Actomyosin contraction of the posterior hemisphere is required for inversion of the Volvox embryo

During inversion of a Volvox embryo, a series of cell shape changes causes the multicellular sheet to bend outward, and propagation of the bend from the anterior to the posterior pole eventually results in an inside-out spherical sheet of cells. We use fluorescent and electron microscopy to study the behavior of the cytoskeleton in cells undergoing shape changes. Microtubules are aligned parallel to the cell's long axis and become elongated in the bend. Myosin and actin filaments are arrayed perinuclearly before inversion. In inversion, actin and myosin are located in a subnuclear position throughout the uninverted region but this localization is gradually lost towards the bend. Actomyosin inhibitors cause enlargement of the embryo. The bend propagation is inhibited halfway and, as a consequence, the posterior hemisphere remains uninverted. The arrested posterior hemisphere will resume and complete inversion even in the presence of an actomyosin inhibitor if the anterior hemisphere is removed microsurgically. We conclude that the principal role of actomyosin in inversion is to cause a compaction of the posterior hemisphere; unless the equatorial diameter of the embryo is reduced in this manner, it is too large to pass through the opening defined by the already-inverted anterior hemisphere.     

Morphogenesis in Volvox: analysis of critical variables.

Inversion, the process by which Volvox embryos turn inside out, was analyzed by a combination of geometrical and experimental techniques. It was shown that simple geometric figures are adequate to represent cell shapes during inversion and that cell volumes remain constant as cell shapes change and the embryo inverts. The first stage of inversion, phialopore opening, results from the release of compressive forces as the embryo withdraws from its surrounding vesicle during a two-stage contraction of each cell around its radial axis. Premature phialopore opening occurs when withdrawal of the embryo from the vesicle is elicited artificially by exposure to either calcium ionophore or hypertonic solutions. The major event of inversion, generation of negative curvature, requires both microtubule-driven elongation of cells (to produce a classical "flask" shape) and cytochalasin-sensitive active migration of cytoplasmic bridges to the outermost ends of flask cells. Colchicine, cyclic GMP and isobutyl methyl xanthine (individually) block both normal elongation and bridge migration; cytochalasin D blocks bridge migration selectively. Flask cell formation and bridge migration are adequate to account for the negative curvature observed. An asymmetric bending of flask cell stalks along the ring of maximum curvature accounts for the fact that the embryo is not constricted in a "purse-string" fashion as negative curvature is generated. Inversion of the posterior hemisphere involves an elastic snap-through resulting from a combination of compressive stresses generated by inversion of the anterior hemisphere and the circumferential restraint imposed by cells at the equator. We conclude that the observed changes in cell shape and the migration of cytoplasmic bridges are the result of an ordered process of membrane-cytoskeletal interactions, and both necessary and sufficient to account for the morphogenetic process of inversion in Volvox.     

INVERSION IN VOLVOX (CHLOROPHYCEAE)1

The asexual embryos of Volvox turn themselves inside out (invert) during development. Data presented indicate that inversion of the embryos is the result of several simple cellular shape changes, coordinated in space and time. Using whole embryos, cell groups and individual embryonic cells isolated by watchmaker's forceps and pressure on the coverslip, it was shown that the phialopore (opening) enlargement and concurrent stretching of the border cells is due to a constriction formed at the equator. However, if the posterior hemisphere is removed, this constriction no longer effects the expansion of the phialopore (which is in the anterior hemisphere) because the equatorial region is no longer anchored and has no base to support the outwardly directed force against the phialopore cells. If the posterior hemisphere is isolated several hours before inversion, the opening resulting from the incision acts as a “phialopore” and the direction of inversion is reversed. Individual cells and cells in groups undergo the same shape changes as corresponding cells in an intact embryo during inversion. This suggests that these cellular deformations are autonomous and inversion is a result of them.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: an ultrastructural study

Inside-out porcine thyroid follicles in culture undergo polarity reversal after being embedded in collagen gel. The newly-formed follicles reexpress some specific thyroid functions lost in inside-out follicles (Chambard et al., 1984. We present here an ultrastructural study of the inversion of polarity in this model system. This process takes place within 24 to 48 hr, without any opening of the original tight junctions, as shown by fixation in the presence of ruthenium red. A general shrinkage of cellular aggregates was noted soon after embedding. At the apical pole, three different modifications were observed: structural changes appeared in the kinocilium, microvilli and underlying cytoskeleton as early as 10 min after embedding, mainly when the apical pole of the cells was in close contact with the collagen fibers; large cytoplasmic lamellipod- or pseudopod-like extensions, covering the adjacent apical domain, protruded from outer apical regions; some other apical areas invaginated and formed channels inside the aggregates. The last two processes prevented close contact between apical cell surfaces and collagen fibers and allowed a persistence of the initial polarity in some of the cells. Newly-formed lumens were closed 24 hr after embedding in gel and the outer surface of the cellular aggregates in close contact with collagen fibers looked like a basal membrane. These mechanisms proceeded at different rates and involved different numbers of cells, but they all appeared to be related to the transformation of inside-out follicles into follicular structures.     

Morphogenesis in the family Volvocaceae: different tactics for turning an embryo right-side out

Green algae of the family Volvocaceae provide an unrivalled opportunity to analyze an evolutionary pathway leading from unicellularity to multicellularity with division of labor. One key step required for achieving multicellularity in this group was the development of a process for turning an embryo inside out: a morphogenetic process that is now known as "inversion," and that is a diagnostic feature of the group. Inversion is essential because at the end of its embryonic cleavage divisions, each volvocacean embryo contains all of the cells that will be present in an adult, but the flagellar ends of all cells are pointed toward the interior, rather than toward the exterior where they will need to be to function in locomotion. Inversion has been studied in greatest detail in Volvox carteri, but although all other volvocacean species have to struggle with the same awkward situation of being wrong-side out at the end of cleavage, they do it in rather different ways. Here, the inversion processes of six different volvocacean species (Gonium pectorale, Pandorina morum, Eudorina unicocca, Volvox carteri, Volvox tertius, and Volvox globator) are compared, in order to illustrate the variation in inversion patterns that exists within this family. The simplest inversion process occurs in the plate-shaped alga Gonium pectorale and the most complicated in the spherical alga Volvox globator. Gonium pectorale goes only from a concave-bowl shape to a slightly convex plate. In Volvox globator, the posterior hemisphere inverts completely before the anterior pole opens and the anterior hemisphere slides over the already-inverted posterior hemisphere; during both halves of this inversion process, the regions of maximum cell-sheet curvature move progressively, as radially symmetrical waves, along the posterior-anterior axis.     

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

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

A kinesin,invA, plays an essential role in volvox morphogenesis

In Volvox carteri adults, reproductive cells called gonidia are enclosed within a spherical monolayer of biflagellate somatic cells. Embryos must "invert" (turn inside out) to achieve this configuration, however, because at the end of cleavage the gonidia are on the outside and the flagellar ends of all somatic cells point inward. Generation of a bend region adequate to turn the embryo inside out involves a dramatic change in cell shape, plus cell movements. Here, we cloned a gene called invA that is essential for inversion and found that it codes for a kinesin localized in the cytoplasmic bridges that link all cells to their neighbors. In invA null mutants, cells change shape normally, but are unable to move relative to the cytoplasmic bridges. A normal bend region cannot be formed and inversion stops. We conclude that the InvA kinesin provides the motile force that normally drives inversion to completion.     

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.     

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.     

Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo

During development Caenorhabditis elegans changes from an embryo that is relatively spherical in shape to a long thin worm. This paper provides evidence that the elongation of the body is caused by the outermost layer of embryonic cells, the hypodermis, squeezing the embryo circumferentially. The hypodermal cells surround the embryo and are linked together by cellular junctions. Numerous circumferentially oriented bundles of microfilaments are present at the outer surfaces of the hypodermal cells as the embryo elongates. Elongation is associated with an apparent pressure on the internal cells of the embryo, and cytochalasin D reversibly inhibits both elongation and the increase in pressure. Circumferentially oriented microtubules also are associated with the outer membranes of the hypodermal cells during elongation. Experiments with the microtubule inhibitors colcemid, griseofulvin, and nocodazole suggest that the microtubules function to distribute across the membrane stresses resulting from microfilament contraction, such that the embryo decreases in circumference uniformly during elongation. While the cytoskeletal organization of the hypodermal cells appears to determine the shape of the embryo during elongation, an extracellular cuticle appears to maintain the body shape after elongation.     

Transfer Cells in Suspensur and Endosperm during Early Embryogeny of Vigna sinensis

During early embryogeny, structural differentiation of the suspensor and endosperm can be observed with the formation of cells with wall ingrowths. In the early proembryo stage, wall ingrowths are seen only on the boundary walls of the embryo sac around the proembryo and at the chalazal end. Later, ingrowths appear in the outer walls of the basal suspensor cells and some wall ingrowths also begin to develop in the outer walls of cellular endospermic cells adjacent to the nucellar cap and the inner integumentary tissues. The suspensor appears to remain active throughout the differentiation stages. Two regions can be clearly distinguished in the suspensor: a basal region and a neck region. Wall ingrowths appear to form only in the cells of the basal region. During the development of the cellular endospermic sheath, its cell number and size both increase slightly. Later, these cells rapidly become separated from each other. Those endospermic cells that abut directly onto the integumentary tissues also develop wall ingrowths. In the region of the fluid endosperm, wall ingrowths are especially abundant in the boundary walls on the ventral side of the embryo sac. The possible pathway of nutrient flow to the developing embryo is discussed.     

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.     

Cell surface changes during preimplantation development in the mouse

Scanning electron microscopy reveals microvilli on all preimplantation stages, indicates that their number and length may be dependent on embryo size, and provides examples of regional alterations in their number. Cellular adherence, as evidence by interactions of microvilli, migration of cellular processes, and junctional complexes, increases during development and is accompanied by changes in the shapes of cells and embryos. Cell surfaces bordering the blastocoel differ markedly from the outer cell surfaces of the embryo.     

Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes

Abstract. Early development and metamorphosis of Reniera sp., a haplosclerid demosponge, have been examined to determine how gastrulation occurs in this species, and whether there is an inversion of the primary germ layers at metamorphosis. Embryogenesis occurs by unequal cleavage of blastomeres to form a solid blastula consisting micro- and macromeres; multipolar migration of the micromeres to the surface of the embryo results in a bi-layered embryo and is interpreted as gastrulation. Polarity of the embryo is determined by the movement of pigment-containing micromeres to one pole of the embryo; this pole later becomes the posterior pole of the swimming larva. The bi-layered larva has a fully differentiated monociliated outer cell layer, and a solid interior of various cell types surrounded by dense collagen. The pigmented cells at the posterior pole give rise to long cilia that are capable of responding to environmental stimuli. Larvae settle on their anterior pole. Fluorescent labeling of the monociliated outer cell layer with a cell-lineage marker (CMFDA) demonstrates that the monociliated cells resorb their cilia, migrate inwards, and transdifferentiate into the choanocytes of the juvenile sponge, and into other amoeboid cells. The development of the flagellated choanocytes and other cells in the juvenile from the monociliated outer layer of this sponge's larva is interpreted as the dedifferentiation of fully differentiated larval cells—a process seen during the metamorphosis of other ciliated invertebrate larvae—not as inversion of the primary germ layers. These results suggest that the sequences of development in this haplosclerid demosponge are not very different than those observed in many cnidarians.     

Polyadenylated RNA of Volvox: isolation and partial characterization

Polyadenylated RNA from Volvox carteri has been isolated and partially characterized. Electrophoretic profiles of total cellular poly(A)-associated RNA of Volvox spheroids indicate a hetero-disperse distribution of size classes with the range extending from an apparent sedimentation value of approximately 10S to greater than 38S. The radioactive labelling kinetics of this material are typical for rapidly-turning-over RNA. The profiles of poly(A) RNA from different cell types show marked differences in average migration rate. Terminally-differentiated somatic cells contain a greater proportion of material of higher molecular weight than either gonidia (germ cells) or cleaving embryos. The poly(A) segments associated with cellular RNA, obtained by selective RNase digestion are heterogeneous in size as determined by gel electrophoresis with the largest tracts estimated to be 75-80 nucleotides long. Gonidia and embryos display the greatest degree of size heterogeneity, while somatic cells show predominantly the largest classes of poly(A) tract. It is apparent that gross changes in poly(A) RNA metabolism accompany development and cellular differentiation in Volvox.     

Urethral chromaffin cells

Summary The urethral mucosa of the rat, rabbit and guinea-pig was examined with both fluorescence and electron microscopy. Employing the former technique, numerous brightly fluorescing flask-shaped cells were observed amongst the basal cells of the urethral epithelium in all three species. In the electron microscope cells with a similar shape and distribution are distinguished by their content of membrane-limited dense granules, extensive Golgi membranes and bundles of filaments. In favourable planes of section short microvilli extend from the apical region of these cells which are joined to neighbouring urethral epithelial cells by zonulae occludentes. These fluorescent, granule-containing cells are classified as urethral chromaffin cells.Fluorescent nerves were not observed in relation to the urethral epithelium although the electron microscope revealed axons lying singly or in groups both beneath and between the urethral epithelial cells. Many of these axons appear varicose and contain small, agranular vesicles, a few large granulated vesicles and numerous mitochondria. Occasionally a vesicle-containing axon lay adjacent to a urethral chromaffin cell. While a direct autonomic innervation of these cells could not be discounted it is concluded that the majority of nerves probably perform a sensory function.     

Scanning electron microscopy of in vitro chemically transformed mouse embryo cells

A cloned nontumorigenic control cell line of C3H mouse embryo cells (C3H/1OT1/2CL8) and two cell lines derived from it by treatment in vitro with 7,12-dimethylbenz(a)anthracene (DMBA) or 3- methylcholanthrene (MCA) were studied by scanning electron microscopy. Confluent control cells were polygonal in shape and extensively flattened with smooth surfaces. Both in vitro transformants were pleomorphic to fusiform in shape, thicker than the control cells, and lacked contact inhibition. Microvilli of variable length and small marginal ruffles were characteristic surface alterations of the MCA- transformed cells, while blebs and numerous cytoplasmic strands extending between cells were typical of the DMBA transformant. Inoculation of the DMBA-transformed cells into C3H mice and re- establishment of cells from one of the subsequent fibrosarcomas in culture revealed an increased number of microvilli on the surface of the cells and an alteration in growth pattern. Other surface characteristics remained the same. A possible relationship between surface topography and outer membrane glycolipids is discussed.     

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in Arabidopsis thaliana

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.