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.     

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.     

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.     

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.     

Modeling the establishment of PAR protein polarity in the one-cell C. elegans embryo

At the one-cell stage, the C. elegans embryo becomes polarized along the anterior-posterior axis. The PAR proteins form complementary anterior and posterior domains in a dynamic process driven by cytoskeletal rearrangement. Initially, the PAR proteins are uniformly distributed throughout the embryo. After a cue from fertilization, cortical actomyosin contracts toward the anterior pole. PAR-3/PAR-6/PKC-3 (the anterior PAR proteins) become restricted to the anterior cortex. PAR-1 and PAR-2 (the posterior PAR proteins) become enriched in the posterior cortical region. We present a mathematical model of this polarity establishment process, in which we take a novel approach to combine reaction-diffusion dynamics of the PAR proteins coupled to a simple model of actomyosin contraction. We show that known interactions between the PAR proteins are sufficient to explain many aspects of the observed cortical PAR dynamics in both wild-type and mutant embryos. However, cytoplasmic PAR protein polarity, which is vital for generating daughter cells with distinct molecular components, cannot be properly explained within such a framework. We therefore consider additional mechanisms that can reproduce the proper cytoplasmic polarity. In particular we predict that cytoskeletal asymmetry in the cytoplasm, in addition to the cortical actomyosin asymmetry, is a critical determinant of PAR protein localization.     

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca²⁺] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal–vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.     

Differential expression of the adhesion molecule Echinoid drives epithelial morphogenesis in Drosophila

Epithelial morphogenesis requires cell movements and cell shape changes coordinated by modulation of the actin cytoskeleton. We identify a role for Echinoid (Ed), an immunoglobulin domain-containing cell-adhesion molecule, in the generation of a contractile actomyosin cable required for epithelial morphogenesis in both the Drosophila ovarian follicular epithelium and embryo. Analysis of ed mutant follicle cell clones indicates that the juxtaposition of wild-type and ed mutant cells is sufficient to trigger actomyosin cable formation. Moreover, in wild-type ovaries and embryos, specific epithelial domains lack detectable Ed, thus creating endogenous interfaces between cells with and without Ed; these interfaces display the same contractile characteristics as the ectopic Ed expression borders generated by ed mutant clones. In the ovary, such an interface lies between the two cell types of the dorsal appendage primordia. In the embryo, Ed is absent from the amnioserosa during dorsal closure, generating an Ed expression border with the lateral epidermis that coincides with the actomyosin cable present at this interface. In both cases, ed mutant epithelia exhibit loss of this contractile structure and subsequent defects in morphogenesis. We propose that local modulation of the cytoskeleton at Ed expression borders may represent a general mechanism for promoting epithelial morphogenesis.     

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo

Morphogenesis of the Drosophila melanogaster embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. We show that the guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. We demonstrate that DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. We propose that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. Our results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors.     

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.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA‐mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.     

Distribution of actin and myosin in mouse trophoblast: Correlation with changes in invasiveness during development in vitro

Mouse trophoblast is an invasive tissue that undergoes conversion to a noninvasive state during normal development. We examined the distribution of actin and myosin during trophoblast development in vitro with double label fluorescence microscopy using fluoresceinated subfragment-1 of myosin to identify actin and indirect immunofluorescence with rhodamine-conjugated antibody to detect myosin. During the outgrowth stage trophoblast spread as a sheet by active movement of the marginal cells. These cells exhibited different patterns of actin and myosin distribution in connection with lamellar extension and fiber formation. Marginal and submarginal cells were packed with overlapping layers of actin fibers, some of which were organized into a lattice that extended throughout the trophoblast. The cytoskeletal function of the fibers appeared to involve maintenance of the cells in a coherent sheet. Cessation of trophoblast spreading was associated with conversion of the cell sheet into a cell network. Cells stained more densely for actin and myosin and contained distinctive actomyosin condensations in the cortex and the cytoplasm. At the same time there was disorganization and then loss of the actin fiber system. These changes in actin and myosin distribution may be associated with mechanisms that control invasiveness by limiting trophoblast expansion.     

Tissue elongation requires oscillating contractions of a basal actomyosin network

Understanding how molecular dynamics leads to cellular behaviours that ultimately sculpt organs and tissues is a major challenge not only in basic developmental biology but also in tissue engineering and regenerative medicine. Here we use live imaging to show that the basal surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or their coupling to extracellular matrix (ECM) blocked elongation of the whole tissue, whereas enhancement of the contractions exaggerated it. Myosin accumulated in a periodic manner before each contraction and was regulated by the small GTPase Rho, its downstream kinase, ROCK, and cytosolic calcium. Disrupting the link between the actin cytoskeleton and the ECM decreased the amplitude and period of the contractions, whereas enhancing cell-ECM adhesion increased them. In contrast, disrupting cell-cell adhesions resulted in loss of the actin network. Our findings reveal a mechanism controlling organ shape and an experimental model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet.     

Implications of a poroelastic cytoplasm for the dynamics of animal cell shape

Two views have dominated recent discussions of the physical basis of cell shape change during migration and division of animal cells: the cytoplasm can be modeled as a viscoelastic continuum, and the forces that change its shape are generated only by actin polymerization and actomyosin contractility in the cell cortex. Here, we question both views: we suggest that the cytoplasm is better described as poroelastic, and that hydrodynamic forces may be generally important for its shape dynamics. In the poroelastic view, the cytoplasm consists of a porous, elastic solid (cytoskeleton, organelles, ribosomes) penetrated by an interstitial fluid (cytosol) that moves through the pores in response to pressure gradients. If the pore size is small (30-60nm), as has been observed in some cells, pressure does not globally equilibrate on time and length scales relevant to cell motility. Pressure differences across the plasma membrane drive blebbing, and potentially other type of protrusive motility. In the poroelastic view, these pressures can be higher in one part of a cell than another, and can thus cause local shape change. Local pressure transients could be generated by actomyosin contractility, or by local activation of osmogenic ion transporters in the plasma membrane. We propose that local activation of Na(+)/H(+) antiporters (NHE1) at the front of migrating cells promotes local swelling there to help drive protrusive motility, acting in combination with actin polymerization. Local shrinking at the equator of dividing cells may similarly help drive invagination during cytokinesis, acting in combination with actomyosin contractility. Testing these hypotheses is not easy, as water is a difficult analyte to track, and will require a joint effort of the cytoskeleton and ion physiology communities.     

Fluorescence visualization of the distribution of microfilaments in gonads and early embryos of the nematode Caenorhabditis elegans

Several intracellular motility events in the Caenorhabditis elegans zygote (pseudocleavage, the asymmetric meeting of the pronuclei, the segregation of germ line-specific granules, and the generation of an asymmetric spindle) appear to depend on microfilaments (MFs). To investigate how MFs participate in these manifestations of zygotic asymmetry, the distribution of MFs in oocytes and early embryos was examined, using both antibodies to actin and the F-actin-specific probe rhodamine-phalloidin. In early-stage zygotes, MFs are found in a uniform cortical meshwork of fine fibers and dots or foci. In later zygotes, concomitant with the intracellular movements that are thought to be MF mediated, MFs also become asymmetrically rearranged; as the zygote undergoes pseudocleavage and as the germ line granules become localized in the posterior half of the cell, the foci of actin become progressively more concentrated in the anterior hemisphere. The foci remain anterior as the spindle becomes asymmetric and the zygote undergoes its first mitosis, at which time fibers align circumferentially around the zygote where the cleavage furrow will form. A model for how the anterior foci of actin may participate in zygotic motility events is discussed. Phalloidin and anti-actin antibodies have also been used to visualize MFs in the somatic tissues of the adult gonad. The myoepithelial cells that surround maturing oocytes are visibly contractile and contain an unusual array of MF bundles; the MFs run roughly longitudinally from the loop of the gonad to the spermatheca. Myosin thick filaments are distributed along the MFs in a periodic manner suggestive of a sarcomere-like configuration. It is proposed that these actin and myosin filaments interact to cause sheath cell contraction and the movement of oocytes through the gonad.     

The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner

The bicoid (bcd) protein in a Drosophila embryo is derived from an anteriorly localized mRNA and comes to be distributed in an exponential concentration gradient along the anteroposterior axis. To determine whether the levels of bcd protein are directly related to certain cell fates, we manipulated the density and distribution of bcd mRNA by genetic means, measured the resultant alterations in height and shape of the bcd protein gradient, and correlated the gradient with the fate map of the respective embryos. Increases or decreases in bcd protein levels in a given region of the embryo cause a corresponding posterior or anterior shift of anterior anlagen in the embryo. The bcd protein thus has the properties of a morphogen that autonomously determines positions in the anterior half of the embryo.     

Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo.

Patterning along the anteroposterior axis is a critical step during animal embryogenesis. Although mechanisms of anteroposterior patterning in the neural tube have been studied in various chordates, little is known about those of the epidermis. To approach this issue, we investigated patterning mechanisms of the epidermis in the ascidian embryo. First we examined expression of homeobox genes (Hrdll-1, Hroth, HrHox-1 and Hrcad) in the epidermis. Hrdll-1 is expressed in the anterior tip of the epidermis that later forms the adhesive papillae, while Hroth is expressed in the anterior part of the trunk epidermis. HrHox-1 and Hrcad are expressed in middle and posterior parts of the epidermis, respectively. These data suggested that the epidermis of the ascidian embryo is patterned anteroposteriorly. In ascidian embryogenesis, the epidermis is exclusively derived from animal hemisphere cells. To investigate regulation of expression of the four homeobox genes in the epidermis by vegetal hemisphere cells, we next performed hemisphere isolation and cell ablation experiments. We showed that removal of the vegetal cells before the late 16-cell stage results in loss of expression of these homeobox genes in the animal hemisphere cells. Expression of Hrdll-1 and Hroth depends on contact with the anterior-vegetal (the A-line) cells, while expression of HrHox-1 and Hrcad requires contact with the posterior-vegetal (the B-line) cells. We also demonstrated that contact with the vegetal cells until the late 32-cell stage is sufficient for animal cells to express Hrdll-1, Hroth and Hrcad, while longer contact is necessary for HrHox-1 expression. Contact with the A-line cells until the late 32-cell stage is also sufficient for formation of the adhesive papillae. Our data indicate that the epidermis of the ascidian embryo is patterned along the anteroposterior axis by multiple inductive influences from the vegetal hemisphere cells and provide the first insight into mechanisms of epidermis patterning in the chordate embryos.