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.     

Vasa expression in a colonial ascidian, Botrylloides violaceus

Evolution of solitary or colonial life histories in tunicates is accompanied by dramatic developmental changes that affect morphology and reproduction. We compared vasa expression in a solitary ascidian and a closely related colonial ascidian, in an effort to uncover developmental mechanisms important during the evolution of these contrasting life histories, including the ability to reproduce by budding. In this study, we explored the origin of germ cells in new buds developing by asexual reproduction in a colonial ascidian, Botrylloides violaceus and compared it to the source of germ cells in a solitary ascidian Boltenia villosa. We studied expression by in situ hybridization of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans. In B. villosa, bv-vasa mRNA was expressed in putative germ cells and oocytes of adult gonads, and was sequestered into a posterior lineage during embryogenesis. In mature colonies of the ascidian B. violaceus, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and in some circulating cells in the zooids and differentiating buds. We propose that expression of vasa in cells other than gonadal germ cells of zooids in a colonial ascidian may serve as a source of germ-line stem cells in the colony.     

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.     

Morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates

Published and original data indicating evolutionary conservation of the morphofunctional organization of reserve stem cells providing for asexual and sexual reproduction of invertebrates are reviewed. Stem cells were studied in representatives of five animal types: archeocytes in sponge Oscarella malakhovi (Porifera), large interstitial cells in colonial hydroid Obelia longissima (Cnidaria), neoblasts in an asexual race of planarian Girardia tigrina (Platyhelmintes), stem cells in colonial rhizocephalans Peltogasterella gracilis, Polyascus polygenea, and Thylacoplethus isaevae (Arthropoda), and colonial ascidian Botryllus tuberatus (Chordata). Stem cells in animals of such diverse taxa feature the presence of germinal granules, are positive for proliferating cell nuclear antigen, demonstrate alkaline phosphatase activity (a marker of embryonic stem cells and primary germ cells in vertebrates), and rhizocephalan stem cells express the vasa-like gene (such genes are expressed in germline cells of different metazoans). The self-renewing pool of stem cells is the cellular basis of the reproductive strategy including sexual and asexual reproduction.     

Stem cells are units of natural selection in a colonial ascidian

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.     

Asexual propagation and regeneration in colonial ascidians

Regeneration is widely distributed among the metazoans. However, clear differences exist as to the degree of regenerative capacity: some phyla can only replace missing body parts, whereas others can generate entirely new individuals. Ascidians are animals that possess a remarkable regenerative plasticity and exhibit a great diversity of mechanisms for asexual propagation and survival. They are marine invertebrate members of the subphylum Tunicata and represent modern-day descendants of the chordate ancestor; in their tadpole stage they exhibit a chordate body plan that is resorbed during metamorphosis. Solitary species grow into an adult that can reach several centimeters in length, whereas colonial species grow by asexual propagation, creating a colony of genetically identical individuals. In this review, we present an overview of the biology of colonial ascidians as a paradigm for study in stem cell and regenerative biology. Focusing on botryllid ascidians, we introduce the potential roles played by multipotent epithelia and multipotent/pluripotent stem cells as source of asexual propagation and regenerative plasticity in the different budding mechanisms, and consider the putative mechanism of body repatterning in a non-embryonic scenario. We also discuss the involvement of intra-colony homeostatic processes in regulating budding potential, and the functional link between allorecognition, chimerism, and regenerative potential.     

Vascular regeneration and angiogenic-like sprouting mechanism in a compound ascidian is similar to vertebrates

Tunicates are useful models for comparing differing developmental processes such as embryogenesis, asexual reproduction, and regeneration, because they are the closest relatives to vertebrates and are the only chordates to reproduce both sexually and asexually. Among them, the ascidian Botryllus schlosseri displays high regenerative potential of the colonial circulatory system (CCS). The CCS runs in the common tunic, forming an anastomized network of vessels defined by simple epithelia and connected to the open circulatory system of the zooids. During asexual propagation, new vessels form by means of a tubular-sprouting mechanism, resembling that occurring in other metazoans, particularly during vertebrate angiogenesis. We studied the regeneration of experimentally ablated CCS by analyzing the general dynamics of reorganization of vessels and tunic, their ultrastructure, cell proliferation, and the immunohistology of regenerating structures using antibodies against vertebrate angiogenic factors-vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), and receptors: VEGFR-1, VEGFR-2, and EGFR. Results show that the regenerative process of CCS occurs by a sprouting mechanism, with participation of angiogenic factors. They also show correspondence between the CCS sprouting of B. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa.     

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.     

Looking at both sides of the invasion: patterns of colonization in the violet tunicate Botrylloides violaceus

Understanding the ecological and evolutionary forces that shape the genetic structure of invasive populations and facilitate their expansion across a large spectrum of environments is critical for the prediction of spread and management of ongoing invasions. Here, we study the dynamics of postestablishment colonization in the colonial ascidian Botrylloides violaceus, a notorious marine invader. After its initial introduction from the Northwest Pacific, B. violaceus spread rapidly along the Pacific and Atlantic coasts of North America, impacting both aquaculture facilities and natural ecosystems. We compare genetic diversity and patterns of gene flow among 25 populations (N=679) from the West and East coasts, and evaluate the contribution of sexual vs. asexual reproduction to this species' invasion success using data from the mitochondrial cytochrome c oxidase subunit I (COI) gene and 13 nuclear polymorphic microsatellite loci. Our results reveal contrasting patterns of spread in the coastal waters of North America. While the West coast was colonized by noncontiguous (long-distance) dispersal, the East coast invasion appears to have occurred through contiguous (stepping-stone) spread. Molecular data further indicate that although dispersal in colonial ascidians is predominantly achieved through sexually produced propagules, aquaculture practices such as high-pressure washing can facilitate fragmentation and potentially exacerbate infestations and spread via asexual propagules. The results presented here suggest that caution should be used against the general assumption that all invasions, even within a single species, exhibit similar patterns of colonization, as highly contrasting dynamics may transpire in different invaded ranges.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.     

PERSPECTIVE: THE EVOLUTIONARY BIOLOGY OF AGING,SEXUAL REPRODUCTION,AND DNA REPAIR

Three recent books on the evolutionary biology of aging and sexual reproduction are reviewed, with particular attention focused on the provocative suggestion by Bernstein and Bernstein (1991) that senescence and genetic recombination are related epiphenomena stemming from the universal challenge to life posed by DNA damages and the need for damage repair. Embellishments to these theories on aging and sex are presented that consider two relevant topics neglected or underemphasized in the previous treatments. The first concerns discussion of cytoplasmic genomes (such as mtDNA), which are transmitted asexually and therefore do not abide by the recombinational rules of nuclear genomes; the second considers the varying degrees of cellular and molecular autonomy which distinguish unicellular from multicellular organisms, germ cells from somatic cells, and sexual from asexual genomes. Building on the Bernsteins' suggestions, two routes to immortality for cell lineages appear to be available to life: an asexual strategy (exemplified by some bacteria), whereby cell proliferation outpaces the accumulation of DNA damages, thereby circumventing Muller's ratchet; and a sexual strategy (exemplified by germlines in multicellular organisms), whereby recombinational repair of DNA damages in conjunction with cell proliferation and gametic selection counter the accumulation of nuclear DNA damages. If true, then elements of both the recombinational strategy (nuclear DNA) and replacement strategy (cytoplasmic DNA) may operate simultaneously in the germ-cell lineages of higher organisms, producing at least some gametes that are purged of the DNA damages accumulated during the lifetime of the somatic parent. For multicellular organisms, production of functionally autonomous and genetically screened gametic cells is a necessary and sufficient condition for the continuance of life.     

文冠果种群繁殖方式及其在种群更新中的作用

以黄土高原丘陵区文冠果天然种群为研究对象,通过样地调查,分析了不同生境文冠果种群的有性和无性繁殖方式及其在种群更新中的地位.结果表明：不同坡向文冠果种群均能进行有性繁殖和无性繁殖,在阳坡、半阴坡生境,文冠果以有性繁殖为主,实生苗数量和占据空间的能力均大于萌生苗,有性繁殖在这2个生境中对种群的更新贡献较大;在半阳坡生境,文冠果以无性繁殖为主,萌生苗数量和占据空间的能力均大于实生苗,无性繁殖对种群更新的贡献较大.在相同生境条件下,文冠果幼苗生长状况均表现为萌生苗＞实生苗.同一起源的文冠果幼苗生长状况均表现为阳坡＞半阳坡＞半阴坡.2种繁殖的瓶颈期在实生苗的种子-幼苗阶段和萌生苗的幼树-成株阶段.黄土丘陵区文冠果种群在新生境定居下来的过程中,首先以实生苗入侵,当水分、光照、养分条件较好时,文冠果又以无性繁殖方式迅速占领空间,繁衍后代.     

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.     

Adaptive molecular evolution of a defence gene in sexual but not functionally asexual evening primroses

Theory predicts that sexual reproduction provides evolutionary advantages over asexual reproduction by reducing mutational load and increasing adaptive potential. Here, we test the latter prediction in the context of plant defences against pathogens because pathogens frequently reduce plant fitness and drive the evolution of plant defences. Specifically, we ask whether sexual evening primrose plant lineages (Onagraceae) have faster rates of adaptive molecular evolution and altered gene expression of a class I chitinase, a gene implicated in defence against pathogens, than functionally asexual evening primrose lineages. We found that the ratio of amino acid to silent substitutions (K(a) /K(s) = 0.19 vs. 0.11 for sexual and asexual lineages, respectively), the number of sites identified to be under positive selection (four vs. zero for sexual and asexual lineages, respectively) and the expression of chitinase were all higher in sexual than in asexual lineages. Our results are congruent with the conclusion that a loss of sexual recombination and segregation in the Onagraceae negatively affects adaptive structural and potentially regulatory evolution of a plant defence protein.     

5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.     

Molecular Cytological Characterization of Somatic Variation in Rice Aneuploids

Aneuploids cannot be stably preserved by sexual propagation, though they can maintain their genetic identity by asexual propagation. However, it is possible for somaclonal variation to occur during asexual propagation. Surveys taken from 2005 to 2007 showed that there were 26 lines with morphological variations from a total of 114 asexually propagated rice lines. Among these, 12 lines were detected that had either increased or decreased chromosome numbers, while the other 14 lines did not show any changes in chromosome number. Lines with increased chromosome numbers include the following four classes: (1) an extra chromosome was retained, and another normal individual chromosome was increased; (2) an extra chromosome was increased, and the normal chromosome numbers did not change; (3) an extra chromosome was lost, and another chromosome was increased; and (4) the genome was doubled. We studied 543 somatic cells from the 12 lines during mitosis and observed abnormal mitotic behaviors such as chromosome lagging, uneven distribution, and chromosome breakage at anaphase. These results show that abnormal mitotic behavior led to the somaclonal variation in chromosome number. However, cytological variation can only explain a minority of the asexual separated lines.     

Internal brooding of clonal propagules by a sea anemone, Anthopleura sp.

Abstract. We investigated the seasonal prevalence of reproductive activities and of the development of brooded propagules in an intertidal sea anemone, Anthopleura sp., on the rocky shore of Mutsu Bay, in northern Japan. A monthly examination of anemones, by dissection and histological techniques, revealed no sign of gonad development, but did reveal that they produce and internally brood propagules throughout the year. Release of propagules was observed in the field. This anemone population appears to be entirely asexual and agametic, and may persist solely through clonal propagation.     

Seasonal changes in the sexualization of the planarian Dugesia ryukyuensis

Asexual individuals in a fissiparous clone of the planarian Dugesia ryukyuensis develop hermaphroditic sexual organs and eventually undergo sexual reproduction instead of asexual reproduction if they are fed with the adults of Bdellocephala brunnea, an oviparous planaria. The experimental sexualization means that the adults of B. brunnea contain a putative sexualizing substance(s), which is the first candidate for the chemical(s) responsible for switching from asexual to sexual reproduction in metazoans. In the present study, the feeding experiment over two consecutive years revealed that the experimental sexualization has seasonal changes. In summer, the asexual individuals were not fully sexualized, though they developed a pair of ovaries. The developing ovaries degenerate if the feeding is stopped. On the contrary, in winter, they developed all the sexual organs. The sexual organs keep developing even if the feeding is stopped after a certain critical point named the point-of-no-return. It was demonstrated that the extreme difference of the sexualization was attributed to the seasonal change of the quality and/or quantity of the sexualizing substance contained in B. brunnea, as well as the minor change of the susceptibility to the sexualizing substance in the asexual individuals. On the other hand, the histological research of B. brunnea revealed that the degree of the maturation of the sexual organs varied extremely through a year. Taking these results into account, we suggest that the production of the sexualizing substance has no direct relation to any particular mature sexual organs.     

Stem cells in reproductive strategy of asexually reproducing invertebrates

Original and literature data supporting the evolutionary conservation of the morphofunctional organization of totipotent cells of germ and stem lineages in metazoan animals are reviewed. We studied stem cells of the colonial rhizocephalans, Peltogasterella gracilis, Polyascus polygenea and Thylacoplethus isaevae, the turbellarian Dugesia tigrina, the colonial hydroid Obelia longissima, and cultured embryonic stem cells of mouse. The typical germinal granules of germ plasm, selective expression of the activity of alkaline phosphatase and of proliferating cell nuclear antigen (PCNA), which are known as markers of stem and primary germ cells of vertebrates, and the specific expression of the protein product of the vasa gene in cells of rhizocephalans, which is a marker of cells of germ and stem lineages of various metazoans, specified the stem cells of invertebrates of such different taxa. The self-renewing pool of totipotent stem cells is the cellular basis of the reproductive strategy, including sexual and asexual reproduction; such cells share morphofunctional features of embryonic stem and germline cells of Metazoa.