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.

Isogamy in large and complex volvocine algae is consistent with the gamete competition theory of the evolution of anisogamy.

Although the gamete competition theory remains the dominant explanation for the evolution of anisogamy, well-known exceptions to its predictions have raised doubts about the completeness of the theory. One of these exceptions is isogamy in large or complex species of green algae. Here, we show that this exception may be explained in a manner consistent with a game-theoretic extension of the original theory: a constraint on the minimum size of a gamete may prevent the evolution of continuously stable anisogamy. We show that in the volvocine algae, both gametes of isogamous species retain an intact chloroplast, whereas the chloroplast of the microgamete in anisogamous species is invariably degenerate. The chloroplast, which functions in photosynthesis and starch storage, may be necessary to provision a gamete for an extended period when gamete encounter rates are low. The single chloroplast accounts for most of the volume of a typical gamete, and thus may constrain the minimum size of a gamete, preventing the evolution of anisogamy. A prediction from this hypothesis, that isogametes should be larger than the microgametes of similar-size species, is confirmed for the volvocine algae. Our results support the gamete competition theory.

The noisy basis of morphogenesis: Mechanisms and mechanics of cell sheet folding inferred from developmental variability.

Variability is emerging as an integral part of development. It is therefore imperative to ask how to access the information contained in this variability. Yet most studies of development average their observations and, discarding the variability, seek to derive models, biological or physical, that explain these average observations. Here, we analyse this variability in a study of cell sheet folding in the green alga Volvox, whose spherical embryos turn themselves inside out in a process sharing invagination, expansion, involution, and peeling of a cell sheet with animal models of morphogenesis. We generalise our earlier, qualitative model of the initial stages of inversion by combining ideas from morphoelasticity and shell theory. Together with three-dimensional visualisations of inversion using light sheet microscopy, this yields a detailed, quantitative model of the entire inversion process. With this model, we show how the variability of inversion reveals that two separate, temporally uncoupled processes drive the initial invagination and subsequent expansion of the cell sheet. This implies a prototypical transition towards higher developmental complexity in the volvocine algae and provides proof of principle of analysing morphogenesis based on its variability.

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 New Morphological Type of Volvox from Japanese Large Lakes and Recent Divergence of this Type and V. ferrisii in Two Different Freshwater Habitats.

Volvox sect. Volvox is characterized by having unique morphological characteristics, such as thick cytoplasmic bridges between adult somatic cells in the spheroids and spiny zygote walls. Species of this section are found from various freshwater habitats. Recently, three species of Volvox sect. Volvox originating from rice paddies and a marsh were studied taxonomically based on molecular and morphological data of cultured materials. However, taxonomic studies have not been performed on cultured materials of this section originating from large lake water bodies. We studied a new morphological type of Volvox sect. Volvox ("Volvox sp. Sagami"), using cultured materials originating from two large lakes and a pond in Japan. Volvox sp. Sagami produced monoecious sexual spheroids and may represent a new morphological species; it could be clearly distinguished from all previously described monoecious species of Volvox sect. Volvox by its small number of eggs or zygotes (5-25) in sexual spheroids, with short acute spines (up to 3 µm long) on the zygote walls and elongated anterior somatic cells in asexual spheroids. Based on sequences of internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA; ITS-1, 5.8S rDNA and ITS-2) and plastid genes, however, the Volvox sp. Sagami lineage and its sister lineage (the monoecious species V. ferrisii) showed very small genetic differences, which correspond to the variation within a single biological species in other volvocalean algae. Since V. ferrisii was different from Volvox sp. Sagami, by having approximately 100-200 zygotes in the sexual spheroids and long spines (6-8.5 µm long) on the zygote walls, as well as growing in Japanese rice paddies, these two morphologically distinct lineages might have diverged rapidly in the two different freshwater habitats. In addition, the swimming velocity during phototaxis of Volvox sp. Sagami spheroids originating from large lakes was significantly higher than that of V. ferrisii originating from rice paddies, suggesting adaptation of Volvox sp. Sagami to large water bodies.

Co-option during the evolution of multicellular and developmental complexity in the volvocine green algae.

Despite its major impact on the evolution of Life on Earth, the transition to multicellularity remains poorly understood, especially in terms of its genetic basis. The volvocine algae are a group of closely related species that range in morphology from unicellular individuals (Chlamydomonas) to undifferentiated multicellular forms (Gonium) and complex organisms with distinct developmental programs and one (Pleodorina) or two (Volvox) specialized cell types. Modern genetic approaches, complemented by the recent sequencing of genomes from several key species, revealed that co-option of existing genes and pathways is the primary driving force for the evolution of multicellularity in this lineage. The initial transition to undifferentiated multicellularity, as typified by the extant Gonium, was driven primarily by the co-option of cell cycle regulation. Further morphological and developmental innovations in the lineage leading to Volvox resulted from additional co-option events involving genes important for embryonic inversion, asymmetric cell division, somatic and germ cell differentiation and the structure and function of the extracellular matrix. Because of their relatively low but variable levels of morphological and developmental complexity, simple underlying genetics and recent evolutionary history, the volvocine algae are providing significant insight into our understanding of the genetics and evolution of major developmental and morphological traits.

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 (8 h light/16 h 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.

Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study show that Volvox photoreceptors are mostly expressed in a cell-type specific manner. This gives reason to believe that cell-type specific light-signaling pathways allow differential regulation of cellular and developmental processes in response to the environmental light cues.

There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator.

BACKGROUND: Epithelial folding is a common morphogenetic process during the development of multicellular organisms. In metazoans, the biological and biomechanical processes that underlie such three-dimensional (3D) developmental events are usually complex and difficult to investigate. Spheroidal green algae of the genus Volvox are uniquely suited as model systems for studying the basic principles of epithelial folding. Volvox embryos begin life inside out and then must turn their spherical cell monolayer outside in to achieve their adult configuration; this process is called 'inversion.' There are two fundamentally different sequences of inversion processes in Volvocaceae: type A and type B. Type A inversion is well studied, but not much is known about type B inversion. How does the embryo of a typical type B inverter, V. globator, turn itself inside out? RESULTS: In this study, we investigated the type B inversion of V. globator embryos and focused on the major movement patterns of the cellular monolayer, cell shape changes and changes in the localization of cytoplasmic bridges (CBs) connecting the cells. Isolated intact, sectioned and fragmented embryos were analyzed throughout the inversion process using light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy techniques. We generated 3D models of the identified cell shapes, including the localizations of CBs. We show how concerted cell-shape changes and concerted changes in the position of cells relative to the CB system cause cell layer movements and turn the spherical cell monolayer inside out. The type B inversion of V. globator is compared to the type A inversion in V. carteri. CONCLUSIONS: Concerted, spatially and temporally coordinated changes in cellular shapes in conjunction with concerted migration of cells relative to the CB system are the causes of type B inversion in V. globator. Despite significant similarities between type A and type B inverters, differences exist in almost all details of the inversion process, suggesting analogous inversion processes that arose through parallel evolution. Based on our results and due to the cellular biomechanical implications of the involved tensile and compressive forces, we developed a global mechanistic scenario that predicts epithelial folding during embryonic inversion in V. globator.

The bending of cell sheets--from folding to rolling.

The bending of cell sheets plays a major role in multicellular embryonic morphogenesis. Recent advances are leading to a deeper understanding of how the biophysical properties and the force-producing behaviors of cells are regulated, and how these forces are integrated across cell sheets during bending. We review work that shows that the dynamic balance of apical versus basolateral cortical tension controls specific aspects of invagination of epithelial sheets, and recent evidence that tissue expansion by growth contributes to neural retinal invagination in a stem cell-derived, self-organizing system. Of special interest is the detailed analysis of the type B inversion in Volvox reported in BMC Biology by Höhn and Hallmann, as this is a system that promises to be particularly instructive in understanding morphogenesis of any monolayered spheroid system.

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.

Volvox: simple steps to developmental complexity?

Volvox, Chlamydomonas, and their close relatives - collectively the volvocine green algae - comprise an excellent system for investigating the origins of developmental complexity. Over a relatively short period of time Volvox evolved an impressive suite of developmental traits, including asymmetric cell division, multicellularity with germ-soma division of labor, embryonic morphogenesis, and oogamy. Recent molecular genetic analyses of important developmental genes and comparative analyses of the fully sequenced Volvox and Chlamydomonas genomes have provided important insights into how these and other traits came to be. Surprisingly, the acquisition of much of the developmental innovation in this family seems to have involved relatively minor tinkering with the ancestral unicellular blueprint.

Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri.

The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are well suited for the investigation of the evolution of multicellularity and development. We sequenced the 138-mega-base pair genome of V. carteri and compared its approximately 14,500 predicted proteins to those of its unicellular relative Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials and few species-specific protein-coding gene predictions. Volvox is enriched in volvocine-algal-specific proteins, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.

[Volvox (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.

Controlled enlargement of the glycoprotein vesicle surrounding a volvox embryo requires the InvB nucleotide-sugar transporter and is required for normal morphogenesis.

Here, we report our analysis of a mutant of Volvox carteri, InvB, whose embryos fail to execute inversion, the process in which each Volvox embryo normally turns itself inside-out at the end of embryogenesis, thereby achieving the adult configuration. The invB gene encodes a nucleotide-sugar transporter that exhibits GDP-mannose transport activity when expressed in yeast. In wild-type embryos, the invB transcript is maximally abundant before and during inversion. A mannoside probe (fluorescent concanavalin A) stains the glycoprotein-rich gonidial vesicle (GV) surrounding wild-type embryos much more strongly than it stains the GV surrounding InvB embryos. Direct measurements revealed that throughout embryogenesis the GV surrounding a wild-type embryo increases in size much more than the GV surrounding an InvB embryo does, and the fully cleaved InvB embryo is much more tightly packed within its GV than a wild-type embryo is. To test the hypothesis that the restraint imposed by a smaller than normal GV directly causes the inversion defect in the mutant, we released InvB embryos from their GVs microsurgically. The resulting embryos inverted normally, demonstrating that controlled enlargement of the GV, by a process in which requires the InvB nucleotide-sugar transporter, is essential to provide the embryo sufficient space to complete inversion.

A gender-specific retinoblastoma-related protein in Volvox carteri implies 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.

[A noneuclidean Volvox, or why it is better not to teach Volvox in the course on zoology].

Empty "spheroid" of Volvox is compared with biomorph "thread", "disk" and solid "sphere" using such characteristics as topological dimensionality, average distance between cells, mutual remoteness of inner and surface cells, contiguity of cells. It is usually supposed that these parameters are significant for physiological gradients that determine cell specialization. One-dimensional "thread" has the longest physiological communications between cells and the average degree of contiguity about 2 (each cell contacts two neighbors). Biological morph "disk" has a degree about 6, two-side frontal physiological gradient inside the cell, and less expressed inter-cell gradient. Biomorph designated as 3-dimensional solid "sphere" has a degree of contiguity about 12-24, strong radial inter-cell gradient (non-equal conditions for surface and inner layers) and short distances between cells. These parameters favor cell specialization and their integration in multicellular organism. The "sphere" corresponds to hypothetical ancestor of Metazoa - "Metschnikoff's Phagocytella", while the "disk" - to "Placula of Bütschli". Biomorph "spheroid" of Volvox has a degree of contiguity about 6 and continuous tangential inter-cell gradient on noneuclidean surface. Radial gradient is absent here. Due to noneuclidean nature of "spheroid" the distances between cells are longer here than in case of "disc" and "sphere". All cells are under the same conditions for specialization and multiple primary integration. The secondary integration in higher Volvocales (differentiation in somatic and generative hemispheres) was probably caused by directed movement of the whole colony. Specialization of cells in lower invertebrates develops in a way which is characteristic for biomorph "sphere" on the basis of 3-dimensionality. The differentiation of animal and vegetal poles is connected with gastrulation (but not with directed movement as in case of Volvox). Gastrulation through invagination does not comparable with inversion of plate-like embryo of Volvox into "spheroid". Invagination is the transformation of a "bent of sphere", whereas the inversion is the "bent of plate". Independently of particular mechanism gastrulation results in 3-dimensionality (as in case of "sphere"). However the integration of cells in Volvox is explained by special peculiarities of 2-dimensional noneuclidean surface. That's why Volvox cannot be considered as model of ancestor of Metazoa.