Cell death in the ‘opaque patch’ in the central mesenchyme of the developing chick limb: a cytological, cytochemical and electron microscopic analysis

Cell death in the ‘opaque patch’ of central mesenchyme of the developing chick forelimb was investigated by a variety of light and electron-microscope cytological and cytochemical techniques. Cell death appears first at stage 23/4 (4 days) and reaches its maximum extent at stages 24 and 25 (4½ and 5 days), at which it separates the ulnar and radial mesenchymal condensations. It then decreases in size to a small area separating the proximal parts of radius and ulna and disappears at stage 28. Cytological studies show the presence of a few isolated dead cells, of mesenchymal cells containing 1–3 ingested dead cells and of macrophages containing up to 18 dead cells in various stages of digestion. These findings are interpreted as showing that isolated dead cells are ingested by neighbouring mesenchymal cells which thus become transformed into macrophages, first ingesting and then digesting further dead cells. Histochemical studies show that isolated dead cells and recently ingested dead cells contain no more acid phosphatase activity, either discrete or diffuse, than either neighbouring living mesenchymal cells, or mesenchymal cells which have ingested 1–3 dead cells. Increased acid phosphatase activity is found within the macrophages, where activity is localized within the digestive vacuoles (‘secondary lysosomes’) containing the dead cells, and within the Golgi apparatus and Golgi vesicles (‘primary lysosomes’) of macrophage cytoplasm. Loss of staining capacity by the dead cell is correlated with high acid phosphatase activity: this is interpreted as indicating the digestion of dead cells within the macrophage by acid hydrolases. There is circumstantial evidence that viable mesenchyme cells in the ‘opaque patch’ area autophagocytose part of their own cytoplasm in secondary lysosomes (1·2–2µm). The role of the ‘opaque patch’ in relation to the pattern of limb chondrogenesis is discussed. It is suggested that cell death may play a role in separation of radius and ulna, and that autophagocytosis may indicate a change in the pathway of differentiation of the mesenchyme cells lying between radius and ulna.