Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Role of mechano-dependent cell movements in the establishment of spatial organization of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

Trajectories of individual cell movements and patterns of differentiation in the axial rudiments in suprablastoporal areas (SBA) in whole embryos of Xenopus laevis artificially stretched in the transverse direction up to 120–200% from the initial length at the early gastrula stage were mapped. We observed the impairment of anisotropic cell movements of longitudinal stretching and latero-medial convergence inherent for SBA. Axial rudiments occurred in all cases, but their location was completely impaired and dramatically different from the normal topology for moderate (120–140%) stretching. Stronger stretching caused a partial ordering of the whole axial complex and its reorientation toward stretching. We concluded that induction factors determine short-range order in their arrangement in SBA, whereas anisotropic cell movements in any direction are needed for long-range order. Moderate transverse stretching destroys normally oriented anisotropy, but it is not enough for establishment of the anisotropy oriented perpendicular to the normal. This explains the disorder at light stretching. The main conclusion of this study is that anisotropic tensions of embryonic tissues play role of long-range order parameters of cell differentiation.     

A Quantitative Study of Regional and Stage Specific Reaction of Xenopus laevisEmbryonic Tissues on Mechanical Load

Residual deformation of fragments of the embryonic tissues preserved after relaxation of the stretching force serve as a criterion of active redistribution of their cells caused by this stretching. We measured residual deformations of the Xenopus laevis ventral and dorsal ectoderm at the early gastrula and lateral ectoderm at the late gastrula-early neurula after stretching of varying time and force. While the samples responded to moderate (up to 40%) short-term stretching as elastic bodies (residual deformations were absent), residual deformation appeared in the early gastrula tissues after 30–60-min stretching, which were more pronounced in the ventral tissues than in the dorsal ones. On the contrary, a contractile reaction developed in the late gastrula-early neurula tissues in response to 60-min stretching, which almost relaxed residual deformation within 20 min after unloading. A conclusion was drawn that gastrulation and neurulation proceed under the conditions of relaxing and nonrelaxing mechanical tensions, respectively. Mechanical bases and morphogenetic role of the described reactions is discussed.     

Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

Polarized Raman spectrum of single crystal uridylyl (3′–5′) adenosine: Local Raman tensors of some functional groups

Polarized Raman scattering measurements have been made of a single crystal of uridylyl(3′–5′)adenosine (UpA) by the use of a Raman microscope with 488.0 nm excitation. The UpA crystal belongs to space group P2₁ (monoclinic), and Raman intensities I_aa, I_bb, and I_c′c′, have been determined for each Raman band. These intensities correspond to the aa, bb, and c′c′ components of the crystal Raman tensor, where c′ is defined as an axis perpendicular to the crystallographic a axis in the ac plane. From these experimental data, and by taking the known crystal structure into account, anisotropic and isotropic molecular Raman tensors have been calculated for the following 11 normal modes: ring stretching modes of the adenine residue (protonated) at 1560, 1516, 1330, and 715 cm⁻¹; ring stretching modes of the uracil residue at 1696, 1657, 1615, 1228, and 790 cm⁻¹; PO⁻₂ symmetric stretching mode at 1080 cm⁻¹; P(—)O single bond stretching mode at 801 cm₋₁. These pieces of information of the Raman tensors are considered to be useful for estimating the orientations of the DNA and RNA strands in a biological complex from a polarized Raman spectroscopic measurement of such a complex. © 1998 John Wiley & Sons, Inc. Biopoly 45: 135–147, 1998     

Simulating in ovulo osmotic potentials and O<Subscript>2</Subscript> tensions normalize growth and pigmentation of immature cotton embryos

Oxygen tensions and osmotic potentials are important physiological factors of plant growth and development. To optimize these variables for cotton (Gossypium hirsutum L.) embryo culture, we quantified dissolved O₂ (dO₂) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O₂ microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO₂ tensions were determined in integuments, nucelli and embryos. Ovular osmotic potentials and pH were determined from extracted ovule sap using vapor pressure osmometers and pH microelectrodes. Dissolved O₂ tensions near or in embryos decreased from 104 mmol m⁻³ at 5 days post-anthesis (DPA) to 83 mmol m⁻³ at 18 DPA. Osmotic potentials of ovule sap decreased from −0.70 megapascals (MPa) at 2 DPA to −1.12 MPa at 8 DPA but then increased to −0.84 MPa by 17 DPA. Ovule sap pH at 5–17 DPA varied inconsistently and ranged from 5.4 to 6.5. Based on these results, a factorial experiment with two osmotic and three O₂ treatments was designed. Immature embryos of cotton cultivar HS-26 were randomly assigned to the treatment combinations and cultured for 33 days. Oxygen treatments did not affect embryo growth, and there were no differences among treatments with regard to percentage of embryos that progressed to a more advanced stage of embryo development. However, cotyledons of embryos grown without osmotic adjustment were abnormally large, and embryos exposed to these treatments were abnormally brown. Browning was less severe for embryos exposed to low O₂ tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O₂ tensions and osmotic potentials that best simulated the observed in ovulo conditions.     

Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed <Emphasis Type="Italic">Xenopus</Emphasis> embryos

Influence of the relaxation of mechanical tensions upon collective cell movements, shape formation, and expression patterns of tissue-specific genes has been studied in Xenopus laevis embryos. We show that the local relaxation of tensile stresses within the suprablastoporal area (SBA) performed at the early-midgastrula stage leads to a complete arrest of normal convergent cell intercalation towards the dorsal midline. As a result, SBA either remains nondeformed or protrudes a strip of cells migrating ventralwards along one of the lateral lips of the opened blastopore. Already, few minutes later, the tissues in the ventral lip vicinity undergo abnormal transversal contraction/longitudinal extension resulting in the abnormal cell convergence toward ventral (rather than dorsal) embryo midline. Within a day, the dorsally relaxed embryos acquire pharyngula-like shapes and often possess tail-like protrusions. Their antero-posterior and dorso-ventral polarity, as well as expression patterns of pan-neural (Sox3), muscular cardiac actin, and forebrain (Otx2) genes substantially deviate from the normal ones. We suggest that normal gastrulation is permanently controlled by mechanical stresses within the blastopore circumference. The role of tissue tensions in regulating collective cell movements and creating pharyngula-like shapes are discussed.     

Excising stem samples underwater at native tension does not induce xylem cavitation

Xylem resistance to water stress‐induced cavitation is an important trait that is associated with drought tolerance of plants. The level of xylem cavitation experienced by a plant is often assessed as the percentage loss in conductivity (PLC) at different water potentials. Such measurements are constructed with samples that are excised underwater at native tensions. However, a recent study concluded that cutting conduits under significant tension induced cavitation, even when samples were held underwater during cutting. This resulted in artificially increased PLC because of what we have termed a ‘tension‐cutting artefact’. We tested the hypothesized tension‐cutting artefact on five species by measuring PLC at native tension compared with after xylem tensions had been relaxed. Our results did not support the tension‐cutting artefact hypothesis, as no differences were observed between native and relaxed samples in four of five species. In a fifth species (Laurus nobilis), differences between native and relaxed samples appear to be due to vessel refilling rather than a tension‐cutting effect. We avoided the tension‐cutting artefact by cutting samples to slightly longer than their measurement length and subsequent trimming of at least 0.5 cm of sample ends prior to measurement.     

PAWS1 controls Wnt signalling through association with casein kinase 1α

The BMP and Wnt signalling pathways determine axis specification during embryonic development. Our previous work has shown that PAWS1 (also known as FAM83G) interacts with SMAD1 and modulates BMP signalling. Here, surprisingly, we show that overexpression of PAWS1 in Xenopus embryos activates Wnt signalling and causes complete axis duplication. Consistent with these observations in Xenopus, Wnt signalling is diminished in U2OS osteosarcoma cells lacking PAWS1, while BMP signalling is unaffected. We show that PAWS1 interacts and co‐localises with the α isoform of casein kinase 1 (CK1), and that PAWS1 mutations incapable of binding CK1 fail both to activate Wnt signalling and to elicit axis duplication in Xenopus embryos.     

Regional cell shape changes control form and function of Kupffer's vesicle in the zebrafish embryo

Cilia-generated fluid flow in an 'organ of asymmetry' is critical for establishing the left-right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry-called Kupffer's vesicle (KV)-ciliated cells are asymmetrically positioned along the anterior-posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as 'KV remodeling' that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left-right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left-right patterning of the embryo.     

Division patterns in the thallus ofPelvetia compressa embryos and the effects of gravity

Summary The pattern of divisions in the thallus ofPelvetia compressa embryos was determined with respect to the embryonic growth axis. To detect all possible division planes, embryos were viewed from two vantages which permitted observations of (1) the thallus pole and (2) the longitudinal embryonic profile. Following formation of rhizoid and thallus cells by any asymmetrical division transverse to the embryonic axis that is established prior to any divisions, the thallus cell divided twice along the embryonic axis (axial divisions) in orthogonal planes, and then divided transverse to the growth axis. This division pattern produced an eight-cell thallus with four cells in each of two layers. The spatial relation between gravity and the first axial division was investigated, and gravity was found to have little effect on the alignment of this division. The reproducible pattern of divisions in the thallus indicates spatial control of spindle positioning.Abbreviations ASW artificial seawater - AF after fertilization     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

Migration-Directing Liquid Properties of Embryonic Amphibian Tissues

Deep ectoderm, mesoderm and endoderm excised from gastrulatingamphibian embryos spontaneously undergo liquid-like movementsin organ culture. Cell populations of these tissues on nonadhesivesubstrata will round up into spheres, spread over one anotherand segregate (sort out) from one another just as immiscibleliquid droplets do. In ordinary liquids, movements like theseare controlled by surface tensions; perhaps surface tensionsalso control the similar movements of liquid-like tissues. Onenecessary condition for tissue surface tension analysis is thatthe tissue must be able (just as ordinary liquids are able)to spontaneously relax internal stretching forces (shear stresses).When cellular aggregates of the germ layers were deformed bygentle compression between parallel glass plates, cells withinthe aggregates were initially stretched. However, the cellssoon returned to their original undistorted shapes. Thus, cellstretching forces were gradually relaxed by cell rearrangements.The in vitro spreading movements of the deep germ layers implythat the surface tension of ectoderm should be greater thanthe surface tension of mesoderm which should be greater thanthe surface tension of endoderm. Quantitative measurements oftissue surface tensions made by parallel plate compression confirmprecisely that relationship. Furthermore, the surface tensionsof these tissues remain constant regardless of the amount ofaggregate flattening—another necessary condition for validsurface tension measurements. These results demonstrate thatamphibian deep germ layers possess fundamental liquid propertieswhich are sufficient to direct their liquid-like rearrangementsin organ culture. Furthermore, I also report that one of theseproperties, surface tension, displays a preliminary correlationwith density of cell surface charge (assessed by electrophoreticmobility) and with the onset of in vivo mesodermal involution.     

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos

SUMMARY Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin‐like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)‐signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV‐induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.     

Multiple steps of leaf thickening during sun‐leaf formation in Arabidopsis

Plant morphological and physiological traits exhibit plasticity in response to light intensity. Leaf thickness is enhanced under high light (HL) conditions compared with low light (LL) conditions through increases in both cell number and size in the dorsoventral direction; however, the regulation of such phenotypic plasticity in leaf thickness (namely, sun‐ or shade‐leaf formation) during the developmental process remains largely unclear. By modifying observation techniques for tiny leaf primordia in Arabidopsis thaliana, we analysed sun‐ and shade‐leaf development in a time‐course manner and found that the process of leaf thickening can be divided into early and late phases. In the early phase, anisotropic cell elongation and periclinal cell division on the adaxial side of mesophyll tissue occurred under the HL conditions used, which resulted in the dorsoventral growth of sun leaves. Anisotropic cell elongation in the palisade tissue is triggered by blue‐light irradiation. We discovered that anisotropic cell elongation processes before or after periclinal cell division were differentially regulated independent of or dependent upon signalling through blue‐light receptors. In contrast, during the late phase, isotropic cell expansion associated with the endocycle, which determined the final leaf thickness, occurred irrespective of the light conditions. Sucrose production was high under HL conditions, and we found that sucrose promoted isotropic cell expansion and the endocycle even under LL conditions. Our analyses based on this method of time‐course observation addressed the developmental framework of sun‐ and shade‐leaf formation.     

Are mineralized tissues open crystal foams reinforced by crosslinked collagen? Some energy arguments

Which of the elementary components (hydroxyapatite (HA) crystals, collagen, non-collagenous organic matter, water) do significantly contribute to the ultrastructural elastic stiffness magnitude and anisotropy of mineralized tissues; and how, i.e. through which shapes and assemblages (which micromechanical morphology)? We suggest answers to these questions by analyzing stiffness-volume fraction relationships of wet and dry tissue specimens in the framework of strain energy considerations. Radial stiffness values of both isotropic and anisotropic tissues are found to depend linearly to quadratically on only the mineral volume fraction. This suggests the isotropic contribution of HA to the ultrastructural stiffness. An energy-based analysis of the difference between the axial and radial stiffness values of anisotropic, collagen-rich tissues allows us to assess the collagen elasticity contribution, which is found to depend linearly on the extra-collagenous mineral concentration. These results suggest that collagen and hydroxyapatite are the elementary components governing the ultrastructural elastic stiffness magnitude and anisotropy of bone and mineralized tendons. The elastic stiffness of water and non-collagenous organic matter does not play a significant role. As for the morphological issue, we suggest that mineralized tissues are isotropic open crystal foams; and that these foams are reinforced unidirectionally by collagen molecules which are mechanically activated through tight links between these molecules and HA-crystals. The HA crystals are mechanically activated through stretching and bending in long bone tissues, they are predominantly stretched in mineralized tendons, and bent in hyperpycnotic tissues.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

A hybrid approach to theoretical analysis of bovine pancreatic trypsin inhibitor.

A static analysis of bovine pancreatic trypsin inhibitor (BPTI) is presented based on a new discrete/continuum approach to modeling the dynamics of biomolecules. This hybrid method utilizes knowledge of the intramolecular potential and molecular configuration to generate a field of elastic modulus tensors. These tensors, which relate the local stress and strain for each atom in the biomolecule, can be used to judge the local rigidity as well as indicate regions of high stress. Comparing the tensor fields for an unrelaxed and a relaxed configuration, the microscopic structure of BPTI is found to be anisotropic and to have regions of stress even when it is relaxed in the potential field. However, when these fields are averaged over the whole protein or over individual residues the structure becomes more isotropic and the stressed regions vanish. Using these averaged tensors, we calculated bulk properties such as Young's modulus and the Lamé constants and they agreed with previously reported values.     

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next‐generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen‐activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single‐mutant plants showed a wrinkled seed coat or a burst‐out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent‐cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin‐like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst‐out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild‐type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal‐mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.     

Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.