Area opaca

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. 

Cut around the perimeter of the area opaca with Vannas scissors, lift out the embryo with a prewetted spatula, and place into fix (usually 4% w/v paraformaldehyde in PBS, but this varies with application). 

Grip the area opaca with a pair of forceps, and gently shake the embryo free of the vitelline membrane and adherent yolk. 

At the time of laying, the chick embryo is a flat disk consisting of about 60,000 cells. It is made up of two layers, the hypoblast ventrally, lying in close association with the yolk, and the epiblast dorsally, facing the vitelline membrane, the membrane which surrounds the yolk. The disk has a translucent iimer core, the area pellucida, from which the embryo will form, and a denser outer ring of mainly yolk-containing cells, the area opaca. Only the cells at the periphery of the blastoderm are attached to the vitelline membrane, and as they migrate radially, the blastoderm expands. 

Fig. 11. Stage 10 (HH), 10 pairs of somites (36-42h) dorsally anterior neuropore closed, prominent optic vesicles, rhombomere boundary 4/5 formed, and neural folds are closed to almost the level of the node. Ventrally, Hensen s node has regressed almost to the end of the primitive streak (the 10th pair of somites has not fully segmented caudally in this illustration), pronephric tubules develop between somites 6 and 10, heart tube turns asymmetrical bulging out to the right and contractions can be seen, and bilateral vitelline veins fan out toward the area opaca, which shows large blood islands to establish circulation.

New (19) already reported the importance of the vitelline membrane for normal growth of the embryo, as well as the special properties of its iimer surface for the attachment of the edge of the extraembryonic region (area opaca) and, therefore, for expansion of the blastoderm. Similarly, inclusion of the vitelUne membrane in agar cultures reduced the developmental anomalies often observed when the embryo was placed directly on agar (20). Chapman and colleagues... 

In the following sections, I consider two examples of operations on Hensen s node excision from a donor quail embryo and transplantation to the inner ring of the area opaca of a host chick embryo to demonstrate embryonic induction, as done by Storey et al. (10), and rotation of the node about its rostrocaudal axis in situ, to demonstrate embryonic regulation as done by Abercrombie (14). 

For embryos operated in New culture (J3, 4] Chapter 18), the glass ring should first be flooded with CMF, and the edges of the area opaca detached from the vitelline membrane. Then, pick up the embryo with a wide-mouthed pipet or with fine forceps (to grasp the extraembryonic membranes) and transfer it to a Sylgard dish for pinning and fixing as just described. 

Embryos can then be visuahzedby subblastodermal injection of ink (about 100 pL) using a 25-gauge needle. Inject the ink outside the area opaca at a shallow angle and minimize the size of the hole. 

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