Cell shape changes and the mechanism of inversion in Volvox

Inversion is a dominant aspect of morphogenesis in Volvox. In this process, the hollow, spheroidal Volvox embryo turns inside-out through a small opening called the phialopore to bring flagella from its inner to its outer surface. Analyses of intact, sectioned, and fragmented embryos by light, scanning electron, and transmission electron microscopy, suggest that shape changes preprogrammed into the cells cause inversion. First, cells throughout the embryo change from pear to spindle shape, which causes the embryo to contract and the phialopore to open. Then cells adjacent to the phialopore become flask-shaped, with long, thin stalks at their outer ends. Simultaneously, the cytoplasmic bridges joining all adjacent cells migrate from the midpoint of the cells to the stalk tips. Together, these changes cause the lips of cells at the phialopore margin to curl outward. Now cells progressively more distal to the phialopore become flask-shaped while the more proximal cells become columnar, causing the lips to curl progressively further over the surface of the embryo until the latter has turned completely inside-out. Fine structural analysis reveals a peripheral cytoskeleton of microtubules that is apparently involved in cellular elongation. Cell clusters isolated before inversion undergo a similar program of shape changes; this suggests that the changes in cellular shape are the cause rather than an effect of the inversion process.     

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.     

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.     

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.     

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.     

Mechanism of formation, ultrastructure, and function of the cytoplasmic bridge system during morphogenesis in Volvox

The cytoplasmic bridge system that links all cells of a Volvox embryo and plays a crucial role in morphogenesis is shown to form as a result of localized incomplete cytokinesis; sometimes bridge formation occurs before other regions of the cell have begun to divide. Vesicles, believed to be derived from the cell interior, align along the presumptive cleavage furrow in the bridge-forming region. Apparently it is where these vesicles fail to fuse that bridges are formed. Conventional and high voltage transmission electron microscopy analyses confirm that bridges are regularly spaced; they possess a constant, highly ordered structure throughout cleavage and inversion. Concentric cortical striations (similar to those observed previously in related species) ring each bridge throughout its length and continue out under the plasmalemma of the cell body to abut the striations of neighboring bridges. These striations are closely associated with an electron-dense material that coats the inner face of the membrane throughout the bridge region and appears to be thickest near the equator of each bridge. In addition to the parallel longitudinal arrays of cortical microtubules that traverse the cells, we observed microtubules that angle into and through the bridges during cleavage; however, the latter are not seen once inversion movements have begun. During inversion, bridge bands undergo relocation relative to the cell bodies without any loss of integrity or change in bridge spacing. Observation of isolated cell clusters reveals that it is the sequential movement of individual cells with respect to a stationary bridge system, and not actual movement of the bridges, that gives rise to the observed relocation.     

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.     

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.     

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.     

Colony formation and inversion in the green algaEudorina elegans

Summary During development of daughter coenobia in the volvocalean algaEudorina a rapid synchronized series of mitotic divisions and cytokineses gives rise to a slightly cup-shaped, patterned array of 16 or 32 cells, the plakea; the nuclei and centrioles of each cell lying at the concave face and the plastids at the convex face. Each cell is connected to its neighbours by cytoplasmic bridges. All cells within a plakea simultaneously elongate and enlarge their nuclear poles; while remaining interconnected by the cytoplasmic bridges at their plastid poles. The result is inversion of the developing coenobia so that the nuclei and centrioles come to lie on the convex, outer surface. Inversion is inhibited by colchicine and cytochalasin B. Both lengthening of the cells and expansion of their nuclear end is apparently mediated by microtubules. Striations on the plasmalemma encircling the bridges are thought to stablize the membrane at these sites during inversion.     

The Protoplasmic Connection in Volvox

SYNOPSIS. Electron-microscopic observations were performed on 2 species of Volvox , one similar to V. globator , the other to V. aureus. The former has distinct protoplasmic connections in the adult coenobium and specific structures, named "medial bodies," in the connections just at the intersection with the middle lamella. The medial body is disk shaped, about 800 mμ in diameter, and is composed of 3 parts, 2 dense outer layers and an intermediate less dense zone. In the latter species, the connection and medial body were not seen. On the other hand, it was commonly seen in both of them that in younger, dividing gonidia neighboring protoplasts were connected with each other by protoplasmic bridges. The bridges are undoubtedly formed due to incomplete cell separation in the division of a gonidium. The structural difference in the adult coen***bium between the 2 species emerges just after inversion of the coenobium. In the globator type the medial body appears just after inversion, and the connection remains unruptured all thru life. In the aureus type, it seems that the connections are withdrawn or degenerate immediately after inversion. It is discussed whether protoplasmic continuity is really maintained by the connection or not in the freeswimming coenobium of Volvox.     

A NEW SPECIES OF VOLVOX SECT. MERRILLOSPHAERA (VOLVOCACEAE,CHLOROPHYCEAE) FROM TEXAS1

Smith (1944) divided the familiar genus Volvox L. into four sections, placing seven species that lacked cytoplasmic bridges between adult cells in the section Merrillosphaera. Herein, we describe a new member of the section Merrillosphaera originating from Texas (USA): Volvox ovalis Pocock ex Nozaki et A. W. Coleman sp. nov. Asexual spheroids of V. ovalis are ovoid or elliptical, with a monolayer of 1,000–2,000 somatic cells that are not linked by cytoplasmic bridges, an expanded anterior region, and 8–12 gonidia in the posterior region. Visibly asymmetric cleavage divisions do not occur in V. ovalis embryos as they do Volvox carteri F. Stein, Volvox obversus (W. Shaw) Printz, and Volvox africanus G. S. West, so the gonidia of the next generation are not yet recognizable in V. ovalis embryos prior to inversion. Molecular phylogenetic analyses of the five chloroplast genes and the internal transcribed spacer (ITS) regions of nuclear rDNA indicated that V. ovalis is closely related to Volvox spermatosphaera Powers ( Powers 1908 , as “spermatosphara”) and/or Volvox tertius Art. Mey.; however, V. ovalis can be distinguished from V. spermatosphaera by its larger gonidia, and from V. tertius by visible differences in gonidial chloroplast morphology.     

葫芦藓精子发生过程中胞间连丝与胞质桥的超微结构

从超微结构水平上对葫芦藓(Funaria hygrometrica Hedw.)精子发生过程中胞间连接系统的结构及其变化动态进行了研究.结果表明,同一区中的相邻生精细胞由大量胞质桥相连,而不同区的细胞之间则不存在胞质桥.胞间连丝存在于套细胞之间以及套细胞与生精细胞之间,但它在生精细胞间不存在.在精子器发生的后期,当精子细胞壁开始降解时,同一个精子器中所有的精子细胞似乎都由扩大的胞质桥相互连接.胞质桥一直保持到精子分化的后期,最终精子细胞同步分化成精子.胞间连丝与胞质桥具有不同的内部结、分布以及生物发生机制,这表明它们在精子器的发育过程中可能扮演着不同的角色.     

Cleavage, incomplete inversion, and cytoplasmic bridges in Gonium pectorale (Volvocales, Chlorophyta)

Multicellularity arose several times in evolution of eukaryotes. The volvocine algae have full range of colonial organization from unicellular to colonies, and thus these algae are well-known models for examining the evolution and mechanisms of multicellularity. Gonium pectorale is a multicellular species of Volvocales and is thought to be one of the first small colonial organisms among the volvocine algae. In these algae, a cytoplasmic bridge is one of the key traits that arose during the evolution of multicellularity. Here, we observed the inversion process and the cytoplasmic bridges in G. pectorale using time-lapse, fluorescence, and electron microscopy. The cytoplasmic bridges were located in the middle region of the cell in 2-, 4-, 8-, and 16-celled stages and in inversion stages. However, there were no cytoplasmic bridges in the mature adult stage. Cytoplasmic bridges and cortical microtubules in G. pectorale suggest that a mechanism of kinesin-microtubule machinery similar to that in other volvocine algae is responsible for inversion in this species.     

葫芦藓精子发生过程中胞间连丝与胞质桥的超微结构

从超微结构水平上对葫芦藓(Funaria hygrometrica Hedw.)精子发生过程中胞间连接系统的结构及其变化动态进行了研究。结果表明,同一区中的相邻生精细胞由大量胞质桥相连,而不同区的细胞之间则不存在胞质桥。胞间连丝存在于套细胞之间以及套细胞与生精细胞之间, 但它在生精细胞间不存在。在精子器发生的后期,当精子细胞壁开始降解时,同一个精子器中所有的精子细胞似乎都由扩大的胞质桥相互连接。胞质桥一直保持到精子分化的后期,最终精子细胞同步分化成精子。胞间连丝与胞质桥具有不同的内部结、分布以及生物发生机制,这表明它们在精子器的发育过程中可能扮演着不同的角色。     

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.     

水稻中央细胞发育期间超微结构变化的观察

本文通过透射电镜对水稻受精前胚囊中央细胞发育过程中超微结构的变化进行观察。结果表明,八核胚囊形成后很快就进行细胞化形成7个细胞,其中刚形成的中央细胞由1个大液泡、2个极核（珠孔端和合点端各1个）和一些含有丰富细胞器的胞质组成。中央细胞以后的发育主要是极核的发育和极核周围胞质的变化。极核发育经历以下过程：a．2个核都膨大呈“椭圆”形。核周围胞质呈不对称分布。b．2个核分别向胚囊中央移动并相互靠近。之后2个极核调整排列方式,由纵排（即与胚囊纵轴平行）变成横排。此时期有细胞质“桥”联结珠孔端卵器、2个极核和合点端反足细胞器。c．横排的极核移向卵器,并排列于卵细胞之上。此时胚囊未明显膨大,但极核相靠近的两边核膜有许多处已形成“融合桥”,核周围的胞质也起较大的变化,如质体内淀粉消失和光面内质网增加等。极核进一步发育直至胚囊成熟期间,极核排列方式及其周围胞质组成未观察到明显的变化,但胚囊体积明显增大。     

Ultrastructural study of a cytoplasmic bridge connecting a pair of erythroblasts in mice

Summary A unique cytoplasmic connection between erythroblasts was studied by electron microscopy in mouse hemopoietic tissues (fetal liver, fetal and neonatal spleen and adult bone marrow). Many pairs of interphase erythroblasts were connected by a cytoplasmic bridge that was very thin and sometimes long in comparison with telophase bridges. The stage of maturation of the cells in a pair was similar. Small numbers of microtubules ran along the cytoplasmic bridge; a mid-body was not seen. The plasma membrane at approximately the middle of the bridge bulged to form a ring-shaped ridge filled with dense amorphous substances; this was called a bulging ring. Thus, the cytoplasmic bridge between erythroblasts did not morphologically correspond to the telophase bridge in the usual cytokinesis. Cytoplasmic bridges were observed in various differentiating stages of erythroblasts, whereas other cell types of the hemopoietic lineage did not have such a bridge. The cytoplasmic bridge is unique to erythroblasts and provides an evidence for the atypical cytokinesis of the erythroblastic lineage.