Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a ‘sticky’ cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles.     

Mechanism of an Alternate Type of Echinoderm Blastula Formation: The Wrinkled Blastula of the Sea Urchin Heliocidaris erythrogramma

While most indirect-developing echinoderms (possessing a feeding larval stage) form a hollow, smooth-walled blastula, most direct-developing species form a wrinkled blastula. The process of wrinkled blastula formation was examined in the direct-developing sea urchin, Heliocidaris erythrogramma . Approximately 5 hrs after fertilization the blastula epithelium contains folds along one, two or three orthogonal planes, which superficially appear like 2-, 4- or 8-cell stages, respectively. Microinjection of fluorescent dye into individual blastomeres of 2-, 4- and 8-cell embryos revealed that the wrinkles correspond with the first, second and third cleavage planes. Two factors appear to generate the wrinkled blastula epithelium. First, blastomeres undergo a partial separation along the first, second and third cleavage planes during early cleavage. Subsequent cell divisions are oriented such that the blastula epithelium is constructed with deep creases along these planes of cell separation. Second, there is no room for the expansion of the developing blastoderm within the tightly fitting fertilization envelope. Prior to hatching from the fertilization envelope, wrinkles in the blastula epithelium disappear, due to an increased packing and elongation of the cells. In addition, a substantial volume of cellular material is removed as lipids are secreted into the blastocoel in an apocrine fashion.     

Involvement of a neutral glycolipid in differential cell adhesion in the Xenopus blastula.

Many different molecular species mediate cell adhesion during embryonic development. These can have either protein or carbohydrate functional groups, which can act in either a homophilic or a heterophilic manner, and often in concert. We report here that a monoclonal antibody, M4B, raised against Xenopus blastomere membranes, inhibits the calcium-dependent adhesion of dissociated blastomeres. M4B maintains its inhibitory effect on adhesion when converted into univalent fragments, and specifically affects calcium-dependent adhesion. The antigen is regulated in both space and time during early development. It is found on cell surfaces throughout the egg to blastula stages, but is more concentrated on cells in the animal and marginal zones of the blastula. It is dramatically downregulated during gastrulation, and becomes largely restricted to gut epithelium by the larval stages. We show also that M4B function is spatially differentiated at the blastula stage, since it inhibits the aggregation of dissociated animal cells to a greater extent than vegetal cells. This membrane antigen may therefore play a role in the differential adhesion observed between different regions of the blastula, and which we presume to underlie the segregation of the primary germ layers during gastrulation. M4B recognizes a complex of plasma membrane glycolipids. Periodate treatment destroys the ability of these glycolipids to react with the antibody, indicating that the epitope resides in the carbohydrate moiety of the glycolipids. Chemical characterization shows that it is a neutral glycolipid, and that the major component is of the glycoglycerolipid, rather than the more common glycosphingolipid class. Blocking experiments with oligosaccharides of defined structure, and antibody crossreactivity show that the M4B antibody does not recognize several known embryonic carbohydrate antigens. These results demonstrate that M4B antibody recognizes a novel group of developmentally regulated glycolipids which function in calcium-dependent cell--cell adhesion in the Xenopus blastula.     

Cancer as a consequence of the rising level of oxygen in the Late Precambrian

The origin of multicelled animal life required collagen-family molecules whose own formation depended on the availability of molecular oxygen. Cancers, by contrast, are characterized by their low use of oxygen. In discussing the relationship between the origin of multicelled life and the origin of cancer, it is useful to think in terms of tissues rather than individual cells or complete animals. When animal tissues are disturbed, their constituent cells may be partially released from the constraints of multicellularity. This permits or obliges cells to reactivate anaerobic metabolic ways used by their single-celled ancestors in the oxygen-deficient Precambrian seas. Inhibition or loss of cell respiration under such circumstances may cause reversion to glycolytic fermentation, a less efficient metabolic style that generates waste products that are retained, thereby producing excess cell-growth. Distortion of tissue architecture may ensue with impairment of cell-to-cell adhesion, thereby liberating individual cells. Cells freed from tissue constraints undergo Darwinian variation which leads to loss of differentiation and produces cell types that are incompatible with the normal functioning of tissues. These steps, which may manifest themselves as carcinogenesis, are not reversible by restoration of oxygen and in effect constitute a demergence from the metazoan state. The existence of cancer among diverse phyla and especially among domesticated animals, suggests that the risk of cancer may be an initial condition of complex multicellular life and that it remains preferentially associated with newly modified designs. If so, there would be therapeutic strategies that have not yet been adequately considered. □ Cambrian explosion, cancer, cell differentiation, collagen, glycolysis, hard parts, metazoan origins.     

Reversine promotes porcine muscle derived stem cells (PMDSCs) differentiation into female germ‐like cells

A small molecular chemical‐Reversine has been shown to promote cell reprogramming and induce dedifferentiation of multiple terminally differentiated mesodermal origin cells, and then differentiate into other cell types within mesodermal lineages as well as neuroectodermal. However, the possibilities of these cells to give rise to germ cell lineages have not been examined. The objective of the current study was to detect the effect of Reversine on PMDSCs differentiation into germ cells. PMDSCs from fetal porcine skeletal muscle and their potential of differentiation into germ cells in vitro were investigated. The phenotype, proliferation potential, characteristic markers of the first adhesion cells (pp1), and the purified 2 times cells (pp3) were analyzed by growth curve, FACS, and RT‐PCR, respectively. Then, the purified cells were induced with 10% or 20% bovine follicular fluid (FF), the results showed that some of the induced pp3 cells were similar as porcine oocyte, and expressed germ cell and oocyte markers analyzed by semi‐quantitative RT‐PCR and immunofluorescent staining. Reversine clearly increased the potentiality of PMDSCs differentiation into large round germ‐like cells in FF induction medium analyzed by morpholgogy, QRT‐PCR and immunofluoresce. The BrdU labeled PMDSCs might differentiate into female germ‐like cells in recipient's kidney capsule, which were positive for germ cell and meiotic markers (Dazl, Vasa, Figla, Stra8, Scp3) and oocyte markers (Zp2, Zp3). These findings provided an efficient model to study the mechanism of cell proliferation and germ cell differentiation in livestock promoted by Reversine. J. Cell. Biochem. 113: 3629–3642, 2012. © 2012 Wiley Periodicals, Inc.     

Cadherin-based cell adhesion in neuromuscular development

The organisation and differentiation of striated skeletal muscles and their innervation is a particularly complex process implicating cells of mesodermic (myoblasts and fibroblasts) and neuroectoderrmic origin (neurons and glial cells). Myogenic and motor neuron precursors, the two major cell types participating in the formation of the neuromuscular axis, migrate, segregate, reassociate and differentiate in a coordinated fashion. The subsequent organisation of muscle cells and the establishment of muscle innervation rely on a complex tissular and cellular architectural organisation, which cannot be understood without taking into account juxtacrine cell interactions, and especially cell adhesion. Cell adhesion receptors of the cadherin family are widely expressed and dynamically regulated in space and time throughout neuromuscular development. A single cell expresses in general more than one cadherin at its surface and it is the combination of these molecules and their level of expression that determine their action within a given cell population. We focused in this review on the expression and roles of classical cadherins in relation to muscle cell and motoneuron differentiation. We also review the latest results on the mode of action of cadherins allowing to propose cellular and molecular cues on the mechanisms by which these cell adhesion receptors control muscle and neuronal cell shape, migration and differentiation.     

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor‐derived gametes

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES‐like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma‐irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor‐derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells. Mol. Reprod. Dev. 53:394–397, 1999. © 1999 Wiley‐Liss, Inc.     

The contribution of adhesion signaling to lactogenesis

The mammary gland undergoes hormonally controlled cycles of pubertal maturation, pregnancy, lactation, and involution, and these processes rely on complex signaling mechanisms, many of which are controlled by cell–cell and cell–matrix adhesion. The adhesion of epithelial cells to the extracellular matrix initiates signaling mechanisms that have an impact on cell proliferation, survival, and differentiation throughout lactation. The control of integrin expression on the mammary epithelial cells, the composition of the extracellular matrix and the presence of secreted matricellular proteins all contribute to essential adhesion signaling during lactogenesis. In vitro and in vivo studies, including the results from genetically engineered mice, have shed light on the regulation of these processes at the cell and tissue level and have led to increased understanding of the essential signaling components that are regulated in temporal and cell specific manner during lactogenesis. Recent studies suggest that a secreted matricellular protein, CTGF/CCN2, may play a role in lactogenic differentiation through binding to β1 integrin complexes, enhancing the production of extracellular matrix components and contributions to cell adhesion signaling.     

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin‐like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re‐expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373‐MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373‐MG cell lines expressing hepaCAM at different levels. The results revealed that high‐level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low‐level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373‐MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM. J. Cell. Biochem. 107: 1129–1138, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.     

All animals develop from a blastula: consequences of an undervalued definition for thinking on development.

An early embryo becomes a blastula at the moment that its constituent cells become organised into a simple epithelium. Epithelial folding and compartmentation are essential elements of animal development. All the different cell types--epithelial and other ones--of which a differentiated organism consists differ in their plasmamembrane-cytoskeletal complex but they are assumed to have an identical genome. The hypothesis is put forward that, perhaps, the basic mechanism underlying differentiation can be defined as the generation of cells which have an identical genome but which differ in their plasmamembrane-cytoskeletal complex and which, because of these differences, can engage in differential protein synthesis-physiology.     

Regulation of heterochromatin remodelling and myogenin expression during muscle differentiation by FAK interaction with MBD2

Focal adhesion kinase (FAK), a major cell adhesion‐activated tyrosine kinase, has an important function in cell adhesion and migration. Here, we report a new signalling of FAK in regulating chromatin remodelling by its interaction with MBD2 (methyl CpG‐binding protein 2), underlying FAK regulation of myogenin expression and muscle differentiation. FAK interacts with MBD2 in vitro, in myotubes, and in isolated muscle fibres. Such an interaction, increased in myotubes exposed to oxidative stress, enhances FAK nuclear localization. The nuclear FAK–MBD2 complexes alter heterochromatin reorganization and decrease MBD2 association with HDAC1 (histone deacetylase complex 1) and methyl CpG site in the myogenin promoter, thus, inducing myogenin expression. In line with this view are observations that blocking FAK nuclear localization by expressing dominant negative MBD2 or suppression of FAK expression by its miRNA in C2C12 cells attenuates myogenin induction and/or impairs muscle‐terminal differentiation. Together, these results suggest an earlier unrecognized role of FAK in regulating chromatin remodelling that is important for myogenin expression and muscle‐terminal differentiation, reveal a new mechanism of MBD2 regulation by FAK family tyrosine kinases, and provide a link between cell adhesion and chromatin remodelling.     

Finite element,occlusal, microwear and microstructural analyses indicate that conodont microstructure is adapted to dental function

Conodonts constitute the earliest evidence of skeletal biomineralization in the vertebrate evolutionary lineage, manifest as a feeding apparatus of tooth‐like elements comprised of enamel‐ and dentine‐like tissues that evolved in parallel with these canonical tissues in other total‐group gnathostomes. As such, this remarkable example of evolutionary parallelism affords a natural experiment in which to explore the constraints on vertebrate skeletal evolution. Using finite element analysis, informed by occlusal and microwear analyses, we tested the hypothesis that coincidence of complex dental function and microstructural differentiation in the enamel‐like tissues of conodonts and other vertebrates is a consequence of functional adaptation. Our results show topological co‐variation in the patterns of stress distribution and crystallite orientation. In regions of high stress, such as the apex of the basal cavity and inner parts of the platform, the crown tissue comprises interwoven prisms, discontinuities between which would have acted to decussate cracks, preventing propagation. These results inform a general occlusal model for platform conodont elements and demonstrate that the complex microstructure of conodont crown tissue is an adaptation to the dental functions that the elements performed.     

Self‐organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self‐organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self‐organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm‐mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro‐structured meshes with limited surface area for cell adhesion. We show that this adhesion‐restriction strategy can trigger a two‐stage self‐organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8–10, then into spherical cysts following differentiation and self‐organization of the sheet‐forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two‐photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E‐cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late‐stage blastocyst. Thus, the mesh culture microenvironment can initiate self‐organization of hiPSCs into trophectoderm‐mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.     

A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering

The gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals.In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors.We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell–cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies.     

The hepatocyte ultrastructure of the Sevan trout (Salmo ischchan gegarkuni Kessel) in ontogeny]

Hepatocytes of Lake Sevan Salmo were examined at several stages in their life cycles which are different from the point of view of the manner of feeding. Salmo were reared at the fish farm, they were fed with the yolk of the chick eggs. It was revealed that hepatocytes of larva, which was sampled immediately after hatching (endogenous feeding) intensively synthesized the proteins; accumulated and secreted the bile product stored glycogen and lipids. The ultrastructure of larva hepatocytes changed on the 5th and 10th day after larva began to accept food (mixed feeding--endogenous and exogenous). Golgi complex became bigger, glycogen disappeared, lipid droplets became smaller (on the 5th day) and disappeared completely (on the 10th day). Morphological differentiation finishes during the fingerling period (exogenous feeding). Cisternae of granular endoplasmic reticulum (GER) and mitochondria are arranged around nucleus, near bile canaliculus and sinusoids. Big areas of glycogen lie between the organelles. Relative volumes of GER, mitochondria, glycogen increased, but the relative volume of Golgi complex diminished.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

NOSTOCACEAN MACRO‐MORPHOGENESIS

A poorly understood feature of nostocacean growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. Using macro‐photography, time‐lapse micro‐cinematography, light and electron microscopy of Nostoc species in pure culture, it has been possible to demonstrate how motility, adhesion and aggregation of photo‐induced hormogonia result in macro‐morphogenesis of dendroid forms. Red‐light induced hormogonia from synchronized cultures aggregate rapidly on agar as tight flowing streams, in patterns responsive to the direction and quality of incident light. Unlike the even textured cell surfaces of heterocystous filaments, the cell walls of swarming hormogonia are covered with a striate mucoid layer containing pili attached to cells of adjacent hormogonia. During differentiation to an aseriate phase, cell wall fusions occur and a gelatinous matrix forms around the enlarging sub‐globose cells. Liquid suspensions of hormogonia aggregate in a solid mass following the net centripetal movement of interlaced loops of curved hormogonia attached by adhesive pili. In darkness or dim white light, compressed hormogonial aggregates form erect tree‐like (dendroid) macro‐structures by photo‐tactic reversal of streaming motility. Hormogonia within the aggregates re‐organize into streams that push upward into the light, forming structured, positively phototropic protuberances, several millimeters in length. Under weak illumination, the structures become branched with crowns of waving hormogonia. The dendroid morphology is stabilized by deposit of gelatinous material derived from successive cycles of cell‐filament development, liberation of heterocysts and formation of dormant cells and trichomes.