Colonial tunicates have hemoblasts, which are undifferentiated coelomic cells that play a key role in tissue renewal during reproduction and regeneration. Some hemoblasts differentiate into somatic lineage cells such as endodermal multipotent epithelial, cardiac and body-wall muscle, and blood cells. There is no well established evidence that somatic hemoblasts are stem cells. Rather, like tissue-restricted progenitor cells, some peripheral hemoblasts give rise to terminally differentiated cells, while other hemoblasts differentiate into germ cells and accessory cells. Unlike somatic lineage cells, germ cells and their precursors express vasa homologues in common. In some colonial tunicates, vasa is indispensable for germ cell development. All vasa-positive hemoblasts appear to differentiate into germ cells, suggesting that most of them are tissue-restricted progenitor cells. When a colony is naturally or experimentally depleted of vasa-expressing cells, vasa and vasa-expressing germ cells can reappear in the colony. We speculate that, in addition to tissue-restricted progenitor cells, highly potent stem cells which regulate the activities of blastogenesis and gametogenesis and eventually cause soma-germ conflict in colonial tunicates may exist in colonial tunicates.
Tunicates (subphylum Urochordata) are sessile or pelagic marine animals. They are characterized by a notochord during their larval stages and by gill slits, an endostyle (thyroid gland homologue), and tunic when adult (Berrill 1950). It is well known that tunicate embryos carry out mosaic development (Satoh 1994), but after metamorphosis some species show remarkable plasticity in their morphogenesis (Berrill 1950). In particular, colonial tunicates exhibit a regeneration potential as great as that of hydrozoans, planarians, and annelids (Berrill 1950; Kawamura et al. 2008a). This regeneration potential is utilized for asexual reproduction, by which whole bodies are periodically reconstructed from tiny body pieces during the life span, resulting in an increase in zooid number.
Many questions concerning hemoblasts await solution. For example, it is uncertain whether each hemoblast is developmentally unipotent or multipotent. We only know that, in vivo, germ line hemoblasts appear not to be interchangeable with somatic hemoblasts (Laird et al. 2005). In invertebrates, multipotent stem cells are believed to exist in the coelomic body cavity (hemocoel). Interstitial cells in hydras, and neoblasts in planarians and annelids, give rise not only to somatic differentiated cells but also to germ line cells (Shibata et al. 1999; Mochizuki et al. 2000, 2001; Rebscher et al. 2007). In vertebrates, developmentally restricted stem cells regulate tissue homeostasis in adult organisms (Lajtha 1979; Hall & Watt 1989). However, over the last decade increasing evidence has shown that, in mammals, there is another type of adult stem cell with loosely restricted or non-restricted developmental potency (Jiang et al. 2002).
Stem cells are generally regarded as having an unlimited capacity for self-renewal by unequal cell division (Hall & Watt 1989). The downstream cells, referred to as committed progenitors, amplify rapidly giving rise to terminally differentiated cells (Lajtha 1979). According to this definition, it is not so easy to determine whether hemoblasts in tunicates are stem cells or progenitor cells. Actually, hemoblasts and hemoblast-like cells are found in many developing tissues in colonial tunicates. In some cases they proliferate so rapidly that they differentiate into different tissues simultaneously (Kawamura et al. 2008b).
This paper reviews the roles played by hemoblasts in the reproduction and regeneration of sessile colonial tunicates. In this context, we discuss the manner in which the multipotent epithelium, body muscle, and gonads are formed. Special attention is paid to the lineage relationship between somatic precursor cells and germ line precursor cells. In general, germ cells are strictly segregated from somatic cells during early or late embryogenesis (Strome & Lehmann 2007). In the lower metazoan, on the other hand, germ cells can regenerate from non-germ line cells even in adulthood (Shibata et al. 1999; Mochizuki et al. 2000, 2001; Torras et al. 2004; Extavour et al. 2005). We would like to reconsider the soma versus germ flexibility of hemoblasts.
Hemoblasts are undifferentiated cells in the hemocoel. They are approximately 5 μm in diameter. Their nucleus is relatively large with a prominent nucleolus. In the cytoplasm, polysomes and a few mitochondria are usually observed (Fig. 2A). At most, they comprise 1–2% of the coelomic cell population (Wright 1981). The hemoblasts that aggregate in the hemocoel are shown in Fig. 2B. These aggregations have been referred to as hematopoietic nodules (Ermak 1976; Kawamura & Sugino 1999) or stem cell niches (Voskoboynik et al. 2008).
Botryllus colonies provide the most remarkable and reliable evidence of hemoblast involvement in tissue renewal. In Botryllus primigenus, coelomic cells aggregate in the test vessel and become a hollow vesicle after a solid mass stage (Fig. 2C). The vesicle then commences morphogenesis (Oka & Watanabe 1957; Kawamura & Sugino 1999). Such cell aggregation occurs periodically at the basal region of the vascular ampullae during the colonial phase B (Fig. 2D–F). Each cell is morphologically undifferentiated and identifiable as a hemoblast (Fig. 2G,I). As shown later, they are distinct from hemoblasts in the gonadal space because of the presence or absence of the vasa product (Sunanaga et al. 2006). The hemoblasts in the vascular ampullae undergo epithelial transformation without unequal cell division to form a multipotent vesicle (Fig. 2H,J). The vesicle then grows and differentiates into the pharynx, digestive tract, atrial (peribranchial) epithelium, and neural complex, until finally it develops into a functional zooid. This mode of tissue renewal is termed vascular budding (Oka & Watanabe 1957). In Botryllus and Botrylloides, vascular budding can also be induced in a vascularized colony by extirpating all the zooids and buds from the colony (Oka & Watanabe 1959; Sabbadin et al. 1975; Rinkevich et al. 1995, 2007).
It has also been suggested that hemoblasts play a role in tissue renewal in Phlebobranchiata. In Perophora, colonies irradiated with X-rays (5000 rads) neither develop stolons nor form buds (Freeman 1964). However, they resume stolonial budding when injected with coelomic cells from an unirradiated colony. It is unclear whether the injected hemoblasts are directly involved in blastogenesis; however, small lymphocytes (hemoblasts) are reported to be the most efficient cells for stolonial growth and bud formation (Freeman 1964). A change from epithelial cells to mesenchymal cells may also be possible in some colonial tunicates. When zooids of Perophora viridis are isolated from a colony and allowed to regress, the resultant cell mass gives rise to new zooids without inducing cell proliferation (Barth & Barth 1966). This suggests that in Perophora, adult tissues, when they disintegrate, might change into formative cells.
In Botryllus schlosseri (B. schlosseri), cells of the cardiac muscle and pericardium differentiate directly from hemoblasts (Nunzi et al. 1979). In Symplegma reptans, a relative of Botryllus, hemoblasts also develop into body muscle cells around the siphon (Fig. 3) (Sugino et al. 2007). They first proliferate actively, exhibiting an undifferentiated configuration (Fig. 3A,B). When associated with the epidermal siphon, the cells become ultrastructurally unique due to the development of an indented nuclear contour (Fig. 3C,D). Interestingly, a similar nuclear configuration is observable in the juvenile oocyte in B. schlosseri (Manni et al. 1994). In any case, thick and thin filaments appear in the cytoplasm facing the epidermal basal lamina (Fig. 3D,E). Myofilaments gradually occupy a large part of the cytoplasm (Fig. 3F), and eventually, sphincter muscle fibers surround the oral and atrial siphons (Fig. 3G) (Sugino et al. 2007). In Botryllus primigenus (B. primigenus), the undifferentiated cells around the siphon are highly labeled with BrdU (Fig. 3A) (labeling index ≥40%), and they all appear to differentiate into body muscle cells without unequal division (Kawamura et al. 2008b). Therefore, the undifferentiated cells with an indented nuclear contour should be regarded as muscle progenitor cells rather than muscle stem cells.
It is difficult to characterize somatic hemoblasts by using histochemistry or molecular biology techniques, and the available literature is very limited. Tunicate calcium-dependent lectin of 14 kDa (TC14) is a calcium-dependent galactose-binding polypeptide (Suzuki et al. 1990). Thus far, four related cDNA and polypeptides that belong to the TC14 family have been found in Polyandrocarpa misakiensis (Shimada et al. 1995). TC14-1 strongly binds to undifferentiated cells which are 4–5 μm in diameter (Kawamura et al. 1991). As shown later, vasa-positive cells are localized only in the ventral hemocoel. It is, therefore, reasonable to assume that the TC14-1-binding cells found scattered around the rest of the body should be non-germ line hemoblasts. TC14-1 is a component of the extracellular matrix (ECM). An anti-TC14-1 polyclonal antibody can block the spread of hemoblasts through the ECM and their movement toward the multipotent epithelium (Kawamura et al. 1991). TC14-3 exhibits cytostatic activity that blocks the proliferation of multipotent cells in vitro (Matsumoto et al. 2001). In the solitary tunicate Pyura stolonifera, lymphocyte-like cells possess receptors for concanavalin A and other lectins (Warr et al. 1977).
Another molecular marker for hemoblasts has been obtained from Botryllus. Hemoblasts express the CD34 antigen, which is an evolutionally conserved molecular marker for undifferentiated hematopoietic stem cells (Ballarin & Cima 2005). In humans, 1–4% of bone marrow cells express the CD34 antigen. This subpopulation of cells includes almost all stem cells (Hall & Watt 1989). In B. primigenus, hemoblasts in the hemocoel express a nanos homologue (BpNos) (Sunanaga et al. 2008).
Recently, a retinoic acid receptor (RAR) homologue has been found to be involved in the process of vascular budding in Botrylloides leachi (Rinkevich et al. 2007). It appears that it is not expressed by free blood cells (including hemoblasts) but by aggregating cells and the differentiating epithelia in vascular buds. Botrylloides RAR may be a marker of the epithelial differentiation of hemoblasts.
Germ line hemoblasts
In some colonial tunicates, hemoblasts contribute to the renewal of the gonads (Mukai & Watanabe 1976a). In B. primigenus, the ovary and testis are located in the gonadal space on each side of the body (Fig. 4A). At the earliest stage of gonad formation, hemoblasts form loose aggregates in the gonadal space (Fig. 4B). They are 4–5 μm in diameter with a high nuclear/cytoplasmic ratio, and are very similar to somatic hemoblasts. In B. schlosseri, however, a small number of hemoblasts injected into a host colony differentiate into either somatic cells or germ cells, suggesting that germ line hemoblasts constitute an independent cell population that is discrete from somatic hemoblasts (Laird et al. 2005).
Loose aggregates of germ line hemoblasts begin to proliferate slowly with a BrdU-labeling index of 19% (Kawamura et al. 2008b). They form compact clumps that proliferate very rapidly (labeling index, LI = 50%). Subsequently, the compact clumps differentiate into male germ cells and testicular epithelium (Mukai & Watanabe 1976a; Sunanaga et al. 2006). In contrast, female germ cells and ovarian follicles develop from loose cell masses. Oocyte development is accompanied by the formation of one or two primary follicle cells (Fig. 4C). The primary follicle cell possesses a large nucleus which lacks a prominent nucleolus (Manni et al. 1993; Sunanaga et al. 2006). Electron-dense materials and mitochondrial assembly first appear in the cytoplasm near the nuclear envelope of the oogonium and young oocyte (Fig. 4D). Germ cells are transmitted from one asexual generation to another until they attain maturity (Izzard 1968; Mukai & Watanabe 1976a; Sabbadin & Zaniolo 1979).
Based on investigations conducted thus far on both solitary and colonial tunicates, embryonic and postembryonic germ line cells strongly express a vasa homologue (Fujimura & Takamura 2000; Takamura et al. 2002; Shirae-Kurabayashi et al. 2006; Sunanaga et al. 2006, 2007; Brown & Swalla 2007; Rosner et al. 2009; Brown et al. 2009). The vasa gene encodes an ATP-dependent RNA helicase belonging to the DEAD box protein family, and it is one of the most reliable markers for germ line cells (Raz 2000). In B. primigenus, the vasa homologue (BpVas) is detected in the gonadal space (Fig. 4E). Loose cell masses, compact clumps, spermatocytes, and young oocytes all strongly express BpVas (Fig. 4F,G) (Sunanaga et al. 2006). In Botrylloides violaceus (B. violaceus), vasa signals are detected not only in germ cells, but also in blood cells and the tunic (Brown & Swalla 2007). As will be discussed later, we recently found that in B. primigenus vasa-positive mobile cells are located in the blood. In Polyandrocarpa misakiensis (P. misakiensis), vasa homologue (PmVas) expression is restricted to germ cells (Sunanaga et al. 2007). The most intense signal is detected in oogonia and young oocytes embedded in the germinal epithelium. Double-stranded PmVas RNA blocks the de novo expression of PmVas as well as gonadal development in P. misakiensis (Sunanaga et al. 2007). Taken together, these results suggest that tunicate vasa homologues may play a decisive role in the formation and/or development of germ cells.
The BpVas transcript completely disappears when the colonies are vascularized (Sunanaga et al. 2006). A few weeks later, when the colonies regenerate by vascular budding, BpVas-positive cells reappear in the gonadal space. In P. misakiensis, no PmVas signals can be detected throughout the course of bud formation. Rather, they first appear in loose aggregates of hemoblasts in one-week-old zooids (Sunanaga et al. 2007). These results suggest that in both B. primigenus and P. misakiensis, germ line hemoblasts are recruited de novo from vasa-negative cells at every zooidal regeneration or asexual reproduction (Sunanaga et al. 2006, 2007).
As mentioned, vasa-positive mobile cells of B. violaceus are scattered in the hemocoel and test vessel. It has been proposed that these cells serve as a source of germ line stem cells (Brown & Swalla 2007). Recently, such vasa-positive coelomic cells have also been found in B. primigenus (Kawamura & Sunanaga, submitted). However, these cells differentiate exclusively into oocytes, a finding which suggests that vasa-positive mobile cells are not germ line stem cells, but female germ line progenitor cells.
In order to understand possible pluripotent stem cells in the hemocoel, it is important to identify gene products shared by both coelomic and germ cells. PL10 is a subfamily of the DEAD box-type RNA helicases. In B. schlosseri, BS-PL10 is expressed by multipotent bud tissues and by proliferating premature germ line cells (Rosner et al. 2006). A specific siRNA depletes the BS-PL10 protein and retards bud development. Therefore, it has been suggested that BS-PL10 is involved in maintaining the undifferentiated and proliferative properties of somatic and germ line stem cells (Rosner et al. 2006). In B. primigenus, most germ line precursor cells and a small number of coelomic cells express BpNos mRNA and protein (Fig. 4H) (Sunanaga et al. 2008). BpMyc also shows a similar expression pattern (Kawamura et al. 2008b). Although they are not specific for hemoblasts, both BpNos and BpMyc may be key molecules which characterize undifferentiated cells and tissues.
Conclusion and perspective
In this paper, we have described two types of undifferentiated free cells in colonial tunicates; somatic hemoblasts and germ line hemoblasts. The somatic hemoblasts have the capacity to differentiate into endodermal multipotent epithelial, cardiac and body-wall muscle, and blood cells. Currently, it is uncertain whether a multipotent group of hemoblasts gives rise to these differentiated cell types, or whether unipotent groups of hemoblasts bring about these respective cell types (Fig. 5, green box). In contrast with somatic hemoblasts, germ line hemoblasts appear to be multipotent. In B. primigenus, they all express first a vasa homologue, and then differentiate into vasa-positive germ cells and vasa-negative accessory cells such as primary follicles and testicular epithelium (Fig. 5, purple box) (Sunanaga et al. 2006).
As shown in Fig. 2, when vascular budding occurs all the aggregating somatic hemoblasts simultaneously develop into epithelial cells without self-renewing. Body muscle cells arise in a similar manner (Fig. 3). In the gonads, there is no evidence that germ line hemoblasts undergo unequal division (Fig. 4). These features are characteristic of tissue progenitor cells rather than stem cells (Fig. 5, green and purple boxes).
In B. primigenus and P. misakiensis, germ line hemoblasts can regenerate from vasa-negative cell populations (Sunanaga et al. 2006, 2007). Recently, we found that the founder cell population expresses a Piwi homologue (Kawamura & Sunanaga, submitted). It is possible that a vasa−/nanos+/c-Myc+/Piwi+ cell population in the hemocoel may serve as the germ line stem cell (Fig. 5 blue box).
We also believe that totipotent stem cells might exist in the hemocoel of colonial tunicates (Fig. 5, red box). This is because in B. primigenus the activities of blastogenesis, such as vascular budding, usually conflict with that of sexual reproduction (Mukai & Watanabe 1976b; our unpublished observations). It is, therefore, reasonable to assume that soma and germ in the colony have scrambled for the offspring of totipotent stem cells. Of course, we need to remember that in B. schlosseri, cell transplantation experiments have indicated that somatic and germ line coelomic cells constitute independent subpopulations (Laird et al. 2005). The hypothetical totipotent stem cells would not exist as a mobile component in the blood, but might be reserved as a fixed component as shown in Fig. 2B (Fig. 5, red box), which would make it practically impossible to harvest them. In order to understand further the conflict and collaboration between soma and germ, we need to elucidate in detail the lineage relationship among hypothetical stem cells, somatic progenitor cells, and germ line cells.
We thank Drs. Hiroshi Watanabe, Mitsuaki Nakauchi, and Hideo Mukai who established colonial tunicate biology in Japan. This work was supported in part by JSPS (No. 19570208, 21570227, 21116507).