Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis

In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis.     

Dynamics of embryonic pancreas development using real-time imaging

Current knowledge about developmental processes in complex organisms has relied almost exclusively on analyses of fixed specimens. However, organ growth is highly dynamic, and visualization of such dynamic processes, e.g., real-time tracking of cell movement and tissue morphogenesis, is becoming increasingly important. Here, we use live imaging to investigate expansion of the embryonic pancreatic epithelium in mouse. Using time-lapse imaging of tissue explants in culture, fluorescently labeled pancreatic epithelium was found to undergo significant expansion accompanied by branching. Quantification of the real-time imaging data revealed lateral branching as the predominant mode of morphogenesis during epithelial expansion. Live imaging also allowed documentation of dynamic beta-cell formation and migration. During in vitro growth, appearance of newly formed beta-cells was visualized using pancreatic explants from MIP-GFP transgenic animals. Migration and clustering of beta-cells were recorded for the first time using live imaging. Total beta-cell mass and concordant aggregation increased during the time of imaging, demonstrating that cells were clustering to form "pre-islets". Finally, inhibition of Hedgehog signaling in explant cultures led to a dramatic increase in total beta-cell mass, demonstrating application of the system in investigating roles of critical embryonic signaling pathways in pancreas development including beta-cell expansion. Thus, pancreas growth in vitro can be documented by live imaging, allowing visualization of the developing pancreas in real-time.     

Reconstructing embryonic development

Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources.     

SnoN in regulation of embryonic development and tissue morphogenesis

SnoN (Ski-novel protein) plays an important role in embryonic development, tumorigenesis and aging. Past studies largely focused on its roles in tumorigenesis. Recent studies of its expression patterns and functions in mouse models and mammalian cells have revealed that SnoN interacts with multiple signaling molecules at different cellular levels to modulate the activities of several signaling pathways in a tissue context and developmental stage dependent manner. These studies suggest that SnoN may have broad functions in the embryonic development and tissue morphogenesis.     

Cellular mechanobiology and cancer metastasis

The primary cause of cancer treatment failure is invasion and metastasis, and invading tumor cells utilize many of the motility patterns that have been documented for normal morphogenesis. Recently, the role of mechanical forces in guiding various tissue and cell movements in embryonic development has been systematically analyzed with new experimental and computational methods. The tissue and cellular mechanobiology approach also holds promise for increasing the understanding of tumor invasion. In fact, the mechanical stiffness of tumors has correlated with invasiveness, and manipulation of extracellular matrix (ECM) stiffness in vitro has suppressed the cancer phenotype. Several important signaling molecules reside on the cytoskeleton, which is affected by external stress imparted by the ECM, and deformation of the nucleus can trigger the activation of certain genes. All these observations suggest that a synthesis of the biology of cancer cell invasion and cellular mechanobiology may offer new targets for the treatment of malignant disease. Accordingly, sensitive and relevant in vivo models and methods to study cancer mechanobiology are needed.     

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.     

Quantitative isolation and in vivo imaging of malaria parasite liver stages

The liver stages of Plasmodium, the causative agent of malaria, are the least explored forms in the parasite's life cycle despite their recognition as key vaccine and drug targets. In vivo experimental access to liver stages of human malaria parasites is practically prohibited and therefore rodent model malaria parasites have been used for in vivo studies. However, even in rodent models progress in the analysis of liver stages has been limited, mainly due to their low abundance and associated difficulties in visualisation and isolation. Here, we present green fluorescent protein (GFP)-tagged Plasmodium yoelii rodent malaria parasite liver infections in BALB/c mice as an excellent quantitative model for the live visualisation and isolation of the so far elusive liver stages. We believe P. yoelii GFP-tagged liver stages allow, for the first time, the efficient quantitative isolation of intact early and late liver stage-infected hepatocyte units by fluorescence activated cell sorting. GFP-tagged liver stages are also well suited for intravital imaging, allowing us for the first time to visualise them in real time. We identify previously unrecognised features of liver stages including vigorous parasite movement and expulsion of 'extrusomes'. Intravital imaging thus reveals new, important information on the malaria parasite's transition from tissue to blood stage.     

Live optical projection tomography

Optical projection tomography (OPT) is a technology ideally suited for imaging embryonic organs. We emphasize here recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse OPT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further developed by merging state-of-the-art mouse embryo culture with the OPT system. We discuss the technological challenges that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.     

Live optical projection tomography

Optical projection tomography (OPT) is a technology ideally suited for imaging embryonic organs. We emphasize here recent successes in translating this potential into the field of live imaging. Live OPT (also known as 4D OPT, or time-lapse OPT) is already in position to accumulate good quantitative data on the developmental dynamics of organogenesis, a prerequisite for building realistic computer models and tackling new biological problems. Yet, live OPT is being further developed by merging state-of-the-art mouse embryo culture with the OPT system. We discuss the technological challenges that this entails and the prospects for expansion of this molecular imaging technique into a wider range of applications.     

Integrative approaches to morphogenesis: lessons from dorsal closure

Although developmental biology has been dominated by the genetic analysis of embryonic development, in recent years genetic tools have been combined with new approaches such as imaging of live processes, automated and quantitative image analysis, mechanical perturbation and mathematical modeling, to study the principles underlying the formation of organisms. Here we focus on recent work carried out on Dorsal Closure, a morphogenetic process during Drosophila embryogenesis, to illustrate how this multidisciplinary approach is yielding new and unexpected insights into how cells organize themselves through the activity of their molecular components to give rise to the stereotyped and macroscopic movements observed during development.     

Avian scale development. X. Dermal induction of tissue-specific keratins in extraembryonic ectoderm

Epidermal-dermal tissue interactions regulate morphogenesis and tissue-specific keratinization of avian skin appendages. The morphogenesis of scutate scales differs from that of reticulate scales, and the keratin polypeptides of their epidermal surfaces are also different. Do the inductive cues which initiate morphogenesis of these scales also establish the tissue-specific keratin patterns of the epidermis, or does the control of tissue-specific keratinization occur at later stages of development? Unlike feathers, scutate and reticulate scales can be easily separated into their epidermal and dermal components late in development when the major events of morphogenesis have been completed and keratinization will begin. Using a common responding tissue (chorionic epithelium) in combination with scutate and reticulate scale dermises, we find that these embryonic dermises, which have completed morphogenesis, can direct tissue-specific statification and keratinization. In other words, once a scale dermis has acquired its form, through normal morphogenesis, it is no longer able to initiate morphogenesis of that scale, but it can direct tissue-specific stratification and keratinization of a foreign ectodermal epithelium, which itself has not undergone scale morphogenesis.     

A driving force for change: interstitial flow as a morphoregulator

Dynamic stresses that are present in all living tissues drive small fluid flows, called interstitial flows, through the extracellular matrix. Interstitial flow not only helps to transport nutrients throughout the tissue, but also has important roles in tissue maintenance and pathobiology that have been, until recently, largely overlooked. Here, we present evidence for the various effects of interstitial flow on cell biology, including its roles in embryonic development, tissue morphogenesis and remodeling, inflammation and lymphedema, tumor biology and immune cell trafficking. We also discuss possible mechanisms by which interstitial flow can induce morphoregulation, including direct shear stress, matrix-cell transduction (as has been proposed in the endothelial glycocalyx) and the newly emerging concept of autologous gradient formation.     

Pulsation and stabilization: Contractile forces that underlie morphogenesis

Embryonic development involves global changes in tissue shape and architecture that are driven by cell shape changes and rearrangements within cohesive cell sheets. Morphogenetic changes at the cell and tissue level require that cells generate forces and that these forces are transmitted between the cells of a coherent tissue. Contractile forces generated by the actin-myosin cytoskeleton are critical for morphogenesis, but the cellular and molecular mechanisms of contraction have been elusive for many cell shape changes and movements. Recent studies that have combined live imaging with computational and biophysical approaches have provided new insights into how contractile forces are generated and coordinated between cells and tissues. In this review, we discuss our current understanding of the mechanical forces that shape cells, tissues, and embryos, emphasizing the different modes of actomyosin contraction that generate various temporal and spatial patterns of force generation.     

The plastic cell: mechanical deformation of cells and tissues

Epithelial cells possess the ability to change their shape in response to mechanical stress by remodelling their junctions and their cytoskeleton. This property lies at the heart of tissue morphogenesis in embryos. A key feature of embryonic cell shape changes is that they result from repeated mechanical inputs that make them partially irreversible at each step. Past work on cell rheology has rarely addressed how changes can become irreversible in a complex tissue. Here, we review new and exciting findings dissecting some of the physical principles and molecular mechanisms accounting for irreversible cell shape changes. We discuss concepts of mechanical ratchets and tension thresholds required to induce permanent cell deformations akin to mechanical plasticity. Work in different systems has highlighted the importance of actin remodelling and of E-cadherin endocytosis. We also list some novel experimental approaches to fine-tune mechanical tension, using optogenetics, magnetic beads or stretching of suspended epithelial tissues. Finally, we discuss some mathematical models that have been used to describe the quantitative aspects of accounting for mechanical cell plasticity and offer perspectives on this rapidly evolving field.     

Extracellular matrices as elastic scaffold and mechanical interaction.

Development of spatial pattern and form is one of the central issues in embryology and is included under the general name of morphogenesis. Recently, many investigations have revealed how does development occurs by each embryonic stem cell or which Genes play a crucial role for morphogenesis. However, still fundamental question is unclear; such as how does each cell recognize spatial information or which kind of information guides each cell to the suitable place. Approximately, we have 6x10(13) cells in our body. If each frame of reference of each cell is included in the gene, gene must have included more than 6x10(13) of information to inform each cell where they are. We could simply suggest this kind of the idea is quite wrong because we know genes are few enough to include such informations. Recently, it has been suggested that interaction between intracellular and extracellular fiber play crucial roll for morphogenesis. The fibers inside cell are quite complicated but well organized the system, and fibers outside of the cell are comparatively very simple fiber. Each of the fibers is well studied, but quantitative investigation of their interaction is lacking although importance is suggested by many researchers. A major problem is lacking of new method or technique. In our topics, we would like to introduce how intracellular and extracellular fiber generate morphogenesis and how we could investigate them using new technique for tissue engineering, one of the promising field of applied cell biology.     

On the evolution of morphogenetic models: mechano-chemical interactions and an integrated view of cell differentiation, growth, pattern formation and morphogenesis

In the 1950s, embryology was conceptualized as four relatively independent problems: cell differentiation, growth, pattern formation and morphogenesis. The mechanisms underlying the first three traditionally have been viewed as being chemical in nature, whereas those underlying morphogenesis have usually been discussed in terms of mechanics. Often, morphogenesis and its mechanical processes have been regarded as subordinate to chemical ones. However, a growing body of evidence indicates that the biomechanics of cells and tissues affect in striking ways those phenomena often thought of as mainly under the control of cell-cell signalling. This accumulation of data has led to a revival of the mechano-transduction concept in particular, and of complexity in general, causing us now to consider whether we should retain the traditional conceptualization of development. The researchers' semantic preferences for the terms 'patterning', 'pattern formation' or 'morphogenesis' can be used to describe three main 'schools of thought' which emerged in the late 1970s. In the 'molecular school', the term patterning is deeply tied to the positional information concept. In the 'chemical school', the term 'pattern formation' regularly implies reaction-diffusion models. In the 'mechanical school', the term 'morphogenesis' is more frequently used in relation to mechanical instabilities. Major differences among these three schools pertain to the concept of self-organization, and models can be classified as morphostatic or morphodynamic. Various examples illustrate the distorted picture that arises from the distinction among differentiation, growth, pattern formation and morphogenesis, based on the idea that the underlying mechanisms are respectively chemical or mechanical. Emerging quantitative approaches integrate the concepts and methods of complex sciences and emphasize the interplay between hierarchical levels of organization via mechano-chemical interactions. They draw upon recent improvements in mathematical and numerical morphogenetic models and upon considerable progress in collecting new quantitative data. This review highlights a variety of such models, which exhibit important advances, such as hybrid, stochastic and multiscale simulations.     

Imaging and modeling growth and morphogenesis in plants

The growth of tissues, organs or organisms derives from the coordinated activities of complex genetic regulatory networks. In addition to its molecular underpinnings, growth also generally involves significant changes in geometry. To fully understand morphogenesis in its molecular and physical contexts the development of an interdisciplinary approach is required associating biology, mathematics, and physics, which held together by computer science. Growth quantitation and digital simulations have been developed to generate and test the plausibilities of complex hypotheses. Increasingly, real-time live imaging protocols are becoming an essential part of this process. In this review, I discuss the evolution of imaging techniques in plant developmental biology and briefly examine the different ways in which these studies have shed light on growth and morphogenesis in plants.     

Convection-Induced Biased Distribution of Actin Probes in Live Cells

Fluorescent markers that bind endogenous target proteins are frequently employed for quantitative live-cell imaging. To visualize the actin cytoskeleton in live cells, several actin-binding probes have been widely used. Among them, Lifeact is the most popular probe with ideal properties, including fast exchangeable binding kinetics. Because of its fast kinetics, Lifeact is generally believed to distribute evenly throughout cellular actin structures. In this study, however, we demonstrate misdistribution of Lifeact toward the rear of lamellipodia where actin filaments continuously move inward along the retrograde flow. Similarly, phalloidin showed biased misdistribution toward the rear of lamellipodia in live cells. We show evidence of convection-induced misdistribution of actin probes by both experimental data and physical models. Our findings warn about the potential error arising from the use of target-binding probes in quantitative live imaging.