ABSTRACT
Amino acid analogues have been observed to give rise to abnormal forms of development of chick and amphibian embryos (Herrmann, 1953; Rothfels, 1954; Waddington & Sirlin, 1954; Feldman & Waddington, 1955; Herrmann, Rothfels-Konigsberg, & Curry, 1955). Assuming that these disturbances may be due to interference with the utilization of amino acids for protein formation, we have attempted an analysis of this effect by comparison of the protein contents and of the uptake of glycine into the proteins of chick embryo explants in the presence and absence of amino acid analogues. The results of such experiments are reported in this paper.
MATERIALS AND METHODS
The chick embryos used for explantation, the explantation technique, and the determination of total protein glycine and of tracer glycine were essentially the same as described previously (Herrmann & Schultz, 1958). The embryos were explanted at the 11–13 somite stage on to the surface of an agar gel containing egg extract as nutrient medium following the procedure given by Spratt (1947) as modified by Rothfels (1954). For the measurement of tracer uptake 20–25 μg. of glycine-l-C14 with a radioactivity of 1 μc. was added to the warm, liquid agarmedium solution and plated out with this mixture. The amino acid analogue used in the present experiments was co-bromoallylglycine (BAG) which is regarded as an analogue of the amino acids leucine and valine. It was added to the medium in the same way as the tracer in a final concentration of 0·15 mg./ml.
Previous experiments (Herrmann & Schultz, 1958) have shown that the uptake of labelled glycine deviates only slightly from a straight line course for the first 4 hours of explantation. Therefore an incubation time of 4 hours was chosen as optimal incubation period throughout the present experiments. After incubation the embryos were collected, washed for 10 seconds in each of three separate portions of 100 ml. saline, the extraembryonic membranes were carefully removed and individual organ primordia were dissected from the embryos and fixed in 5 per cent, trichloracetic acid, under the dissecting microscope. Nucleic acids and lipoids were removed by extraction with hot trichloracetic acid and with alcohol-ether followed by alcohol-chloroform respectively. Possibly adhering tracer glycine was removed by washing with a 1 per cent, glycine solution followed by six washings with distilled water. The proteins were hydrolysed and the protein glycine was isolated chromatographically as dinitrofluorobenzene derivative (Krol, 1952). The quantity of the intensely yellow glycine derivative was determined spectrophotometrically and the radioactivity of the isolated samples was measured by counting the glycine compound as an infinitely thin layer in a gas-flow counter with about 20 per cent, efficiency. The ratio of counts/ min./ μg. glycine in the tracer glycine was used as conversion factor for calculation of microgrammes of tracer glycine in the samples. Specific activities are given as the ratio of tracer glycine/total glycine.
Calculations of the means and standard errors of specific activities, tracer protein glycine and total protein glycine were carried out in the conventional way. The differences between specific activities of the organ primordia listed in Table 1 and between the contents of tracer protein glycine or total protein glycine, obtained for one and the same set of embryos in the presence or absence of the analogue, have been calculated for each separate experiment. The means and standard errors for these ‘paired’ difference values were used for evaluation of their statistical significance.
RESULTS
From the data compiled in Table 1 it is apparent that the uptake of tracer glycine into the proteins of the tissues of the embryo explants differs widely. In terms of specific activities the highest values were found in the proteins of the primitive knot, followed closely by the values obtained for the spinal cord, with somewhat lower values in the somites and the segmental plate,1 and the lowest figures for the brain, heart, and notochord. With the number of determinations carried out so far no statistically significant differences could be established between the specific activities of the primitive knot, the spinal cord, and the somites. The specific activities of these tissues are, however, significantly higher than those of the brain, the heart, and the notochord. Of these latter tissues the brain is significantly higher in activity than the heart, and the heart, in turn, has a significantly higher activity than the notochord. One can see that in one case morphogenetically closely related tissues, like segmental plate and somites, have an uptake of the same order of magnitude although their state of differentiation has to be assumed to differ considerably. On the other hand, brain and spinal cord, tissues of a similar morphogenetic relationship, show widely differing incorporation rates.
From the data recorded in Table 2, it can be seen that the sum of the total protein glycine values of the dissected primordia is only 10 per cent, lower than the corresponding value of the embryo as a whole. In contrast, the sum of the values for the protein glycine-l-C14 of the separate primordia is only about 30 per cent, of the tracer content of the whole embryo. This must mean that a tissue which accounts for less than 10 per cent, of the protein content contains about 70 per cent, of tracer glycine of the whole embryo. Since the endoderm is not included among the dissected primordia, and since it is the tissue in direct contact with the tracer-containing medium, it is considered as the possible site of such a high tracer incorporation.
Comparison of protein glycine content of the whole embryo determined directly with that derived from measurements of individual primordial

The wide range of incorporation rates found in the organs of explanted chick embryos by our quantitative measurements was not observed in radioautographic estimations by Feldman & Waddington (1955) obtained in chick embryos in vivo. This absence of larger differences could be due to a greater uniformity of the considerably higher growth rates of the tissues when developing in the egg as compared to the residual growth prevailing during cultivation in vitro under conditions of the present experiments. However, the long incubation in the presence of tracer glycine for periods of 32–42 hours as used in the experiments of Feldman & Waddington (1955) may eliminate differences observable after shorter incubations. In particular it has to be considered that under Feldman & Waddington’s conditions, and in the absence of time curves for tracer incorporation, some tissues may still show an increasing tracer accumulation while in others the tracer content may already be declining. Thus apparently similar tracer values may represent entirely different stages of incorporation. It should also be pointed out that radioautographic analyses do not lend themselves readily to the establishment of determinations of specific activities. A higher grain count may represent, at least in part, a difference in the protein concentration in the observed tissues. Such considerations may explain the discrepancy between the large differences of incorporation between notochord and neural tube found in our experiments as compared to the apparently similar incorporations for the same tissues apparent in the radioautographs of Pantelouris & Mulherkar (1957).
On addition of the bromoallylglycine to the medium statistically significant changes in the measured parameters were found in the case of the somites and of the primitive knot (Table 3). In the former the total protein glycine decreased on cultivation of the embryos in the presence of the analogue while the tracer glycine content remained apparently unchanged. In the case of the primitive knot tissue, both the total protein glycine and the tracer glycine show a significant decrease. No changes of statistically unequivocal significance could be observed in the other organ primordia.
DISCUSSION
It was pointed out in another paper (Herrmann & Schultz, 1958) that compared with embryos developing in the egg the growth rate of the explanted embryos is low. On the other hand, differentiation as measured, for example, by the formation of somites, of the heart tube, or of the brain ventricles and eye stalks, seems to proceed at a practically undiminished rate. At the present time it is not possible to state whether the observed differential rates of incorporation of tracer amino acids into the proteins of the different organ primordia are connected with the residual growth or with the formation of new proteins during differentiation. If related to residual growth, incorporation rates and growth rates (protein accumulation) in the different primordia should run parallel. In explants, however, growth measured in terms of protein accumulation is so slow that differences in the growth rates of the primordia could not be established under the conditions of the present experiments. Even measurements of growth rates of individual organs at early stages of chick embryogenesis in vivo are as yet too incomplete to allow a closer comparison of incorporation rates in explants and the actual growth rates observed in the embryo developing in the egg. From data obtained by Schmalhausen (1926) it would seem that the fresh weight of the heart and of the brain increases about 4–5 times from the third to the fourth day of incubation. In comparison, the protein nitrogen of an average somite (Herrmann et al., 1951) increases by about the same ratio during the development from the second to the third day and a doubling of the somite proteins was found during the third to fourth day period. Tracer incorporation, however, was found to be significantly higher in the somite proteins than in the protein moiety of the heart and the brain. If Schmalhausen’s (1926) measurements can be substantiated, the growth rates, at least when observed in vivo, cannot be related to incorporation rates found in explants.
In this context it should be mentioned that according to Schmalhausen (1926) growth and differentiation during early stages of development occur in alternating spurts of abruptly increasing or decreasing rates. For the comparison of incorporation rates of different organ primordia assessment of their state with respect to these developmental phases may be desirable. Further investigation of the relations of incorporation and protein accumulation (growth) and differentiation will be made by attempting to vary the growth rates of the explants and by comparison of incorporation rates and protein accumulation under these conditions.
It is noteworthy that bromoallylglycine at the concentration used in the present experiments (about 0·001 M) affects the measured parameters only in two primordia, the somites and the primitive knot. In both instances addition of the analogue to the medium leads to a lower total protein glycine content. Since no significant net protein increase could be detected in whole explanted embryos, the assumption of an inhibition of protein accumulation would seem less likely than the assumption of a protein deficit due to increased protein degradation. This can lead apparently to a lowering of both the tracer glycine and the total protein glycine as in the case of the primitive knot. In the somites, loss of total protein glycine occurs while the content of tracer glycine is practically the same in explants cultivated with or without analogues. In this case one can postulate that the loss in total protein content is partially compensated by newly formed proteins containing tracer. As an alternative interpretation one can assume that the proteins in the somites may have a finite life span, like haemoglobin (Shemin & Rittenberg, 1946) or muscle proteins (Dreyfus et al., 1956). In this case protein loss could occur by degradation of the ‘older’ unlabelled proteins without decrease of the tracer content confined to the ‘younger’ proteins, which would remain undegraded during the experimental explantation period.
If these conclusions are correct, the increased protein degradation found in the presence of bromoallylglycine would corroborate observations by Jensen, Lehmann, & Weber (1956), who found an increased catheptic activity in amphibian tail tissue after administration of leucine analogues. To what extent the protein loss in the present experiments can be attributed to an increased proteolysis by cathepsin will be determined in future experiments. It should be pointed out that o-fluorophenylalanine, an analogue of phenylalanine, was found to be without effect on the catheptic activity of liver homogenates. In liver slices, it inhibited both the protein synthesis and protein degradation (Steinberg & Vaughan, 1956). However, bromoallylglycine gives rise to far-reaching degenerative changes in the cells of the somites which may be related to the more extensive protein degradation discussed above. The correlation of protein loss and the degenerative processes in the somites will be discussed in a separate paper (P. W. Schultz, to be published).
SUMMARY
The incorporation of glycine-l-C14 into the chromatographically isolated protein glycine of the primordia of explanted chick embryos was determined quantitatively.
According to decreasing tracer uptake, the following order of the tested primordia was found: primitive knot, spinal cord, somites, segmental plate, brain, heart, and notochord.
Presence of co-bromoallylglycine in the medium (0·15 mg./ml.) leads to a lowered total protein content in the somites and in the primitive knot and a lowered tracer glycine content in the latter.
The possible relation of these results to the development of the organ primordia and their protein metabolism is discussed.
ACKNOWLEDGEMENTS
The authors are greatly indebted to Dr. Karl Dittmer, Department of Chemistry, the Florida State University, Tallahassee, Florida, for supplying co-bromoallylglycine and to Dr. Daniel Steinberg, National Institute of Health, for a critical discussion of the manuscript. The work reported in this paper was supported by grants from the Association for the Aid of Crippled Children and the United States Public Health Service (Grant No. B·549).
The material reported in this paper is part of a thesis submitted by P. W. Schultz to the Department of Biology, University of Wisconsin, in partial fulfilment of the requirements for a Ph.D. degree.
REFERENCES
The axial mesoderm caudal to its segmented portion as defined by Spratt (1955).