Operations on Paraxial Mesoderm in Embryos in New Culture

When the aim of the experiments is to investigate the mechanisms that set up the paraxial mesoderm, or when the fates and movements of presumptive somitic cells at or shortly after gastrulation are to be investigated, operations in the egg are very difficult. It is therefore generally necessary to resort to a method of whole embryo culture. For this, set up cultures of embryos at the desired stage (between HH stage 3 [mid-primitive streak], and HH stage 8 [4 somites]), following the protocol in Chapter 15 of this volume. Leave the embryo completely submerged in saline during the operation.

The region of the primitive streak embryo (stages 3-4) that contributes to the somites lies in the most anterior (cranial) one-third or so of the primitive streak and in the ectoderm to either side of this. By stage 4-5, some of the somite progenitors have already left the streak and lie in the middle layer next to the anterior streak and gradually migrate cranially as more cells are added from the streak and node regions.

To operate on these cultured embryos, it is usually better to use fine mounted needles (entomological or sharpened tungsten) rather than knives. Trypsin is generally not necessary, but may be used (at 0.1% w/v) to facilitate separation of tissues if the specific manipulation desired turns out to be difficult because of adherence of the tissues to one another. Operations on the mesoderm are usually easier in the absence of trypsin because the middle layer readily separates into cells in the presence of enzyme. However, for each type of operation, the sequence in which the cuts are made is very important. In some regions of the embryo it is easier to begin with a medial cut and proceed laterally, whereas for some other regions an anterior cut, proceeding caudally, is more effective. You should investigate the relative merits of different ways to dissect the tissue of interest before starting a set of experiments.

As described for operations on Hensen's node, it is important to avoid damaging the vitelline membrane at all costs. Any leakage of the albumen culture fluid to the inside of the ring will diminish survival and may cause the grafted tissue to fall out of its site.

After the operation, follow the steps described for Hensen's node grafts (see Chapter 16). Transfer the ring with the embryo to a plastic dish, seal it, place it in a humid chamber, and incubate 38°C for 24-48 h. If cultures are set up using large glass rings (about 30 mm; see ref. 5) or other methods for extended cul ture (see ref. 8), embryos operated at stages 3-6 should survive to stage 15 or even longer.

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