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The effect of an amino acid analog on catheptic activity in somite mesoderm of the chick embryo
The Effect of an Amino Acid Activity in Somite Mesoderm E.
M.
Analog on Catheptic of the Chick Embryo
DEUCHAR
1NTROI)UCTION
The connection between tissue differentiation and protein synthesis in developing tissues is a subject of fundamental interest, but a very complex one to investigate experimentally. Recently, amino acid analogs (substances with a close structural resemblance to certain amino acids) have been found to be useful experimental tools with which to gain preliminary insight into some aspects of this sublect. If an amino acid analog is substituted for the normal amino acid in the nutrient medium on which an embryo or organ is growing, the analog becomes incorporated into the tissue proteins in place of its normal homolog, so that abnormal proteins, usually incompatible with growth, are produced. Occasionally this leads to quite clear-cut abnormalities in a particular developing tissue. If these abnormalities can be prevented merely by adding excess of the normal amino acid at the same time as its analog, one has clear evidence that the particular developmental process that was affected depended on the presence of the amino acid or some larger molecule into which it was rapidly incorporated. There may, however, arise secondarily a quite widespread dislocation of protein synthesis, with a consequent lowered utilization of other amino acids. For instance, Schultz and Herrmann (1958) found that W-bromoallylglycine an analog of leucine, reduced the uptake of radioactive (BAG), glycine into proteins of the primitive knot of chick embryos. The most striking effect of BAG on chick embryos, however, was that it blocked specifically the segmentation of the somites (Herrmann et al., 1955). When embryos of stage 11 (Hamburger and Hamilton, 1951) were explanted on an agar medium containing 129
130
E.
M.
DEUCHAR
BAG, no turther somites were formed during the period of treatment, but other tissues were unaffected, and control explants developed perfectly normally. Schultz and Herrmann (1958) went on to investigate the effects of BAG on protein synthesis in the explanted embryos, using the rate of uptake of W-labeled glycine as a measure ot the rate of synthesis of new protein. They found that only the segmented mesoderm, and no other tissue, had a lower total glycine content in BAG-treated embryos than in controls. Somewhat unexpectedly, though, both treated and control embryos showed similar concentrations of C4-glycine in the somite proteins, so there was no evidence of a lowered rate of protein synthesis as the result of treatment. To explain these results, the authors suggested that there had indeed been a lowered rate of incorporation of C”-glycine after BAG treatment, but that this had been masked, and the relative C”-glycine content maintained high, because of an accelerated breakdown of pre-existing protein in the treated tissue, which resulted in a proportionately greater loss of nonradioactive glycine. That enhanced protein breakdown may result from treatment with a leucine analog has been shown by von Hahn and Lehmann (1958) in work on Xenopus larvae. They found that the leucine aminoketone “E9” raises the activity of cathepsins (which are the predominant and most widespread intracellular proteolytic enzymes) in regenerating tail tissue of these larvae. In view of these findings, it was decided in the present study to measure catheptic activity in somite tissues of BAG-treated and control chick embryos at the stages used by Herrmann and his collaborators: to see whether there was evidence of enhanced proteolysis in the treated tissue. MATERIAL
AND
METHODS
Eggs (Light Sussex X Rhode Island for 32 hours (embryos to be explanted) series 1 that were not explanted).
Red) were incubated either or for 45 hours (controls of
Explantation Essentially the same procedure was used as described in another 1960). Blastoderms of 32 hours’ incubation were paper (Deuchar, trimmed just outside the limit of the area vasculosa, and after removal of the vitelline membrane and any adhering yolk they were placed, dorsal surface downward, on clots of agar medium in watch-
CATHEPTIC
ACTIVITY
IN
SOMITE
MESODERM
131
glass culture dishes. The medium was made up by mixing doublestrength Pannett-Compton saline containing 2% glucose with an equal volume of 1.5% agar solution which had previously been boiled and allowed to cool to about 40° C. The mixture was immediately poured into sterile watch glasses in petri-dish culture chambers, and allowed to set. In the experimental series, BAG was dissolved in the saline immediately before it was mixed with the agar, and its final concentration in the culture medium was 0.15 mg/ml (cf. Herrmann et al., 1955). The embryos were cultured on the medium for 10 hours at 37.5” C, then removed in order to dissect out the somite tissue. Cathepsin
Assays
The somite mesoderm was dissected out in ice-cold saline, all other tissue being removed except for the epidermis, which could not be removed successfully. The segmented and unsegmented portions were collected separately, from about twenty embryos per experiment, in saline kept at 2O C. The tissues were then washed free of saline by transferring them through two changes of chilled glassdistilled water, and immediately homogenized in approximately 60 ~1 of this fluid, in chilled Perspex microhomogenizers. Homogenate ( 11-J samples) was added to tubes containing 35.6 ,LL~of buffered 1% casein substrate (Duspiva, 1939) at pH 4.56, and the tubes were incubated on a water bath at 3S” C for 10 hours. (The optimal pH, enzyme concentration, and incubation time for the assays had been determined in preliminary experiments, and the time curve for the reaction had been found to be linear up to 12 hours at 3&O C.) At the end of 10 hours the reaction was stopped by adding 64.5 ~1 of 10% trichloroacetic acid to each tube, which precipitated the undigested casein. The tubes were then centrifuged in the cold for 15 minutes at 3500 rpm on a Baird and Tatlock semimicro angle centrifuge. To 100 ~1 aliquots of supernatant, 100 ~1 aliquots of first 13.5% sodium carbonate, then 20% (v/v) Folin-Ciocalteu reagent, were added, producing a blue color by reaction with the soluble casein products. The intensity of color developed was measured as an extinction value at 780 rnp on a Hilger Uvispek spectrophotometer, using microcells. Under the conditions of the assay, 0.3 gm casein (i.e., 0.1% concentration) gave an extinction reading of 0.14. Reagent blanks (distilled water instead of homogenate) were incubated with each experimental series, and the extinc-
1%
E.
hf.
IXXJCHAH
tion values of the blanks were subtracted from those of the experimental samples. From a standard curve obtained with known concentrations of casein solution, the final extinction values could be converted into micrograms of casein rendered soluble by the catheptic activity. The total nitrogen in samples of the homogenates was also estimated, by the ultramicro-Kjeldahl method of Boell and Shen (1954). Hence the results could be expressed as micrograms casein digested per microgram total nitrogen in the homogenate samples. RESULTS
1. Catheptic Activity Control Embryos
in Somite Mesoderm
of 45Hour
catheptic activity was measured In a first series of experiments, in somite mesoderm dissected out from embryos that had not been explanted but had simply been incubated in ovo for 45 hours. As Table 1 shows, there was always a higher catheptic activity per unit nitrogen in the unsegmented tissue than in the tissue that had already segmented into somite blocks.
CATHEPTIC
ACTIVITY"
lhpt.
IN
DIWEREST
11”.
PARTS
SOMITE
Segmented mesoderm
1 2 3 4 5 ~IWII Standard
on.
0. 23X 0. 370 0. 291 0.15:! 0. 399 ~ .~~ 0 292 *0.044
error
(1 Activity sample.
expressed
as micrograms
2. Effects
of BAG on Catheptic
soluble
caseiu
MESOT~ERM
4~
STAGF:
11
nit.rogcn
in
Unsegmenbed mesodcrm
0.397 0.021 0. 374 0, YOX 0, 710 0. as1 kO.077 per microgram
total
Activity
Catheptic activity in somite mesoderm taken from embryos that had been explanted for 10 hours in medium containing O.l5mg/ml of BAG was compared with the activity in mesoderm from embryos explanted in medium without RAG. For this series of determina-
(:ATHEF’TI(:
ACTIVITY
IN
SOMITE
MESODEHhl
1:3:3
tions, both segmented and unsegmented mesoderm were taken together, to conserve material, since the results of the first few experiments had shown essentially the same rise of catheptic activity in both parts after treatment. The final results (Table 2) show a variable, but always marked, increase in catheptic activity in the somite mesoderm of BAG-treated embryos as compared with controls (p < 0.01 for the differences between experimentals and controls ) .
DISCUSSION
There can be no doubt from the foregoing data that treatment of chick embryos with W-bromoallylglycine produces a marked increase in the activity of catheptic enzymes in the somite mesoderm. This confirms the tentative conclusion already reached by Schultz and Herrmann (1958) that the deficiency in total protein glycine in these treated tissues resulted from an abnormally high rate of protein breakdown. It has not been possible in the present series of experiments to estimate catheptic activity in other tissues as well as in the somite mesoderm, owing to the time limit imposed by the instability of proteins in operated tissues, making speed of dissection essential for consistent results. Rut with regard to the somites in particular, it was frequently observed that the cells of posterior somites in the treated embryos, which had been normal prior to treatment, became more loosely packed and spherical, so that they resembled the cells of unsegmented mesoderm. If this change in appearance indeed represents a reversion to the presegmented condi-
134
E.
M.
DEUCHAH
tion, the increase in catheptic activity in these cells is perhaps also to be interpreted as a reversion to the type of metabolism characteristic of unsegmented mesoderm, for this normally has a higher catheptic activity than segmented mesoderm (Table 1). But the increase in catheptic activity resulting from BAG-treatment is very large, and is probably better considered as a degenerative change, since it leads to arrest of development. It is not strictly comparable to the “dedifferentiation” characteristic of, for instance, the cells forming a regeneration blastema. One may remark here that some protein breakdown certainly occurs in the wounded areas of stump tissue that may fringe a regenerate, and it was possibly partly because of this that Xenopus tail regenerates were found to have an extremely high catheptic activity in the initial phases of regeneration (Deuchar et al., 1957). It cannot be ruled out, however, that some of the healthy blastema cells may also carry out active proteolysis. If so, this must provide raw materials, such as peptides and amino acids, which can then be built up into new tissue proteins. In fact, following the high catheptic activity there was an increase in concentration of free amino acids in the tail regenerates (Deuchar et al., 1957). Lehmann (1959) has emphasized the delicate balance that must exist between protein breakdown and protein synthesis in rapidly growing tissues of all kinds, and he believes, as does Herrmann (personal communication), that cathepsins play an important role as regulators of growth. If one thinks of animal development in these terms, one is struck with the number of instances where one tissue degenerates, to be replaced by another that may well use for its growth some products of proteolysis from the degenerated cells. To give just a few examples: the fetal cortex of the mammalian adrenal gland degenerates to give place to adult cortex; the embryonic notochord in vertebrates degenerates as the centra of the vertebrae develop. In insects, at the onset of pupation larval organs degenerate wholesale, to be replaced by imaginal organs or their primordia. Shatoury and Waddington (1957) have shown that in Drosophila the developing imaginal gut has its lumen filled with degenerating cells from the former larval intestine. Some proteolysis products of these cells must undoubtedly pass into the newly forming gut tissue, On a much larger scale, there is almost certainly a provision of protein breakdown products to the metamorphosing amphibian larva
CATHEPTIC
ACTIVITY
IN
SOMITE
MESODERM
135
by the regressing tail tissue, which is resorbed into the body at this stage. We have no direct biochemical evidence demonstrating this, but at least it has been shown that extensive proteolysis occurs in the resorbing tail of Xenopus larvae, for the catheptic activity of the tail tissue at this stage shows a spectacular increase ( Weber, 1957). Perhaps the dominant biochemical feature of early development in multicellular organisms is the continuous breakdown of reserve proteins (e.g., yolk) into materials that are then converted by synthesis into an array of more varied proteins, distributed among the various tissues. We know as yet very little about rates and sites of protein breakdown in embryos, but from the meager data available it seems that catheptic activity is specially high in certain regions and at certain times in which nutritive proteins are being digested most actively. For instance, the yolk sac of the chick embryo shows high c;itheptic activity (Borger and Peters, 1933) ; yolk-rich region; of Xenopus embryos (Deuchar, 1958)) and the yolk-absorption stage of frog tadpoles (Urbani, 1955) show maximal catheptic activity also. There is therefore good reason to suppose that in embryos cathepsins play the general, long-term role of assisting in the breakdown of storage proteins. There is an obvious need for studies with radioactive tracers to follow the course of this breakdown and the fates of the raw materials so produced. During relatively local and short-term steps in differentiation, however, such as the bl’ocking out of a pair of somites, the significance of fluctuations in catheptic activity may be much more subtle. In the present experiments, the rise in catheptic activity after treatment with BAG is certainly to be regarded as indicating an upset of protein metabolism due to the entry of BAG into molecules that would normally have contained leucine. Since no further somites were formed in the presence of BAG, it may be inferred that somite segmentation in chick embryos is dependent on the presence of leucine. The leucine analog, “E9,” used by von Hahn and Lehmann ( 1958) produced an even more dramatic cathepsin rise (100%) in the tail tissue of Xenopus larvae, where it also strongly inhibited tail regeneration. One may possibly explain the extreme sensitivity to Ieucine analogs of these two processes, somite formation in chicks and tail formation in Xenoms, by the fact that the tissues concerned normally contain high concentrations of leucine. Mammalian muscle proteins are particularly rich in leucine (Block and Rolling, 1951),
136
E. ht.
DEUCHAR
and both the skin and the blood of Xenopus larvae contain large amounts of free leucine (Deuchar, 1956). These particular tissues (the early tail regenerate consists at first of little more than skin and blood or lymph) would therefore be expected to suffer most acutely from blockage to the uptake of leucine, because their leucine requirements are high. The possibility that abundant supplies of particular amino acids may be essential for particular steps in tissue differentiation merits further investigation. One instance that has been especially well worked out by Wilde (1955) is the relation between phenylalanine and the differentiation of neural crest cells in urodele embryos. He was able to show that different analogs of phenylalanine, in which different parts of the molecule were modified in structure, altered the path of neural crest differentiation by suppressing one or other type of neural crest derivative. It is to be hoped that as more amino acid analogs become available for use by developmental biologists, more examples of this type may come to light, so that we may acquire broader glimpses of the pattern of protein synthesis in differentiating tissues. SUMMARY
1. In the somite mesoderm of 45hour chick embryos (Hamburger and Hamilton’s stage 11) there is a higher catheptic activity in the unsegmented region than in the region that has already undergone segmentation. 2. The somite mesoderm of embryos cultured for 10 hours on an agar-glucose medium containing 0.15 mg/ml of the leucine analog W-bromoallylglycine (BAG) has a markedly higher catheptic activity than somite mesoderm of control embryos cultured without BAG. 3. These results confirm the indications from Schultz and Herrmann’s study (1958) of the rate of uptake of radioactive glycine into these tissues, which were that there is an abnormally high rate of protein breakdown in BAG-treated somite mesoderm. 4. The findings are compared with those of von Hahn and Lehmann (1958) using a leucine analog on Xenopus larvae. The role of cathepsins in embryonic development, and the significance of individual amino acids for particular steps in differentiation, are discussed.
CATHEPTIC
ACTIVITY
IN
SOMITE
MESODERM
137
I am very grateful to Dr. H. Herrmann for his advice and interest in this tine. I should also like to thank Miss work, and for supplies of W-bromoallylgly J. Rees for the organization of technical assistance. REFERENCES BLOCK, R. J., and BOLLING, D. ( 1951). “The Amino Acid Composition of Pro2nd ed. C. C Thomas, Springfield, Illinois. teins and Foods,” BOELL, E. J., and SHEN, S. C. (1954). An improved ultramicro-Kjeldahl technique. Exptl. Cell Reseurch 7, 147-152. Untersuchmlgen uber BOXER, G., and PETERS, T. ( 1933). Ch emisch-biologisch Wachstumsfordernde Stoffe. 1. Die Enzyme des Extraktes aus Hiihnerembryonen. Z. physiol. Chem. Hoppe-Seyler’s 214, 91-103. DEUCHAR, E. M. (1956). Amino acids in developing tissues of Xenopus 1uevi.s. J. Emhrtyol. Exptl. Moqphol. 4, 327-346. DEUCHAH, E. M. (1958). Regional differences in catheptic activity in Xenopus laevis embryos. J. Embryol. Exptl. Morphol. 6, 223-237. DEUCHAR, E. M. (1960). Relation between somite segmentation rate and ATPase activity in early chick embryos. J. Embryol. Exptl. Morphol. 8, in press. DEUCHAR, E. M., WEBER, R., and LEHMANN, F. E. (1957). Differential changes of catheptic activity in regenerating tails of Xenopus larvae related to protein breakdown and total nitrogen. Helv. Physiol. Acta 15, 212-229. DUSPIVA, F. (1939). Beitrage zur Histophysiologie des Insektendarmes. I. Protoplasmu 32, 211-250. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HERRMANN, H., ROTHFELS-K~NIGSBERG, V., and CURRY, M. F. (1955). A comparison of the effects of antagonists of leucine and methionine on the chick embryo. J. Exvtl. 2001. 128, 339-378. LEHMAXN, F. E. (1959). Chemisch gehemmtes Wachstum van Regeneraten und Tumoren und die Dynamik gewebseigener Proteasen. Verb. naturforsch. Ges. Basel 70, 45-80. SCHULTZ, P. W., and HERRMANN, H. (1958). Effect of a leucine analogue on incorporation of glycine into the proteins of explanted chick embryos. J. Embryol. Exptl. Morphol. 6, 262-269. SHATOURY, H. H. EL, and WADDINGTON, C. H. ( 1957). Development of the intestinal tract during the larval period of Drosophila. 1. Embryol. Exptl. Morphol. 5, 134-142. URBANI, E. ( 1955 ). Gli enzimi proteolitici nella cellula, e nell’embrione. Experientiu 11, 209-218. voN HAHN, H. P., and LEHMANN, F. E. (1958). Die Veranderung der Kathepsinaktivitat im regenerierenden Schwanz der Xenopuslarve unter dem Einffuss morphostatischer Hemmstoffe. Helu. Physiol. A& 16. 107-126. WERER, R. (1957). On the biological function of cathepsin in tail tissue of Xenopus larvae. Exnerientia 13, 153. WILDE, C. E. (1955). The Urodele neuroepithelium. II. The relationship between phenyl alanine metabolism and the differentiation of neural crest cells 1. Morphol. 97, 313-344.
M.
Analog on Catheptic of the Chick Embryo
DEUCHAR
1NTROI)UCTION
The connection between tissue differentiation and protein synthesis in developing tissues is a subject of fundamental interest, but a very complex one to investigate experimentally. Recently, amino acid analogs (substances with a close structural resemblance to certain amino acids) have been found to be useful experimental tools with which to gain preliminary insight into some aspects of this sublect. If an amino acid analog is substituted for the normal amino acid in the nutrient medium on which an embryo or organ is growing, the analog becomes incorporated into the tissue proteins in place of its normal homolog, so that abnormal proteins, usually incompatible with growth, are produced. Occasionally this leads to quite clear-cut abnormalities in a particular developing tissue. If these abnormalities can be prevented merely by adding excess of the normal amino acid at the same time as its analog, one has clear evidence that the particular developmental process that was affected depended on the presence of the amino acid or some larger molecule into which it was rapidly incorporated. There may, however, arise secondarily a quite widespread dislocation of protein synthesis, with a consequent lowered utilization of other amino acids. For instance, Schultz and Herrmann (1958) found that W-bromoallylglycine an analog of leucine, reduced the uptake of radioactive (BAG), glycine into proteins of the primitive knot of chick embryos. The most striking effect of BAG on chick embryos, however, was that it blocked specifically the segmentation of the somites (Herrmann et al., 1955). When embryos of stage 11 (Hamburger and Hamilton, 1951) were explanted on an agar medium containing 129
130
E.
M.
DEUCHAR
BAG, no turther somites were formed during the period of treatment, but other tissues were unaffected, and control explants developed perfectly normally. Schultz and Herrmann (1958) went on to investigate the effects of BAG on protein synthesis in the explanted embryos, using the rate of uptake of W-labeled glycine as a measure ot the rate of synthesis of new protein. They found that only the segmented mesoderm, and no other tissue, had a lower total glycine content in BAG-treated embryos than in controls. Somewhat unexpectedly, though, both treated and control embryos showed similar concentrations of C4-glycine in the somite proteins, so there was no evidence of a lowered rate of protein synthesis as the result of treatment. To explain these results, the authors suggested that there had indeed been a lowered rate of incorporation of C”-glycine after BAG treatment, but that this had been masked, and the relative C”-glycine content maintained high, because of an accelerated breakdown of pre-existing protein in the treated tissue, which resulted in a proportionately greater loss of nonradioactive glycine. That enhanced protein breakdown may result from treatment with a leucine analog has been shown by von Hahn and Lehmann (1958) in work on Xenopus larvae. They found that the leucine aminoketone “E9” raises the activity of cathepsins (which are the predominant and most widespread intracellular proteolytic enzymes) in regenerating tail tissue of these larvae. In view of these findings, it was decided in the present study to measure catheptic activity in somite tissues of BAG-treated and control chick embryos at the stages used by Herrmann and his collaborators: to see whether there was evidence of enhanced proteolysis in the treated tissue. MATERIAL
AND
METHODS
Eggs (Light Sussex X Rhode Island for 32 hours (embryos to be explanted) series 1 that were not explanted).
Red) were incubated either or for 45 hours (controls of
Explantation Essentially the same procedure was used as described in another 1960). Blastoderms of 32 hours’ incubation were paper (Deuchar, trimmed just outside the limit of the area vasculosa, and after removal of the vitelline membrane and any adhering yolk they were placed, dorsal surface downward, on clots of agar medium in watch-
CATHEPTIC
ACTIVITY
IN
SOMITE
MESODERM
131
glass culture dishes. The medium was made up by mixing doublestrength Pannett-Compton saline containing 2% glucose with an equal volume of 1.5% agar solution which had previously been boiled and allowed to cool to about 40° C. The mixture was immediately poured into sterile watch glasses in petri-dish culture chambers, and allowed to set. In the experimental series, BAG was dissolved in the saline immediately before it was mixed with the agar, and its final concentration in the culture medium was 0.15 mg/ml (cf. Herrmann et al., 1955). The embryos were cultured on the medium for 10 hours at 37.5” C, then removed in order to dissect out the somite tissue. Cathepsin
Assays
The somite mesoderm was dissected out in ice-cold saline, all other tissue being removed except for the epidermis, which could not be removed successfully. The segmented and unsegmented portions were collected separately, from about twenty embryos per experiment, in saline kept at 2O C. The tissues were then washed free of saline by transferring them through two changes of chilled glassdistilled water, and immediately homogenized in approximately 60 ~1 of this fluid, in chilled Perspex microhomogenizers. Homogenate ( 11-J samples) was added to tubes containing 35.6 ,LL~of buffered 1% casein substrate (Duspiva, 1939) at pH 4.56, and the tubes were incubated on a water bath at 3S” C for 10 hours. (The optimal pH, enzyme concentration, and incubation time for the assays had been determined in preliminary experiments, and the time curve for the reaction had been found to be linear up to 12 hours at 3&O C.) At the end of 10 hours the reaction was stopped by adding 64.5 ~1 of 10% trichloroacetic acid to each tube, which precipitated the undigested casein. The tubes were then centrifuged in the cold for 15 minutes at 3500 rpm on a Baird and Tatlock semimicro angle centrifuge. To 100 ~1 aliquots of supernatant, 100 ~1 aliquots of first 13.5% sodium carbonate, then 20% (v/v) Folin-Ciocalteu reagent, were added, producing a blue color by reaction with the soluble casein products. The intensity of color developed was measured as an extinction value at 780 rnp on a Hilger Uvispek spectrophotometer, using microcells. Under the conditions of the assay, 0.3 gm casein (i.e., 0.1% concentration) gave an extinction reading of 0.14. Reagent blanks (distilled water instead of homogenate) were incubated with each experimental series, and the extinc-
1%
E.
hf.
IXXJCHAH
tion values of the blanks were subtracted from those of the experimental samples. From a standard curve obtained with known concentrations of casein solution, the final extinction values could be converted into micrograms of casein rendered soluble by the catheptic activity. The total nitrogen in samples of the homogenates was also estimated, by the ultramicro-Kjeldahl method of Boell and Shen (1954). Hence the results could be expressed as micrograms casein digested per microgram total nitrogen in the homogenate samples. RESULTS
1. Catheptic Activity Control Embryos
in Somite Mesoderm
of 45Hour
catheptic activity was measured In a first series of experiments, in somite mesoderm dissected out from embryos that had not been explanted but had simply been incubated in ovo for 45 hours. As Table 1 shows, there was always a higher catheptic activity per unit nitrogen in the unsegmented tissue than in the tissue that had already segmented into somite blocks.
CATHEPTIC
ACTIVITY"
lhpt.
IN
DIWEREST
11”.
PARTS
SOMITE
Segmented mesoderm
1 2 3 4 5 ~IWII Standard
on.
0. 23X 0. 370 0. 291 0.15:! 0. 399 ~ .~~ 0 292 *0.044
error
(1 Activity sample.
expressed
as micrograms
2. Effects
of BAG on Catheptic
soluble
caseiu
MESOT~ERM
4~
STAGF:
11
nit.rogcn
in
Unsegmenbed mesodcrm
0.397 0.021 0. 374 0, YOX 0, 710 0. as1 kO.077 per microgram
total
Activity
Catheptic activity in somite mesoderm taken from embryos that had been explanted for 10 hours in medium containing O.l5mg/ml of BAG was compared with the activity in mesoderm from embryos explanted in medium without RAG. For this series of determina-
(:ATHEF’TI(:
ACTIVITY
IN
SOMITE
MESODEHhl
1:3:3
tions, both segmented and unsegmented mesoderm were taken together, to conserve material, since the results of the first few experiments had shown essentially the same rise of catheptic activity in both parts after treatment. The final results (Table 2) show a variable, but always marked, increase in catheptic activity in the somite mesoderm of BAG-treated embryos as compared with controls (p < 0.01 for the differences between experimentals and controls ) .
DISCUSSION
There can be no doubt from the foregoing data that treatment of chick embryos with W-bromoallylglycine produces a marked increase in the activity of catheptic enzymes in the somite mesoderm. This confirms the tentative conclusion already reached by Schultz and Herrmann (1958) that the deficiency in total protein glycine in these treated tissues resulted from an abnormally high rate of protein breakdown. It has not been possible in the present series of experiments to estimate catheptic activity in other tissues as well as in the somite mesoderm, owing to the time limit imposed by the instability of proteins in operated tissues, making speed of dissection essential for consistent results. Rut with regard to the somites in particular, it was frequently observed that the cells of posterior somites in the treated embryos, which had been normal prior to treatment, became more loosely packed and spherical, so that they resembled the cells of unsegmented mesoderm. If this change in appearance indeed represents a reversion to the presegmented condi-
134
E.
M.
DEUCHAH
tion, the increase in catheptic activity in these cells is perhaps also to be interpreted as a reversion to the type of metabolism characteristic of unsegmented mesoderm, for this normally has a higher catheptic activity than segmented mesoderm (Table 1). But the increase in catheptic activity resulting from BAG-treatment is very large, and is probably better considered as a degenerative change, since it leads to arrest of development. It is not strictly comparable to the “dedifferentiation” characteristic of, for instance, the cells forming a regeneration blastema. One may remark here that some protein breakdown certainly occurs in the wounded areas of stump tissue that may fringe a regenerate, and it was possibly partly because of this that Xenopus tail regenerates were found to have an extremely high catheptic activity in the initial phases of regeneration (Deuchar et al., 1957). It cannot be ruled out, however, that some of the healthy blastema cells may also carry out active proteolysis. If so, this must provide raw materials, such as peptides and amino acids, which can then be built up into new tissue proteins. In fact, following the high catheptic activity there was an increase in concentration of free amino acids in the tail regenerates (Deuchar et al., 1957). Lehmann (1959) has emphasized the delicate balance that must exist between protein breakdown and protein synthesis in rapidly growing tissues of all kinds, and he believes, as does Herrmann (personal communication), that cathepsins play an important role as regulators of growth. If one thinks of animal development in these terms, one is struck with the number of instances where one tissue degenerates, to be replaced by another that may well use for its growth some products of proteolysis from the degenerated cells. To give just a few examples: the fetal cortex of the mammalian adrenal gland degenerates to give place to adult cortex; the embryonic notochord in vertebrates degenerates as the centra of the vertebrae develop. In insects, at the onset of pupation larval organs degenerate wholesale, to be replaced by imaginal organs or their primordia. Shatoury and Waddington (1957) have shown that in Drosophila the developing imaginal gut has its lumen filled with degenerating cells from the former larval intestine. Some proteolysis products of these cells must undoubtedly pass into the newly forming gut tissue, On a much larger scale, there is almost certainly a provision of protein breakdown products to the metamorphosing amphibian larva
CATHEPTIC
ACTIVITY
IN
SOMITE
MESODERM
135
by the regressing tail tissue, which is resorbed into the body at this stage. We have no direct biochemical evidence demonstrating this, but at least it has been shown that extensive proteolysis occurs in the resorbing tail of Xenopus larvae, for the catheptic activity of the tail tissue at this stage shows a spectacular increase ( Weber, 1957). Perhaps the dominant biochemical feature of early development in multicellular organisms is the continuous breakdown of reserve proteins (e.g., yolk) into materials that are then converted by synthesis into an array of more varied proteins, distributed among the various tissues. We know as yet very little about rates and sites of protein breakdown in embryos, but from the meager data available it seems that catheptic activity is specially high in certain regions and at certain times in which nutritive proteins are being digested most actively. For instance, the yolk sac of the chick embryo shows high c;itheptic activity (Borger and Peters, 1933) ; yolk-rich region; of Xenopus embryos (Deuchar, 1958)) and the yolk-absorption stage of frog tadpoles (Urbani, 1955) show maximal catheptic activity also. There is therefore good reason to suppose that in embryos cathepsins play the general, long-term role of assisting in the breakdown of storage proteins. There is an obvious need for studies with radioactive tracers to follow the course of this breakdown and the fates of the raw materials so produced. During relatively local and short-term steps in differentiation, however, such as the bl’ocking out of a pair of somites, the significance of fluctuations in catheptic activity may be much more subtle. In the present experiments, the rise in catheptic activity after treatment with BAG is certainly to be regarded as indicating an upset of protein metabolism due to the entry of BAG into molecules that would normally have contained leucine. Since no further somites were formed in the presence of BAG, it may be inferred that somite segmentation in chick embryos is dependent on the presence of leucine. The leucine analog, “E9,” used by von Hahn and Lehmann ( 1958) produced an even more dramatic cathepsin rise (100%) in the tail tissue of Xenopus larvae, where it also strongly inhibited tail regeneration. One may possibly explain the extreme sensitivity to Ieucine analogs of these two processes, somite formation in chicks and tail formation in Xenoms, by the fact that the tissues concerned normally contain high concentrations of leucine. Mammalian muscle proteins are particularly rich in leucine (Block and Rolling, 1951),
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and both the skin and the blood of Xenopus larvae contain large amounts of free leucine (Deuchar, 1956). These particular tissues (the early tail regenerate consists at first of little more than skin and blood or lymph) would therefore be expected to suffer most acutely from blockage to the uptake of leucine, because their leucine requirements are high. The possibility that abundant supplies of particular amino acids may be essential for particular steps in tissue differentiation merits further investigation. One instance that has been especially well worked out by Wilde (1955) is the relation between phenylalanine and the differentiation of neural crest cells in urodele embryos. He was able to show that different analogs of phenylalanine, in which different parts of the molecule were modified in structure, altered the path of neural crest differentiation by suppressing one or other type of neural crest derivative. It is to be hoped that as more amino acid analogs become available for use by developmental biologists, more examples of this type may come to light, so that we may acquire broader glimpses of the pattern of protein synthesis in differentiating tissues. SUMMARY
1. In the somite mesoderm of 45hour chick embryos (Hamburger and Hamilton’s stage 11) there is a higher catheptic activity in the unsegmented region than in the region that has already undergone segmentation. 2. The somite mesoderm of embryos cultured for 10 hours on an agar-glucose medium containing 0.15 mg/ml of the leucine analog W-bromoallylglycine (BAG) has a markedly higher catheptic activity than somite mesoderm of control embryos cultured without BAG. 3. These results confirm the indications from Schultz and Herrmann’s study (1958) of the rate of uptake of radioactive glycine into these tissues, which were that there is an abnormally high rate of protein breakdown in BAG-treated somite mesoderm. 4. The findings are compared with those of von Hahn and Lehmann (1958) using a leucine analog on Xenopus larvae. The role of cathepsins in embryonic development, and the significance of individual amino acids for particular steps in differentiation, are discussed.
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I am very grateful to Dr. H. Herrmann for his advice and interest in this tine. I should also like to thank Miss work, and for supplies of W-bromoallylgly J. Rees for the organization of technical assistance. REFERENCES BLOCK, R. J., and BOLLING, D. ( 1951). “The Amino Acid Composition of Pro2nd ed. C. C Thomas, Springfield, Illinois. teins and Foods,” BOELL, E. J., and SHEN, S. C. (1954). An improved ultramicro-Kjeldahl technique. Exptl. Cell Reseurch 7, 147-152. Untersuchmlgen uber BOXER, G., and PETERS, T. ( 1933). Ch emisch-biologisch Wachstumsfordernde Stoffe. 1. Die Enzyme des Extraktes aus Hiihnerembryonen. Z. physiol. Chem. Hoppe-Seyler’s 214, 91-103. DEUCHAR, E. M. (1956). Amino acids in developing tissues of Xenopus 1uevi.s. J. Emhrtyol. Exptl. Moqphol. 4, 327-346. DEUCHAH, E. M. (1958). Regional differences in catheptic activity in Xenopus laevis embryos. J. Embryol. Exptl. Morphol. 6, 223-237. DEUCHAR, E. M. (1960). Relation between somite segmentation rate and ATPase activity in early chick embryos. J. Embryol. Exptl. Morphol. 8, in press. DEUCHAR, E. M., WEBER, R., and LEHMANN, F. E. (1957). Differential changes of catheptic activity in regenerating tails of Xenopus larvae related to protein breakdown and total nitrogen. Helv. Physiol. Acta 15, 212-229. DUSPIVA, F. (1939). Beitrage zur Histophysiologie des Insektendarmes. I. Protoplasmu 32, 211-250. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HERRMANN, H., ROTHFELS-K~NIGSBERG, V., and CURRY, M. F. (1955). A comparison of the effects of antagonists of leucine and methionine on the chick embryo. J. Exvtl. 2001. 128, 339-378. LEHMAXN, F. E. (1959). Chemisch gehemmtes Wachstum van Regeneraten und Tumoren und die Dynamik gewebseigener Proteasen. Verb. naturforsch. Ges. Basel 70, 45-80. SCHULTZ, P. W., and HERRMANN, H. (1958). Effect of a leucine analogue on incorporation of glycine into the proteins of explanted chick embryos. J. Embryol. Exptl. Morphol. 6, 262-269. SHATOURY, H. H. EL, and WADDINGTON, C. H. ( 1957). Development of the intestinal tract during the larval period of Drosophila. 1. Embryol. Exptl. Morphol. 5, 134-142. URBANI, E. ( 1955 ). Gli enzimi proteolitici nella cellula, e nell’embrione. Experientiu 11, 209-218. voN HAHN, H. P., and LEHMANN, F. E. (1958). Die Veranderung der Kathepsinaktivitat im regenerierenden Schwanz der Xenopuslarve unter dem Einffuss morphostatischer Hemmstoffe. Helu. Physiol. A& 16. 107-126. WERER, R. (1957). On the biological function of cathepsin in tail tissue of Xenopus larvae. Exnerientia 13, 153. WILDE, C. E. (1955). The Urodele neuroepithelium. II. The relationship between phenyl alanine metabolism and the differentiation of neural crest cells 1. Morphol. 97, 313-344.