The technique for the localization of β-glucuronidase, based on the precipitation of ferric 8-hydroxyquinoline from quinolyl-8-glucuronide has been applied to explanted chick embryos. When the enzyme is first detectable, at the head-process stage, it appears to be widely diffused in the dorsal side of the embryo and in the adjacent area opaca. In later stages the histochemical reaction is confined to the region of the developing somites and neural tube.

In vertebrate tissues there appears to be a connexion between β-glucuronidase and the proliferation of cells. This connexion was first noticed by Levvy, Kerr, & Campbell (1948) when they were investigating the effect of toxic compounds on mouse-liver glucuronidase. The early stages of embryonic development are characterized by rapid proliferations of cells and it is, therefore, of some interest to study the localization of β -glucuronidase during these stages. Small amounts of β-glucuronidase can be detected in the early embryos of Xenopus laevis (Billett, 1956) and in those of Drosophila melanogaster (Billett & Counce, unpublished). In these embryos no marked increase in the enzyme can be associated with the proliferation of cells. The large amount of yolk in the Xenopus and Drosophila embryos was a complicating factor in the above experiments. It was not possible to localize the enzyme in these embryos with a histochemical method. An embedding technique suited to the nature of the embryos and the requirements of the histochemical test could not be devised.

It has, however, proved relatively simple to apply a modification of the Friedenwald & Becker (1948) technique to whole chick embryos cultivated in vitro. This technique, because of the diffuse nature of the reaction and the destruction of cytological detail which it entails, can only demonstrate the presence of β -glucuronidase in particular cells or groups of cells (Billett & McGee-Russell, 1956). Intracellular localization of the enzyme is impossible. The method appears to be well suited to the present purpose, aimed at a histological rather than a cytological localization of the enzyme in the early chick embryo.

The modification of the Friedenwald & Becker technique, used in these experiments, has been described elsewhere (Billett & McGee-Russell, 1955).

Chick embryos, which varied in age from the primitive streak to 10 or more somites, were transferred to culture chambers according to the method described by New (1955). About an hour after explantation the albumen, which is in contact with the dorsal side of the embryos, was removed and the embryos were completely surrounded by a saturated solution of ferric 8-hydroxyquinoline and quinolyl-8-glucuronide in 0·1 M acetate buffer at pH 4·5. The embryos were incubated in the substrate mixture for 24 hours at 37° C. Development was arrested by the substrate, and at the end of incubation the embryonic tissues appeared to have suffered little distortion. After this treatment the embryos were removed from the culture chambers, washed in water, and the vitelline membranes removed. The embryos were then washed in oxalate buffer, again in water, and then placed for half an hour in neutral formaldehyde solution (4 per cent, w/v). After a final washing in water the embryos were mounted in Farrant’s medium. The formation of brown crystals of ferric 8-hydroxyquinoline, embedded in the tissue, was taken to indicate the presence of the enzyme.

Controls were set up in three ways: (1) substrate mixture was prepared without the addition of glucuronide; (2) embryos were heated to 80–90° C. for 5 minutes; (3) substrate was used containing 0·001 M potassium hydrogen saccharate. The latter is a specific inhibitor for the enzyme (Lewy, 1952).

Our findings are summarized in Table 1. Typical preparations are shown in the Plate. The enzyme could not be detected in the primitive streak stage. In 5 out of 9 cases the head-process stage gave a strongly positive reaction. At a slightly later stage, formation of the head-fold, only 1 positive reaction out of 10 was recorded. These observations suggest that the enzyme disappears at this stage. Alternatively, a technical failure of the histochemical reaction would account for the result. However, the results for the head-fold embryos were obtained from four different batches of embryos which contained earlier and later stages which gave positive results. Once the somites had formed, a strong positive reaction was usually obtained.

Table 1.
graphic
graphic

An examination of the preparations gave a general impression that the enzyme was at first widely diffused on the dorsal side of the embryo, and that it later became more strictly localized in the mid-dorsal line in the region of the neural tube and somites. Little or no enzyme could be detected on the ventral side of the embryo. Crystals were only deposited in the embryos and in the extra-embryonic tissue immediately surrounding the embryo. At the headprocess stage crystals were deposited in the region of the primitive streak. In the later stages crystals were rarely deposited in the retreating streak, in marked contrast with the remainder of the embryo (Plate, figs. B-D).

Some of the treated embryos were embedded in 10 per cent, w/v gelatin and sectioned. Satisfactory embedding was not achieved because the crystals of ferric 8-hydroxyquinoline dissolved if left in the aqueous gelatin at 37° C. for more than 6 hours. Only a short embedding time, of approximately 2 hours, could be used, and this made sections difficult to cut. Examination of the sections showed crystals of ferric 8-hydroxyquinoline embedded in the neural folds and adjacent ectoderm, and in the somites.

The effect of potassium hydrogen saccharate on the development of the embryos

Although a 0·001 M solution of potassium hydrogen saccharate prevented the formation of ferric 8-hydroxyquinoline in the histochemical test, a similar concentration of the inhibitor did not prevent the normal development of embryos explanted at a primitive streak stage. This observation suggests that the enzyme is either inactive or inaccessible in the intact embryo. In this connexion it is to be noted that Karunairatnum & Levvy (1949) failed to influence liver regeneration and growth in mice by the administration of large doses of saccharic acid.

Using the method described above, βglucurinodase cannot be detected in chick embryos until the tissues have begun to differentiate. These results do not support a general hypothesis that the enzyme is specifically associated with the proliferation of cells.

Hollinger & Rossiter (1952), studying Wallerian degeneration of nerve, observed that an increase in β-glucuronidase occurred after cellular proliferation had taken place in the degenerating tissue. In the liver of the rat, regenerating after sub-total hepatectomy, the peak of β-glucuronidase activity also occurs after the phase of rapid cellular proliferation (Mills et al., 1950).

These results for regenerating tissue, and the present findings for the chick embryo, suggest that β-glucuronidase is often associated with tissues which are in the process of differentiation following an initial phase of cellular proliferation. Changes in β-glucuronidase activity may in fact reflect changes in cell type, as was suggested by Mills & Smith (1951). Raised β-giucuronidase levels, such as occur in certain neoplasms (Fishman et al., 1947, 1950) may indicate a type of tissue whose cells have reverted to, and remain in, a state which is characteristic of those found in tissues in the early stages of differentiation.

We wish to thank Professor C. H. Waddington for the help and advice he has given us in our work. The work was assisted by a grant from the Advisory Committee for Medical Research for Scotland. One of us (L. M.) is indebted to the Indian Government for an overseas scholarship.

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The photographs are of whole chick embryos seen from the dorsal side. The crystals of ferric 8-hydroxyquinoline appear as black spots. The preparations are unstained.

Fig. A. Head-process. Test. Deposits of ferric 8-hydroxyquinoline in the dorsal side of embryo and in the adjacent area pellucida.

Fig. B. Three pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline in dorsal side of embryo and in area pellucida on each side of the embryo.

Fig. C. About ten pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline concentrated in mid-dorsal line, in neural tube and somites.

Fig. D. About twelve pairs of somites. Similar to fig. C.

Fig. A. Head-process. Test. Deposits of ferric 8-hydroxyquinoline in the dorsal side of embryo and in the adjacent area pellucida.

Fig. B. Three pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline in dorsal side of embryo and in area pellucida on each side of the embryo.

Fig. C. About ten pairs of somites. Test. Deposits of ferric 8-hydroxyquinoline concentrated in mid-dorsal line, in neural tube and somites.

Fig. D. About twelve pairs of somites. Similar to fig. C.