Genetic basis for soma is present in undifferentiated volvocine green algae

Somatic cellular differentiation plays a critical role in the transition from unicellular to multicellular life, but the evolution of its genetic basis remains poorly understood. By definition, somatic cells do not reproduce to pass on genes and so constitute an extreme form of altruistic behaviour. The volvocine green algae provide an excellent model system to study the evolution of multicellularity and somatic differentiation. In Volvox carteri, somatic cell differentiation is controlled by the regA gene, which is part of a tandem duplication of genes known as the reg cluster. Although previous work found the reg cluster in divergent Volvox species, its origin and distribution in the broader group of volvocine algae has not been known. Here, we show that the reg cluster is present in many species without somatic cells and determine that the genetic basis for soma arose before the phenotype at the origin of the family Volvocaceae approximately 200 million years ago. We hypothesize that the ancestral function was involved in regulating reproduction in response to stress and that this function was later co‐opted to produce soma. Determining that the reg cluster was co‐opted to control somatic cell development provides insight into how cellular differentiation, and with it greater levels of complexity and individuality, evolves.     

EVOLUTION OF DEVELOPMENTAL PROGRAMS IN VOLVOX (CHLOROPHYTA)1

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).     

Stable nuclear transformation of Gonium pectorale

Background  

Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of ~16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri.     

Genetic control of somatic cell differentiation in Volvox analysis of somatic regenerator mutants

The somatic regenerator (reg) mutants of Volvox carteri affect the ability of the normally terminally differentiated somatic cells to establish and/or maintain the differentiated state. Thirty-nine reg mutants of four phenotypic classes have been mapped to two, unlinked genes, regA and regB. Mutants at the regA locus have one of three phenotypes: All somatic cells regenerate new spheroids, somatic cells in the spheroid posterior region regenerate while those in the anterior region differentiate as somatic cells, or regenerating and nonregenerating cells are randomly intermixed. The regB mutant has a random intermixture of regenerating and nonregenerating cells. Somatic cells regenerate new Volvox spheroids in two ways; the cells lose their characteristic shape, become immotile, enlarge and undergo cleavage similar to that of normal reproductive cells or undergo cell division without prior enlargement or loss of cell shape. Temperature shift experiments on a cold-sensitive reg mutant suggest that the gene product acts after the somatic cell initials are formed at the end of cleavage.     

EARLY EVOLUTION OF THE GENETIC BASIS FOR SOMA IN THE VOLVOCACEAE

To understand the hierarchy of life in evolutionary terms, we must explain why groups of one kind of individual, say cells, evolve into a new higher level individual, a multicellular organism. A fundamental step in this process is the division of labor into nonreproductive altruistic soma. The regA gene is critical for somatic differentiation in Volvox carteri, a multicellular species of volvocine algae. We report the sequence of regA‐like genes and several syntenic markers from divergent species of Volvox. We show that regA evolved early in the volvocines and predict that lineages with and without soma descended from a regA‐containing ancestor. We hypothesize an alternate evolutionary history of regA than the prevailing “proto‐regA” hypothesis. The variation in presence of soma may be explained by multiple lineages independently evolving soma utilizing regA or alternate genetic pathways. Our prediction that the genetic basis for soma exists in species without somatic cells raises a number of questions, most fundamentally, under what conditions would species with the genetic potential for soma, and hence greater individuality, not evolve these traits. We conclude that the evolution of individuality in the volvocine algae is more complicated and labile than previously appreciated on theoretical grounds.     

Evolution of reproductive development in the volvocine algae

The evolution of multicellularity, the separation of germline cells from sterile somatic cells, and the generation of a male–female dichotomy are certainly among the greatest innovations of eukaryotes. Remarkably, phylogenetic analysis suggests that the shift from simple to complex, differentiated multicellularity was not a unique progression in the evolution of life, but in fact a quite frequent event. The spheroidal green alga Volvox and its close relatives, the volvocine algae, span the full range of organizational complexity, from unicellular and colonial genera to multicellular genera with a full germ–soma division of labor and male–female dichotomy; thus, these algae are ideal model organisms for addressing fundamental issues related to the transition to multicellularity and for discovering universal rules that characterize this transition. Of all living species, Volvox carteri represents the simplest version of an immortal germline producing specialized somatic cells. This cellular specialization involved the emergence of mortality and the production of the first dead ancestors in the evolution of this lineage. Volvocine algae therefore exemplify the evolution of cellular cooperation from cellular autonomy. They also serve as a prime example of the evolution of complex traits by a few successive, small steps. Thus, we learn from volvocine algae that the evolutionary transition to complex, multicellular life is probably much easier to achieve than is commonly believed.     

Differentiation of germinal and somatic cells in Volvox carteri

Volvox carteri is a spherical alga with a complete division of labor between around 2000 biflagellate somatic cells and 16 asexual reproductive cells (gonidia). It provides an attractive system for studying how a molecular genetic program for cell-autonomous differentiation is encoded within the genome. Three types of genes have been identified as key players in germ-soma differentiation: a set of gls genes that act in the embryo to shift cell-division planes, resulting in asymmetric divisions that set apart the large-small sister-cell pairs; a set of lag genes that act in the large gonidial initials to prevent somatic differentiation; and the regA gene, which acts in the small somatic initials to prevent reproductive development. Somatic-cell-specific expression of regA is controlled by intronic enhancer and silencer elements.     

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 with the 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.     

Comparative Analysis of Embryonic Inversion in Algae of the Genus <Emphasis Type="Italic">Volvox</Emphasis> (Volvocales,Chlorophyta)

Recent literary data on inversion (turning inside out) in the embryos of flagellated algae of the genus Volvox are critically analyzed. In this process, active changes in the shape of embryonic cells and the displacement of intercellular cytoplasmic bridges play an important role. After inversion, the flagella appear on the outer side of the young colony and provide its motility. Within the genus Volvox, two main modes of embryo inversion have been recently established during the asexual developmental cycle—inversion of type A and inversion of type B—represented by the two species most thoroughly studied, respectively, Volvox carterif. nagariensis and V. globator. However, the published opinion that the inversion of V. aureus embryos is of the type B seems to be doubtful. Comparative and evolutionary aspects of embryonic inversion in Volvox are discussed with the use of data on other genera of colonial volvocine algae.     

<Emphasis Type="Italic">Volvox</Emphasis> (Chlorophyta,Volvocales) as a model organism in developmental biology

Model systems based on two or more related species with different types of development are finding increasing use in current comparative embryology. Green algae of the genus Volvox offer an interesting opportunity to study sex pheromones, morphogenesis as well as the formation of a somatic cell line undergoing terminal differentiation, senescence, and death as well as a line of reproductive cells, which at first grow and then undergo a series of consecutive divisions that give rise to new organisms. However, almost all studies of the recent years were conducted on a single species, Volvox carteri f. nagariensis. The goal of this publication was to advertise the cosmopolitan alga V. aureus as a model species in developmental biology. Published data on V. aureus are briefly reviewed in comparison with the development of V. carteri and outlooks of further studies are specified. In particular, the expediency of collecting new V. aureus strains from nature to study their development in clonal culture is outlined.     

Protein synthesis in a new system for the study of senescence

In asexual individuals of the green alga Volvox carteri, more than 99% of the cells are somatic cells which undergo synchronous programmed senescence and cell death every generation. Only a small number of reproductive cells survive to produce the next generation. The specific activity of pulse-labelled somatic cell protein preparations declines sharply during senescence, but no decline is seen in the nonageing reproductive cells. Two-dimensional polyacrylamide gel electrophoresis reveals that somatic and reproductive cells synthesize very different patterns of polypeptides. During the period when observable senescent changes are first evident in somatic cells, there is a change in the pattern of polypeptides being synthesized. Our results suggest that senescence in Volvox somatic cells is triggered by a change in the pattern of gene expression and are consistent with theories of programmed cell senescence.     

THE FLAGELLAR PHOTORESPONSE IN VOLVOX SPECIES (VOLVOCACEAE,CHLOROPHYCEAE)1

Steering their swimming direction toward the light is crucial for the viability of Volvox colonies, the larger members of the volvocine algae. While it is known that this phototactic steering is achieved by a difference in behavior of the flagella on the illuminated and shaded sides, conflicting reports suggest that this asymmetry arises either from a change in beating direction or a change in beating frequency. Here, we report direct observations of the flagellar behavior of various Volvox species with different phyletic origin in response to light intensity changes and thereby resolve this controversy: Volvox barberi W. Shaw from the section Volvox sensu Nozaki (2003) changes the direction of the flagellar beating plane, while species encompassed in the group Eudorina (Volvox carteri F. Stein, Volvox aureus Ehrenb., and Volvox tertius Art. Mey.) decrease the flagellar beating frequency, sometimes down to flagellar arrest.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

Sexual reproduction and sex determination in green algae

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt⁺) and mating type minus (mt^?), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt⁺ and mt^? mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.     

A microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox

Nuclear division immediately follows nuclear DNA doubling in all stages of the life cycle examined in the green alga Volvox; fluorescence microfluorometry of individual cells revealed no evidence of prolonged accumulation of nuclear DNA prior to mitosis in reproductive cells. Somatic cell nuclear DNA quantity is unaffected by developmental events in gonidia of the same spheroid; it remains constant from the end of cleavage until the death of the cell. In reproductive cells, chloroplast DNA replication precedes nuclear replication. The sites of plastid DNA accumulation, made visible by use of the fluorochrome 4′,6-diamidino-2-phenylindole, increase in number during the prolonged growth phase of the V. carteri gonidium. Microspectrofluorometry of fluorochrome-stained DNA in situ shows that plastid DNA increases exponentially throughout this phase. The continuous plastid DNA accumulation during gonidial growth appears to represent a prokaryote-like instead of a eukaryote-like control of DNA synthesis. Most somatic cells contain plastid DNA, and this does not increase in amount during colony growth and reproduction. Most sperm cells also contain plastid DNA, although approximately 5% of somatic cells and up to 20% of sperm cells have no discernable plastid DNA. This is the second group of organisms in which DNA-free plastids have been observed.     

Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

Molecular and cellular dynamics of early embryonic cell divisions in Volvox carteri

Cell division is fundamental to all organisms and the green alga used here exhibits both key animal and plant functions. Specifically, we analyzed the molecular and cellular dynamics of early embryonic divisions of the multicellular green alga Volvox carteri (Chlamydomonadales). Relevant proteins related to mitosis and cytokinesis were identified in silico, the corresponding genes were cloned, fused to yfp, and stably expressed in Volvox, and the tagged proteins were studied by live-cell imaging. We reveal rearrangements of the microtubule cytoskeleton during centrosome separation, spindle formation, establishment of the phycoplast, and generation of previously unknown structures. The centrosomes participate in initiation of spindle formation and determination of spindle orientation. Although the nuclear envelope does not break down during early mitosis, intermixing of cytoplasm and nucleoplasm results in loss of nuclear identity. Finally, we present a model for mitosis in Volvox. Our study reveals enormous dynamics, clarifies spatio-temporal relationships of subcellular structures, and provides insight into the evolution of cell division.

Analysis of cell divisions of the microalga Volvox reveals enormous dynamics of cytoskeletal and membranous structures with coordination of intranuclear spindle formation by cytosolic centrosomes.

IN A NUTSHELLBackground: Mitosis, a type of cell division, is fundamental to all eukaryotic life and must be carried out very accurately. Even though the process of mitosis itself is highly conserved among eukaryotes, there are significant differences between animals, fungi, plants, and algae. From an evolutionary point of view, the green alga Volvox carteri used here possesses both key animal and plant functions and it exhibits important features of the last common eukaryotic ancestor that have been lost in other lineages. Prior to our work, a comprehensive in vivo analysis of the entire process of cell division in green algae was lacking.Question: How exactly does cell division work in green algae? How do the cytosolic centrosomes deal with the persistent nuclear envelope in this process? What is the relationship between different microtubular structures?Findings: Our study reveals enormous dynamics during mitosis, clarifies spatio-temporal relationships of subcellular structures, and provides insights into evolution of cell division. Although the nuclear envelope does not break down during early mitosis of Volvox, it becomes permeable and the nucleus temporarily loses its identity. Two microtubule-organizing centers, the centrosomes, located immediately outside the nuclear envelope participate in initiation of the mitotic spindle formation inside the nuclear envelope. This process also defines the orientation of the mitotic spindle. In cytokinesis, an algae-specific microtubule structure, the phycoplast, replaces the spindle. The microtubules of the phycoplast may play a direct role in promoting the cell membrane invagination of the cleavage furrow.Next steps: How are the massive rearrangements of subcellular structures regulated? What happens at the nuclear pores when the nuclear envelope becomes permeable at the onset of mitosis? What determines in later embryogenesis which cells then divide asymmetrically rather than symmetrically?