Embryo development and chick growth in a helium - oxygen atmosphere

Embryo development and chick growth in helium- oxygen atmospher

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology

The chicken embryo and its micro environment during egg storage and early incubation

When egg storage periods are prolonged (>7 days), hatchability and chick quality declines. The reason for this decline has been investigated, but is still not completely understood. At oviposition the developmental stage of the chicken embryo varies and so do the total number of viable cells. During storage, changes can occur in the embryo. Embryo viability at the end of storage seems to be dependent on the number of viable cells and the developmental stage of the embryo at oviposition. When the hypoblast is completely formed (during the quiescent developmental stage), the embryo seems to be more able to endure prolonged storage periods than embryos that are less or more advanced. During storage, changes also occur in egg characteristics such as albumen viscosity, albumen pH and yolk pH. There appears to be an interaction between albumen pH and embryo viability during early incubation and perhaps also during storage. An albumen pH of 8.2 seems to be optimal for embryo development. Albumen pH may influence embryo viability, but embryo viability may in turn, affect albumen pH. It has been hypothesised that an embryo in which the hypoblast is completely formed is better able to provide an effective barrier between the internal embryo and the exterior (yolk and albumen) and/or is better able to produce sufficient amount of carbon dioxide, which will reduce the pH level in the micro environment of the embryo to the optimal pH of 8.2. It appears that, to maintain hatchability and chick quality after prolonged storage periods, embryonic development should be advanced to the stage in which the hypoblast is completely formed or the atmosphere during storage and early incubation should be altered in such a way that albumen pH is maintained at the optimal level of 8.2

Excess Imidacloprid Exposure Causes the Heart Tube Malformation of Chick Embryos

As a neonicotinoid pesticide, imidacloprid is widely used to control sucking insects on agricultural planting and fleas on domestic animals. However, the extent to which imidacloprid exposure has an influence on cardiogensis in early embryogenesis is still poorly understood. In vertebrates, the heart is the first organ to be formed. In this study to address whether or not imidacloprid exposure affects early heart development, the early chick embryo has been used as an experimental model because of the accessibility of chick embryo at its early developmental stage. The results demonstrate that exposure of the early chick embryo to imidacloprid caused malformation of heart tube. Furthermore，the data reveal that down-regulation of GATA4, Nkx2.5 and BMP4 and up-regulation of Wnt3a led to aberrant cardiomyocyte differentiation. In addition, imidacloprid exposure interfered with basement membrane (BM) breakdown, E-cadherin/Laminin expression and mesoderm formation during the epithelial-mesenchymal transition (EMT) in gastrula chick embryos. Finally, the DiI-labeled cell migration trajectory indicated that imidacloprid restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. A similar observation was also obtained from the cell migration assay of scratch wounds in vitro. Additionally, imidacloprid exposure negatively affected the cytoskeleton structure and expression of corresponding adhesion molecules. Taken together, these results reveal that the improper EMT, cardiac progenitor migration and differentiation are responsible for imidacloprid exposure-induced malformation of heart tube during chick embryo development

The early stages of heart development: insights from chicken embryos

The heart is the first functioning organ in the developing embryo and the detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to human. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulations and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic or biochemical approaches, is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analyses of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mouse and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding of early heart development using the chicken model

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29–37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut

Neural tube-ectoderm interactions are required for trigeminal placode formation

Cranial sensory ganglia in vertebrates develop from the ectodermal placodes, the neural crest, or both. Although much is known about the neural crest contribution to cranial ganglia, relatively little is known about how placode cells form, invaginate and migrate to their targets. Here, we identify Pax-3 as a molecular marker for placode cells that contribute to the ophthalmic branch of the trigeminal ganglion and use it, in conjunction with DiI labeling of the surface ectoderm, to analyze some of the mechanisms underlying placode development. Pax-3 expression in the ophthalmic placode is observed as early as the 4-somite stage in a narrow band of ectoderm contiguous to the midbrain neural folds. Its expression broadens to a patch of ectoderm adjacent to the midbrain and the rostral hindbrain at the 8- to 10-somite stage. Invagination of the first Pax-3-positive cells begins at the 13-somite stage. Placodal invagination continues through the 35-somite stage, by which time condensation of the trigeminal ganglion has begun. To challenge the normal tissue interactions leading to placode formation, we ablated the cranial neural crest cells or implanted barriers between the neural tube and the ectoderm. Our results demonstrate that, although the presence of neural crest cells is not mandatory for Pax-3 expression in the forming placode, a diffusible signal from the neuroectoderm is required for induction and/or maintenance of the ophthalmic placode

Formation of plaques in chick embryo tissue cultures by the virus of herpes simplex

Thesis (M.A.)--Boston UniversityStudies on the virus of herpes simplex have been done in t he past by using t he embryonated egg and the newborn mouse. Only comparatively recently has attention been directed to the use of the tissue culture method for this purpose. Howe ver, relatively little has been accomplished up to the present time by this method in the quantitative studies of this virus. This can probably be attributed, for the most part, to the lack of adequate and accurate tissue culture techniques. An interesting approach to the development of a more accurate tissue culture technique was made by Dulbecco in 1952, who devised a new tissue culture technique by which he succeeded in obtaining focal necrotic areas (plaques) in chick embryo monolayer tissue cultures by the viruses of Western equine encephalomyelitis and Newcastle Disease. Thus the possibility arose for the study of certain animal viruses along lines similar to the ones used in the related field of bacterial viruses. Using this new technique, Dulbecco and Vogt have recently proved that, indeed, it can be successfully used for as saying poliomyelitis virus. The purpose of this work was to investigate the possibility of applying; this "plaque-production" method to the assay of herpes simplex virus and to simultaneously compare its plaque producing capacity in tissue culture with that on the chorioallantoic membrane of the developing chick embryo. The currently employed techniques were modified for the sake of simplicity and convenience. The tissue cultures were set up as follows, in principle following the techniques devised by Dulbecco and Noyes: Five to six 11-day-old chick embryos were washed in Hanks' balanced salt solution (HBSS) and pressed through a 30-mesh wire screen into 15 ml of HBSS by means of a 50-ml syringe, and this material centrifuged at 800 r.p.m. for 30 seconds. The sediment was resuspended with 15 ml of BBSS and after vigorous pipetting centrifuged at 800 r.p.m. for 20 seconds. The supernatant was removed and allowed to stand for 20 minutes. The sediment of this was discarded and the cell concentration of the suspension determined in a hemocytometer. An appropriate amount of this cell suspension was added to the nutrient medium, which consisted of 40% HBSS (with indicator), 40% unheated horse serum and 20% chick embryo extract in order to give a final concentration of 8 x 10^6 cells per ml, the pH of the suspension being 7.6. 3 ml of this were then introduced into each 50-mm Petri dish, coated with chicken plasma, and the dishes sealed with Parafilm. The cultures were incubated at 37 C for 48 hours and at that time the nutrient medium removed, the cultures washed with HBSS, and 0.1 ml or more of the virus inoculated with a hypodermic syringe, gauge 27 needle, and well distributed all over the cell layer. The virus used was the Z strain of herpes simplex which had undergone 3 tissue culture and subsequently 5 egg passages since isolation from primary sources. Following inoculation the cultures were incubated at 37 C for 45 minutes and then covered with a thin layer of plasma, or agar incorporated in the nutrient medium (baiting a final concentration of 0.75%). If plasma overlay was used, 3 ml of nutrient medium were added per culture. The petri dishes were sealed then and incubated at 37 C. At the time of examination of the cultures they were stained with neutral red in order to facilitate the visualization of the plaques, the nutrient fluid or agar overlay removed, and the plaques counted and examined microscopically. The observations and results were as follows: Soon after incubation the cells settled out and began to grow, forming a continuous monolayer of cells covering the entire bottom of the Petri dish. They also exhibited good metabolic activity. At 48 hours the pH of the nutrient medium was down to 7.0-7.2 and a uniform cell layer had formed consisting mainly of healthy fibroblast-type cells. No satisfactory cell layer could be obtained without use of the plasma undercoating. Following inoculation of the virus, plaques could be already detected in the cell layer after a 48-hour incubation period in all the experiments carried out. They appeared as round to slightly elongated, colorless, necrotic areas standing out in the red background of stained living cells, and had a diameter of 1-2 mm on the third day. Microscopically they appeared as bounded areas of cellular debris and if examined earlier the gradual disintegration of the cells was apparent. No plaques were produced in control cultures without virus or virus inactivated by heat and their number increased with the concentration of the virus. The endpoint for plaque production of the 10-fold dilutions of this virus was at 10^-4. In cultures with undiluted virus all the cells were completely destroyed, while with 10^-1 inoculum, semi-confluent plaques were produced. At dilutions of 10^-2 , 10^-3, and 10^-4 discrete and separated plaques were formed. The discreteness of the plaques was dependent on the presence of plasma or agar overlay. Either one could be efficiently used and there was no difference between them in regard to plaque formation. A comparative study with the developing chick embryo, inoculated on the 11th day on the C A membrane, showed that the number of foci produced on the membrane correlates with the number of foci produced on the cell layer in tissue culture although the titer was approximately 10-fold lower in vitro with this particular herpes simplex specimen. The results of these experiments have shown that the Z strain of herpes simplex virus produces plaques in monolayer chick embryonic tissue cultures and this assumption was based on the facts that - (1) No plaques were formed in cultures where the virus was absent or inactivated by heat, (2) the number of plaques increased in proportion to the concentration of the virus inoculum, and (3) there is a correlation between the number of plaques formed in tissue culture and number of pox produced on the CA membrane of the developing chick embryo. The technique employed throughout the experiments described in this paper includes some modifications of the ones already practiced, which simplify and economize, the procedure and makes it easily adaptable for use in any bacteriological laboratory. Further more detailed work is needed to evaluate the aspects and adequacy of this method for quantitative study of the virus of herpes simplex. If satisfactory, it would seem to offer some advantages over certain others currently used

Violation of cell lineage restriction compartments in the chick hindbrain

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development