Culturing of avian embryos for time-lapse imaging

Monitoring morphogenetic processes, at high resolution over time, has been a longstanding goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h

The ECM Moves during Primitive Streak Formation—Computation of ECM Versus Cellular Motion

Galileo described the concept of motion relativity—motion with respect to a reference frame—in 1632. He noted that a person below deck would be unable to discern whether the boat was moving. Embryologists, while recognizing that embryonic tissues undergo large-scale deformations, have failed to account for relative motion when analyzing cell motility data. A century of scientific articles has advanced the concept that embryonic cells move (“migrate”) in an autonomous fashion such that, as time progresses, the cells and their progeny assemble an embryo. In sharp contrast, the motion of the surrounding extracellular matrix scaffold has been largely ignored/overlooked. We developed computational/optical methods that measure the extent embryonic cells move relative to the extracellular matrix. Our time-lapse data show that epiblastic cells largely move in concert with a sub-epiblastic extracellular matrix during stages 2 and 3 in primitive streak quail embryos. In other words, there is little cellular motion relative to the extracellular matrix scaffold—both components move together as a tissue. The extracellular matrix displacements exhibit bilateral vortical motion, convergence to the midline, and extension along the presumptive vertebral axis—all patterns previously attributed solely to cellular “migration.” Our time-resolved data pose new challenges for understanding how extracellular chemical (morphogen) gradients, widely hypothesized to guide cellular trajectories at early gastrulation stages, are maintained in this dynamic extracellular environment. We conclude that models describing primitive streak cellular guidance mechanisms must be able to account for sub-epiblastic extracellular matrix displacements

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time-dependent rearrangements of a primordial biomaterial. We conclude that the ECM microenvironment changes markedly in time and space during the most important period of amniote morphogenesis—as determined by fluctuating textural properties

Microplate-based chromatin immunoprecipitation method, Matrix ChIP: a platform to study signaling of complex genomic events

The chromatin immunoprecipitation (ChIP) assay is a major tool in the study of genomic processes in vivo. This and other methods are revealing that control of gene expression, cell division and DNA repair involves multiple proteins and great number of their modifications. ChIP assay is traditionally done in test tubes limiting the ability to study signaling of the complex genomic events. To increase the throughput and to simplify the assay we have developed a microplate-based ChIP (Matrix ChIP) method, where all steps from immunoprecipitation to DNA purification are done in microplate wells without sample transfers. This platform has several important advantages over the tube-based assay including very simple sample handling, high throughput, improved sensitivity and reproducibility, and potential for automation. 96 ChIP measurements including PCR can be done by one researcher in one day. We illustrate the power of Matrix ChIP by parallel profiling 80 different chromatin and transcription time-course events along an inducible gene including transient recruitment of kinases

Refuerzo del puente puangue en Chile mediante configuración arco tipo network. Comportamiento sísmico.

Actualmente se está estudiando una nueva técnica de reforzar puentes existentes de vigas continuas en Chile debido a la existencia de socavación en las pilas intermedias ubicadas en el cauce. Este método se basa en un cambio estático de la estructura utilizando un arco tipo network para sustentar el tablero. El tablero existente se mantiene y se utiliza como tirante para el arco con tendones pretensados. Durante el proceso constructivo el tablero se levanta y cuando actúa como puente en arco, se suprimen las pilas, generando un único vano y evitándose los problemas de socavación. La técnica está planteada para puentes de luz pequeña y mediana de varios vanos con sistema apoyo continuo. Este trabajo profundiza el concepto general del cambio estático mediante el estudio de los efectos sísmicos en la estructura modificada. Se analiza el comportamiento general del puente y se identifica los elementos críticos bajo dicha acción que puedan ser importantes para los criterios del método de refuerzo. Para este estudio se ha considerado como ejemplo el puente Puangue, ubicado en la provincia Melipilla en el Región Metropolitana de Chile. Esta región se caracteriza por presentar una alta sismicidad. Corresponde a un puente de hormigón armado de vigas continuas con 4 vanos, el cual presenta importantes daños debido a la socavación. No existe información sobre la armadura del puente (planos), solo dimensiones geométricas exteriores. La metodología del trabajo corresponde a la modelación del puente original en un programa de análisis estructural, comparando los resultados sísmicos con los del modelo del puente modificado con refuerzo de arco y péndolas verticales y network. Los resultados indican que este nuevo método de refuerzo, aplicado al puente Puangue, es una opción factible desde la perspectiva técnica, frente efectos sísmicos, debido a que no existen problemas graves en el tablero al someterse al sismo. Sin embargo es importante considerar estas acciones en la verificación del arco, por lo tanto es imprescindible incorporar criterios sísmicos en la verificación global de este método de refuerzo. Finalmente, es posible apoyar que este tipo de refuerzo es una solución factible y atractiva

Refuerzo del puente puangue en Chile mediante configuración arco tipo network. Comportamiento sísmico.

Actualmente se está estudiando una nueva técnica de reforzar puentes existentes de vigas continuas en Chile debido a la existencia de socavación en las pilas intermedias ubicadas en el cauce. Este método se basa en un cambio estático de la estructura utilizando un arco tipo network para sustentar el tablero. El tablero existente se mantiene y se utiliza como tirante para el arco con tendones pretensados. Durante el proceso constructivo el tablero se levanta y cuando actúa como puente en arco, se suprimen las pilas, generando un único vano y evitándose los problemas de socavación. La técnica está planteada para puentes de luz pequeña y mediana de varios vanos con sistema apoyo continuo. Este trabajo profundiza el concepto general del cambio estático mediante el estudio de los efectos sísmicos en la estructura modificada. Se analiza el comportamiento general del puente y se identifica los elementos críticos bajo dicha acción que puedan ser importantes para los criterios del método de refuerzo. Para este estudio se ha considerado como ejemplo el puente Puangue, ubicado en la provincia Melipilla en el Región Metropolitana de Chile. Esta región se caracteriza por presentar una alta sismicidad. Corresponde a un puente de hormigón armado de vigas continuas con 4 vanos, el cual presenta importantes daños debido a la socavación. No existe información sobre la armadura del puente (planos), solo dimensiones geométricas exteriores. La metodología del trabajo corresponde a la modelación del puente original en un programa de análisis estructural, comparando los resultados sísmicos con los del modelo del puente modificado con refuerzo de arco y péndolas verticales y network. Los resultados indican que este nuevo método de refuerzo, aplicado al puente Puangue, es una opción factible desde la perspectiva técnica, frente efectos sísmicos, debido a que no existen problemas graves en el tablero al someterse al sismo. Sin embargo es importante considerar estas acciones en la verificación del arco, por lo tanto es imprescindible incorporar criterios sísmicos en la verificación global de este método de refuerzo. Finalmente, es posible apoyar que este tipo de refuerzo es una solución factible y atractiva

Refuerzo del puente puangue en Chile mediante configuración arco tipo network. Comportamiento sísmico.

Actualmente se está estudiando una nueva técnica de reforzar puentes existentes de vigas continuas en Chile debido a la existencia de socavación en las pilas intermedias ubicadas en el cauce. Este método se basa en un cambio estático de la estructura utilizando un arco tipo network para sustentar el tablero. El tablero existente se mantiene y se utiliza como tirante para el arco con tendones pretensados. Durante el proceso constructivo el tablero se levanta y cuando actúa como puente en arco, se suprimen las pilas, generando un único vano y evitándose los problemas de socavación. La técnica está planteada para puentes de luz pequeña y mediana de varios vanos con sistema apoyo continuo. Este trabajo profundiza el concepto general del cambio estático mediante el estudio de los efectos sísmicos en la estructura modificada. Se analiza el comportamiento general del puente y se identifica los elementos críticos bajo dicha acción que puedan ser importantes para los criterios del método de refuerzo. Para este estudio se ha considerado como ejemplo el puente Puangue, ubicado en la provincia Melipilla en el Región Metropolitana de Chile. Esta región se caracteriza por presentar una alta sismicidad. Corresponde a un puente de hormigón armado de vigas continuas con 4 vanos, el cual presenta importantes daños debido a la socavación. No existe información sobre la armadura del puente (planos), solo dimensiones geométricas exteriores. La metodología del trabajo corresponde a la modelación del puente original en un programa de análisis estructural, comparando los resultados sísmicos con los del modelo del puente modificado con refuerzo de arco y péndolas verticales y network. Los resultados indican que este nuevo método de refuerzo, aplicado al puente Puangue, es una opción factible desde la perspectiva técnica, frente efectos sísmicos, debido a que no existen problemas graves en el tablero al someterse al sismo. Sin embargo es importante considerar estas acciones en la verificación del arco, por lo tanto es imprescindible incorporar criterios sísmicos en la verificación global de este método de refuerzo. Finalmente, es posible apoyar que este tipo de refuerzo es una solución factible y atractiva

Transient Increase in VEGF-A Leads to Cardiac Tube Anomalies and Increased Risk of Congenital Heart Malformations.

Vascular Endothelial Growth Factor (VEGF) plays a critical role during early heart development. Clinical evidence shows that conditions associated with changes in VEGF signaling in utero are correlated with an increased risk of congenital heart defects (CHD) in newborns. However, how malformations develop after abnormal VEGF exposure is unknown. During embryogenesis, a primitive heart, consisting of an endocardial tube enveloped by a myocardial mantle, is the first organ to function. This tubular heart ultimately transforms into a four-chambered heart. To determine how a transient increase in VEGF prior to heart tube formation affects heart development leading to CHD, we applied exogenous VEGF or a control (vehicle) solution to quail embryos in ovo at Hamburger-Hamilton (HH) stage 8 (28-30 hrs of incubation), right before heart tube formation. Light microscopy analysis of embryos re-incubated after treatment for 13 hrs (to approximately HH11/HH12) showed that increased VEGF leads to impaired heart tube elongation accompanied by diameter expansion. Micro-CT analysis of embryos re-incubated for 9 days (to approximately HH38), when the heart is fully formed, showed that VEGF treatment increased the rate of cardiac malformations in surviving embryos. However, despite no sex differences in survival, female embryos were more likely to develop cardiac malformations. Our results further suggest that heart tube malformations after a transient increase in VEGF right before heart tube formation may be reversible, leading to normal hearts. This article is protected by copyright. All rights reserved