The asymmetric division of the Arabidopsis zygote: from cell polarity to an embryo axis

During plant embryogenesis, a simple body plan consisting of shoot and root meristem that are connected by the embryo axis is set up by the first few rounds of cell divisions after fertilization. Postembryonically, the elaborate architecture of plants is created from stem cell populations of both meristems. Here, we address how the main axis (apical-basal) of the plant embryo is established from the single-celled zygote and the role that the asymmetric division of the zygote plays in this process. We will mainly draw on examples from the model plant Arabidopsis, for which several key regulators have been identified during the last years.     

Gene expression patterns in the maize caryopsis: clues to decisions in embryo and endosperm development.

We will describe gene expression patterns in the maize caryopsis, which provide clues to developmental decisions and questions in the embryo and endosperm. The emphasis will be on the development of the root/shoot axis, which is the main achievement of plant embryogenesis. Data obtained in the vegetative seedling are included as far as they may be relevant to the elaboration of the shoot/root axis. Development of the embryo will be briefly compared to endosperm as both seed compartment exhibit pronounced differences.     

Growth and development of embryo parts during the germination of caryopses of the wild oat (Avena fatua L.)

Wild oat (Avena fatua L.) caryopses were germinated on moist filter paper and under water in the presence and absence of hydrogen peroxide (H₂O₂). The sequential growth and development of embryo parts were studied. Germination, as indicated by radicle emergence, was least and slowest in caryopses submerged in deoxygenated water. The coleorhiza in such caryopses elongated much earlier than the root, in contrast to the other treatments where the coleorhiza and the root emerged at about the same time. In caryopses incubated on moist filter paper all embryo parts showed considerable growth. In H₂O₂ treated caryopses only the epicotyl showed substantial growth over the experimental period. In all treatments the first mitotic peaks were noticed at the same period. The occurrence of these early nuclear divisions may be due to release of 4 C nuclei from inhibition by the uptake of water during caryopsis imbibition. The mitosis continued in the radicle of the embryo in those caryopses germinating on moist filter paper, indicating occurrence of DNA synthesis. In the other two treatments, however, few divisions were detected. Here the early growth of the root, causing caryopsis germination, was due to cell elongation, especially in the proximal part of the root.     

Key divisions in the early Arabidopsis embryo require POL and PLL1 phosphatases to establish the root stem cell organizer and vascular axis

Arabidopsis development proceeds from three stem cell populations located at the shoot, flower, and root meristems. The relationship between the highly related shoot and flower stem cells and the very divergent root stem cells has been unclear. We show that the related phosphatases POL and PLL1 are required for all three stem cell populations. pol pll1 mutant embryos lack key asymmetric divisions that give rise to the root stem cell organizer and the central vascular axis. Instead, these cells divide in a superficially symmetric fashion in pol pll1 embryos, leading to a loss of embryonic and postembryonic root stem cells and vascular specification. We present data that show that POL/PLL1 drive root stem cell specification by promoting expression of the WUS homolog WOX5. We propose that POL and PLL1 are required for the proper divisions of shoot, flower, and root stem cell organizers, WUS/WOX5 gene expression, and stem cell maintenance.     

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Structure and development of stomata on the primary root of Ceratonia siliqua L

Stomata of various sizes are produced on the primary root of Ceratonia siliqua L. Most are generated during embryogenesis, prior to seed desiccation. They can be detected on the dry embryo in a wide zone just above the root tip. Initially, large stomata are formed. These have the ability to induce divisions of their neighbouring cells, creating particular cell patterns around them. Later, small perigenous stomata are generated. As the root grows following seed germination, the stomatal zone overlaps with that of the root hairs. Although root stomata of C. siliqua undergo a structural differentiation that seems almost identical to that of the elliptical stomata formed on leaves, they are unable to move and remain permanently open. Polarizing microscopy of fully differentiated stomata and young stomata at the stage of stomatal pore formation revealed deposition of radial cellulose microfibril systems on their periclinal walls. However, these systems were less developed than those on leaf stomata, a feature that might be responsible for their inactivity. Besides, plastids of the root guard cells (GCs) do not differentiate into chloroplasts but function solely as amyloplasts. Root stomata have a short life span. During rapid and intense root growth, GCs cannot keep pace with the elongation of their neighbouring rhizodermal cells. They therefore split in their mid-region, transversely to the stoma axis. The two parts of the transversely torn stoma are dragged apart and a large opening is formed on the root surface, just above the substomatal cavity. The root stomata, together with these openings, may facilitate increased gaseous exchange during respiration and/or an increased transfer of some nutrients and water in the rapidly growing primary root.     

WOX gene phylogeny in Poaceae: a comparative approach addressing leaf and embryo development

The phylogeny based on the homeodomain (HD) amino acid sequence of the WOX (WUSCHEL-related homeobox gene family) was established in the 3 major radiations of the Poaceae family: Pooideae (Brachypodium distachyon), Bambusoideae (Oryza sativa), and Panicoideae (Zea mays). The genomes of all 3 grasses contain an ancient duplication in the WOX3 branch, and the cellular expression patterns in maize and rice indicate subfunctionalization of paralogues during leaf development, which may relate to the architecture of the grass leaf and the encircling of the stem. The use of maize WOX gene family members as molecular markers in maize embryo development for the first time allowed us to visualize cellular decisions in the maize proembryo, including specification of the shoot/root axis at an oblique angle to the apical-basal polarity of the zygote. All molecular marker data are compatible with the conclusion that the embryonic shoot/root axis comprises a discrete domain from early proembryo stages onward. Novel cell fates of the shoot and the root are acquired within this distinct morphogenic axis domain, which elongates and thus separates the shoot apical meristem and root apical meristem (RAM) anlagen in the maize embryo.     

Early development and axis specification in the sea anemone Nematostella vectensis

We investigated the early development of the sea anemone Nematostella vectensis, an emerging model system of the Cnidaria. Early cleavage stages are characterized by substantial variability from embryo to embryo, yet invariably lead to the formation of a coeloblastula. The coeloblastula undergoes a series of unusual broad invaginations-evaginations which can be blocked by cell cycle inhibitors suggesting a causal link of the invagination cycles to the synchronized cell divisions. Blastula invagination cycles stop as cell divisions become asynchronous. Marking experiments show a clear correspondence of the animal-vegetal axis of the egg to the oral-aboral axis of the embryo. The animal pole gives rise to the concave side of the blastula and later to the blastopore of the gastrula, and hence the oral pole of the future polyp. Asymmetric distribution of granules in the unfertilized egg suggest an animal-vegetal asymmetry in the egg in addition to the localized position of the pronucleus. To determine whether this asymmetry reflects asymmetrically distributed determinants along the animal-vegetal axis, we carried out blastomere isolations and embryonic divisions at various stages. Our data strongly indicate that normal primary polyps develop only if cellular material from the animal hemisphere is included, whereas the vegetal hemisphere alone is incapable to differentiate an oral pole. Molecular marker analysis suggests that also the correct patterning of the aboral pole depends on signals from the oral half. This suggests that in Nematostella embryos the animal hemisphere contains organizing activity to form a normal polyp.     

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

Summary Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division. divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all.     

The homeodomain-containing gene Xdbx inhibits neuronal differentiation in the developing embryo

The development of the vertebrate nervous system depends upon striking a balance between differentiating neurons and neural progenitors in the early embryo. Our findings suggest that the homeodomain-containing gene Xdbx regulates this balance by maintaining neural progenitor populations within specific regions of the neuroectoderm. In posterior regions of the Xenopus embryo, Xdbx is expressed in a bilaterally symmetric stripe that lies at the middle of the mediolateral axis of the neural plate. This stripe of Xdbx expression overlaps the expression domain of the proneural basic/helix-loop-helix-containing gene, Xash3, and is juxtaposed to the expression domains of Xenopus Neurogenin related 1 and N-tubulin, markers of early neurogenesis in the embryo. Xdbx overexpression inhibits neuronal differentiation in the embryo and when co-injected with Xash3, Xdbx inhibits the ability of Xash3 to induce ectopic neurogenesis. One role of Xdbx during normal development may therefore be to restrict spatially neuronal differentiation within the neural plate, possibly by altering the neuronal differentiation function of Xash3.     

Confocal Microscopy Study of <Emphasis Type="Italic">Arabidopsis</Emphasis> Embryogenesis Using GFP:mTn

Embryogenesis in transgenic Arabidopsis plants with GFP:mTn, a chimeric fusion of soluble shifted green fluorescent protein and a mouse actin binding domain, was studied. Confocal laser scanning microscopy was used to determine patterns of formation and cellular responses during asymmetric cell division. Before such cells divide, the nucleus moves to the position where new cell walls are to be formed. The apical–basal axis of the embryo develops mainly at the zygote to octant stage, and these events are associated with asymmetric divisions of the zygote and hypophyseal cells. Formation of the radial axis is established from the dermatogen to the globular-stage embryo via tangential cell division within the upper tiers. Bilateral symmetry of the embryo primarily happens at the triangular stage through zig-zag cell divisions of initials of the cotyledonary primordia. All stages of embryogenesis are described in detail here.     

Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions

Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires that some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern, whether it is lineage-dependent or occurs at random, or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos, individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and thus embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together, our results suggest that two steps influence the allocation of cells to the blastocyst. The first step, involving orientation of 2- to 4-cell divisions along the animal-vegetal axis, can affect the second step, the establishment of inside and outside cell populations by asymmetric 8- to 32-cell divisions.     

Drosophila neural progenitor polarity and asymmetric division

In the Drosophila embryonic central nervous system, the neural precursor cells called neuroblasts undergo a number of asymmetric divisions along the apical-basal axis to give rise to different daughter cells of distinct fates. This review summarizes recent progress in understanding the mechanisms of these asymmetric cell divisions. We discuss proteins that are localized at distinct domains of cortex in the neuroblasts and their role in generating asymmetry. We also review uniformly cortical localized factors and actin cytoskeleton-associated motor proteins with regard to their potential role to serve as a link between distinct cortical domains in the neuroblasts. In this review, asymmetric divisions of sensory organ precursor and larval neuroblasts are also briefly discussed.     

The First Appearance of Polyploid Nuclei in Primary Roots of Two Diploid Angiosperms

In Anchusa capensis, where the polyploid divisions are restrictedto a particular time and place in germination, cytophotometricanalysis showed that the polyploid cells arise at the time ofconversion of the globular-shaped embryo to the heart-shapedembryo. In Spinacia oleracea, where the divisions occur throughoutthe growth of the root, these polyploid cells do not arise duringembryogenesis but during germination and subsequent growth. Anchusa capensis Thunb., summer forget-me-not, Spinacia oleracea L., spinach, root meristem, polyploid divisions, determinate divisions, indeterminate divisions, germination, embryogenesis, cytophotometry     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.