Dissection chick

Grunwald GB, Pratt RS, Lilien J. 1982. Enzymic dissection of embryonic cell adhesive mechanisms. III. Immunological identification of a component of the calcium-dependent adhesive system of embryonic chick neural retina cells. J Cell Sci 55 69-83. 

Figure 6.9 (a) Three views of an embryonic chick face (stage 27, 144h) stained with ethidium bromide the images were taken under a fluorescence dissecting microscope. The white lines in the image indicate, approximately, the plane of the sectioning ... 

The complexity and plasticity of BBB properties called for experimental dissection of the disrnption process in both in vitro and in vivo conditions. Multiple cell and organ cnltnres, animal models, and measurement techniques have been developed, each of which addresses some of the issues involved. The development of research into BBB characteristics was initially approached in avian embryos, where transplanted endothelial quail cells invaded a developing chick chimera. A simpler cell culture model of the BBB was developed by Rubin and co-workers. More recently, an immortalized cell line created from vascular endothelial cells was used to develop another model of the BBB in co-cultures with glioma cells and was used to demonstrate nitric oxide-induced perturbations of these cells. hi another cell culture model, hypoxia was shown to increase the susceptibility to oxidative stress and intercellular permeability. ... 

A preliminary series of experiments with chick embryo explants cul-tered on dextran medium suggested that all regions of the embryos were not affected to the same extent by protein starvation. In order to quantitate this response, procedures were developed which would allow the dissection of embryos into brain, neural tube, somite, heart, and extraembryonic membrane (area opaca plus area pellucida) in a reproducible manner so that the quantities of DNA, RNA, and protein in these regions could be determined. Relative to embryo explants cultured on whole egg homogenate medium, in those cultured on protein starvation medium the accumulation of macromolecules in the brain region was most restricted and the heart least. 

As an initial approach to an analysis of the basis for the differences between embryo regions in their apparent sensitivity to protein starvation the breakdown of protein was studied (Klein et al., 1971). Chick embryos of 11-13 somites were exposed to C-labeled amino acids for 3 hours in buffered chick Ringer s salt solution. Explants were next cultured for 6 hours on semi-solid medium to reduce the level of free [ C]amino acids and to allow protein labeling to increase. They were then transferred to either whole egg homogenate growth medium or protein starvation medium. Explants were cultured for various periods up to 48 hours and dissected regions were analyzed for protein radioactivity and the radioactivity soluble in 5% trichloroacetic acid. Protein and DNA contents were also determined. 

Dissect embryos from the decidua or chick embryos from the eggshell into ice-cold L15 air (Note 1). Dissect embryos away from membranes and adhering yolk and assess developmental stage. 

Fig. 2. In-ovo electroporation. A pair of electrodes held by a manipulator (A) is inserted intom a window opened on the shell (B). The electrode is placed on the vitelline membrane overlying the embryo (C), and a 25-V 50-ms pulse is charged five times. The entire procedure is monitored under a dissection microscope. Plasmid solution is injected to the E2 (HH stage 10) chick neural tube (D) prior to the pulse charge.The dimensions of the electrode are shown in E. Most of the electrode is insulated (black in the figure) so that only the tip is exposed (white area). One hour after electroporation, some embryos were fixed, processed for paraffin sectioning, and observed with a Nomarski interference microscope (F). The right-hand side of the figure corresponds to the right of the embryo, where injected plasmid was transfected. The morphology of the cells and the structure of neural tube were almost normal. The blue deposit inside the neural tube is a complex of plasmid and the color substrates not removed by washing in dimethylformamide after whole-mount in-situ hybridization. Twenty-four hours after electroporation, the development of yolk sac plexus, vitelline veins, and vitelline arteries are retarded in the area contacted on the electrodes (arrows in G). Bar is 2mm (C) 50 pm (F) 4mm (G). Source (3).

Dissect out embryos in ice-cold PBS (mouse, chick) or Howard s Ringer (chick) and open cavities. 

This protocol works well on intact embryos up to at least mouse embryonic d 12, chick Hamburger and Hamilton (3) stage 24, and zebrafish to 72h (see also Note 7 for zebrafish modifications). For older embryos, it is likely that some block dissection is required prior to fixation to keep background levels low. Alternatively, some tissues, such as the neural tube, can be dissected after performing the procedure on intact older embryos. 

Dissect out the embryos into an appropriate physiological salt solution, such as PBS, for mouse embryos or Howard s Ringer for chick embryos, and process directly. 

From work on chick embryo muscle cells the transient appearance of single-strand breaks in DNA, regulated by nuclear ADP-ribosyltransferase (ADPRT), has been proposed as part of a general mechanism for eukaryotic differentiation [1,2]. Investigation of the stimulation of quiescent lymphocytes has provided concordant results supporting this hypothesis, and has allowed a preliminary dissection of the molecular events of lymphocyte activation underlying immune responses. 

Fixation times might be reduced for very small embryos. Young mouse embryos, if left inside the decidua, can be fixed overnight safely. Fixation can be reduced to 2-4 h for embryos that are dissected out, although overnight fixation is not obviously detrimental. Chick embryos below stage 17, however, should be fixed only for 2 h (S Smith, personal communication). 

---