Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Unusual electron-dense dome associates with compound plasmodesmata in the embryo-suspensor of genus Sedum (Crassulaceae)

Plasmodesmata ensure the continuity of cytoplasm between plant cells and play an important part in the intercellular communication and signal transduction. During the development of the suspensor of both Sedum acre L. and Sedum hispanicum L., changes in the ultrastructure of plasmodesmata and adjoining cytoplasm are observed. Numerous simple plasmodesmata are present in the inner wall of the two-celled embryo separating the basal cell from the apical cell. From the early-globular to the torpedo stage of embryo development, the part of the wall separating the basal cell from the first layer of the chalazal suspensor cells is perforated by unusual, compound plasmodesmata. The role and the sort of transport through these plasmodesmata are discussed.     

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.     

DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila

The Drosophila protein Bazooka is required for both apical-basal polarity in epithelial cells and directing asymmetric cell division in neuroblasts. Here we show that the PDZ-domain protein DmPAR-6 cooperates with Bazooka for both of these functions. DmPAR-6 colocalizes with Bazooka at the apical cell cortex of epithelial cells and neuroblasts, and binds to Bazooka in vitro. DmPAR-6 localization requires Bazooka, and mislocalization of Bazooka through overexpression redirects DmPAR-6 to ectopic sites of the cell cortex. In the absence of DmPAR-6, Bazooka fails to localize apically in neuroblasts and epithelial cells, and is distributed in the cytoplasm instead. Epithelial cells lose their apical-basal polarity in DmPAR-6 mutants, asymmetric cell divisions in neuroblasts are misorientated, and the proteins Numb and Miranda do not segregate correctly into the basal daughter cell. Bazooka and DmPAR-6 are Drosophila homologues of proteins that direct asymmetric cell division in early Caenorhabditis elegans embryos, and our results indicate that homologous protein machineries may direct this process in worms and flies.     

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis

The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.     

Cell polarity in sea urchin embryos: reorientation of cells occurs quickly in aggregates

Four apical components were used as markers for the apical end of the cell in studies centering on cell polarity in the early blastula stage of sea urchin embryos and in aggregates of cleavage stage cells. Cells were observed to maintain their polarity for several hours if dissociated and cultured in suspension. Orientation of cells in aggregates initially is random; however, within 3 hr the cells have reoriented so that their apical-basal axis corresponds to the correct inside-outside position in the aggregate. This reorientation occurs before formation of a basal lamina or a new hyalin layer in the aggregate, and appears to take place by a rotation or other movement of individual cells. The polarity within each cell is maintained during reorientation. An apical surface antigen is colocalized with concentrations of filamentous actin. Treatment of isolated cells with cytochalasin B causes the antigen to lose its apical position and eventually become distributed around the outside of the cell. Microtubules are visible radiating from two foci closely associated with the nucleus in untreated cells. Treatment of isolated cells with nocodazole leaves the apical cell surface marker and its associated actin undisturbed, but causes the nucleus to lose its apical position. Cytochalasin B and colchicine both prevent reorientation of cells in aggregates. Thus polarity appears to be a constant for the cells, and their reorientation in aggregates occurs prior to the polarized release of extraembryonic matrix and basal lamina.     

Intracellular distribution of yolk droplets, lipid bodies and Golgi apparatus in the chick neuroepithelial cells during neurulation

Vitelline and lipidic inclusions which are present in the neuroepithelial cells during chick embryo neurulation show a typical intracellular localization in the apical zone of the cell. In the same cellular zone the Golgi apparatus can be seen during the successive stages of neurulation. These patterns of inclusion and organelle polarity during chick embryo neurulation may be related to active consumption of the reserves contained in inclusions during this morphogenetic process. Such an active consumption would imply a close relationship between the vitelline and lipidic inclusions and the Golgi apparatus. On the other hand, the apical position of the Golgi apparatus in the neuroepithelial cells reveals the remarkable apicobasal polarity of these cells which remains unchanged during chick embryo neurulation.     

Dedifferentiation and microtubule reorganization in the apical cell protoplast ofSphacelaria (Phaeophyceae)

Summary The apical cell ofSphacelaria, a tip-growing filamentous brown alga, and its protoplast constitute a model for the investigation of the consequences of cell wall removal on microtubular cytoskeletal organization and cell polarity. In the apical cell, the microtubular cytoskeleton is strongly polarized and, in most cases, extends from two centrosomes to the cortex where it constitutes a fine meshwork. Observations of microtubule dynamics throughout the cell cycle emphasize the coincidence between orientation of the mitotic axis and cell polarity. Just after protoplast isolation, dramatic alterations of initial polarity are observed, whatever the mitotic stage. In particular, the coincidence between cytoplasmic polarity and polarity of the system nucleus-centrosomes is lost in most cases. 12–24 h after protoplast isolation, the cell shows a more symmetrical organization while a dense cortical microtubular network spreads out concomitantly with wall reformation. Our discussion emphasizes the possible relationship between cell polarity and cell totipotency, and the relevance of such a model for higher plant studies.     

Ontogenesis in the Fucophyceae: case studies and comparison of fucoid zygotes and Sphacelaria apical cells

In multicellular eukaryotes, the zygote, a single cell, gives rise to the different cell types of the organism. The study of the mechanisms involved is a key point of developmental biology. Generally, the first stages are characterized by an orderly sequence of asymmetrical divisions resulting from an initial developmental polarity. The establishment of this initial polarity has been the subject of numerous studies in animals, but not in higher plants since the zygote is encased in several layers of tissues that prevent experimental approaches. Moreover, plant development is characterized by two successive ontogenetic steps: the construction of the embryonic apico-basal axis and the establishment of meristems in charge of organogenesis. Members of the Fucophyceae provide good models for the investigation of these processes. Any inferred homology of mechanisms must take into account the polyphyletic nature of the algae. This paper is a tentative review of two case studies: fucoid zygotes and Sphacelaria apical cells, and deals respectively with the two successive ontogenetic steps characteristic of higher plant development. The first part concerns development of the fucoid zygotes. Fucoid zygotes, including those of different species, are considered as model systems in plants for studying the establishment of the polarity axis because, at the moment of fertilization, they do not have any morphological or biochemical polarity. This report concerns progress in the identification of some cellular or molecular mechanisms involved in the settlement and/or stabilization of the polarity axis, and the consequence of this polar organisation for the control of asymmetrical divisions and the building of a functional embryo. The second part concerns the apical cell of Sphacelaria as a model for establishing and maintaining a meristematic cell. The apical cell exhibits a permanent polarized organisation throughout repetitive asymmetric divisions and can be comparatively analysed in situ and isolated as a protoplast. This allowed us to investigate the evolution of the cytoplasmic cytoskeleton, centrosomes and the mitotic apparatus during the cell cycle in relation to the cell polarity; particularly the interactions between the cytoskeleton and cell wall. For the two models, the results are compared with mechanisms involved in the development of other multicellular organisms, and their value in gaining an insight into higher plant ontogenesis is assessed.     

Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusualin that oblique cell divisions result in the formation of severalsuspensor cells prior to the development of the embryo proper.Characteristic changes in microtubular distribution can be foundwithin the zygote and the proembryo during their development.After fertilization, the ellipsoid-shaped zygote has randomlydistributed microtubules within its cytoplasm. As the zygotetakes on a more rounded appearance, microtubules organize intoa dense meshwork. Furthermore, microtubule bundles appear atthe chalazal region of the cell prior to the first mitotic divisionof the zygote. At the preprophase stage of mitosis, a preprophaseband of microtubules appears in the cytoplasm of the zygote.The zygote divides obliquely and unequally and gives rise toan apical cell and a slightly larger basal cell. Many randomly-alignedmicrotubules can be found in the cortex of the basal cell. Theincrease in the abundance of microtubules coincides with theisotropic expansion of the basal cell. The early division ofthe basal cell and subsequent division of the apical cell resultsin the formation of a four-celled embryo, of which three cellsnear the micropylar pole develop as suspensor cells. In thesuspensor cells, the microtubules tend to orient in the samedirection as the long axis of the cell. In addition, prominentmicrotubules can also be found near the adjoining cell wallsof the four-celled embryo. The terminal cell is highly cytoplasmicwith abundant microtubules within the cell. Subsequent divisionsof the terminal cell give rise to additional suspensor cellsand the embryo proper. In the mature embryo, five suspensorcells are usually present; one eventually grows through themicropyle of the inner integument and four grow towards thechalazal pole. The cortical microtubules of suspensor cellsredistribute from a longitudinal to a transverse direction asthey grow towards their respective poles.Copyright 1998 Annalsof Botany Company Embryogenesis, endosperm, microtubules, preprophase band, suspensor cells,Cymbidium sinense(Andr.) Willd.     

Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.     

Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila beta-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dalpha-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.     

Development of cellular polarity of hamster embryos during compaction.

Development of cellular polarity is an important event during early mammalian embryo development and differentiation. Blastomeres of hamster embryos at various stages were examined by scanning electron microscopy (SEM) and immunocytochemical staining. SEM observations revealed that 1- to 7-cell-stage embryos showed a uniform distribution of microvilli throughout the cell surface. Microvillous polarization was initially noted in the blastomeres (10-35%) of 8-cell-stage embryos. The polarized microvilli were observed mostly in the basal region of cell-cell contact and occasionally at the apical, outward-facing surface of the blastomere. Fluorescein-isothiocyanate-conjugated concanavalin A failed to reveal any polarity in the blastomeres regardless of the stages of the embryos. Actin staining showed that microfilaments were present beneath the cell surface, and in addition, areas of cell contact were more heavily stained, indicating a thick microfilament domain. Microtubules were located throughout the cytoplasm and were heavily concentrated near the nucleus during interphase, although they became redistributed in the region of the mitotic spindle during karyokinesis. The position of nucleus changed from the cell center to the apical, outward-facing surface of the cell, and it distanced itself from the basal microvillous pole. It is suggested that the changes in the cell surface and nuclear position are the first manifestations of cell polarity in peri-compacted hamster embryos, which appear as early as the 8-cell stage; furthermore, the outward migration of the nuclei may parallel the redistribution of microtubules in the cytoplasm.     

PIN polarity maintenance by the cell wall in Arabidopsis

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.     

Planar polarity of ependymal cilia

Ependymal cells, epithelial cells that line the cerebral ventricles of the adult brain in various animals, extend multiple motile cilia from their apical surface into the ventricles. These cilia move rapidly, beating in a direction determined by the ependymal planar cell polarity (PCP). Ciliary dysfunction interferes with cerebrospinal fluid circulation and alters neuronal migration. In this review, we summarize recent studies on the cellular and molecular mechanisms underlying two distinct types of ependymal PCP. Ciliary beating in the direction of fluid flow is established by a combination of hydrodynamic forces and intracellular planar polarity signaling. The ciliary basal bodies' anterior position on the apical surface of the cell is determined in the embryonic radial glial cells, inherited by ependymal cells, and established by non-muscle myosin II in early postnatal development.