Contractile and relaxing reactivity in carotid and femoral arteries of chicken embryos

In the embryo, hypoxemia causes redistribution of cardiac output from the periphery toward the heart and the brain. In view of this, we investigated developmental changes in the contractile and relaxing properties of the peripheral femoral artery (Fem) and the more central carotid artery (Car) at 0.7, 0.8, and 0.9 of the chicken embryo incubation time. Isolated arteries were studied in myographs and were exposed to norepinephrine or phenylephrine. High K(+) (125 mM) and electrical field stimulation (0.25-16 Hz) were used to induce receptor-independent and neurogenic contractions. Relaxing responses to ACh were evaluated in the absence and presence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and before and after endothelium removal. alpha(1)-Adrenergic contractile responses increased in a time-dependent manner and were significantly larger in Fem than in Car. Neurogenic contractions and adrenergic nerves could only be demonstrated in Fem at 0.9 incubation. ACh caused relaxation in both Fem and Car at 0.7, 0.8, and 0.9 incubation. The NO-independent part of the relaxation was more pronounced in Car than in Fem at all developmental stages. We conclude that the chicken embryo is a useful model to investigate the development of vasomotor control and vascular heterogeneity. The observed regional vascular differences may contribute to cardiac output redistribution during hypoxia in the embryo and might result from endothelial and neurogenic influences on vascular smooth muscle differentiation.     

In ovo electroporations of HH stage 10 chicken embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology. With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function. By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function. Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements. Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive. This video demonstrates first how to make a window in the eggshell to manipulate the embryo. Next, the embryo is visualized with a dilute solution of India ink injected below the embryo. A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. Finally, the egg is sealed with tape and placed back into an incubator for further development. Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.     

In ovo transfection of chicken embryos using cationic liposomes

It is reported that cationic liposomes are capable of transfecting embryos in unincubated fertile chicken eggs and that the cationic liposome, TransfectAce^TM, has superior properties to Lipofectin^TM. In order to determine the duration of expression of genes introduced in this way, embryos were transfected with an expression vector encoding the firefly luciferase cDNA under the control of the Rous sarcoma virus long terminal repeat (LTR). Luciferase activity could be observed consistently in day 3 embryos and activity was detectable up to day 8 of incubation. The relative expression of luciferase under the control of different viral promoters was compared in transfected chicken embryo fibroblasts and day 3 embryos. The cytomegalovirus immediate early promoter and the SV40 early promoter directed the highest amount of expression in fibroblasts while the Rous sarcoma virus LTR caused the highest amount of expression in embryos. Chicken embryo fibroblasts were transfected with the luciferase vector in order to examine duration of reporter gene expressionin vitro. Luciferase expression was decreased exponentially over a 24-day period after which point luciferase activity could no longer be detected. These data suggest that stable integration of transfected DNA using liposomes is a rare event. Nevertheless, liposome-mediated transfection of embryos is suitable for the examination of promoter activityin vivo and may be a useful method to transfect genes to study embryonic development.     

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin‐containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross‐talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus‐mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication‐Competent Avian sarcoma‐leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back‐transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)⁺Neo^r+‐electroporated melanocytes from Hypeco nera were selectively grown in G418‐supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS‐mOrange, mOrange⁺ keratinocytes and transplanted EGFP⁺ melanocytes were frequently juxtaposed each other. High‐resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.     

鸡胚胎在生命科学研究中的应用

鸡作为优秀的模式动物在生命科学研究中的作用日益受到人们重视,其中以鸡胚胎的贡献尤为突出。本文简要综述了鸡胚胎在发育生物学、肿瘤、再生、神经生物学和胚胎干细胞等研究领域中的应用,同时对鸡基因组和鸡胚胎的研究方法也进行了简单探讨。     

Chick embryos exposed to trichloroethylene in an ex ovo culture model show selective defects in early endocardial cushion tissue formation

BACKGROUND: Formation of the primitive heart is a critical step for establishing a competent circulatory system necessary for continued morphogenesis, and as such has significant potential as a target for environmental insult. The goal of this study was to identify the initial cellular events that precede more superficially observable abnormalities resulting from exposing early chick embryos to trichloroethylene (TCE). METHODS: A whole embryo culture method was used to assess the susceptibility of endocardial epithelial-mesenchymal transformation in the early chick heart to TCE. This method has the benefits of maintaining the anatomical relationships of developing tissues and organs, instantaneously exposing precisely staged embryos to quantifiable levels of TCE in a protein-free medium, and the ability to directly monitor developmental morphology. RESULTS: A minority of embryos (Hamburger and Hamilton [HH] stage 13-14) exposed to TCE (10-80 ppm) were not viable after 24 hr in culture and exhibited a variety of gross malformations in a dose-dependent fashion. However, the majority of treated embryos remained viable and developed into HH stage 17 embryos that were superficially indistinguishable from vehicle-treated controls. Further analysis of the hearts of these superficially normal embryos by whole-mount confocal microscopy revealed selective reduction in the number of atrioventricular canal mesenchymal cells. Additionally, those mesenchymal cells that did develop migrated abnormally as long thin cords of adherent cells. CONCLUSIONS: The regional selectivity of these effects in the chick heart suggests a critical window of susceptibility to TCE in the epithelial-mesenchymal transformation of atrioventricular canal endocardium.     

The developmental stage of chicken embryos modulates the impact of in ovo olfactory stimulation on food preferences

Like mammals, bird embryos are capable of chemosensory learning, but the ontogeny of their feeding preferences has not been examined. We tested if the timing of stimulation in chicken embryos modulates the impact of in ovo olfactory stimulation on later food preferences. We exposed chicken embryos to an olfactory stimulus for a 4-day period in the middle or toward the end of the incubation period. The chicks were tested for their preference between foods with and without the olfactory stimulus in 3-min choice tests and on a 24-h time scale. Regardless of the type of food (familiar or novel) or the duration of the test, the control chicks not exposed to the olfactory stimulus consistently showed significant preferences for non-odorized foods. Chicks that were exposed in ovo to the olfactory stimulus did not show a preference for odorized or non-odorized foods. Only those chicks that were exposed to the olfactory stimulus toward the end of the incubation period differed from the controls and incorporated a higher proportion of odorized food into their diets on a 24-h time scale. This result indicates that olfactory stimulation at the end of embryonic development has a stronger impact on later feeding preferences. Our findings contribute to the growing pool of recent data appreciating the impact of olfactory signals on behavior regulation in avian species.     

Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos

The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off", works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes.     

Magnetic resonance microscopy of chick embryos in ovo

Magnetic resonance imaging (MRI) of the live 11-day chick embryo with special radiofrequency coils and 3-D imaging methods has produced contiguous 1.25-mm-thick slices with 200-microns pixel resolution, permitting definition of cardiac chambers, cerebral ventricles, spinal cord, liver, and lungs. It was the objective of this study to image younger chick embryos in ovo with higher spatial resolution through the application of implanted radiofrequency coils. Fertilized Arbor Acre eggs were windowed at 9, 6, and 4 days. Circular coils 18 mm in diameter tuned to 85.5 MHz were suspended around the developing embryo. The eggs were sealed with tape and maintained at 37 degrees C during the imaging procedure. MRI was performed in a 2.0-Tesla GE system utilizing a 3-D Fourier transform acquisition in sagittal and axial planes with a partial saturation sequence (TR = 400 ms, TE = 27 ms). Approximately 1 hour of imaging time was required to obtain 16 contiguous 600-microns-thick slices with 50-microns pixel resolution. Embryos remained viable through the imaging procedure. Embryos were photographed, fixed, and cleared for correlative anatomical study. Vitelline vessels, dorsal aorta, aortic arches, cardinal veins, and cardiac chambers were identified as areas of decreased signal intensity. Cerebral ventricles and the vitreous portion of the eye have signal intensities that are less than adjacent neural, scleral, and lens tissue. Further refinements in MR instrumentation and imaging sequences promise improvements in resolution and offer the potential for sequential observations of the intact embryo.     

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.     

Bulk elastic properties of chicken embryos during somitogenesis

We present measurements of the bulk Young's moduli of early chick embryos at Hamburger-Hamilton stage 10. Using a micropipette probe with a force constant k ~0.025 N/m, we applied a known force in the plane of the embryo in the anterior-posterior direction and imaged the resulting tissue displacements. We used a two-dimensional finite-element simulation method to model the embryo as four concentric elliptical elastic regions with dimensions matching the embryo's morphology. By correlating the measured tissue displacements to the displacements calculated from the in-plane force and the model, we obtained the approximate short time linear-elastic Young's moduli: 2.4 ± 0.1 kPa for the midline structures (notocord, neural tube, and somites), 1.3 ± 0.1 kPa for the intermediate nearly acellular region between the somites and area pellucida, 2.1 ± 0.1 kPa for the area pellucida, and 11.9 ± 0.8 kPa for the area opaca.     

Thermal effect on heart rate and hemodynamics in vitelline arteries of stage 18 chicken embryos

We investigated the thermal effects on heart rate, hemodynamics, and response of vitelline arteries of stage-18 chicken embryos. Heart rate was monitored by a high-speed imaging method, while hemodynamic quantities were evaluated using a particle image velocimetry (PIV) technique. Experiments were carried out at seven different temperatures (36–42 °C with 1 °C interval) after 1 h of incubation to stabilize the heart rate. The heart rate increased in a linear manner (r=0.992). Due to the increased cardiac output (or heart rate), the hemodynamic quantities such as mean velocity (U_mean), velocity fluctuation (U_fluc), and peak velocity (U_peak) also increased with respect to the Womersley number (Ω) in the manner r=0.599, 0.693, and 0.725, respectively. This indicates that the mechanical force exerting on the vessel walls increases. However, the active response (or regulation) of the vitelline arteries was not observed in this study.     

Introduction of DNA into chick embryos by in ovo electroporation

Gene transfer by in ovo electroporation has been applied to the study of developmental biology, especially to central nervous system (CNS) development. Plasmids are injected into the neural tube of stage 10 chick embryos, and a 25-V 25-msec square pulse is applied five times. Since DNA moves toward the anode, the cathode side of the neural tube is transfected, and the cathode side is used as the control. Expression of translation product of the introduced DNA is observed 2 h after electroporation, peaks around 20 h after electroporation and then weakens. Expression is transient when plasmids are used as expression vectors, but they are very suitable for studying early developmental events (e.g., gene expression cascades or interactions). Misexpression of Pax-5 is shown as an example.     

Vascular reactivity and baroreflex function during hyperthermia in conscious rats

The hemodynamic responses to vasoconstrictor agents are blunted during heating in anesthetized rats. It is unknown whether reflex neural responses to these agents are also altered during hyperthermia. Therefore, the purpose of this study was to determine the effect of hyperthermia on the hemodynamic and baroreflex-mediated sympathetic neural responses to vasoactive agents in conscious, unrestrained rats. The splanchnic sympathetic nerve activity (SpNA) and systemic and regional hemodynamic responses to injections of phenylephrine and sodium nitroprusside were measured during normothermia (37 degrees C) and hyperthermia (41.5 degrees C). The hemodynamic responses to phenylephrine and sodium nitroprusside were blunted with heating, whereas the SpNA responses to both agents were augmented or unchanged. At 41.5 degrees C, the baroreflex curves relating heart rate (HR) and SpNA to mean arterial blood pressure were shifted to the right. The operating range and gain of the blood pressure (BP)-HR reflex were significantly reduced during heating, whereas the operating range of the BP-SpNA reflex was augmented at 41.5 degrees C. These results indicate that heating alters the cardiovascular and sympathetic neural responses to vasoactive agents in vivo. Furthermore, the data suggest that heating differentially affects arterial baroreflex control of HR and SpNA, shifting both curves toward higher BP values but selectively attenuating baroreflex control of HR.     

Vascular reactivity of the efferent branchial arteries of the lemon shark,Negaprion queenslandicus

1. Efferent branchial artery tissue rings from Negaprion queenslandicus were used to examine the effects of adrenergic and cholinergic drugs on the vasculature.2. The presence of α- and β-adrenoceptors were demonstrated.3. Adrenaline and noradrenaline produced variable, including biphasic, responses.4. Acetylcholine produced concentration-dependent contractions in the presence of a predominantly intact endothelium.     

Vascular abnormalities due to hyperthermia in chick embryos

Intraembryonic vascular abnormalities were studied in chick embryos exposed to temperatures 3 degrees C and 4 degrees C above normal temperature (38 degrees C) from the beginning of incubation. The average duration of hyperthermia was 54 and 53 hours, respectively. Immediately after exposure, the embryos were examined with FITC-Dextran microangiography in vivo. Following hyperthermia various abnormalities in the heart, ventral aortae, aortic arches, omphalomesenteric arteries, and the distal dorsal aortae frequently occurred. There were also significant microvascular changes in the head, in the lateral and caudal parts of the embryos, and in the pellucid area of the yolk sac. In another series incubation at 41 degrees C, hyperthermia of 3 degrees C during the first 3 days of development produced several extraembryonic vascular abnormalities. These included duplication and abnormal branching of the cranial vitelline vein, absence or abnormal course of the omphalomesenteric vessels, aneurysmatic dilatation or abnormal course of the caudal vitelline vein, and aneurysmatic dilatation or occlusion of the abdominal venous sinus. Most frequent findings were blind, congested, and dilated microvascular segments in the pellucid area, commonly associated with an irregular microvascular pattern and perivascular swelling. The abnormalities described are assumed to be caused by the direct effects of hyperthermia upon the developing vessels resulting in microvascular insufficiencies, pathological leakage, and perivascular oedema. Such disturbances may have serious consequences for embryonic vascular development and microcirculation, which in turn may have adverse effects on further embryonic growth and development.     

Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo

In the chicken embryo, acute hypoxemia results in cardiovascular responses, including an increased peripheral resistance. We investigated whether local direct effects of decreased oxygen tension might participate in the arterial response to hypoxemia in the chicken embryo. Femoral arteries of chicken embryos were isolated at 0.9 of incubation time, and the effects of acute hypoxia on contraction and relaxation were determined in vitro. While hypoxia reduced contraction induced by high K(+) to a small extent (-21.8 +/- 5.7%), contractile responses to exogenous norepinephrine (NE) were markedly reduced (-51.1 +/- 3.2%) in 80% of the arterial segments. This effect of hypoxia was not altered by removal of the endothelium, inhibition of NO synthase or cyclooxygenase, or by depolarization plus Ca(2+) channel blockade. When arteries were simultaneously exposed to NE and ACh, hypoxia resulted in contraction (+49.8 +/- 9.3%). Also, relaxing responses to ACh were abolished during acute hypoxia, while the vessels became more sensitive to the relaxing effect of the NO donor sodium nitroprusside (pD(2): 5.81 +/- 0.21 vs. 5.31 +/- 0.27). Thus, in chicken embryo femoral arteries, acute hypoxia blunts agonist-induced contraction of the smooth muscle and inhibits stimulated endothelium-derived relaxation factor release. The consequences of this for in vivo fetal hemodynamics during acute hypoxemia depend on the balance between vasomotor influences of circulating catecholamines and those of the endothelium.     

Telomere length in Agave tequilana Weber plants during the in vitro to ex vitro transition

Acclimatization ex vitro is a key stage of the micropropagation process, in which the vitro plants leave the sterile, high humidity environment in which they originated and form new leaves and roots, during which they suffer different types of stress. Changes in the telomere length (shortening and lengthening) have been associated with age, the development of tissue, loss of cell replication and the ability of regeneration in different plant species. However, the genetic and biological factors that are involved in the process of shortening of telomeres across the ageing of plant species are still unknown. In this study, we used terminal restriction fragments (TRF) to examine the changes of telomere length during the in vitro to ex vitro transition in vitro plants of Agave tequilana, and their relationships with age in plants grown in commercial plantations. The results showed that in vitro grown plants present the longest telomeres and that a shortening occurs during the first 6 months of ex vitro acclimatization, (compared to the plantlets that were kept in vitro). A lengthening of the telomeres was observed in the acclimatized 1-year-old plants and that this was maintained in 2 and 3-year-old plants. We also observed TRF variations in the different tissues (leaves, stems and roots) of acclimatized plants. In field plants, we did not observe any important changes in the length of the telomeres. We suggest that agaves have a mechanism that maintains telomere length at the non-critical stages during development.