Multicellular Bodies Research Articles

Ion Signaling in Cell Motility and Development in Dictyostelium discoideum.

Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.

A Mutant of Dictyostelium discoideum, KI-Cell, as a Model of Collective Cell Migration Independent of Chemotaxis.

Collective cell migration occurs when the orientation of cell polarity is aligned with each other in a group of cells. Such collective polarization depends on a reciprocal process between cell intrinsic mechanisms such as cell-cell adhesion and extracellular guidance mechanism such as wound healing and chemotaxis. As part of its development life cycle, individual single cells of Dictyostelium discoideum exhibit chemotaxis toward cAMP, which is secreted from a certain population of cells. During the formation of multicellular body by chemotaxis-dependent cell aggregation, D. discoideum is also known to relay on multiple cell-cell adhesion mechanisms. In particular, tail-following behavior at the contact site, called contact following of locomotion (CFL), plays a pivotal role on the formation of the multicellular body. However, whether and how CFL alone can lead to a formation of collective behavior was not well understood. KI cell is a mutant of D. discoideum that lacks all chemotactic activity. Yet, it can exhibit the CFL activity and show nontrivial collective cell migration. This mutant provides an excellent model system to analyze the mechanism of the CFL and the macroscopic phenomena brought by the CFL. This chapter describes protocols for using KI cell to understand the biophysics and cell biology behind the collective cell migration induced by CFL.

RHO of plant signaling was established early in streptophyte evolution

The algal ancestors of land plants underwent a transition from a unicellular to a multicellular body plan.1 This transition likely took place early in streptophyte evolution, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage.2 How this transition was brought about is unknown; however, it was likely facilitated by the evolution of novel mechanisms to spatially regulate morphogenesis. In land plants, RHO of plant (ROP) signaling plays a conserved role in regulating polarized cell growth and cell division orientation to orchestrate morphogenesis.3,4,5,6,7,8 ROP constitutes a plant-specific subfamily of the RHO GTPases, which are more widely conserved throughout eukaryotes.9,10 Although the RHO family originated in early eukaryotes,11,12 how and when the ROP subfamily originated had remained elusive. Here, we demonstrate that ROP signaling was established early in the streptophyte lineage, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage. This period corresponds to when the unicellular-to-multicellular transition likely took place in the streptophytes. In addition to being critical for the complex morphogenesis of extant land plants, we speculate that ROP signaling contributed to morphological evolution in early streptophytes.

A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues.

Parallel evolution of the G protein-coupled receptor GrlG and the loss of fruiting body formation in the social amoeba Dictyostelium discoideum evolved under low relatedness.

Aggregative multicellularity relies on cooperation among formerly independent cells to form a multicellular body. Previous work with Dictyostelium discoideum showed that experimental evolution under low relatedness profoundly decreased cooperation, as indicated by the loss of fruiting body formation in many clones and an increase of cheaters that contribute proportionally more to spores than to the dead stalk. Using whole-genome sequencing and variant analysis of these lines, we identified 38 single nucleotide polymorphisms in 29 genes. Each gene had 1 variant except for grlG (encoding a G protein-coupled receptor), which had 10 unique SNPs and 5 structural variants. Variants in the 5' half of grlG-the region encoding the signal peptide and the extracellular binding domain-were significantly associated with the loss of fruiting body formation; the association was not significant in the 3' half of the gene. These results suggest that the loss of grlG was adaptive under low relatedness and that at least the 5' half of the gene is important for cooperation and multicellular development. This is surprising given some previous evidence that grlG encodes a folate receptor involved in predation, which occurs only during the single-celled stage. However, non-fruiting mutants showed little increase in a parallel evolution experiment where the multicellular stage was prevented from happening. This shows that non-fruiting mutants are not generally selected by any predation advantage but rather by something-likely cheating-during the multicellular stage.

Unicellular and multicellular developmental variations in algal zygotes produce sporophytes.

The emergence of sporophytes, that is, diploid multicellular bodies in plants, facilitated plant diversification and the evolution of complexity. Although sporophytes may have evolved in an ancestral alga exhibiting a haplontic life cycle with a unicellular diploid and multicellular haploid (gametophyte) phase, the mechanism by which this novelty originated remains largely unknown. Ulotrichalean marine green algae (Ulvophyceae) are one of the few extant groups with haplontic-like life cycles. In this study, we show that zygotes of the ulotrichalean alga Monostroma angicava, which usually develop into unicellular cysts, exhibit a developmental variation producing multicellular reproductive sporophytes. Multicellular development likely occurred stochastically in individual zygotes, but its ratio responded plastically to growth conditions. Sporophytes showed identical morphological development to gametophytes, which should reflect the expression of the same genetic programme directing multicellular development. Considering that sporophytes were evolutionarily derived in Ulotrichales, this implies that sporophytes emerged by co-opting the gametophyte developmental programme to the diploid phase. This study suggests a possible mechanism of sporophyte formation in haplontic life cycles, contributing to the understanding of the evolutionary transition from unicellular to multicellular diploid body plans in green plants.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A.thaliana and the single LRL/DROP protein MpLRL in M.polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A.thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M.polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.

Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation

BackgroundLand plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium (Physcomitrella) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. patens, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. patens.ResultsHere, we investigated the effect of nitrogen conditions on the growth and development of P. patens. We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space.ConclusionsOur more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens.

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene

BackgroundAstrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections.ResultsTo establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36–33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest “MPN survival” [5.5 ± 5.9% (0.12–12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES.ConclusionsThe optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Numbers of Mutations within Multicellular Bodies: Why It Matters

Multicellular organisms often start life as a single cell. Subsequent cell division builds the body. Each mutational event during those developmental cell divisions carries forward to all descendant cells. The overall number of mutant cells in the body follows the Luria–Delbrück process. This article first reviews the basic quantitative principles by which one can understand the likely number of mutant cells and the variation in mutational burden between individuals. A recent Fréchet distribution approximation simplifies calculation of likelihoods and intuitive understanding of process. The second part of the article highlights consequences of somatic mutational mosaicism for understanding diseases such as cancer, neurodegeneration, and atherosclerosis.

Heterogeneous individual motility biases group composition in a model of aggregating cells

Aggregative life cycles are characterized by alternating phases of unicellular growth and multicellular development. Their multiple, independent evolutionary emergence suggests that they may have coopted pervasive properties of single-celled ancestors. Primitive multicellular aggregates, where coordination mechanisms were less efficient than in extant aggregative microbes, must have faced high levels of conflict between different co-aggregating populations. Such conflicts within a multicellular body manifest in the differential reproductive output of cells of different types. Here, we study how heterogeneity in cell motility affects the aggregation process and creates a mismatch between the composition of the population and that of self-organized groups of active adhesive particles. We model cells as self-propelled particles and describe aggregation in a plane starting from a dispersed configuration. Inspired by the life cycle of aggregative model organisms such as Dictyostelium discoideum or Myxococcus xanthus, whose cells interact for a fixed duration before the onset of chimeric multicellular development, we study finite-time configurations for identical particles and in binary mixes. We show that co-aggregation results in three different types of frequency-dependent biases, one of which is associated to evolutionarily stable coexistence of particles with different motility. We propose a heuristic explanation of such observations, based on the competition between delayed aggregation of slower particles and detachment of faster particles. Unexpectedly, despite the complexity and non-linearity of the system, biases can be largely predicted from the behavior of the two corresponding homogenous populations. This model points to differential motility as a possibly important factor in driving the evolutionary emergence of facultatively multicellular life-cycles.

Competition for finite resources as coordination mechanism for morphogenesis: An evolutionary algorithm study of digital embryogeny

The standard view of embryogenesis is one of cooperation driven by the cells' shared genetics and evolutionary interests. However, numerous examples from developmental biology and agriculture reveal a surprising amount of competition among body cells, tissues, and organs for both metabolic and informational resources. To explain the existence of such competition we had hypothesized that evolution uses limiting “reservoirs” of resource molecules as a communication medium - a global scratchpad, to enable tissues across the body to coordinate growth. Here, we test this hypothesis via an evolutionary simulation of embryogeny in silico. Genomes encode state transition rules for cells, such as proliferation, differentiation, and resource use, enabling virtual embryos to develop a specific large-scale morphology. An evolutionary algorithm operates over the genomes, with fitness defined as a function of specific morphological requirements for the final embryo shape. We found that not only does such an algorithm rapidly discover rules for cellular behavior that reliably make embryos with specific anatomical properties, but that it discovers the strategy of using finite resources to coordinate development. Given the option of using finite or infinite reservoirs (which determine cells’ ability to carry out specific actions), evolution preferentially uses finite reservoirs, which results in higher fitness and increased consistency (without needing direct selection for morphological invariance). We report aspects of anatomical, physiological/transcriptional, and genomic analysis of evolved virtual embryos that help understand how evolution can use competition among genetically identical subunits within a multicellular body to coordinate reliable, complex morphogenesis. Our results suggest that under some conditions, composite multi-scale systems will promote conflict and artificial scarcity for their components.

The greening ashore.

More than half a billion years ago a streptophyte algal lineage began terraforming the terrestrial habitat and the Earth's atmosphere. This pioneering step enabled the subsequent evolution of all complex life on land, and the past decade has uncovered that many traits, both morphological and genetic, once thought to be unique to land plants, are conserved across some streptophyte algae. They provided the common ancestor of land plants with a repertoire of genes, of which many were adapted to overcome the new biotic and abiotic challenges. Exploring these molecular adaptations in non-tracheophyte species may help us to better prepare all green life, including our crops, for the challenges precipitated by the climate change of the Anthropocene because the challenges mostly differ by the speed with which they are now being met.

Evolution of fungal phenotypic disparity.

Organismal-grade multicellularity has been achieved only in animals, plants and fungi. All three kingdoms manifest phenotypically disparate body plans but their evolution has only been considered in detail for animals. Here we tested the general relevance of hypotheses on the evolutionary assembly of animal body plans by characterizing the evolution of fungal phenotypic variety (disparity). The distribution of living fungal form is defined by four distinct morphotypes: flagellated; zygomycetous; sac-bearing; and club-bearing. The discontinuity between morphotypes is a consequence of extinction, indicating that a complete record of fungal disparity would present a more homogeneous distribution of form. Fungal disparity expands episodically through time, punctuated by a sharp increase associated with the emergence of multicellular body plans. Simulations show these temporal trends to be non-random and at least partially shaped by hierarchical contingency. These trends are decoupled from changes in gene number, genome size and taxonomic diversity. Only differences in organismal complexity, characterized as the number of traits that constitute an organism, exhibit a meaningful relationship with fungal disparity. Both animals and fungi exhibit episodic increases in disparity through time, resulting in distributions of form made discontinuous by extinction. These congruences suggest a common mode of multicellular body plan evolution.

Exosome mediated biological functions within skeletal microenvironment.

Exosomes are membranous lipid vesicles fused with intracellular multicellular bodies that are released into the extracellular environment. They contain bioactive substances, including proteins, RNAs, lipids, and cytokine receptors. Exosomes in the skeletal microenvironment are derived from a variety of cells such as bone marrow mesenchymal stem cells (BMSCs), osteoblasts, osteoclasts, and osteocytes. Their biological function is key in paracrine or endocrine signaling. Exosomes play a role in bone remodeling by regulating cell proliferation and differentiation. Genetic engineering technology combined with exosome-based drug delivery can therapy bone metabolic diseases. In this review, we summarized the pathways of exosomes derived from different skeletal cells (i.e., BMSCs, osteoblasts, osteocytes, and osteoclasts) regulate the skeletal microenvironment through proteins, mRNAs, and non-coding RNAs. By exploring the role of exosomes in the skeletal microenvironment, we provide a theoretical basis for the clinical treatment of bone-related metabolic diseases, which may lay the foundation to improve bone tumor microenvironments, alleviate drug resistance in patients.

Calcium responses to external mechanical stimuli in the multicellular stage of Dictyostelium discoideum

Calcium acts as a second messenger to regulate many cellular functions, including cell motility. In Dictyostelium discoideum, the cytosolic calcium level oscillates synchronously, and calcium waves propagate through the cell population during the early stages of development, including aggregation. In the unicellular phase, the calcium response through Piezo channels also functions in mechanosensing. However, calcium dynamics during multicellular morphogenesis are still unclear. Here, live imaging of cytosolic calcium revealed that calcium wave propagation, depending on cAMP relay, disappeared at the onset of multicellular body (slug) formation. Later, other forms of occasional calcium bursts and their propagation were observed in both anterior and posterior regions of migrating slugs. This calcium signaling also occurred in response to mechanical stimuli. Two pathways—calcium release from the endoplasmic reticulum via IP3 receptor and calcium influx from outside the cell—were involved in calcium signals induced by mechanical stimuli. These data suggest that calcium signaling is involved in mechanosensing in both the unicellular and multicellular phases of Dictyostelium development using different molecular mechanisms.

A look at early human embryonic development and the importance of darkness for embryogenesis

Human development is a highly complex process where a single cell called zygote forms a multicellular body made up of trillions of cells. Human pregnancy takes about 10 lunar months or 40 weeks. Prenatal life is divided into three stages namely germinal, embryonic and fetal stages. All stages are critical, but the most important is the embryonic stage through which embryogenesis occurs. It normally occurs in the upper segment of the uterus into its endometrium, called decidua after occurrence of conception. Many factors can affect embryogenesis including the mother's use of medications and even the mother's psychological state. Of these factors, light may be. The embryo is surrounded by three regions of darkness These areas from the inside out are the amniotic cavity, the uterine cavity, and the abdominal cavity. Light could affect embryonic development either by direct toxic effect on the embryo, or indirectly by photo-oxidation of components in media. Therefore, it is necessary to study them in future studies to investigate the potentially harmful effect of light exposure on embryonic development. This may be useful to avoid potential obstacles to the IVF process as embryos can be exposed to light sources of varying intensity.

Spontaneous and Driven Growth of Multicellular Lipid Compartments to Millimeter Size from Porous Polymer Structures**

AbstractThis report describes a method to obtain multicellular shaped compartments made by lipids growing from a sponge‐like porous structure. Each compartment is several tens of micrometers in diameter and separated by membranes comprised of phospholipid and amphipathic molecules. The multi‐compartment structure spontaneously grew to a millimeter scale, driven by an ionic concentration difference between the interior and exterior environments of the sponge. These compartments can also easily incorporate hydrophilic species as a well as smaller materials such as liposomes. Additionally, we showed that mechanical squeezing of the sponge was also effective in producing multicellular bodies. These simple methods to obtain large‐scale multicellular compartment of lipid membrane will help future designs and trials of chemical communications on artificial cells.

Social selection within aggregative multicellular development drives morphological evolution.

Aggregative multicellular development is a social process involving complex forms of cooperation among unicellular organisms. In some aggregative systems, development culminates in the construction of spore-packed fruiting bodies and often unfolds within genetically and behaviourally diverse conspecific cellular environments. Here, we use the bacterium Myxococcus xanthus to test whether the character of the cellular environment during aggregative development shapes its morphological evolution. We manipulated the cellular composition of Myxococcus development in an experiment in which evolving populations initiated from a single ancestor repeatedly co-developed with one of several non-evolving partners—a cooperator, three cheaters and three antagonists. Fruiting body morphology was found to diversify not only as a function of partner genotype but more broadly as a function of partner social character, with antagonistic partners selecting for greater fruiting body formation than cheaters or the cooperator. Yet even small degrees of genetic divergence between distinct cheater partners sufficed to drive treatment-level morphological divergence. Co-developmental partners also determined the magnitude and dynamics of stochastic morphological diversification and subsequent convergence. In summary, we find that even just a few genetic differences affecting developmental and social features can greatly impact morphological evolution of multicellular bodies and experimentally demonstrate that microbial warfare can promote cooperation.

Study on Transorgan Regulation of Intervertebral Disc and Extra-Skeletal Organs Through Exosomes Derived From Bone Marrow Mesenchymal Stem Cells.

Exosomes are membranous lipid vesicles fused with intracellular multicellular bodies and then released into the extracellular environment. They contain various bioactive substances, including proteins, mRNA, miRNAs, lncRNAs, circRNAs, lipids, transcription factors, and cytokine receptors. Under certain conditions, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts, chondrocytes, adipocytes, and biological functions. This study provides a theoretical basis for the application of exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) in osteology, exploring different sources of exosomes to improve bone microenvironment and resist bone metastasis. We also provided new ideas for the prevention and rehabilitation of human diseases by exosomes.