Identification of emergent motion compartments in the amniote embryo

Abstract

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ～400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen's node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen's node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

Mechanics of head fold formation: investigating tissue-level forces during early development

During its earliest stages, the avian embryo is approximately planar. Through a complex series of folds, this flat geometry is transformed into the intricate three-dimensional structure of the developing organism. Formation of the head fold (HF) is the first step in this cascading sequence of out-of-plane tissue folds. The HF establishes the anterior extent of the embryo and initiates heart, foregut and brain development. Here, we use a combination of computational modeling and experiments to determine the physical forces that drive HF formation. Using chick embryos cultured ex ovo, we measured: (1) changes in tissue morphology in living embryos using optical coherence tomography (OCT); (2) morphogenetic strains (deformations) through the tracking of tissue labels; and (3) regional tissue stresses using changes in the geometry of circular wounds punched through the blastoderm. To determine the physical mechanisms that generate the HF, we created a three-dimensional computational model of the early embryo, consisting of pseudoelastic plates representing the blastoderm and vitelline membrane. Based on previous experimental findings, we simulated the following morphogenetic mechanisms: (1) convergent extension in the neural plate (NP); (2) cell wedging along the anterior NP border; and (3) autonomous in-plane deformations outside the NP. Our numerical predictions agree relatively well with the observed morphology, as well as with our measured stress and strain distributions. The model also predicts the abnormal tissue geometries produced when development is mechanically perturbed. Taken together, the results suggest that the proposed morphogenetic mechanisms provide the main tissue-level forces that drive HF formation.     

Developmental imaging: Insights into the avian embryo

The study of embryonic events using different animal model systems is crucial for gaining insights into human development and birth defects. Biological imaging plays a major role in this effort by providing a spatiotemporal framework to link complex cell movements with molecular data. However, depending on the age of the embryo and the location of a morphogenetic event, visualization often requires the design of novel culture and imaging techniques. One of the primary model systems for biological imaging is the avian embryo, due to its accessibility to manipulation, relatively two-dimensional morphogenesis early on, and viability when grown in culture. Significant work in avian embryo culture and cell labeling, together with advances in imaging technology, now make it possible to monitor many developmental events within the period from egg laying to hatching. Here, we present the latest in avian developmental imaging, focusing on cell labeling, embryo culture, and imaging technologies.     

Identification of emergent motion compartments in the amniote embryo

The tissue scale deformations (≥1mm) required to form an amniote embryo are poorly understood. Here, we studied ∼400 μm-sized explant units from gastrulating quail embryos. The explants deformed in a reproducible manner when grown using a novel vitelline membrane-based culture method. Time-lapse recordings of latent embryonic motion patterns were analyzed after disk-shaped tissue explants were excised from three specific regions near the primitive streak: 1) anterolateral epiblast, 2) posterolateral epiblast, and 3) the avian organizer (Hensen''s node). The explants were cultured for 8 hours—an interval equivalent to gastrulation. Both the anterolateral and the posterolateral epiblastic explants engaged in concentric radial/centrifugal tissue expansion. In sharp contrast, Hensen''s node explants displayed Cartesian-like, elongated, bipolar deformations—a pattern reminiscent of axis elongation. Time-lapse analysis of explant tissue motion patterns indicated that both cellular motility and extracellular matrix fiber (tissue) remodeling take place during the observed morphogenetic deformations. As expected, treatment of tissue explants with a selective Rho-Kinase (p160ROCK) signaling inhibitor, Y27632, completely arrested all morphogenetic movements. Microsurgical experiments revealed that lateral epiblastic tissue was dispensable for the generation of an elongated midline axis— provided that an intact organizer (node) is present. Our computational analyses suggest the possibility of delineating tissue-scale morphogenetic movements at anatomically discrete locations in the embryo. Further, tissue deformation patterns, as well as the mechanical state of the tissue, require normal actomyosin function. We conclude that amniote embryos contain tissue-scale, regionalized morphogenetic motion generators, which can be assessed using our novel computational time-lapse imaging approach. These data and future studies—using explants excised from overlapping anatomical positions—will contribute to understanding the emergent tissue flow that shapes the amniote embryo.     

Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila

During development tissue deformations are essential for the generation of organs and to provide the final form of an organism. These deformations rely on the coordination of individual cell behaviours which have their origin in the modulation of subcellular activities. Here we explore the role endocytosis and recycling on tissue deformations that occur during dorsal closure of the Drosophila embryo. During this process the AS contracts and the epidermis elongates in a coordinated fashion, leading to the closure of a discontinuity in the dorsal epidermis of the Drosophila embryo. We used dominant negative forms of Rab5 and Rab11 to monitor the impact on tissue morphogenesis of altering endocytosis and recycling at the level of single cells. We found different requirements for endocytosis (Rab5) and recycling (Rab11) in dorsal closure, furthermore we found that the two processes are differentially used in the two tissues. Endocytosis is required in the AS to remove membrane during apical constriction, but is not essential in the epidermis. Recycling is required in the AS at early stages and in the epidermis for cell elongation, suggesting a role in membrane addition during these processes. We propose that the modulation of the balance between endocytosis and recycling can regulate cellular morphology and tissue deformations during morphogenesis.     

Seeing through Musculoskeletal Tissues: Improving In Situ Imaging of Bone and the Lacunar Canalicular System through Optical Clearing

In situ, cells of the musculoskeletal system reside within complex and often interconnected 3-D environments. Key to better understanding how 3-D tissue and cellular environments regulate musculoskeletal physiology, homeostasis, and health is the use of robust methodologies for directly visualizing cell-cell and cell-matrix architecture in situ. However, the use of standard optical imaging techniques is often of limited utility in deep imaging of intact musculoskeletal tissues due to the highly scattering nature of biological tissues. Drawing inspiration from recent developments in the deep-tissue imaging field, we describe the application of immersion based optical clearing techniques, which utilize the principle of refractive index (RI) matching between the clearing/mounting media and tissue under observation, to improve the deep, in situ imaging of musculoskeletal tissues. To date, few optical clearing techniques have been applied specifically to musculoskeletal tissues, and a systematic comparison of the clearing ability of optical clearing agents in musculoskeletal tissues has yet to be fully demonstrated. In this study we tested the ability of eight different aqueous and non-aqueous clearing agents, with RIs ranging from 1.45 to 1.56, to optically clear murine knee joints and cortical bone. We demonstrated and quantified the ability of these optical clearing agents to clear musculoskeletal tissues and improve both macro- and micro-scale imaging of musculoskeletal tissue across several imaging modalities (stereomicroscopy, spectroscopy, and one-, and two-photon confocal microscopy) and investigational techniques (dynamic bone labeling and en bloc tissue staining). Based upon these findings we believe that optical clearing, in combination with advanced imaging techniques, has the potential to complement classical musculoskeletal analysis techniques; opening the door for improved in situ investigation and quantification of musculoskeletal tissues.     

Mechanics of blastocyst morphogenesis

During pre‐implantation development, the mammalian zygote transforms into the blastocyst, the structure that will implant the embryo in the maternal uterus. Consisting of a squamous epithelium enveloping a fluid‐filled cavity and the inner cell mass, the blastocyst is sculpted by a succession of morphogenetic events. These deformations result from the changes in the forces and mechanical properties of the tissue composing the embryo. Here, I review the recent studies, which, for the first time, informed us on the mechanics of blastocyst morphogenesis.     

Multicolor protein labeling in living cells using mutant β-lactamase-tag technology

Protein labeling techniques using small molecule probes have become important as practical alternatives to the use of fluorescent proteins (FPs) in live cell imaging. These labeling techniques can be applied to more sophisticated fluorescence imaging studies such as pulse-chase imaging. Previously, we reported a novel protein labeling system based on the combination of a mutant β-lactamase (BL-tag) with coumarin-derivatized probes and its application to specific protein labeling on cell membranes. In this paper, we demonstrated the broad applicability of our BL-tag technology to live cell imaging by the development of a series of fluorescence labeling probes for this technology, and the examination of the functions of target proteins. These new probes have a fluorescein or rhodamine chromophore, each of which provides enhanced photophysical properties relative to coumarins for the purpose of cellular imaging. These probes were used to specifically label the BL-tag protein and could be used with other small molecule fluorescent probes. Simultaneous labeling using our new probes with another protein labeling technology was found to be effective. In addition, it was also confirmed that this technology has a low interference with respect to the functions of target proteins in comparison to GFP. Highly specific and fast covalent labeling properties of this labeling technology is expected to provide robust tools for investigating protein functions in living cells, and future applications can be improved by combining the BL-tag technology with conventional imaging techniques. The combination of probe synthesis and molecular biology techniques provides the advantages of both techniques and can enable the design of experiments that cannot currently be performed using existing tools.     

Morphogenesis as a macroscopic self-organizing process

We start from reviewing different epistemological constructions used for explaining morphogenesis. Among them, we explore the explanatory power of a law-centered approach which includes top-down causation and the basic concepts of a self-organization theory. Within such a framework, we discuss the morphomechanical models based upon the presumption of feedbacks between mechanical stresses imposed onto a given embryo part from outside and those generated within the latter as a kind of active response. A number of elementary morphogenetic events demonstrating that these feedbacks are directed towards hyper-restoration (restoration with an overshoot) of the initial state of mechanical stresses are described. Moreover, we show that these reactions are bound together into the larger scale feedbacks. That permits to suggest a reconstruction of morphogenetic successions in early Metazoan development concentrated around two main archetypes distinguished by the blastopores geometry. The perspectives of applying the same approach to cell differentiation are outlined. By discussing the problem of positional information we suggest that the developmental pathway of a given embryo part depends upon its preceded deformations and the corresponding mechanical stresses rather than upon its static position at any moment of development.     

Four-color, 4-D time-lapse confocal imaging of chick embryos

Our understanding of the molecular mechanisms that direct cell motility, cell division, and cell shaping has benefited from innovations in cell labeling and the ability to resolve intracellular dynamics with multispectral, high-resolution imaging. However, due to difficulties with in vivo cell marking and monitoring, most studies have been restricted to fixed tissue or cells in culture. Here, we report the delivery of multiple (up to four), multicolor fluorescent protein (FP) constructs and four-dimensional (4-D), multispectral time-lapse confocal imaging of cell movements in living chick embryos. Cell cytoskeletal components are fluorescently tagged after microinjection and electroporation of a cocktail of FP constructs into specific regions of chick embryos. We tested 11 different FP constructs in various two-, three-, and four-color combinations using multispectral imaging and linear unmixing to limit the crosstalk between different emission spectra. We monitored intracellular dynamics in individual multicolored migrating cells in vivo and developed a set of advantageous imaging parameters for 4-D time-lapse confocal microscopy. We find that the number of four-color labeled cells in a typical embryo is approximately 10% of the total number of fluorescently labeled cells; this value consistently increases showing that approximately 50% of the total labeled cells have only one-color. We find that multicolored cells are photostable for time-lapses of approximately 2-3 h. Thus, cell labeling with up to four FP color schemes combined with multispectral, 4-D confocal time-lapse imaging offers a powerful tool to simultaneously analyze cellular and molecular dynamics during chick embryogenesis.     

Origin of somatic embryos from cultured shoot tips ofSorghum bicolor (L.) moench

Summary Histologic examination of shoot-tip explants, 1 wk after culture initiation on Murashige and Skoog medium with 2.5 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/liter kinetin, reveals active meristematic centers inside cultured tissue. Clusters of cells in these meristematic centers exhibit remarkable resemblance to the initial three divisions in the zygotic embryo. Several such meristematic groups of cells are observed in the cultured explant at this stage. Embryogenesis is obviously initiated very early in this tissue in the presence of 2,4-D. A well-defined, white globular embryogenic callus develops in culture in about 4 wk, and it consists of clusters of embryoids with large cells characterized by thick cell walls, numerous lipoidal vesicles, and localized areas of carbohydrate storage. These cells resemble the scutellar tissue of the embryo. However, there are cells within this tissue that themselves appear embryogenic. They undergo cell division giving rise to small clusters of cells. As long as 2,4-D is present in the medium, the cells apparently retain the capacity to proliferate and to produce more cells capable of embryogenesis. Embryogenesis seems to occur via two processes, initiation of somatic embryos early in culture and secondary embryogensis from the scutellar tissue that forms in vitro.     

Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice.     

Ultrastructural identification of apoptotic nuclei using the TUNEL technique

Summary We describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor α. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as the associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.     

Multiple topological labeling for imaging single plasmids

Sequence-specific labeling methods for double-stranded DNA are required for mapping protein binding sites or specific DNA structures on circular DNA molecules by high-resolution imaging techniques such as electron and atomic force microscopies. Site-specific labeling can be achieved by ligating a DNA fragment to a stem-loop-triplex-forming oligonucleotide, thereby forming a topologically linked complex. The superhelicity of the plasmid is not altered and the process can be applied to two different target sites simultaneously, using DNA fragments of different sizes. Observation of the labeled plasmids by electron microscopy revealed that, under conditions where the triple helices were stable, the two labels were located at 339+/-34 bp from one another, in agreement with the distance between the two target sequences for triple helix formation (350 bp). Under conditions where the triple helices were not stable, the short DNA fragments could slide away from their target site. The concomitant attachment of two different stable labels makes it possible, for the first time to our knowledge, to label a circular DNA molecule and obtain information on its direction. In addition to its potential applications as a tool for structural investigations of single DNA molecules and their interactions with proteins, this DNA labeling method may also prove useful in biotechnology and gene therapy.     

A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−¹ casein hydrolysate, 9 μ M 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.2 μ M kinetin. An efficient two-step, two-medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μ M 2,4-D and 4.7 μ M kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4-D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata -based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa , with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.     

植物蛋白质组学研究若干重要进展

植物蛋白质组学近年来正从定性向精确定量蛋白质组学的方向发展。国际上近两年发表的约160篇研究论文报道了利用不断改进的双向电泳结合生物质谱技术、多维蛋白质鉴定技术, 以及包括双向荧光差异凝胶电泳、15N体内代谢标记、同位素标记的亲和标签、同位素标记相对和绝对定量等在内的第2代蛋白质组学技术, 对植物组织(器官)与细胞器、植物发育过程和植物响应环境胁迫的蛋白质组特征, 以及植物蛋白质翻译后修饰和蛋白质相互作用等方面的研究成果。该文对上述报道进行总结, 综述了2007年以来植物蛋白质组学若干重要问题研究的新进展。     

Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.     

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.     

Parallels between tissue repair and embryo morphogenesis

Wound healing involves a coordinated series of tissue movements that bears a striking resemblance to various embryonic morphogenetic episodes. There are several ways in which repair recapitulates morphogenesis. We describe how almost identical cytoskeletal machinery is used to repair an embryonic epithelial wound as is involved during the morphogenetic episodes of dorsal closure in Drosophila and eyelid fusion in the mouse foetus. For both naturally occurring and wound-activated tissue movements, JNK signalling appears to be crucial, as does the tight regulation of associated cell divisions and adhesions. In the embryo, both morphogenesis and repair are achieved with a perfect end result, whereas repair of adult tissues leads to scarring. We discuss whether this may be due to the adult inflammatory response, which is absent in the embryo.     

Effect of 2,4-dichlorophenoxyacetic Acid on the expression of embryogenic program in carrot

Embryogenesis in a wild carrot cell line, W001C, can start and progress up to the first morphogenetic stage (the globular-stage embryo) in 2,4-dichloropenoxyacetic acid (2,4-D). To clarify the quantitative effect of 2,4-D on this cell line, morphological and biochemical criteria have been used to monitor embryogenesis in the presence of increasing concentrations of 2,4-D. The biochemical criteria are the ability to inactivate cycloheximide and the expression of an embryogenic polypeptide E₁. The results show that 2,4-D can affect embryogenesis in a quantitative manner but never fully suppresses embryogenesis unless it is coupled with high cell density.