Experimentally induced changes in glutamotransferase activity in embryonic tissue

DEVELOPMENTAL

7, 192-206

BIOLOGY,

Experimentally

Induced Activity

( 1963)

Changes

in Glutamotransferase

in Embryonic

Tissue’

A. A. MOSCONA AND J. L. HUBBY With the technical assistance of Nilda Saenz Depurtment

of Zoology,

University

Accepted

of Chicago,

October

Chicago,

Illinois

18, 1962

INTRODUCTION

A basic aspect of differentiation in embryonic systems is the sequential appearance of new enzymatic patterns in cells and tissues, resulting in specialized metabolisms and characteristic products. The mechanisms involved in the appearance of enzymes in embryonic differentiation are therefore of major interest to students of development, as frequently emphasized by Weiss (1953). Progress in this area hinges upon the availability of suitable systems in which the mechanisms involved in the appearance and maintenance of characteristic enzymes might be experimentally analyzed. To be useful, such a system must be suitable for in vitro analysis; it must be based on a tissue which can be readily and cleanly isolated from embryos at different developmental stages, and in which the diversity of structural components, differences in their developmental origins, and the presence of transient cells are minimal; and it must involve a readily assayable enzyme system with a pattern of activity characteristic of that tissue throughout its development. The purpose of this paper is to report briefly on an experimental situation which meets these requirements: the activity of glutamotransferase in embryonic chick neural retina maintained in vitro. The detection and estimation of the enzyme glutamotransferase is based on its ability to catalyze the exchange of the amide group of ’ Research supported by grants C-4272 from the United States Public Health Service, National Cancer Institute (A.A.M.), and NSF-17640 from the National Science Foundation (J.L.H.). 192

CHANGES

IN

GLUTAMOTRANSFERASE

ACTIVITY

193

glutamine for hydroxylamine to yield glutamohydroxamic acid (GHA ) (Waelsch, 1952; Rudnick et al., 1954; see review by Meister, 1962). The precise function of this enzyme in the retina is at present unknown. The neural retina of the chick embryo is derived solely from the optic vesicle. It it an avascular tissue-therefore uncontaminated by endothelial and blood cells. It can be readily and cleanly isolated from the eye by simple dissection. Embryonic retina tissue and dissociated retinal cells can be maintained in vitro. It was previously found (Rudnick and Waelsch, 1955) that glutamotransferase activity in the retina of the chick embryo starts to rise above the minimal tissue level at about 17 days’ incubation, when it increases sharply concurrently with the functional maturation of the corollary to eye (Wald and Zussman, 1938). The present work-a previous studies on retinal differentiation (Moscona, 1957)-began with an attempt to chart glutamotransferase activity in retina grown in vitro; it was soon found that when early embryonic neural retina, devoid of detectable glutamotransferase activity, was placed in organ culture, activity appeared rapidly and reached high levels days in advance of its normal developmental chronology. Certain of the basic parameters involved in this experimental situation were examined and are presented here. MATERIALS

AND

METHODS

Neural retinas were obtained from White Leghorn chick embryos at stages of development ranging from 7 days’ incubation to hatching; from chicks 7,8,33, and 34 days old; and from roosters aged 16 and 72 weeks. Isolated eyes, handled with sterile precautions, were bisected in Tyrode’s solution, the vitreous humor was removed, and the halves were left in the saline for about 5 minutes at room temperature. The neural retina detached itself readily from the tapetum and by gentle prodding was made to come off as an intact sheet. The isolated sheets of pure neural retina tissue were rinsed in Tyrode’s solution and prepared either for culture or enzyme assay (see below). Organ Culture Flask cultures and liquid culture medium were used. Retinas from two eyes were cut up into l-3 mm” fragments which were suspended in 24 ml culture medium in a 125-ml Erlenmeyer flask. Each culture

194

MOSCONA

AND

HUBBY

series consisted of up to 10 flasks. The flasks were gassed with a mixture of 5% CO, in air, sealed, and placed on a gyratory shaker rotating 80 rpm (diameter of rotation ?i inch) at 38°C for varying lengths of time. Culture medium was changed every second or third day, depending on the harvest schedule. Cultures were harvested at 2- or 3-day intervals; at these times samples were taken for histological examination and the bulk of the tissue was rinsed 3 times by short centrifugation in phosphate buffer (0.01 M, pH 7.1) and assayed for enzyme activity. More than 120 cultures were assayed. The standard culture medium consisted of Eagle’s basal medium with 0.025% (Le., 1.75 millimoles ) glutamine, 10% horse serum, 2% chick embryo extract ( lo-day embryos), and 1% penicillin-streptomycin (50 units each per milliliter). Modifications of this medium are described in the text. Cell Aggregates Suspensions of retina cells were prepared by our standard trypsinization procedure ( Moscona, 1961a). The dissociated cells were dispersed in culture medium and dispensed in 24-ml aliquots into 125-ml Erlenmeyer flasks. The equivalent of one pair of retinas in dissociated cells was put into each flask. According to counts obtained by means of a Coulter counter, one pair of ‘i--day retinas yielded on the average 36 x 10’; dissociated cells; one pair of lo-day retinas yielded 187 X 10” cells. Cell aggregates were obtained by the rotation-mediated cell aggregation procedure (for details see Moscona, 1961a). The flasks were rotated 80 rpm at 38°C for various lengths of time. Culture medium was changed every 2 days. The harvested aggregates were washed 3 times by brief centrifugation in phosphate buffer and assayed for glutamotransferase activity. Samples were examined histologically. Cell Cultures Trypsin-dissociated retina cells were dispersed in standard culture medium and dispensed in Urn1 aliquots into T-60 flat tissue-culture flasks; these were gassed with a 5% CO,-air mixture, sealed, and incubated at 38°C. The equivalent in cells of a quarter of one retina was placed in each flask. Medium was changed every 3 days. After various periods of cultivation the cells were scraped off, washed 3 times in phosphate buffer by centrifugation, and assayed for enzyme activity.

CHANGES

IN

GLUTAMOTRANSFERASE

195

ACTIVITY

Enzyme Assay Procedure The method of assay for glutamotransferase, utilizing the detection of glutamohydroxamic acid with ferric chloride, was essentially from Rudnick et al. (1954). In our system the reaction mixture consisted 5 ;tmoles of PO,-- and Mn++, 0.05 pmole of 90 pmoles glutamine, adenosine triphosphate, 50 pmoles acetate buffer at pH 6.4, and the enzyme solution. This mixture was incubated for 10 minutes at 37°C. Fifteen micromoles of hydroxylamine were added to give a final volume of 1 ml. The reaction mixture was incubated at 37°C for the minimum amount of time required for accurate estimation of the hydroxamic acid formed. Material with low activity was incubated usually for 1 hour, and maximal amounts of the enzyme preparation were used; incubation of material with high activity was 10 or 20 minutes, and smaller amounts of enzyme preparation were used. Control reactions were prepared for each assay and consisted of all the above ingredients except hydroxylamine. At the termination of the reaction period 0.75 ml of a solution containing equal parts of 2.5N HCI, 15% trichloroacetic acid, and 5% ferric chloride in 0.1 N HCl was added to the mixture. This solution was centrifuged, and the optical density at 500 rnp was measured on a Beckman DU spectrophotometer. The protein was estimated with Folin’s reagent after the method of Lowry et al. (1951). The specific activity of the enzyme was defined as the number of micromoles of GHA produced per hour per milligram of protein. One micromole glutamohydroxamic acid per milliliter was calculated to give an optical density of 0.450 at 500 mp. Under our conditions an activity of 0.08 or greater could be detected per milligram of protein in the assay. RESULTS

Glutamotransferase

Activity

in Embryonic

and Adult

Retina

Freshly dissected retinas from 7-day chick embryos had no detectable glutamotransferase activity even when the equivalent of three whole retinas was assayed under standard conditions for an hour. Ten-day retina gave no values higher than specific activity of 0.1. From the tenth day on specific activity increased slightly, but erratically, up to the sixteenth day, when a sharp rise began which continued after hatching. Figure 1 and Table 1 show the development of

196

MOSCONA

AND

120 100 -

IIUBI~Y

.

80 -

.

60 -

. I

40.

20 t l5 F 2 IO 0 i; E w ti

.

l

. .

l .

8 6_

. .

4-

. . 2-

.

72

DAYS

WEEKS AiE

FIG. 1. Specific and after hatching.

activity

of glutamotransferase

in the retina

of chick

embryos

specific activity of glutamotransferase in the retina. Highest activities were obtained in 5-week-old chicks. Retinas from older animals gave lower values; however, since only two older roosters were examined, these values must be taken with some reservation. Thus, the development of glutamotransferase activity in the retina is characterized by very low activity up to the sixteenth day of embryonic development, and by a sharp increase thereafter. This corresponds closely to and confirms the results obtained by Rudnick and Waelsch (1955). Our higher values may be explained, in part, by our use of greater substrate concentration in the assay. Glutamotransferase

Activity

in Organ Cultures

of Embryonic

Retina

Retina from IO-day embryos. As stated above, at this stage of de-

Average specific activity

Age:

<0.06

7

0.07

10

0.06

11

0.06

12

GLUTAYOTRANSFERASE

0.09

13

0.14

15

0.37

16

IN

TABLE Embryos

ACTIVITY

1

0.58

17

CHICK

1.38

18

RETINA

1.78

19

21

7.7 6.7

20

OF VARIOUS

14.5

Hatch.

AGES

56.0

1 Wk

85.0

5 Wk

49.0

16 Wk

Chicks and edlIlts

41.1

72 Wk

198

MOSCONA

AND

HUBBY

velopment enzyme activity in the retina is at a very low level and does not go up significantly for another 7 days. However, when the lo-day embryonic retina is isolated and grown in organ culture in standard medium, activity rises rapidly and continues to increase as shown in Fig. 2 and Table 2. This very striking and relatively rapid enhamement TABLE GLIJTAMOTRANSFERASE

ACTIVITY

Preparation

Retina,

Retina,

FMina,

‘?-day embryos

l&day

emhryos

16-day embryos

Other embryonic Skin

RETINAS

IS ORGAN

CULTURE Average specitic activity

Days in culture

Protein (mg/retina)

0

4 6 8

0.44 1 .3 2 0 1.9

10

1.2

0 2

1.9 2.1

4 6 8 10

2.1 x.2 1.6 0.85

0.07 1.20 5.2 8.3 13.9 22.5

0 2 4 6 8 10

2.2 2.9 2.2 1.8 2.35 2.15

0.37 5.9 10.35 11.05 19.9 18.1

2 3 2 4

0.8 0.9 0.22 0.32


<0.06" 0.19 1.10

2.80 7.10

t,issues, lo-day

Lung

a No activity assay conditions.

2

OF CHICK

detected.

The figure represents

the maximum

value possible under

of the activity of this enzyme in the isolated retina of IO-day chick embryos was found to take place also in culture media without embryo extract and glutamine; it occurred also in a minimal maintenance

CHANGES

IN

GLUTAMOTRANSFERASE

199

ACTIVITY

22

2c

18

16

14

8

6

A, L

9

4-DAYS I I-

AGE

IN CULTUREIN DAYS -

IO 17

FIG. 2. Specific activity of glutamotransferase in organ cultures embryos of different ages compared with the in uiuo values.

19

21

of retina

from

200

MOSCONA

AND

HUBBY

medium consisting of Tyrode’s and 10% horse serum. In sharp contrast with the retina, there was no increase in glutamotransferase activity in organ cultures of skin or lung from these embryos, grown under identical conditions (Table 2). Retina from 7- and S-day embryos. An increase in glutamotransferase activity was obtained also in cultures of retinas from 7- and 16day embryos (Fig. 2; Table 2). In cultures of 18day retina enzyme activity rises very rapidly, at a rate similar to that in the retina in viuo. Activity in cultures of retina from ‘I-day embryos rises slowly and does not reach values attained by lo-day retina cultured for a similar length of time; however, it reaches higher values than in embryos of comparable age. Particularly noteworthy is the 3- to 4-day lag period in the “i-day retina cultures before a significant increase in enzyme activity becomes detectable. This differs from the much sharper increase in enzyme activity in lo-day retinas in culture and the even more rapid increase in cultured 16-day retinas. Evidently, under the conditions tested, the rate of increase in specific activity of the explanted retina varies significantly with the age of the donor embryo. Other factors, particularly the amount of tissue per culture, may conceivably be involved. Glutamotransferase

Activity

in Aggregates of Retina Cells

Cells from lo-day embryos. To determine whether the increase in enzyme activity was dependent upon the original integrity of the retina, studies on cell aggregates were made. Cell aggregates were harvested every 2 days in one series of experiments, and every 3 days in others. Protein and glutamotransferase activity data are summarized in Table 3. Figure 3 presents the changes in enzyme activity plotted against duration of culture. It is evident that retinal tissue reconstructed from dissociated and aggregated cells showed a striking rise in glutamotransferase activity and behaved, in this respect, like cultures of intact retina. The rate of increase in aggregates was slower than in organ cultures maintained for a comparable length of time and lower final values were reached. Cells from 7-day embryos. Aggregates of 7-day retina cells (Table 3) harvested after 6 days in culture showed a rate of increase in enzyme activity comparable to that of 7-day retina in organ culture for 6 days. The increase in enzyme activity and the values reached after 6 days were lower than those for aggregates of IO-day retina cells cultured for the same length of time.

CHANGES

IN

GLUTAMOTRANSFERASE

GL~TAM~TRANsFERAsE DISSOCIATEU DOllOr

OF

Specific activity

0.44 0.59


0 2 3 4

1 9 0.72 2.4 I ‘I!)

0.07 1.09 0.96

5 ti 8 9

I.16 2.29 1 .35 1.6

7

10

ACTIVITY Is AGGREGATES CHICK RETINA CELLS IIg. protein per retina

WC

201

ACTIVITY

IO

1.8

1.53 2 .3tj 2.28 fi.05 5.0 7.48

Since cells dissociated from retinas of 16-day embryos do not form sizable aggregates (Moscona, 1961b), no attempt was made to obtain comparable data from this stage of development. Glutamotransferase

Activity

in Retina Cell Cultures

To determine whether continuous increase in enzyme activity was displayed by cells not associated in multicellular frameworks, cultures of dissociated cells were examined. Satisfactory “monolayer” cultures of dissociated neural retina cells from lo-day embryos are difficult to prepare because of the tendency of these cells to aggregate. Within a day, small clusters form, and these might be expected to behave, with respect to enzyme development, like regular small aggregates. Subsequently, many of these clusters flatten out into sheets of cells, while others persist. For enzyme assay, cells from equivalent flasks were pooled. The data are summarized in Fig. 3. It is evident that in the first days, concurrent with formation of cell clusters, specific activity of the enzyme increased to a level of 2.0; it remained at this level throughout a prolonged period of cultivation. The low activity and its leveling off in these cell cultures are in contrast with the continuous increase in glutamotransferase activity in aggregates and in organ cultures of retina. The initial increase in glutamotransferase activity in these cultures and the level maintained could, perhaps, be related to the clustering of the dissociated cells and persistence of

202

MOSCONA

AND

HUBBY

20

16

6

DAYS

IN CULTURE

FIG. 3. Specific activity of glutamotransferase in organ cultures, gates, and cell cultures of chick retina from lo-day embryos.

cell aggre-

CHANGES

IS

GLUTAMOTRANSFERASE

ACTIVITY

203

small aggregates. The data strongly suggest, but do not prove, that a relationship exists between the state of cell aggregation in the retina and continuous increase in the activity of this enzyme. Histological

Obseroations

The histology of the retina in chick embryos was reviewed by Coulombre (1961). On the seventh day the neural retina consists of a relatively thick zone of cells which will form the nuclear layers, and of an inner thin nerve-fiber layer. On the tenth day differentiation has progressed to a point where a clear distinction can be made between the outer and the inner nuclear layers, the outer and inner plexiform layers, the ganglion cell layer, and the nerve fiber layer. From then on development consists mainly of changes in the relative thicknesses of these layers, cell size, shape, and increase in number of arborized connections. It is thought that from the fifteenth day on, with the appearance of the visual pigments (Wald and Zussman, 1938), the functional differentiation of the retina proceeds rapidly. Histological examination of retina cultured as fragments and as cell aggregates provides no evidence that the striking increase in enzyme activity in culture is paralleled by accelerated histological changes. In organ cultures of retina from 7-day embryos maintained for 6 days, tissue differentiation proceeded to a stage comparable structurally with that of lo- to 12-day retina in the embryo, In organ cultures of retina from lo-day embryos the structural integrity of the tissue was retained throughout the period of cultivation except for indications of degenerative changes in the ganglionic layer. There was no evidence of a correlation between degree of ganglionic degeneration and glutamotransferase activity ( Rudnick, 1959). In aggregates the cells became arranged in typical rosettes and subsequently formed masses of neural retina tissue and nerve fibers. It seems thus, at present, that there are no morphologically detectable changes that can be convincingly related to the increase in the activity of glutamotransferase in the cultured retina. DISCUSSION

It was shown that glutamotransferase activity in the neural retina of the chick embryo can be markedly and consistently enhanced by the simple expedient of isolating the tissue and maintaining it in uitro.

204

MOSCONA

AND

HUBBY

It was further shown that, under the conditions tested, the rate of increase in specific activity of this enzyme was dependent upon the age of the donor embryo. It was also found that increase in activity took place in suboptimal culture media, including a maintenance medium consisting of Tyrode’s solution and 10% horse serum. No increase in glutamotransferase activity was detected in other tissues (lung, skin) similarly treated and maintained in organ culture. This indicates that the effect does not represent a nonspecific response to explantation or culture conditions and that the enhancement of activity in explanted embryonic chick retina represents a modification of the developmental pattern typical of this tissue. The fact that the rates of increase in enzyme activity and the values reached after a comparable length of cultivation were highest in organ cultures and cell aggregates, and lowest in cell cultures, indicates that the effect, while independent of the original integrity of the tissue, is dependent on multicellular association of the cells. These differences suggest a number of obvious possibilities toward further experimental analysis of initiation and development of activity of this enzyme in the retina. Since the purpose of this paper is to briefly present factual information, only the following remarks will be made. Since we are measuring specific activity of the enzyme there is, at present, no information whether the changes observed represent a net increase in number of enzyme molecules or activation of preexisting enzyme molecules. Since the exact role of glutamotransferase in metabolism is not clear, speculations on the relationship between increase in enzyme activity and the differentiation of the retina would be out of place. Histologically there is, at present, no evidence indicative of any gross changes that might be directly correlated with the increase in glutamotransferase activity in the cultured tissue or in cell aggregates. The possibility remains that the increase in enzyme activity is related to changes at the fine structural or molecular level in the cells, involved in their functional maturation. It seems of considerable interest that the mere isolation of the retina from the embryonic environment caused such a marked and rapid increase in glutamotransferase activity. It suggests that, in embryonic development this enzyme might be controlled by repressor of suitable substrates. In or inhibitory mechanisms, or availability these terms, isolation in vitro of the retina might be viewed as conducive to a reduction in the inhibitory influences, or as presentation

CHANGES

of activating visaged, and present, the in that they characteristic manipulated

IN

GLUTAMOTRANSFERASE

ACTIVITY

205

can readily be ensubstrates. Other interpretations some of the possibilities are being examined. Thus, at main significance of the reported findings is, primarily, introduce a system in which the development of a enzyme in an embryonic tissue can be experimentally with the aid of controllable variables. SUMMARY

Glutamotransferase activity in the retina of chick embryos at different stages of development was assayed in freshly isolated tissue and, after cultivation in &To, as organ cultures, cell aggregates, and cell cultures. In the embryo, specific activity of this enzyme is low until the 16th day of development, then rises rapidly. In cultures of retinal tissue from early embryos there is a rapid, sharp, and continuous increase in the specific activity of glutamotransferase. The values reached greatly exceed those attained in viva in embryos of comparable age. There is a similar continuous increase in specific activity of this enzyme in aggregates of retina cells from early embryos. In cell cultures, the rise is slight and this low level persists upon prolonged cultivation, owing, perhaps, to the presence of cell clusters. Under the conditions studied, the rate of increase in specific activity in retina cultures depended on the age of donor embryo. NO increase in activity was obtained in two other tissues examined under identical in vitro conditions. Some of the implications and possibilities suggested by these findings are mentioned. REFERENCES COULOMBRE, A. J. ( 1961). Cytology of the developing eye. Intern. Reo. Cytol. 11, 161-194. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-

275. MEISTER, A. ( 1962). Glutamine synthesis. In “The Enzymes” (P. D. Boyer, H. Lardy, and K. MyrbBck, eds. ), pp. 443-468. Academic Press, New York. MOSCONA, A. ( 1957). Formation of lentoids by dissociated retinal cells of the chick embryo. Science 125, 598-599. MOSCONA, A. ( 1961a). Rotation mediated histogenetic aggregation of dissociated cells. Erptl. Cell Research 22, 455-475.

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A. ( 19611,). Tissue reconstruction from dissociated cells. In “Growth Systems” (M. X. Z arrow, ed.), pp. 197-220. Basic Books, New York. RUDNICK, D. ( 1959 ). Glutamotransferase and histogenesis in the transplanted chick retina. J. Erpfl. Zool. 142, 643-666. RUDNICK, D., and WAELSCH, H. ( 1955). D evelopment of glutamotransferase and glutamine synthetase in the nervous system of the chick. 1. Exptl. 2001. 129, 309-326. RUDNICK, D., htEL.4, P., and WAELSCH, H. (1954). Enzymes of glutamine metabolism in the developing chick embryo. A study of glutamotransferase and glutamine synthetase. J. Exptl. Zool. 126, 297-321. WAELSCH, H. ( 1952). Certain aspects of intermediary metabolism of glutamine, asparagine and glutathione. Advances in Enzymol. 13, 237-319. WALD, C., and ZUSSMAN, H. (1938). Carotenoids of the chicken retina. J. BioE. Chem. 122, 449-460. WEISS, P. ( 1953). Some introductory remarks on the cellular basis of differentiation. J. Embryol. Expfl. Morphol. 1, 181-211. MOSCONA,

in Living