Asexual Reproductive Cells Research Articles

Volvox as a Model for Studying Cell Death and Senescence

The spherical green alga Volvox consists of several hundred or thousand of somatic cells that undergo terminal differentiation, senescence and death, and a small number of gonidia (asexual reproductive cells) that give rise to the next generation. In the first part of this paper, the ontogenetic diversity of the genus Volvox is briefly considered, as well as the mechanisms of differentiation into the two types of cells mentioned above, which have been thoroughly studied during recent years in Volvox carteri. Then, a detailed critical analysis of the literature and some of my own data on senescence and cell death (mainly in V. carteri and, to a lesser extent, in V. aureus) was carried out, and it was noted that this aspect of Volvox developmental biology has not been sufficiently studied. Some perspectives of further research of the processes of cell death and senescence in representatives of the genus Volvox in a comparative aspect are indicated.

Quantification of laminarialean zoospores in seawater by real‐time PCR

SUMMARYLaminarialean plants undergo heteromorphic alternation of generations between the macroscopic diploid sporophyte and the microscopic haploid gametophyte. The generations change through the formation and release of asexual or sexual reproductive cells. It is difficult to monitor the release and diffusion of zoospores into the environment. Furthermore, even if zoospores can be detected, species identification remains difficult. This study attempted to develop a sensitive and fast identification method for laminarialean zoospores using quantitative PCR. In addition, we aimed to estimate the density of zoospores in natural seawater. Specific primers for Saccharina japonica and Undaria pinnatifida were designed and used to estimate the quantity of zoospores in seawater. DNA samples (environmental DNA) were collected from seawater once or twice each month for 2 years at the same area, and seasonal variations in the release of zoospores was monitored. The estimated maturation period based on the number of released zoospores in this study was comparable with those of the previously reported maturation periods of S. japonica and U. pinnatifida sporophytes. This supports the validity of our method in estimating zoospore release from laminarialean plants. The method will be a useful tool for ecological studies on these commercially relevant species.

Major ontogenetic transitions during Volvox (Chlorophyta) evolution: when and where might they have occurred?

This paper represents an attempt to unify data from various lines of Volvox research: developmental biology, biogeography, and evolution. Several species (such as Volvox carteri and Volvox spermatosphaera) are characterized by rapid divisions of asexual reproductive cells, which may proceed in darkness. By contrast, several other species (such as Volvox aureus, Volvox globator, and Volvox tertius) exhibit slow and light/dependent divisions. The transition from the former pattern of asexual life cycle to the latter one has occurred in three lineages of the genus Volvox. Since V. aureus (unlike V. carteri) is able to complete the life cycle at a short photoperiod (8h light/16h dark regime), it is reasonable to suggest that the abovementioned evolutionary transitions might have occurred as adaptations to short winter days in high latitudes under warm climate conditions in the deep past. In the case of the lineage leading to V. tertius + Volvox dissipatrix, the crucial reorganizations of asexual life cycle might have occurred between about 45 and 60 million years ago in relatively high latitudes of Southern Hemisphere.

Ethical Issues of Embryo Genetic Manipulation

Ethical Issues of Embryo Genetic Manipulation

Ontogenetic diversity of colonies and intercellular cytoplasmic bridges in the algae of the genus Volvox

In all representatives of the genus Volvox, cells of cleaving embryos are connected by cytoplasmic bridges, which play an important role in the process of young colony inversion. However, during subsequent development, the intercellular bridges are retained not in all species of Volvox; the occurrence of the bridges in an adult colony correlates withthe small size of mature gonidia (asexual reproductive cells) and with the presence of cell growth in the intervals between divisions. This complex of ontogenetic features is derived and arises independently in three evolutionary lineages of colonial volvocine algae. A putative role of the syncytial state of adult colonies for the evolution of developmental cycles in Volvox is discussed.

EVOLUTION OF DEVELOPMENTAL PROGRAMS INVOLVOX(CHLOROPHYTA)

The volvocine green algal genus Volvox includes ∼20 species with diverse sizes (in terms of both diameter and cell number), morphologies, and developmental programs. Two suites of characters are shared among distantly related lineages within Volvox. The traits characteristic of all species of Volvox—large (>500) numbers of small somatic cells, much smaller numbers of reproductive cells, and oogamy in sexual reproduction—have three or possibly four separate origins. In addition, some species have evolved a suite of developmental characters that differs from the ancestral developmental program. Most multicellular volvocine algae, including some species of Volvox, share an unusual pattern of cell division known as palintomy or multiple fission. Asexual reproductive cells (gonidia) grow up to many times their initial size and then divide several times in rapid succession, with little or no growth between divisions. Three separate Volvox lineages have evolved a reduced form of palintomy in which reproductive cells are small and grow between cell divisions. In each case, these changes are accompanied by a reduction in the rate of cell division and by a requirement of light for cell division to occur. Thus, two suites of characters—those characteristic of all Volvox species and those related to reduced palintomy—have each evolved convergently or in parallel in lineages that diverged at least 175 million years ago (mya).

Channelrhodopsins ofVolvox carteriAre Photochromic Proteins That Are Specifically Expressed in Somatic Cells under Control of Light, Temperature, and the Sex Inducer

Channelrhodopsins are light-gated ion channels involved in the photoresponses of microalgae. Here, we describe the characterization of two channelrhodopsins, Volvox channelrhodopsin-1 (VChR1) and VChR2, from the multicellular green alga Volvox carteri. Both are encoded by nuclear single copy genes and are highly expressed in the small biflagellated somatic cells but not in the asexual reproductive cells (gonidia). Expression of both VChRs increases after cell cleavage and peaks after completion of embryogenesis, when the biosynthesis of the extracellular matrix begins. Likewise, expression of both transcripts increases after addition of the sex-inducer protein, but VChR2 is induced much more than VChR1. The expression of VChR1 is specifically promoted by extended dark periods, and heat stress reduces predominantly VChR1 expression. Expression of both VChRs increased under low light conditions, whereas cold stress and wounding reduced expression. Both VChRs were spectroscopically studied in their purified recombinant forms. VChR2 is similar to the ChR2 counterpart from Chlamydomonas reinhardtii with respect to its absorption maximum (460 nm) and photocycle dynamics. In contrast, VChR1 absorbs maximally at 540 nm at low pH (D540), shifting to 500 nm at high pH (D500). Flash photolysis experiments showed that after light excitation, the D540 dark state bleaches and at least two photoproducts, P600 and P500, are sequentially populated during the photocycle. We hypothesize that VChR2 is a general photoreceptor that is responsible for the avoidance of blue light and might play a key role in sexual development, whereas VChR1 is the main phototaxis photoreceptor under vegetative conditions, as it is more specifically adapted to environmental conditions and the developmental stages of Volvox.

A Gender-Specific Retinoblastoma-Related Protein inVolvox carteriImplies a Role for the Retinoblastoma Protein Family in Sexual Development

Here, we describe the cloning and characterization of RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development.

Cell proliferation dynamics of somatic and germline tissues during zooidal life span in the colonial tunicateBotryllus primigenus

Botryllus primigenus is a colonial tunicate in which three successive generations develop synchronously. To identify proliferation centers and possible adult stem cells during asexual reproduction, somatic and germline cells were labeled with 5-bromo-2'-deoxyuridine (BrdU). In the youngest generation, multipotent epithelial cells exhibited an average labeling index (LI) of 30% 24 hr after BrdU injection. In the middle generation, the LI of organ rudiments decreased gradually and reached zero by the beginning of the eldest generation. Exceptionally, cells of specialized tissues such as the pharyngeal inner longitudinal vessel and the posterior end of the endostyle continued DNA synthesis and mitosis even in the eldest generation. Proliferating somatic and germline cells of younger generations expressed a Botryllus myc homolog (BpMyc), but adult tissues did not. This result strongly suggests that in B. primigenus undifferentiated progenitor cells are discernible from possible adult stem cells by the presence or absence of BpMyc.

Germ cell specification in Volvox carteri.

Volvox carteri illustrates with diagrammatic clarity Weismann's concept of an immortal germline that produces a mortal soma that will carry it for a time, but then perish. Each V. carteri adult consists of about 16 asexual reproductive cells (gonidia) in the interior of a sphere that consists at its surface of about 2000 biflagellate somatic cells. When mature, each gonidium divides to form a juvenile with this same cellular composition. Half-way through their maturation, juveniles hatch out of the parenteral spheroid, whereupon parental somatic cells undergo programmed death while juvenile gonidia prepare for a new round of reproduction. The first visible step in V. carteri germ-soma differentiation is asymmetric cleavage, which sets apart large gonidial initials from small somatic initials. Experimental analysis indicates that it is a difference in size, not any difference in cytoplasmic quality, that determines whether a cell will become germinal or somatic. Mutational and molecular studies lead to the following model for the genetic control of the germ-soma dichotomy: first, the gls locus acts to cause asymmetric division; then large cells activate a set of lag loci that suppress expression of somatic genes, while small cells activate the regA locus that suppresses gonidial genes.

The influence of UV-B radiation on the reproductive cells of the intertidal macroalga, Enteromorpha intestinalis

The relative sensitivity of the reproductive cells of the seaweed Enteromorpha intestinalis to UV-B was assessed by measuring in vivo chlorophyll fluorescence ( F v variable fluorescence), germination success and growth rates. Zoospores (the asexual reproductive cells) exhibit up to a 6 fold higher sensitivity to UV-B exposure than the mature thalli (measured as chlorophyll fluorescence, F v), and differences in growth rates were also found. Consistent patterns emerged throughout these experiments in variable fluorescence, germination success and growth rates, indicating a greater sensitivity in the sexual reproductive phase of the life cycle compared with the asexual phase. Inhibition of germination success (up to 50%) and growth rates (up to 16.4%) of settled gametes and zoospores after 1-h exposures to elevated levels of UV-B (equivalent to 27 and 31% ozone depletion) showed that damage to the reproductive cells was irreversible. In conclusion, the ecological significance of elevated UV-B exposure in the marine environment may be seriously under-estimated if effects on the early lifestages of algae are not considered.

Two enhancers and one silencer located in the introns of regA control somatic cell differentiation in Volvox carteri.

The regA gene plays a central role in germ-soma differentiation of Volvox carteri by suppressing all reproductive functions in somatic cells. Here we show that the minimal promoter of regA consists of only 42 bp immediately upstream of the transcription start site, and that it contains no discernible regulatory elements. However, introns 3 and 5 are both required for regA expression in somatic cells, and intron 7 is essential for silencing regA in gonidia (asexual reproductive cells). A regA gene lacking intron 7 rescues the normal phenotype of mutant somatic cells, but also results in gonidia that reproduce only weakly and soon die out. The same phenotype is observed when a regA gene containing intron 7 is placed under control of a constitutive promoter, suggesting that the silencing activity of intron 7 is promoter specific. Intron 7 is unusual in that it contains a potential ORF that is in frame with exons 7 and 8, and some transcripts are produced in which intron 7 is retained. However, a regulatory role for the intron 7 translation product can be ruled out, because a construct in which intron 7 must be translated, and one in which it cannot be translated, both result in wild-type development of both cell types. Furthermore, intron 7 is unable to act in trans to silence regA, but is able to exert its normal effect when placed in a different location within the gene. Therefore, it appears that intron 7 functions in gonidia as a classical cell-type-specific and promoter-specific enhancer, of the inhibitory type that is often referred to as a silencer.

Volvox as a Model System for Studying the Ontogeny and Phylogeny of Multicellularity and Cellular Differentiation

Volvox carteri, a spherical alga with a complete division of labor between approximately 2000 biflagellate somatic cells and 16 asexual reproductive cells called gonidia, provides a very attractive system for analyzing how a molecular-genetic program for cell-autonomous cellular differentiation may be encoded within a genome. Then, when considered in combination with a group of closely related volvocine algae that includes unicellular Chlamydomonas plus a series of colonial forms of increasing cell number and complexity, it also provides an attractive model system for analyzing how such a program for multicellularity and cytodifferentiation may have evolved. It is proposed that the following were some of the key steps in this evolutionary pathway: (1) The Chlamydomonas cell wall was transformed into an extracellular matrix (ECM) that joined sister cells into a colonial unit. (2) Larger organisms with more abundant ECM were favored because of the role the ECM plays in storing limiting nutrients. (3) In the V. carteri lineage the ancestral biphasic first biflagellate and then reproductive pathway of development became converted to a dichotomous pathway by introduction of two kinds of cell-type-specific negative regulators: one that blocked growth and reproduction in presumptive somatic cells and one that blocked somatic development in presumptive gonidia. Progress has been made in cloning and characterizing genes that are involved in setting apart the two cell lineages of V. carteri and in subsequently controlling their dichotomous differentiation. The strengths and weaknesses of V. carteri and its relatives as a model system for studying the evolution of multicellularity are discussed.

Aspergillus SteA (sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction.

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63-75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12alphap. SteAp and Ste12alphap lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A DeltasteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.

The genetic program for germ-soma differentiation in Volvox.

Volvox carteri possesses only two cell types: mortal somatic cells and potentially immortal asexual reproductive cells called gonidia. Mutational analysis indicates that three categories of genes play central roles in programming this germ-soma division of labor: First the gls genes function during embryogenesis to cause asymmetric divisions that produce large and small cells. Then the lag genes act in the large cells (gonidial initials) to repress functions required for somatic development while the regA locus acts in the small cells (somatic initials) to repress functions required for reproductive development. Transposon tagging and DNA transformation have recently been used to recover and characterize the glsA and regA genes, and the sequences of these genes lead to testable hypotheses about how they play their roles in germ-soma differentiation.

Asymmetric division, cell size and germ-soma specification in Volvox

Volvox carteri is a spheroidal green alga in which each young adult has ∼2000 small, terminally differentiated somatic cells at the surface and ∼16 large, potentially immortal asexual reproductive cells, or gonidia, on the interior. When mature, each gonidium initiates a stereotyped cleavage program, during which prospective gonidia and somatic cells of the next generation are set apart by asymmetric divisions. At the end of cleavage, prospective gonidia are ∼30 X the volume of prospective somatic cells, and a variety of studies lead to the conclusion that it is this difference in size, not an accompanying difference in cytoplasmic quality, that triggers entirely different programs of gene expression and differentiation in the two cell lineages. Genes that appear to play key roles in differentiation include gls , which is required for asymmetric division, regA , which suppresses reproductive development in small cells, and the lag genes which suppress somatic development in large cells. The prospects for analysing the nature of these genes and their actions have been brightened by recent molecular-genetic developments, but the mechanism by which differences in cell size are transduced into differences in gene expression remains obscure.

The relationship between cell size and cell fate in Volvox carteri.

In Volvox carteri development, visibly asymmetric cleavage divisions set apart large embryonic cells that will become asexual reproductive cells (gonidia) from smaller cells that will produce terminally differentiated somatic cells. Three mechanisms have been proposed to explain how asymmetric division leads to cell specification in Volvox: (a) by a direct effect of cell size (or a property derived from it) on cell specification, (b) by segregation of a cytoplasmic factor resembling germ plasm into large cells, and (c) by a combined effect of differences in cytoplasmic quality and cytoplasmic quantity. In this study a variety of V. carteri embryos with genetically and experimentally altered patterns of development were examined in an attempt to distinguish among these hypotheses. No evidence was found for regionally specialized cytoplasm that is essential for gonidial specification. In all cases studied, cells with a diameter > approximately 8 microns at the end of cleavage--no matter where or how these cells had been produced in the embryo--developed as gonidia. Instructive observations in this regard were obtained by three different experimental interventions. (a) When heat shock was used to interrupt cleavage prematurely, so that presumptive somatic cells were left much larger than they normally would be at the end of cleavage, most cells differentiated as gonidia. This result was obtained both with wild-type embryos that had already divided asymmetrically (and should have segregated any cytoplasmic determinants involved in cell specification) and with embryos of a mutant that normally produces only somatic cells. (b) When individual wild-type blastomeres were isolated at the 16-cell stage, both the anterior blastomeres that normally produce two gonidia each and the posterior blastomeres that normally produce no gonidia underwent modified cleavage patterns and each produced an average of one large cell that developed as a gonidium. (c) When large cells were created microsurgically in a region of the embryo that normally makes only somatic cells, these large cells became gonidia. These data argue strongly for a central role of cell size in germ/soma specification in Volvox carteri, but leave open the question of how differences in cell size are actually transduced into differences in gene expression.

Cellular Mechanisms of the Evolution of Ontogenesis in Volvox

Summary The green flagellates of the genus Volvox can be conditionally subdivided into two groups according to the size of gonidia (asexual reproductive cells) at the onset of cleavage. In Volvox carteri and several other species the gonidium undergoes an extended period of hypertrophied growth, after which a series of rapid fissions occurs. Embryonic cells do not grow during the intervals between consecutive divisions. In representatives of the second group (e. g., V. aureus) the period of gonidial enlargement is comparatively nondurable; thus the cleavage begins when the gonidium is relatively small, and each division is followed by a period of cellular growth. In this paper the evolutionary relationships between two types of asexual life cycle in Volvox are analysed on the basis of literary and our own data. It is supposed that the V. aureus type of development with slow divisions of small gonidia is more advanced in the evolutionary respect than the V. carteri type of development with rapid divisions of large gonidia. Experimental analysis of the role of light and dark for embryonic cleavage progression in V. aureus, V. carteri f. nagariensis and V. tertius as well as the experiments with several metabolic inhibitors (aminopterin, actinomycin D, cycloheximide and streptomycin) have enabled us to elucidate cellular mechanisms of the evolution of ontogenesis in Volvox.

The program for cellular differentiation in Volvox carteri as revealed by molecular analysis of development in a gonidialess/somatic regenerator mutant

Development of a 'gonidialess'/'somatic regenerator' double mutant of Volvox carteri was analyzed with a number of cell-type-specific cDNA probes that had been identified in a previous study. Whereas in wild-type strains somatic cells and gonidia (asexual reproductive cells) constitute two distinct cell lineages, in this mutant all cells first differentiate as somatic cells and then redifferentiate as gonidia. During the initial period of somatic differentiation, we found that both gonidial and 'early' somatic transcripts were accumulated in the mutant, consistent with the idea that it is the regA gene product (which is defective in this mutant) that normally acts to suppress gonidial gene expression in somatic cells. Later in development, levels of early somatic transcripts fell abruptly, levels of the late somatic transcripts remained extremely low, and levels of gonidial transcripts rose as the cells redifferentiated. Thus it appears that in the mutant cells the gonidial program of development takes over and somatic differentiation is aborted before the stage at which late somatic genes are normally activated. These results provide molecular genetic support for a model which postulates that three types of genes (including the two that are defective in the strain studied here) are crucial for converting the sequential program of differentiation seen in more primitive volvocalean algae to the dichotomous program of germ-soma differentiation that occurs in wild-type V. carteri.

Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle

Volvox carteri possesses two morphologically and functionally distinct cell types: somatic cells and gonidia (asexual reproductive cells). To define the developmental programs involved in the differentiation of these two cell types, we have isolated 31 nonhomologous cDNA clones that hybridized to RNAs that were significantly more abundant in one cell type than the other. Details of the cell-type- and stage-specificity of expression of the transcripts detected by these cDNAs (plus five genes previously characterized by others) were examined by Northern-blot analysis. Accumulation patterns for the 19 gonidial transcripts fell into two distinct classes: transcripts of one gene were maximally abundant in very early cleavage, whereas transcripts of the other 18 did not reach maximal abundance until quite late in gonidial development. Similarly, the 12 somatic-cell-specific transcripts fell into two categories: transcripts of 5 “early” somatic genes became abundant soon after the completion of embryogenesis, whereas transcripts of 7 “late” somatic genes were not detected until later developmental stages. Expression of 3 other genes (two involved in flagellar development and one that encodes an extracellular matrix component) was also found to be restricted largely to somatic cells. These studies indicate that phenotypic differences between somatic cells and gonidia can be at least partially explained by differential regulation of RNA accumulation, and that there appear to be multiple patterns of accumulation of cell-type-specific transcripts within each cell type.