fraction following trypsin digestion. The substance causing the activity was heat stable and proteinase K resistant. It was identified as phospholipids and their derivatives; lysophospholipids and unsaturated free fatty acids (Fig. 5) (Arai et al. 2004).
Other factors related to regeneration and asexual reproduction
A gene, Athena, has been identified in Botryllus schlosseri (Laird et al. 2005). Athena antisense morpholino oligos, double-stranded RNA, and siRNA induce retardation or defects in bud growth and organogenesis (Laird et al. 2005), although the putative product of this gene exhibits no significant similarity to known proteins.
A mitogenic polypeptide was isolated from P. misakiensis (Kawamura et al. 2006). P-trefoil is a member of the trefoil growth factor family that has been known to play a role in tissue repair of the intestinal epithelium (Lefebvrfe et al. 1996; Mashimo et al. 1996; Playford et al. 1996). It is associated with a serine protease inhibitor, P-serpin that has been identified previously as budding-specific cDNA (Kawamura et al. 1998). Both P-trefoil (45K component) and P-serpin are specifically induced during budding by coelomic cells (Fig. 4J), and they stimulate in vitro proliferation of the epithelial culture cells and induce the expression of gut-specific alkaline phosphatase (Kawamura et al. 2006).
Tunicates occupy the highest position on the phylogenetic tree among animals that carry out asexual reproduction. In pelagic tunicates, all species except Appendiculata propagate by means of strobilation or budding. In sessile tunicates, all species in Aplousobranchiata and some groups of species in Phlebobranchiata and Stolidobranchiata carry out asexual reproduction. These animals are also known to have high potency for regeneration. Correctly speaking, this power of regeneration causes asexual reproduction. In this paper, we reviewed for the first time cytological features of formative cells and tissues in sessile tunicates. It has turned out that epithelial formative tissues are in general differentiated to some extent. During regeneration or asexual reproduction, they undergo dedifferentiation via exocytosis of organelles, activation of the nucleolus, alteration of the cel–cell adhesion mode, etc.
This paper has also shown that coelomic cells usually take part in histogenesis in collaboration with undifferentiated epithelial cells. They are squamous, fibroblast-like cells in Perophora and Polyandrocarpa. In Polycitor, coelomic cells are associated with the epicardium. At strobilation, cell aggregates near the cut surface increase conspicuously in number. They may be the precursor of formative tissues. A detailed report will be published elsewhere. Recently, we reviewed coelomic multipotent cells, hemoblasts, in colonial tunicates (Kawamura et al. submitted). Hemoblasts of somatic lineage give rise to epithelial cells, cardiac muscle cells, body muscle cells, and blood cells. Germline hemoblasts, on the other hand, differentiate into germ cells and their accessory cells. Epithelial precursor cells in Phlebobranchiata and Stolidobranchiata dealt with in this paper differ from those hemoblasts in several points: Hemoblasts are spherical in shape. The nucleus is large, the nucleolus is prominent, and the cytoplasm has fewer organelles. In contrast, epithelial precursor cells take squamous shape, the nucleus is ellipsoidal in general, and the cytoplasm is abundant in organelles derived from pinocytized granules, phagocytized vacuoles, etc.
In conclusion, regenerative power of colonial tunicates is based on dedifferentiation of epithelial formative tissue, a reservoir of epithelial precursor cells, and hemoblasts. The coordinated suppression and induction of dedifferentiation enables tissue remodeling in colonial tunicates.
We thank Drs Hiroshi Watanabe, Mitsuaki Nakauchi, and the late Hideo Mukai who established colonial tunicate biology in Japan. This work was supported in part by JSPS (No. 19570208).