Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC. Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.     

Method for Treating and Processing Whole Chick Embryos for Autoradiography, Immunocytochemistry and other Techniques

A method is presented for In situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions “in ovo.” The chick embryo is placed in a “shell-less” culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo + membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

Method for Treating and Processing Whole Chick Embryos for Autoradiography,Immunocytochemistry and other Techniques

A method is presented for In situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions “in ovo.” The chick embryo is placed in a “shell-less” culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo + membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

流感疫苗中间品-鸡胚尿囊病毒液质量控制的研究

为建立流感疫苗中间品-鸡胚尿囊病毒液质量控制点提供依据。用鲎试剂法、微生物限度检查法、沙门菌检查法检测流感疫苗中间品一鸡胚尿囊病毒液的细菌内毒素含量、微生物限度及沙门菌。在300份鸡胚尿囊病毒液中,细菌内毒素含量大于5EU／mL的阳检率为5％;微生物限度检查中小于lO个／mL细菌菌落数的占78．67％,小于10个／mL霉菌菌落数的占80．67％,小于10个／mL酵母菌菌落数的占88．67％。沙门菌属的阳检率为4％,其中未检出A～F群的沙门菌。对流感疫苗中间品-鸡胚尿囊病毒液进行细菌内毒素,微生物限度及沙门菌检测可避免不合格尿囊病毒液进入后续生产,污染后续中间品。     

Microdissection by Ultrasonication: Application to Early Chick Embryos

A technique utilizing microdissection by ultrasonication was applied to scanning electron microscopy of chick embryos during the first three days of incubation. Using a tank cleaner operating at 80 kHz, whole embryos immersed in pure acetone were sonicated until fragmentation became evident. At 12 hr incubation disintegration occurred by one second or less. At 18 hr, three sonic bursts of one second each produced only partial fragmentation. All three germ layers retained their original relationships to each other. During the second day of incubation, large pieces of integument were removed and somites began to microdissect after 10-20 seconds of sonication. Late in the third day of incubation, sonication for 1 min or more was required to produce significant microdissection. Living embryos exposed to 0.1% collagenase for 10 min prior to standard fixation fragmented in a different manner. Lamellipodia and filopodia were most sensitive and were largely destroyed. The three major germ layers (ectoderm, endoderm, mesoderm), however, retained their structural integrity and original relationships to each other. Factors contributing to the results reported here include: 1) extracellular fibrils of varying chemical composition, 2) primitive cell junctions, 3) biomechanical stability in the nonfibrillar portions of the extracellular matrix, and 4) effects of technical procedures performed prior to sonication. Sonicated tissues of early embryos reveal features that are difficult to demonstrate in other ways and may be unrecognized in conventional preparations.     

Chick embryo retina development in vitro: The effect of insulin

In this paper we study the development of chick embryo retina culturedin vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum-and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31–37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retinal cells were organized in identifiable layers, nevertheless the typical organization equivalent to 14-day in ovo retina was absent. The addition of insulin in the medium markedly increased the wet weight of cultured retinas, their protein content and the level of tubulin pools, particularly that of non-assembled fraction. Nevertheless insulin did not modify DNA synthesis and did not induce the increment of both neuron specific enolase and actin. Morphological observations show that insulin markedly increased the number and the thickening of the fiber layers. These results, together with the facts that retina synthesizes and secretes insulin and possesses specific insulin receptors suggest that insulin can have autocrine or paracrine regulatory functions in retinal development by exerting a general effect on retinal growth and a more specific one on tubulin production.     

Magnesium Deficiency Enhances Hydrogen Peroxide Production and Oxidative Damage in Chick Embryo Hepatocyte In Vitro

Magnesium deficiency and oxidative stress have been identified as correlative factors in many diseases. The origin of free radicals correlated with oxidative damage resulting from Mg-deficiency is unclear at the cellular level. To investigate whether hydrogen peroxide (H₂O₂) is associated in the oxidative stress induced by Mg-deficiency, the effect of Mg²⁺ deficiency (0, 0.4, 0.7 mM) on the metabolism of H₂O₂ was investigated in cultured chick embryo hepatocytes. After being cultured in the media with various concentrations of Mg²⁺ for 1, 2, 4, 6 and 10 days, parameters of H₂O₂ production, catalase activity, lipid peroxidation, intracellular total Mg and cell viability were analyzed. Results demonstrated that long-term incubation of chick embryo hepatocyte in extracellular Mg²⁺-deprivative and Mg²⁺-deficient (0.4 mM) states significantly enhanced the production of H₂O₂ (approximately twofold, respectively) and lipid peroxidation in the cell cultures, while decreasing the cell viability. Additionally, the reversing action of Mg²⁺ re-added to 1.0 mM and the partial reversing action of dimethylthiourea suggested that (i) [Mg²⁺]_e deficiency induced the increase of H₂O₂ production, (ii) [Mg²⁺]_e deficiency decreased catalase activity in chick embryo hepatocyte in vitro, subsequently causing oxidative stress and cell peroxidative damage.     

Developmental Pattern of Plasminogen Activator Activity in Chick Brain Hemispheres

Plasminogen activators play key roles in several developmental events. In previous works we demonstrated the existence of typical developmental patterns of protease activity in the chick optic lobe and cerebellum. The aim of this work is to study the temporal pattern of development of plasminogen activator activity in the brain hemispheres. Plasminogen activator activity was assayed in soluble fractions derived by ultracentrifugation from Triton X-100 treated membrane fractions by using a radial fibrinolytic assay. Employing different inhibitors and anti-plasminogen activators antibodies we showed that developing brain hemispheres express only one type of enzyme which corresponds to the urokinase-type. Other results indicate that the protease activity displays a temporal pattern which completely differs from those of general parameters of development. This suggests that the plasminogen activator activity is developmentally regulated and could display specific functions during particular stages of development.     

Ganglioside Expression During Differentiation of Chick Retinal Cells In Vitro

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunoflourescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.     

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.     

Chick embryos can form teratomas from microinjected mouse embryonic stem cells

We examined whether chick embryos are a suitable experimental model for the evaluation of pluripotency of stem cells. Mouse embryonic stem cells (mESCs) expressing the reporter gene, LacZ or GFP were injected into the subgerminal cavity of blastoderms (freshly oviposited) or the marginal vein of chick embryos (2 days of incubation). Injected mESCs were efficiently incorporated into the body and extra‐embryonic tissues of chick embryos and formed small clusters. Increased donor cell numbers injected were positively associated with the efficiency of chimera production, but with lower viability. A single mESC injected into the blastoderm proliferated into 34.7 ± 3.8 cells in 3 days, implying that the chick embryo provides an optimal environment for the growth of xenogenic cells. In the embryo body, mESCs were interspersed as small clustered chimeras in various tissues. Teratomas were observed in the yolk sac and the brain with three germ layers. In the yolk sac, clusters of mESCs gradually increased in volume and exhibited varied morphology such as a water balloon‐like or dark‐red solid mass. However, mESCs in the brain developed into a large soft tissue mass of whitish color and showed a tendency to differentiate into ectodermal lineage cells, including primitive neural ectodermal and neuronal cells expressing the neurofilament protein. These results indicate that chick embryos are useful for the teratoma formation assays of mESCs and have a broad‐range potential as an experimental host model.     

Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.5, CM-Sephadex C–50 ion exchange and Sephadex G-75 gel filtration. Judging from the dose-response analyses, the factor was purified by some hundred-fold when EE was used as the starting material. The activity was associated with a macro-molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts.
Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF-related.     

Chick embryos have the same pattern of hypoxic lower‐brain activation as fetal mammals

cFos expression (indicating a particular kind of neuronal activation) was examined in embryonic day (E) 18 chick embryos after exposure to 4 h of either normoxia (21% O₂), modest hypoxia (15% O₂), or medium hypoxia (10% O₂). Eight regions of the brainstem and hypothalamus were surveyed, including seven previously shown to respond to hypoxia in late‐gestation mammalian fetuses (Breen et al., 1997; Nitsos and Walker, 1999b). Hypoxia‐related changes in chick embryo brain activation mirrored those found in fetal mammals with the exception of the medullary Raphe, which showed decreased hypoxic activation, compared with no change in mammals. This difference may be explained by the greater anapyrexic responses of chick embryos relative to mammalian fetuses. Activation in the A1/C1 region was examined in more detail to ascertain whether an O₂‐sensitive subpopulation of these cells containing heme oxygenase 2 (HMOX2) may drive hypoxic brain responses before the maturation of peripheral O₂‐sensing. HMOX2‐positive and ‐negative catecholaminergic cells and interdigitating noncatecholaminergic HMOX2‐positive cells all showed significant changes in cFos expression to hypoxia, with larger population responses seen in the catecholaminergic cells. Hypoxia‐induced activation of lower‐brain regions studied here was significantly better correlated with activation of the nucleus of the solitary tract (NTS) than with that of HMOX2‐containing A1/C1 neurons. Together, these observations suggest that (1) the functional circuitry controlling prenatal brain responses to hypoxia is strongly conserved between birds and mammals, and (2) NTS neurons are a more dominant driving force for prenatal hypoxic cFos brain responses than O₂‐sensing A1/C1 neurons. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 64–74, 2016     

Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo

Replication-competent retroviral vectors based on Rous sarcoma virus (RSV) are becoming increasingly popular for expressing genes in both primary cell cultures and embryonic chick tissuesin ovo. In this article, we review the features of RSV and its life cycle that make it suitable for use as a vector. We describe the design and use of the RCAS and RCAS(BP) series of vectors, which are currently the most widely used RSV-based vectors, illustrating both their strengths and weaknesses. Finally, we outline laboratory protocols suitable for the handling of these retroviral vectors.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

雏鸡听觉印记学习

雏鸡听觉印记学习是研究新生个体在早期发育阶段学习与记忆形成的神经机制的良好实验模型.听觉刺激对印记学习有很好的强化作用,作用效果依赖于频率、强度和持续时间等声音特征.从声音的选择、听觉刺激所引起的分子水平和组织水平改变等方面综述雏鸡听觉印记学习的研究进展.