Telomerase maintained in self-renewing tissues during serial regeneration of the urochordate Botryllus schlosseri

Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6-15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2-100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Stem cells in asexual reproduction of the colonial ascidian <Emphasis Type="Italic">Botryllus tubaratus</Emphasis> (Tunicata: Ascidiacea)

Histological, cytochemical and ultrastructure research on the budding of the colonial ascidian Botryllus tuberatus aimed at searching for stem cells was performed. A dense mass of undifferentiated cells and the connection of the outer epidermal and inner atrial epithelia were revealed for the first time in the early buds of B. tuberatus. Undifferentiated cells revealed in the early buds and vascular system of the colony had morphological features of the stem and primary germ cells of metazoans. Intensive expression of alkaline phosphatase, the cytochemical marker of embryonal stem and primary germ cells of vertebrate animals, was revealed in developing buds and in some cells of the hematocyte population. Based on the literature and the author’s data it is hypothesized that the self-renewing pool of stem cells of the colonial ascidian B. tuberatus is the cellular basis of its reproductive strategy, including both sexual and asexual reproduction.     

Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates

We investigated the mechanism by which germline cells are recruited in every asexual reproductive cycle of the budding tunicate Polyandrocarpa misakiensis using a vasa homolog (PmVas) as the germline-specific probe. A presumptive gonad of Polyandrocarpa arose as a loose cell aggregate in the ventral hemocoel of a 1-week-old developing zooid. It developed into a compact clump of cells and then separated into two lobes, each differentiating into the ovary and the testis. The ovarian tube that was formed at the bottom of the ovary embedded the oogonia and juvenile oocytes, forming the germinal epithelium. PmVas was expressed strongly by loose cell aggregates, compact clumps, and peripheral germ cells in the testis and germinal epithelium. No signals were detected in growing buds and less than 1-week-old zooids, indicating that germ cells arise de novo in developing zooids of P. misakiensis. Cells of the loose cell aggregates were 5–6 μm in diameter. They looked like undifferentiated hemoblasts in the hemocoel. To examine the involvement of PmVas in the germline recruitment at postembryonic stages, both growing buds and 1-week-old developing zooids were soaked with double-stranded PmVas RNA. The growing buds developed into fertile zooids expressing PmVas, whereas the 1-week-old zooids developed into sterile zooids that did not express PmVas. In controls (1-week-old zooids) soaked with double-stranded lacZ RNA, the gonad developed normally. These results strongly suggest that in P. misakiensis, PmVas plays a decisive role in switching from coelomic stem cells to germ cells.     

vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala)

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.     

Programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian Botryllus schlosseri

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.     

中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

A new mode of colony multiplication by modified budding in the ascidian Clavelina gemmae n. sp. (Clavelinidae)

Abstract. A new mechanism of asexual multiplication of colonies is described in a colonial ascidian of the genus Clavelina (Aplousobranchiata). The mechanism consisted of the production of star-shaped buds that originate from a basal vessel that bends anteriorly and extends along the dorsal region of the zooids. Once they are well developed, the buds detached easily and were dispersed by water movement. Analysis of the fine structure of the buds revealed that they were a modification of the stolonic budding common in this genus. Time-lapse video recordings showed that released buds required several days to develop, allowing for a potentially significant dispersal range. The buds underwent organogenesis during which the central part gave rise to a new blastozooid with a defined polarity; the arms of the star gave rise to stolons. A new species is defined based on the presence of this type of budding and on other morphological features. The significance of these findings, which adds to the known mechanisms of asexual reproduction in ascidians, is discussed in relation to the biology and distribution of the species.     

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.     

Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration

Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).     

Pattern of cell proliferation during budding in the colonial ascidian Diplosoma listerianum

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells.     

Germ lineage properties in the urochordate Botryllus schlosseri – From markers to temporal niches

The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or β-estradiol administration affirmed the BS-Vasa⁺BS-DDX1⁺BS-cadherin⁺γ-H2AX⁺phospho-Smad1/5/8⁺ population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived ‘germ line niches’ that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.     

Haemocytes and blastogenetic cycle in the colonial ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>: a matter of life and death

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change. This research was supported by the Italian M.I.U.R. (PRIN 2006)     

From Skin Cells to Ovarian Follicles?

Generating oocytes from cells derived from skin in vitro may provide a valuable modelfor identifying factors involved in germ cell formation and oocyte differentiation. In addition, the“oocytes” produced could potentially be useful for therapeutic cloning, and thus offer newpossibilities for tissue therapy. We recently reported the differentiation of cells derived fromporcine fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cellsexpressed germ cell markers and formed aggregate like oocyte-cumulus complexes that secretedovarian steroid hormones and responded to gonadotropin stimulation. Some of these aggregatesextruded large oocyte-like cells that expressed markers appropriate to oocytes. We now showfurther evidence of germ cell marker expression during differentiation. We have also comparedthe oocyte-like cells with natural oocytes for their expression levels of Oct4, growthdifferentiation factor-9b (GDF9b), the deleted in azoospermia -like (DAZL) gene, vasa, zonapellucida (ZP), and the meiosis marker synaptonemal complex protein 3 (SCP3), and haverevealed interesting similarities and differences.     

Two isoforms of vasa homologs in a teleost fish: their differential expression during germ cell differentiation.

Two isoforms of vasa mRNA and protein are present in a teleost fish, tilapia. One (vas-s) lacks a part of the N-terminal region found in the other isoform (vas). Both isoforms are expressed in oocytes through the embryonic stage when primordial germ cells (PGCs) localize in the lateral plate mesoderm. After PGC localization in the gonadal anlagen, vas-s expression increased and vas expression became undetectable. Expression of both isoforms was observed again after morphological gonadal sex differentiation, irrespective of genotypic sex. In ovary, compared with vas expression vas-s expression predominated throughout oogenesis. In testis, vas expression was predominant compared with vas-s during spermatogenesis. These results indicate that relative expression of two vasa isoforms is dependent upon germ cell differentiation and sex.     

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea)

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.