Cellular organization in lab-evolved and extant multicellular species obeys a maximum entropy law.

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri. We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing.

Minimal model of the hydrodynamical coupling of flagella on a spherical body with application to Volvox.

Flagella are hairlike appendages attached to microorganisms that allow the organisms to traverse their fluid environment. The algae Volvox are spherical swimmers with thousands of individual flagella on their surface, and their coordination is not fully understood. In this work, a previously developed minimal model of flagella synchronization is extended to the outer surface of a sphere submerged in a fluid. Each beating flagellum tip is modeled as a small sphere, elastically bound to a circular orbit just above the spherical surface and a regularized image system for Stokes flow outside of a sphere is used to enforce the no-slip condition. Biologically relevant distributions of rotors results in a rapidly developing and robust symplectic metachronal wave traveling from the anterior to the posterior of the spherical Volvox body.

Morphological and molecular identification of the dioecious "African species Volvox rousseletii (Chlorophyceae) in the water column of a Japanese lake based on field-collected and cultured materials.

Volvox rousseletii is a dioecious species belonging to Volvox sect. Volvox that has previously only been found in Africa. During field surveys in a large dam lake (Lake Sagami) in Kanagawa Prefecture, central Japan, we encountered a Volvox sect. Volvox species that produces dioecious sexual spheroids in the water column. Although sexual induction of this species in culture did not produce adequately well-developed sexual spheroids for species identification, molecular data directly obtained from field-collected sexual spheroids verified the identity of field-collected male and female sexual spheroids as well as cultured materials. Based on molecular and morphological data, the species was identified as V. rousseletii. This is the first record of a dioecious species of Volvox sect. Volvox in Japan.

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.

Cell-Type Transcriptomes of the Multicellular Green Alga Volvox carteri Yield Insights into the Evolutionary Origins of Germ and Somatic Differentiation Programs.

Germ-soma differentiation is a hallmark of complex multicellular organisms, yet its origins are not well understood. Volvox carteri is a simple multicellular green alga that has recently evolved a simple germ-soma dichotomy with only two cell-types: large germ cells called gonidia and small terminally differentiated somatic cells. Here, we provide a comprehensive characterization of the gonidial and somatic transcriptomes of V. carteri to uncover fundamental differences between the molecular and metabolic programming of these cell-types. We found extensive transcriptome differentiation between cell-types, with somatic cells expressing a more specialized program overrepresented in younger, lineage-specific genes, and gonidial cells expressing a more generalist program overrepresented in more ancient genes that shared striking overlap with stem cell-specific genes from animals and land plants. Directed analyses of different pathways revealed a strong dichotomy between cell-types with gonidial cells expressing growth-related genes and somatic cells expressing an altruistic metabolic program geared toward the assembly of flagella, which support organismal motility, and the conversion of storage carbon to sugars, which act as donors for production of extracellular matrix (ECM) glycoproteins whose secretion enables massive organismal expansion. V. carteri orthologs of diurnally controlled genes from C. reinhardtii, a single-celled relative, were analyzed for cell-type distribution and found to be strongly partitioned, with expression of dark-phase genes overrepresented in somatic cells and light-phase genes overrepresented in gonidial cells- a result that is consistent with cell-type programs in V. carteri arising by cooption of temporal regulons in a unicellular ancestor. Together, our findings reveal fundamental molecular, metabolic, and evolutionary mechanisms that underlie the origins of germ-soma differentiation in V. carteri and provide a template for understanding the acquisition of germ-soma differentiation in other multicellular lineages.

Evolution of cytokinesis-related protein localization during the emergence of multicellularity in volvocine green algae.

BACKGROUND: The volvocine lineage, containing unicellular Chlamydomonas reinhardtii and differentiated multicellular Volvox carteri, is a powerful model for comparative studies aiming at understanding emergence of multicellularity. Tetrabaena socialis is the simplest multicellular volvocine alga and belongs to the family Tetrabaenaceae that is sister to more complex multicellular volvocine families, Goniaceae and Volvocaceae. Thus, T. socialis is a key species to elucidate the initial steps in the evolution of multicellularity. In the asexual life cycle of C. reinhardtii and multicellular volvocine species, reproductive cells form daughter cells/colonies by multiple fission. In embryogenesis of the multicellular species, daughter protoplasts are connected to one another by cytoplasmic bridges formed by incomplete cytokinesis during multiple fission. These bridges are important for arranging the daughter protoplasts in appropriate positions such that species-specific integrated multicellular individuals are shaped. Detailed comparative studies of cytokinesis between unicellular and simple multicellular volvocine species will help to elucidate the emergence of multicellularity from the unicellular ancestor. However, the cytokinesis-related genes between closely related unicellular and multicellular species have not been subjected to a comparative analysis. RESULTS: Here we focused on dynamin-related protein 1 (DRP1), which is known for its role in cytokinesis in land plants. Immunofluorescence microscopy using an antibody against T. socialis DRP1 revealed that volvocine DRP1 was localized to division planes during cytokinesis in unicellular C. reinhardtii and two simple multicellular volvocine species T. socialis and Gonium pectorale. DRP1 signals were mainly observed in the newly formed division planes of unicellular C. reinhardtii during multiple fission, whereas in multicellular T. socialis and G. pectorale, DRP1 signals were observed in all division planes during embryogenesis. CONCLUSIONS: These results indicate that the molecular mechanisms of cytokinesis may be different in unicellular and multicellular volvocine algae. The localization of DRP1 during multiple fission might have been modified in the common ancestor of multicellular volvocine algae. This modification may have been essential for the re-orientation of cells and shaping colonies during the emergence of multicellularity in this lineage.

Efficient single-pixel multispectral imaging via non-mechanical spatio-spectral modulation.

Combining spectral imaging with compressive sensing (CS) enables efficient data acquisition by fully utilizing the intrinsic redundancies in natural images. Current compressive multispectral imagers, which are mostly based on array sensors (e.g, CCD or CMOS), suffer from limited spectral range and relatively low photon efficiency. To address these issues, this paper reports a multispectral imaging scheme with a single-pixel detector. Inspired by the spatial resolution redundancy of current spatial light modulators (SLMs) relative to the target reconstruction, we design an all-optical spectral splitting device to spatially split the light emitted from the object into several counterparts with different spectrums. Separated spectral channels are spatially modulated simultaneously with individual codes by an SLM. This no-moving-part modulation ensures a stable and fast system, and the spatial multiplexing ensures an efficient acquisition. A proof-of-concept setup is built and validated for 8-channel multispectral imaging within 420~720 nm wavelength range on both macro and micro objects, showing a potential for efficient multispectral imager in macroscopic and biomedical applications.

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.

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.

Volvox: A simple algal model for embryogenesis, morphogenesis and cellular differentiation.

Patterning of a multicellular body plan involves a coordinated set of developmental processes that includes cell division, morphogenesis, and cellular differentiation. These processes have been most intensively studied in animals and land plants; however, deep insight can also be gained by studying development in simpler multicellular organisms. The multicellular green alga Volvox carteri (Volvox) is an excellent model for the investigation of developmental mechanisms and their evolutionary origins. Volvox has a streamlined body plan that contains only a few thousand cells and two distinct cell types: reproductive germ cells and terminally differentiated somatic cells. Patterning of the Volvox body plan is achieved through a stereotyped developmental program that includes embryonic cleavage with asymmetric cell division, morphogenesis, and cell-type differentiation. In this review we provide an overview of how these three developmental processes give rise to the adult form in Volvox and how developmental mutants have provided insights into the mechanisms behind these events. We highlight the accessibility and tractability of Volvox and its relatives that provide a unique opportunity for studying development.

Dynamics of a Volvox embryo turning itself inside out.

Deformations of cell sheets are ubiquitous in early animal development, often arising from a complex and poorly understood interplay of cell shape changes, division, and migration. Here, we explore perhaps the simplest example of cell sheet folding: the "inversion" process of the algal genus Volvox, during which spherical embryos turn themselves inside out through a process hypothesized to arise from cell shape changes alone. We use light sheet microscopy to obtain the first three-dimensional visualizations of inversion in vivo, and develop the first theory of this process, in which cell shape changes appear as local variations of intrinsic curvature, contraction and stretching of an elastic shell. Our results support a scenario in which these active processes function in a defined spatiotemporal manner to enable inversion.

Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga Volvox carteri and their potential role in cellular differentiation.

The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in multicellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation.

Effects of culturing media on hepatocytes differentiation using Volvox sphere as co-culturing vehicle.

Volvox sphere is a unique design to mimic natural volvox consists of a large outer-sphere that contains smaller inner-spheres, which provide three-dimensional (3D) environment to culture cells. The purpose of this study is to co-culture mesenchymal stem cells (MSCs) and AML12 liver cells in Volvox spheres and to evaluate the effects of two media, DMEM and DMEM/F12 on the cultured cells. The results of this study shows that the 3D Volvox sphere can successfully be applied for co-culture of MSCs and AML12 liver cells, and the MSCs are able to differentiate into hepatocyte-like cells expressing hepatocyte-specific markers including albumin (ALB), alpha feto-protein (AFP) and cytokeratin 18 (CK18) mRNA expressions and producing CK18 and ALB proteins. Interestingly, the MSCs expressed higher ALB, AFP and CK18 mRNA expression at the initial 7-day culture by using DMEM, whereas, the MSCs expressed more mRNA expressions from 7-day to 14-day by the usage of DMEM/F12. The result demonstrated that DMEM and DMEM/F12 media could affect MSCs behaviors during a 14-day culture.

Flagellar synchronization through direct hydrodynamic interactions.

Flows generated by ensembles of flagella are crucial to development, motility and sensing, but the mechanisms behind this striking coordination remain unclear. We present novel experiments in which two micropipette-held somatic cells of Volvox carteri, with distinct intrinsic beating frequencies, are studied by high-speed imaging as a function of their separation and orientation. Analysis of time series shows that the interflagellar coupling, constrained by lack of connections between cells to be hydrodynamical, exhibits a spatial dependence consistent with theory. At close spacings it produces robust synchrony for thousands of beats, while at increasing separations synchrony is degraded by stochastic processes. Manipulation of the relative flagellar orientation reveals in-phase and antiphase states, consistent with dynamical theories. Flagellar tracking with exquisite precision reveals waveform changes that result from hydrodynamic coupling. This study proves unequivocally that flagella coupled solely through a fluid can achieve robust synchrony despite differences in their intrinsic properties.DOI: http://dx.doi.org/10.7554/eLife.02750.001.

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.

[Ontogenetic diversity of colonies and intercellular cytoplasmic bridges in the algae of the genuis 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.

Swimming like algae: biomimetic soft artificial cilia.

Cilia are used effectively in a wide variety of biological systems from fluid transport to thrust generation. Here, we present the design and implementation of artificial cilia, based on a biomimetic planar actuator using soft-smart materials. This actuator is modelled on the cilia movement of the alga Volvox, and represents the cilium as a piecewise constant-curvature robotic actuator that enables the subsequent direct translation of natural articulation into a multi-segment ionic polymer metal composite actuator. It is demonstrated how the combination of optimal segmentation pattern and biologically derived per-segment driving signals reproduce natural ciliary motion. The amenability of the artificial cilia to scaling is also demonstrated through the comparison of the Reynolds number achieved with that of natural cilia.

Amidic and acetonic cryoprotectants improve cryopreservation of volvocine green algae.

A number of volvocalean green algae species were subjected to a two-step cryopreservation protocol with various cryoprotectants. Potential cryoprotectants were methanol (DMSO), N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylformamide, and hydroxyacetone (HA). We confirmed prior reports that MeOH was effective for cryopreserving Chlamydomonas, but did not work well for larger volvocaleans such as Volvox. In contrast, DMF and HA were effective for both unicellular and multicellular representatives. When we used a cold-inducible transposon to probe Southern blots of Volvox DNA samples taken before and after storage for one month in LN, we could detect no differences, indicating that the genome had remained relatively stable and that the transposon had not been induced by the cryopreservation procedure. We believe these methods will facilitate long-term storage of several volvocine algal species, including Volvox strains harboring transposon-induced mutations of developmental interest.