nucleic acid and protein synthesis and pattern regulation ... · proposed axial gradients and of...

/ . Embryol. exp. Morph. Vol. 21, J, pp. 33-54, February 1969 3 3

Printed in Great Britain

Nucleic acid and protein synthesis and patternregulation in hydra

I. Regional patterns of synthesis and changes in synthesisduring hypostome formation

By S. G. CLARKSON1

From the Department of Biology as Applied to Medicine,Middlesex Hospital Medical School, London

Hydra provides a convenient system for the study of the regulation of alinear pattern of organization. Almost any region is capable of being recon-stituted into the whole organism and regulation is always polarized, i.e. distalstructures (hypostome and tentacles) are formed from distal ends and proximalstructures (peduncle and basal disk) from proximal ends.

Two models have recently been proposed to account for polarized regulationin hydra. Both incorporate axial gradients, but they differ radically from eachother and some controversy exists as to which formulation is correct. In themodel of Webster (Webster, 1966tf, b; Webster & Wolpert, 1966) regulationoccurs as the result of the interaction of three factors, namely time for hypo-stome determination, inhibition of hypostome determination, and threshold forinhibition. No suggestions are made with regard to the physiological basis ofthese factors. On the other hand, Burnett (1961, 1966) claims a close causalrelationship between growth and form. In his model, regulation is determinedby the local balance of two factors, a growth stimulator arising from thehypostome, and a growth inhibitor produced by regions of active growth.

One approach towards an understanding of the physiological basis of theproposed axial gradients and of the factors involved in regulation is thedetermination of nucleic acid and protein synthesis in intact and regeneratinghydra. While the axial distributions of DNA, RNA, and protein have beenstudied in a few hydroid species, e.g. Hydra littoralis (Li & Lenhoff, 1961) andTubularia larynx (Tardent, 1963), no reports exist of the quantitative measure-ment of their synthesis. This provides the subject of this paper. Essentially twoquestions were asked: (1) Does hydra possess axial gradients of DNA, RNA, or

1 Author's address: Department of Molecular, Cellular and Developmental Biology,University of Colorado, Boulder, Colorado 80302, U.S.A.

3 JEEM 21

34 S. G. CLARKSON

protein synthesis? (2) Is the formation of a hypostome during regulationaccompanied by radical changes in DNA, RNA, or protein synthesis?

MATERIALS AND METHODS

Hydra Httoralis were used for all experiments. Details with regard to culturemethods, selection of animals, and operative procedures are as given in Webster& Wolpert (1966).

Radioactivity labelling procedures. Radioactive precursors were administeredby incubating intact or regenerating hydra with precursor in culture medium' M ' (Muscatine, 1961). In some experiments precursor was administered in thepresence of 10~5M reduced glutathione (GSH) in 'M' , because GSH at thisconcentration causes the hypostome to open wide for 35 min (Loomis, 1955),thereby giving endodermal cells better access to the precursor. The hydra wereincubated at 26 °C as batches of five or six in 0-2 ml or ten in 0-5 ml of labelledmedium.

[3H]thymidine (3 or 5 c/mM), [3H]uridine (3-33 c/mM), and [14C]algal proteinhydrolysate (640^c/mg) were obtained from the Radiochemical Centre,Amersham.

Radioactivity determination. Incubations were terminated by washing thehydra four times with an excess of unlabelled precursor in ' M ' solution pre-chilled to 4 °C. Debris was removed from the basal disks and, in regionalincorporation studies, the animals were cut in the unlabelled precursor mediumand identical regions pooled. The samples were disrupted by sonication underice (Dawe Soniprobe, setting no. 3,5 s burst), aliquots removed for DNA, RNA,or protein estimation, and cold 10 % trichloroacetic acid added to the remaininghomogenate. In studies utilizing [14C]algal protein hydrolysate the samples wereheated at 90 °C for 20 min to hydrolyse nucleic acids, and then cooled by theaddition of cold 5 % trichloroacetic acid. Acid-insoluble radioactive materialwas assayed by collecting the precipitate on a Millipore filter (type GS, 25 mm,pore diameter 0-22 /*), washing thoroughly with cold 5 % trichloroacetic acid,briefly rinsing with a 2:1 mixture of chloroform:isopropanol and then ether,and counting in a Packard TriCarb scintillation counter in a toluene-basedscintillant containing PPO (2,5-diphenyloxazole), 5 g/1., and dimethyl-POPOP(4-bis-[4-methyl-5-phenyloxazoyl-2]-benzene), 0-3 g/1. Corrections were madefor 3H self-absorption, but 14C self-absorption was negligible. All radioactivityis reported as counts/min per jug nucleic acid or protein. This is based on theassumption that each radioactive precursor was incorporated into the relevantportion of the acid-insoluble fraction.

Protein and nucleic acid estimation. Protein determinations were madedirectly on aliquots of the homogenate by the method of Lowry, Rosebrough,Farr & Randall (1951). Wellcome Chemical Control Serum (Burroughs Well-come and Co., London) was used as standard.

Pattern regulation in hydra. I 35

Nucleic acids were extracted by the addition of an equal volume of cold 10%trichloroacetic acid to aliquots of the homogenate, followed by centrifugatjon at4 °C. DNA was extracted from the precipitate either by a single treatment with1 N-NaOH for 1 h at 37 °C, or by two 20 min extractions with 1-8N perchloricacid at 70 °C. DNA determinations were made directly on the alkali digest, or onthe combined acid extracts, by the indole-HCl method of Ceriotti (1955) asmodified by Bonting & Jones (1957). Calf thymus DNA (Sigma Chemical Co.London) was used as standard.

RNA was extracted from the precipitate by hydrolysis in 0-3N-KOH for 1 h at37 °C. After cooling under ice, each sample was acidified with an equal volumeof cold 0-4N perchloric acid and allowed to stand for 10 min at 0 °C. Followingcentrifugation, the precipitate was washed with 1-vol. of cold 0-2N perchloricacid and centrifuged again. The two supernatants were combined and estimatedfor RNA by u.v. absorption at 260 m/.t. Yeast RNA (Sigma Chemical Co.,London) was used as standard.

RESULTS

1. Conditions of radioactive precursor incorporation

It has generally been thought that hydra are impermeable to most exogenousfree compounds. Burnett, Baird & Diehl (1962) were forced to cut hydra inorder to obtain incorporation of [3H]thymidine, and Campbell (1965) devised amicroinjection procedure for the same purpose. However, initial experimentsinvolving incubation of intact hydra with [3H]thymidine administered as acomponent of the normal culture medium indicated that this precursor could beincorporated into the acid-insoluble fraction to quite a large extent, the greatmajority of the radioactive material presumably being incorporated into DNA.In view of this discrepancy the incorporation of [3H]thymidine, [3H]uridine, and[l4C]algal protein hydrolysate was investigated.

(a) Incorporation with time

To establish the kinetics of [3H]thymidine incorporation, triplicate batches ofsix intact hydra were incubated in 50/*c/ml [3H]thymidine (5 c/raivi) in ' M ' forthe times indicated (Fig. 1A). Similar experiments were performed with[3H]uridine (3-33 c/raM) at 15 /*c/ml in ' M ' (Fig. 1B) and with [14C]algal proteinhydrolysate (640/^c/mg) at 10//c/ml in ' M ' (Fig. 1C). Regression lines havebeen calculated for the results shown in Fig. 1; in each case the relationship islinear (correlation coefficient, r < 0-001) and the intercepts on the x and y axesare not significantly different from zero. Non-specific absorption of any of thethree precursors is considered negligible, for animals incubated for 5 minpossessed specific activities which were no higher than the values predicted fromthe equation of their respective regression line for a 5 min incubation. It istherefore concluded that hydra can incorporate [3H]thymidine, [3H]uridine, and

3-2

36 S. G. CLARKSON

[14C]algal protein hydrolysate from the surrounding medium at a constant ratefor at least the times investigated.

(b) Incorporation at different precursor concentrations

To determine how the rate of incorporation of [3H]thymtdine varies as afunction of the concentration of externally supplied [3H]thymidine of constantspecific activity, duplicate batches of five intact hydra were incubated for 2\ h in

3 6 9 18Incubation time (h)


24

120

| 90o

^ 6 0'p

Co

un

ts/i

o.

-c

-

/

I 1 I

/ _

/

/

i i i


Fig. 1. Kinetics of (A) [3H]thymidine incorporation into DNA, (B) [3H]uridineincorporation into RNA, and (C) [14C]algal protein hydrolysate incorporation intoprotein.

[3H]thymidine (5 c/mM) in 10~5M GSH in ' M ' at various concentrations between3-125 and 50/tc/ml. Similar experiments were performed with [3H]uridine(3-33 c/mM) in ' M ' over the range 3-125-50/^c/ml, and with [14C]algal proteinhydrolysate (640 ̂ c/mg) in ' M ' over the range 3-20 /ic/mi. The results are shownin Fig. 2, together with the regression lines (r < 0-001 in each case). The inter-


cepts on the x and y axes are not significantly different from zero, and it isevident that the rate of incorporation of each precursor is a linear function ofthe external precursor concentration over the range investigated.

140

7 120 -

7-5 15 30Concentration of

[3H]thymidine (/<c/m

60 6-25125 25Concentration of[3H]uridine (/ic/m\)

o

M 60

I 4 5c

3 30

15

c1

-

-

-

•'' 1

1

1

1

//

1

•

-

-

-

-

1

3 6 12 20Concentration of [14C]algalprotein hydrolysate (//c/ml)

Fig. 2. Influence of (A) [3H]thymidine concentration on its incorporation into DNA,(B) [3H]uridine concentration on its incorporation into RNA, and (C) [14C]algalprotein hydrolysate concentration on its incorporation into protein.

2. Reduced glutathione as a specific means to increase radioactive precursorincorporation

Preliminary experiments involving incubation of intact hydra with [3H]thy-midine in 10~5M GSH in ' M ' indicated that incorporation could be increasedsome threefold over that obtained by incubating hydra with [3H]thymidine in' M ' alone. The following experiments were designed to test if this increasedincorporation was due specifically to the glutathione feeding reflex.

38 S. G. CLARKSON

(a) DNA synthesis

In the first experiment, four groups of duplicate batches of five animals wereincubated for 2^h in [3H]thymidine (5 C/IUM) at 50/tc/ml. Groups (1) and (2)comprised intact animals; groups (3) and (4) comprised animals cut at thesubhypostomal level immediately prior to incubation: both distal and proximalparts were retained for incubation. Groups (1) and (3) were incubated in[3H]thymidine in ' M ' alone; groups (2) and (4) were incubated in [3H]thymidinein 10~5M GSH in ' M \ The results are shown in Table 1.

Table 1. Reduced glutathione as a specific means to increasethe incorporation of [3H]thymidine into DNA

Specific activity Mean specificTreatment and materials (c.p.m.//*g DNA) activity

286

1019

845

992

A significant threefold increase was obtained by incubating intact hydra inthe presence of GSH compared to that obtained by incubation in [3H]thymidinein ' M ' alone: applying Student's t test, P < 0-05 for the difference between themeans of groups (1) and (2). Reduced glutathione presumably could cause thisincrease by allowing the cells of the endoderm better access to the thymidine bymeans of the feeding reflex and/or by establishing a complex biochemicalsituation which somehow allowed more incorporation of the precursor. Byremoving the hypostome and tentacles just before incubation, however, thefeeding reflex would be prevented while still allowing endodermal cells betteraccess to the precursor. If the latter were the major factor responsible for theenhanced incorporation using GSH, then animals cut at the subhypostomal levelshould show no significant difference between their specific activities whenincubated in the presence or absence of GSH. This was in fact found to be thecase: P > 0-10 for the difference between the means of groups (3) and (4). It istherefore concluded that the increased incorporation obtained when hydra areincubated in the presence of GSH is not due to a non-specific biochemical effect,but rather that it reflects better access given to the cells of the endoderm to the[3H]thymidine by means of the GSH feeding reflex.

In addition, it is evident from Table 1 that cutting at the subhypostomal levelleads to a very significant increase in incorporation: P < 0-02 for the difference

(1) Intact hydra[3H]TDR alone

(2) Intact hydra[ 3 H ] T D R + 1 0 - 5 M G S H

(3) Subhypo. cut hydra[3H]TDR alone

(4) Subhypo. cut hydra[3H]TDR + 1 0 - 5 M G S H

277,296/

1122j916/886 \805/968 \

1015/

Pattern regulation in hydra. I 39between the means of groups (1) and (3). Presumably this could be due to astimulation of DNA synthesis on cutting and/or the fact that endodermal cellsare again given better access to the precursor. Incubation of intact animals in thepresence of GSH, group (2), produces similar specific activities to those ofanimals cut at the subhypostomal level incubated in [3H]thymidine in ' M ' alone,group (3). However, an increase in the availability of precursor to endodermalcells is considered the major factor for the increased incorporation with GSHand, on this basis, it would therefore seem unlikely that cutting stimulates DNAsynthesis. This conclusion is further borne out by a consideration of groups (2)and (4), for animals cut at the subhypostomal level incubated in the presence ofGSH do not show a significant increase in incorporation when compared tointact animals similarly incubated: P > 0-80 for the difference between themeans of groups (2) and (4).

The most obvious difficulty in evaluating the above results is that one of thelikely consequences of the feeding reflex is mechanical stretching of cells of thehypostome. This, in turn, could lead to localized permeability changes andcould possibly explain the slightly higher incorporation obtained by intactanimals incubated in GSH, group (2), compared to that obtained by incubatinganimals cut at the subhypostomal level in GSH, group (4). If this were the case,then changes in permeability of the cells of the hypostome would be relativelyunimportant. A more serious alternative, however, is the possibility that cuttingdoes stimulate DNA synthesis and that this stimulation is balanced by that dueto permeability changes. The present experiment does not allow us to distinguishbetween these alternatives, and the conclusion that cutting does not stimulateDNA synthesis should therefore be accepted, for the moment, with somecaution.

(b) RNA synthesisThe procedure in this experiment was exactly the same as that in the previous

one except that [3H]uridine (3-33 c/mM) at 10/ic/ml was substituted for[3H]thymidine. The specific activities of the four groups are shown in Table 2.

Using the same rationale as was applied to the previous experiment, thesignificantly increased incorporation obtained by incubating intact animals inthe presence of GSH (P < 0-01 for the difference between the means of groups(1) and (2)) is considered to be due to the GSH feeding reflex, for there is nosignificant difference between the incorporation obtained for animals cut at thesubhypostomal level incubated in the presence or absence of GSH {p > 0-70 forthe difference between the means of groups (3) and (4)).

Table 2 also demonstrates that animals cut at the subhypostomal level show amarked increase in [3H]uridine incorporation (P < 0-01 for the differencebetween the means of groups (1) and (3)) which may reflect a stimulation ofRNA synthesis on cutting and/or an increased availability of [3H]uridine toendodermal cells. Since animals cut at the subhypostomal level incubated in

40 S. G. CLARKSON

[3H]uridine in ' M' alone, group (3), possess much higher specific activities thando intact animals incubated in the presence of GSH, group (2), and since betteraccess of endodermal cells to the precursor is a feature common to both groups,it may be suggested that cutting does stimulate RNA synthesis. However, thereremains the possibility that cutting leads to an even greater availability of theprecursor than is attained by the GSH feeding reflex through, for example, the[3H]uridine entering the cells at the cut surface and traversing the mesoglea.This could account perhaps for the difference between groups (2) and (4):animals cut at the subhypostomal level incubated in the presence of GSH

Table 2. Reduced glutathione as a specific means to increasethe incorporation of[3H]uridine into RNA

Treatment and materials

(1) Intact hydra[3H]UDR alone

(2) Intact hydra[ 3 H ] U D R + 1 0 - 5 M G S H

(3) Subhypo. cut hydra[3H]UDR alone

(4) Subhypo. cut hydra[ 3 H ] U D R + 1 0 - 5 M G S H

Specific activity(c.p.m.//*g RNA)

23-1 \25-5/62-3i58-5/

212-6 \239-7 /

253-6 \222-0 /

Mean specificactivity

24-3

60-4

226-2

237-8

possess a significant (P < 0-02) increase in incorporation compared to intactanimals similarly incubated. However, if an incorporation pathway via the cutsurface were a major factor, it is surprising that there was no significant differ-ence between groups (2) and (4) of the comparable experiment with [3H]thy-midine (Table 1), for uridine and thymidine have similar molecular weights.Moreover, the difference between groups (2) and (4) in the present experimentrepresents nearly a 300% increase in [3H]uridine incorporation. In view of themagnitude of this increase, it is considered likely that cutting does stimulate

. RNA synthesis.

(c) Protein synthesis

An experiment identical to the previous one was performed except that theincorporation of [14C]algal protein hydrolysate (640/^c/mg) at 10/*c/ml intoprotein of the four groups was measured. Results are shown in Table 3.

The results demonstrate a significant difference (P < 0-01) between the meansof groups (1) and (2), but not (P > 0-30) between the means of groups (3) and(4). It is therefore concluded that the enhanced incorporation obtained as aresult of incubating intact animals in the presence of GSH is due primarily to thefact that cells of the endoderm are given better access to the precursor by meansof the GSH feeding reflex.

Pattern regulation in hydra. I 41This experiment further demonstrates a significant difference (P < 0-02)

between the means of groups (1) and (3), and a slightly significant difference(P < 0-10) between the means of groups (2) and (4). The latter differencerepresents only a 25 % increase in the incorporation of [14C]algal proteinhydrolysate and, while this might indicate that cutting does stimulate proteinsynthesis, the possibility cannot be excluded that part or all of this increasedincorporation is due to the easier entry of the precursor. It would be surprising,however, if the large increase in RNA synthesis obtained under identicalconditions (Table 2) were not accompanied by some increase in protein synthesis,and for this reason it is felt that the apparent stimulation of protein synthesisfollowing cutting is probably genuine.

Table 3. Reduced glutathione as a specific means to increase theincorporation of [uC]algal protein hydrolysate into protein

Treatment and materials

(1) Intact hydra[14C]hydrolysate alone

(2) Intact hydra[14C]hydrolysate+10-5 M GSH

(3) Subhypo. cut hydra[14C]hydrolysate alone

(4) Subhypo. cut hydra[14C]hydroylsate+10~5 M GSH

Specific activity(c.p.m.//*g protein)

36-7 \33-6/

92-7»860/

96-2 \105-4/

107-81114-7/

Mean specificactivity

35-2

89-4

100-8

111-3

3. Regional patterns of nucleic acid and protein synthesis

(a) DMA synthesisIn the first experiment, ten intact hydra were incubated for 24 h in [3H]thy-

midine (3 c/mM) at 12-5 /*c/ml in 'M ' , cut into regions which were pooled andtheir specific activities determined. Results of three experiments are shown inTable 4. Since the information required is the pattern of incorporation ratherthan the absolute activities, the mean specific activity of each region has beendetermined from the three experiments and each one is expressed as a percentageof the maximum mean specific activity of the five regions.

The pattern which emerges from these experiments is a broad distribution ofactivity with a very slight peak in the bud. The hypostome and tentacles and thepeduncle and basal disk possess a somewhat lower activity, but the activity of thegastric region is approximately constant.

One difficulty in evaluating these results is that the digestive zone is of fargreater size and contains many more cells than any of the other four regions. It ispossible therefore that the over-all activity of the digestive zone measured inthese experiments could be masking a significant difference between a 'sub-

42 S. G. CLARKSON

hypostomal growth zone' and the remainder of the digestive zone. The followingexperiment was designed to investigate this possibility.

Twelve intact hydra were incubated in [3H]thymidine under conditionsidentical to the previous experiment. At the end of 24 h the digestive zones wereisolated and cut into thirds, identical regions pooled, and their specific activitiesdetermined. Results of three experiments are shown in Table 5.

Table 4. Regional incorporation of [3H]thymidineinto DNA of budding hydra

Regions

Hypostome andtentacles

Digestive zoneBudding zoneBudPeduncle and

basal disk

Specific

Exp. 1

16511941196-5202-7

133-5

activity (c.p.m.//<A

Exp. 2

260-5280-5260-5281-9

226-4

tg DNA)

Exp. 3

2280230-6231-3254-2

185-6

Meanspecificactivity

217-92351229-4246-3

181-8

% o fmaximum

meanspecificactivity

889593

100

74

Table 5. Incorporation of[3H]thymidine into DNAwithin the digestive zone

Regions of thedigestive zone

Distal thirdMiddle thirdProximal third

Specific activity (c.p

Exp. 1

484-1473-4521-6

Exp. 2

473-53941493-4

.m.//*g DNA)

Exp. 3

3951420-9326-3


450-9429-5447-1

% o fmaximum


1009599

It is evident that there are no major variations in the pattern of thymidineincorporation within the digestive zone. Thus, unless there is a highly localized'subhypostomal growth zone', the over-all activity of the digestive zonemeasured in the previous experiments does reflect an approximately uniformincorporation of [3H]thymidine along its length.

One criticism that can be levelled against both experiments, however, is thatan incubation time of 24 h may be too long, and that this length of time allowsthe [3H]thymidine to be 'soaked up ' by cells of all regions, thus resulting in theapparently broad distribution of activity. One way of circumventing thisproblem is to use a shorter incubation period in conjunction with another means

Pattern regulation in hydra. I 43of administering the precursor. The following experiment was thereforeperformed.

Twenty animals were incubated for \ h in [3H]thymidine (3 c/misi) at 50 /tc/mlin 10~5M GSH in 'M' , cut into regions which were pooled and their specificactivities determined. Results are shown in Table 6.

Table 6. Regional incorporation of [3H]thymidine into DNA ofhydra incubated in the presence of reduced glutathione

Regions


Digestive zoneBudding zone*Peduncle and

basal disk

* Twenty buds were also

Specific activity(c.p.m.//*g DNA)

61-9759-985510

38-44

% o fmaximum

specific activity

1009789

62

isolated but were lost during centrifugation.

The results from this pulse-labelling experiment indicate an approximatelyconstant activity within the gastric region and a somewhat lower activity in thepeduncle and basal disk. Comparison with the data from a 24 h incubationperiod without GSH shows that the only change in the pattern of specificactivities is that the hypostome and tentacles possess greater activity than thedigestive zone during a brief label with GSH (Tables 4, 6). The simplest way ofexplaining this slightly altered pattern is to postulate that the increased activityat the distal end reflects some localized permeability changes associated with thefeeding reflex. This, of course, would indicate that such changes are not majorcontributory factors to the increased incorporation obtained by incubatingintact animals in the presence of GSH.

These three experiments indicate therefore that DNA synthesis is almostuniformly distributed throughout the body column.

(b) RNA synthesis

Ten intact hydra were incubated for 2 h in [3H]uridine (3-33 c/mivi) at25 /fc/ml in 'M' , cut into regions which were pooled and their specific activitiesdetermined. The results of three experiments are shown in Table 7.

The pattern emerging from these experiments is an axial gradient of incorpora-tion of [3H]uridine. In all three experiments the maximum specific activity isfound in the hypostome and tentacles, the second highest activity in the digestivezone, and the lowest in the peduncle and basal disk. The relative activities of thebud and budding zone are less clear: in two experiments the incorporation bythe bud is somewhat higher than that of the budding zone, but in the third

44 S. G. CLARKSON

experiment the situation is reversed to such an extent that the pooled data fromthe three experiments indicate greater incorporation by the budding zone thanby the bud.

Table 7. Regional incorporation of [3H]uridine into RNA ofbudding hydra

Regions



basal disk

Specific activity (c.p.m.//tg

(Exp. 1

65-447-935-437-6

26-2

A

Exp. 2

62-245033-634-7

29-5

RNA)

Exp. 3

61-748-246136-4

32-8


63147038-436-2

29-5

% o fmaximum


100746157

47

Table 8. Regional incorporation of [uC]algalprotein hydrolysateinto protein of budding hydra

Regions



basal disk

Specific activity (c.p.m

Exp. 1

43120-220-920-9

230

K

Exp. 2

38-722-426-319-9

17-4

.//*g protein)

Exp. 3

43 021-817-423-5

19-5


41-621-521-521-4

200

% o fmaximum


100525251

48


The procedure in this experiment was exactly the same as that in the previousone except that [3H]uridine was replaced by [14C]algal protein hydrolysate(640 /*c/mg) at 7-5 /jc/ml. Results are shown in Table 8.

The only feature common to all three experiments is that the maximumspecific activity is found in the hypostome and tentacles. More proximal regionsvary as to which region possesses the second highest specific activity, but theextent of this variation is small. When the data from the three experiments arepooled, the results indicate an approximately uniform incorporation of [14C]algalprotein hydrolysate into all regions proximal to the hypostome and tentacles.Thus, an axial gradient in incorporation exists only between the hypostome and

Pattern regulation in hydra. I 45tentacles and the digestive zone, and no major quantitative differences existbetween the more proximal regions.

4. Regional nucleic acid and protein content

The preceding data on the regional patterns of syntheses involved chemicalanalyses of the nucleic acid and protein content of the five regions in order to

Table 9. Regional nucleic acid and protein content of budding hydra

DNARegions Og/region)

Hypostome and tentacles 0-24Digestive zone 0-38Budding zone 014Bud ('medium bud'stage) 017Peduncle and basal disk 009

Total/tg/hydra 102

O-10-i

cO)

oo. 005 -<fZQ

O-30-i

^ 0 1 5 -<TZQ

0-50-

ein

o£. 0-25 -

Z

Hypostomeand

tentacles

|

Digestive I

zone |

I I

Bud hBudding

zone

RNAOg/region)

0-861-830-590-820-40

4-50

rPeduncle and

basal diskL

ProteinOg/region)

2-645-662-451-891-32

13 96

-Mean

0073

Mean

023

Mean

032

l

J

Fig. 3. DNA/protein, DNA/RNA, and RNA/protein ratios of budding hydra.

46 S. G. CLARKSON

express radioactivity in terms of specific activities. The results of a typicalanalysis are shown in Table 9, where each value for the regional nucleic acid orprotein content is the mean of three determinations, each of which was made onten pooled regions. It should be noted that the DNA values were recorded from2-day starved hydra, whereas the RNA and protein values were obtained fromanimals 17-20 h after feeding.

In Fig. 3 are shown the regional DNA/protein, DNA/RNA, and RNA/proteinratios obtained from the data in Table 9. Some minor chemical differencesbetween the regions are revealed, e.g. the relatively high DNA/protein andDNA/RNA ratios of the hypostome and tentacles, and the relatively highDNA/protein and RNA/protein ratios of the bud. The extent of these differencesis not very large, however, and the DNA/protein ratio of the bud is only 27 %higher than that of the parent, nowhere near the threefold difference found byLi&Lenhoff(1961).

5. Nucleic acid and protein synthesis during hypostome formation

(a) DNA synthesis

To determine whether the formation of a hypostome during regeneration isaccompanied by a localized increase in DNA synthesis, use was made of thespecificity of the GSH feeding reflex (Table 1) and the fact that a new hypostomeis determined, in the majority of cases, within 6 h of cutting at the subhypo-stomal level (Webster & Wolpert, 1966).

Table 10. Incorporation of[3H]thymidine into DNA of intact andcut subhypostomes incubated in the presence of reduced glutathione

Specific activityTreatment of subhypostomes (c.p.m./^g DNA) Mean specific activity

Control: Intact 1 230-5]2 228-8 \ 240-43 261-8J

Subhypo. cut 1 268-7)2 258-3 [ 256-33 2420j

Animals were incubated for 6 h in [3H]thymidine (3 c/mM) at 25 jnc/ml in10~5M GSH in 'M' . One batch comprised ten intact hydra, and the other, tenanimals cut at the subhypostomal level immediately before incubation. After 6 hthe subhypostomal regions of the two batches were isolated, pooled separately,and their specific activities determined. Results are shown in Table 10.

A slight increase in mean specific activity is evident in subhypostomes cutprior to incubation compared to those incubated intact; the difference betweenthese means is not, however, significant (P > 0-30). These findings confirm the

Pattern regulation in hydra. I 47earlier experiment on the incorporation by whole animals incubated for 2\ h(Table 1), and it is clear that incorporation measurements on whole animals donot mask localized areas of high DNA synthetic activity. Although permeabilitychanges associated with the feeding reflex probably account for some of theincorporation obtained by intact animals incubated in the presence of GSH, thedata shown in Tables 4 and 6 indicate that this contribution is relatively smallduring a \ h pulse label. Over a 6 h incubation period this effect is considerednegligible because the GSH feeding reflex ceases after approximately 35 min(Loomis, 1955).

It is therefore concluded that cutting does not stimulate DNA synthesis, andthat during the determination of a hypostome from the subhypostomal regionthere is no major increase in DNA synthesis over that occurring in a similarregion left in situ.

(b) RNA synthesis

The procedure in this experiment was exactly the same as that in the previousone except that [3H]thymidine was replaced by [3H]uridine (3-33 c/mM) at10//c/ml. Results are shown in Table 11.

Table 11. Incorporation of z[H]uridine into RNA of intact and cutsub-hypostomes incubated in the presence of reduced glutathione

Specific activityTreatment of subhypostomes (c.p.m.//*g RNA) Mean specific activity

Control: Intact 1 26-7]2 33-9 \ 32-33 36-3J

Subhypo. cut 1 158-3]2 164-2 \ 151-53 132-OJ

The results demonstrate a highly significant (P > 0-001) increase in [3H]uri-dine incorporation into subhypostomes cut prior to incubation compared tothose incubated intact. These findings confirm the experiment on the incorpora-tion by whole animals incubated for 2\ h (Table 2), although both are subjectto the criticism that at least part of the increased incorporation followingcutting may be due to the easier entry of the precursor. It is considered unlikely,however, that such a factor could account for the 370 % increase in incorpora-tion of [3H]uridine into RNA obtained in the present experiment. It is suggestedtherefore that cutting does stimulate RNA synthesis, and that during thedetermination of a hypostome from the subhypostomal region there is a markedincrease in RNA synthesis compared to that occurring in a similar region leftin situ.

48 S. G. CLARKSON


The results of a similar experiment involving the incorporation of [14C]algalprotein hydrolysate (640/*c/mg) at 5/^c/ml into protein of intact and cutsubhypostomes are shown in Table 12.

The present results support the data shown in Table 3 and demonstrate thatcutting at the subhypostomal level leads to a very significant (P < 0-01) 34%increase in [14C]algal protein hydrolysate incorporation. It is suggested thereforethat there is a significant increase in protein synthesis during the determinationof a hypostome from the subhypostomal region.

Table 12. Incorporation of [uC]algal protein hydrolysate into protein of intact andcut hypostomes incubated in the presence of reduced glutathione

Specific activityTreatment of subhypostomes (c.p.m.//*g protein) Mean specific activity

Control: Intact 1 420]2 40-4 [ 40-33 38-6J

Subhypo. cut 1 51-9)2 57-3 \ 53-93 52-6J

DISCUSSION

The experiments have been concerned with nucleic acid and protein synthesisin intact and regenerating hydra, and the following points are important for theinterpretation of the results.

(1) Radioactivity was determined in total acid-insoluble material so thepossibility cannot be excluded of some incorporation of [3H]thymidine intoRNA, or of [3H]uridine into DNA.

(2) RNA measurements were made without any separation of RNA into itsconstituent classes.

(3) The regional patterns of incorporation may not reflect the regionalpatterns of syntheses, but rather regional differences in permeability to therelevant precursor. There is no simple means of resolving this difficulty on thepresent evidence since no measurements were made of the specific activity of theimmediate precursor in each of the regions. While permeability is evidently animportant factor in determining the extent of incorporation, as witnessed by thethreefold increase in incorporation of all three precursors when administered inthe presence of reduced glutathione, the similarity in the regional patterns of[3H]thymidine incorporation obtained after a 24 h incubation and a \ h pulselabel with reduced glutathione indicate, however, that regional permeabilitydifferences are slight.


(4) No antibiotics were employed and the possibility therefore remains thatan intracellular bacterial population might have significantly contributed to theradioactivity measurements. This is considered unlikely, however, in view of thedistinctly different regional patterns of incorporation of the three precursors.Moreover, they are incorporated at very different rates following removal of thehypostome and tentacles, and it is hardly likely, for example, that bacteria couldaccount for the large increase in [3H]uridine incorporation under theseconditions. With these possible doubts in mind, it is assumed that the incorpora-tion of each of the three precursors employed in this work is a reasonableestimate of the relevant synthetic activity in hydra.

The data on the regional pattern of DNA synthesis (Tables 4-6) are in accordwith other quantitative data dealing with the growth pattern of hydra. Mitoticcells have been found to be generally distributed throughout the body column ofH. littomlis (Campbell, 1967 a; Clarkson & Wolpert, 1967), and this mitoticdistribution is similar to the distribution of [3H]thymidine-labelled nuclei(Campbell, 1967«). Similar studies on the pattern of mitotic figures in H.pseudoligactis (Shostak, Patel & Burnett, 1965; Campbell, 19676; H. D. Park,1967, personal communication), Clytia (Hale, 1964) and Campanularia(Crowell, Wyttenbach & Suddith, 1965) further indicate that growth is notrestricted to particular zones within hydroids. The present results support thisevidence from histology and radioautography, and it would appear that hydrapossesses neither an axial gradient in DNA synthesis nor any region which canbe termed a localized growth zone.

The suggestion of Ham & Eakin (1958) that a burst of mitosis occurs shortlyafter removal of the hypostome and tentacles is not borne out by the experimentsinvolving reduced glutathione (Tables 1, 10). Rather, they indicate that cuttingdoes not stimulate DNA synthesis and that during the formation of a newhypostome from the subhypostomal region there is no major increase in DNAsynthesis over that occurring in an intact subhypostome. This is consistent withthe finding that there is no significant difference between the number of mitoticfigures in intact and 6 h regenerating subhypostomal regions (Webster, 1964).Moreover, recent histological evidence (Park, Ortmeyer & Blankenbaker, 1967)indicates that mitotic activity is not increased during any stage of distal regenera-tion in H. pseudoligactis. It is concluded that the determination of the hypostomeis not accompanied by a localized increase in growth, and the suggestion ofBurnett (1961, 1966) that a new hypostome arises from activation of growthsubstances and the formation of a new growth centre is therefore considereduntenable.

The experiments dealing with the regional incorporation of [3H]uridine(Table 7) indicate a disto-proximal gradient of RNA synthesis, and hence apossible correlation between metabolic and morphogenetic activities. Incontrast, the data shown in Table 8 are consistent with the concept of an axialgradient of protein synthesis only in so far as the hypostome and tentacles

4 J E E M 2 I

50 S. G. CLARKSON

possess greater activity then the remaining body regions. In general terms, theseresults bear out the suggestion of Child (1941) that 'high physiological activity'is associated with a region possessing organizing properties, but of course theydo not establish a causal link between RNA or protein synthesis and the factorsresponsible for the formation and localization of the hypostome to the distal end.The experiments can be interpreted only in terms of total RNA and totalprotein synthesis and they indicate that RNA synthesis is more likely to beimportant than protein synthesis in the control of polarized regulation in hydra.Despite the limitations of this level of analysis, it is of interest to consider theseresults in relation to the axial variations in the time-threshold and inhibitoryfactors proposed by Webster (1966a, b). Since it is assumed that the inhibitorneed only be produced by the hypostome whereas the time-threshold propertiesof a region are determined by its position on the linear axis (Webster, 1966 a, b),it is tempting to relate the time-threshold properties, rather than the level ofinhibition, to the axial variations in RNA synthesis. The assumption that thethreshold of a region is more stable than the level of inhibition (Webster,1966a, b) would also lend support to this idea. On the other hand, it is far fromclear why such gross regional differences in total RNA synthesis should exist,and more basic facts are required—for example, the classes of RNA involved—before definite conclusions can be drawn with regard to the significance of theaxial variations in RNA and protein synthesis.

The experiments in which reduced glutathione was employed to increase[3H]uridine and [14C]algal protein hydrolysate incorporation suggest that theformation of a hypostome is accompanied by a very large increase in RNAsynthesis (Tables 2, 11) and a slight but significant increase in protein synthesis(Tables 3, 12). While reduced glutathione allows an estimate to be made of theamount of incorporation due to the greater availability of precursor to endo-dermal cells via the coelenteron, the possibility cannot be excluded that part, ifnot all, of the apparent stimulation of RNA and protein synthesis is due to theprecursor entering the cut surface and traversing the mesoglea. However, thelack of stimulation of [3H]thymidine incorporation under identical conditions(Tables 1,10) would suggest that precursor entry via the cut surface plays eithera minor or highly variable role in determining the extent of incorporationobtained after cutting. Of the two, the former is considered more likely and it istherefore suggested that the apparent stimulation of RNA and protein synthesisduring distal regeneration is genuine.

These results raise some interesting possibilities with regard to the sequence ofmetabolic events that occur during hypostome formation. Following a 6 hincubation of cut subhypostomes, RNA synthesis is increased by 370%(Table 11), while a 2\ h incubation of whole animals results in a 290 % increasein [3H]uridine incorporation in the cut samples (Table 2). The RNA content ofthe subhypostomal region represents approximately 14% of the total RNAcontent of hydra (Table 9). Thus, if the 290 % increase in incorporation obtained

Pattern regulation in hydra. I 51in the latter experiment is due to a stimulation of RNA synthesis only in thesubhypostomal region, the extent of this stimulation would have to be of theorder of 2100%. While this possibility cannot be entirely eliminated, it seemsmore likely that RNA synthesis is stimulated throughout the whole animal, or atleast stimulated not merely within the subhypostomal region, during the first2\ h of distal regeneration.

In the same way, comparison of the data in Tables 3 and 12 might suggest thatprotein synthesis is slightly stimulated throughout the whole animal within 2\ hof cutting at the subhypostomal level. This suggestion is less easily justified,however, in view of the small increase in protein synthesis obtained in theseexperiments. It should be noted that at least some of the increased incorporationobtained in the experiments shown in Tables 2 and 3 is likely to be due to theeasier access of the precursors to the explanted hypostomes and tentacles sinceboth proximal and distal parts were retained for incubation in these experiments.The low values for RNA and protein content of this region suggest, however,that such contributions would be relatively small.

The very large increase in RNA synthesis obtained in these experimentssuggests once more that this is the metabolic activity initially and primarilyinvolved in pattern regulation in hydra. Moreover, the results support thetentative suggestion that RNA synthesis is likely to be related to the time-threshold properties of a region for, although hypostome formation is accom-panied both by the restoration of the level of inhibition to that of intact hydraand by a rise in the threshold for inhibition, it is assumed that the inhibitor needonly be produced by the hypostome, whereas threshold must rise throughout thesystem for regulation to occur (Webster, 1966&). Thus it appears likely that thestimulation of RNA synthesis within the presumptive hypostomal regionrepresents in part the synthesis of RNA molecules coding for the inhibitor, whilethe apparent rise in RNA synthesis throughout the system is a manifestation ofchanges in threshold properties. This is of importance in relation to the possi-bility that the gradient in threshold could be the factor responsible for the axialdifferences in time for hypostome formation, and a question of interest now wouldbe whether there are qualitative differences in the populations of RNA moleculessynthesized during hypostome formation at different body levels, or whetherthere are simply differences in the rates of synthesis of the same populations.

The results reported in this paper provide additional evidence for the earlierconclusion (Clarkson & Wolpert, 1967) that the factors determining growth inhydra are quite different from those determining its form. In contrast, bothRNA and protein synthesis appear to have important roles in pattern regulationin hydra, but it is clear that an understanding of the precise nature of these roleswill require much further investigation. One approach towards this goal is theexperimental alteration of pattern with compounds that primarily affect onlyone type of metabolic activity. This will be investigated in the following paper(Clarkson, 1969).

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52 S. G. CLARKSON

SUMMARY

1. Biochemical techniques were used to determine (a) whether axial gradientsin DNA, RNA or protein synthesis exist in hydra, and (b) whether the formationof the hypostomal region is accompanied by radical changes in these metabolicactivities. A new technique for administering radioactive precursors wasdevised to help answer the second question.

2. [3H]thymidine incorporation is approximately constant within the gastricregion, very similar within the digestive zone, and somewhat lower in thehypostome and tentacles, and peduncle and basal disk.

3. The regional pattern of [3H]uridine incorporation follows a disto-proximalgradient with the hypostome and tentacles possessing the highest incorporation.

4. [14C]algal protein hydrolysate incorporation is maximal in the hypostomeand tentacles, but lower and approximately uniform in the remaining bodyregions.

5. The formation of a new hypostome appears to be accompanied by noincrease in DNA synthesis, a large increase in RNA synthesis, and a slightincrease in protein synthesis. The stimulation of RNA synthesis probably occursthroughout the whole animal, and not merely within the presumptive hypostomalregion, during the first 2\ h of distal regeneration.

6. The significance of the results is discussed in relation to concepts developedby other workers to explain polarized regulation in hydra.

RESUME

La synthese des acides nucleiques et des proteines et la regulation de la morpho-genese chez Vhydre. I. La topographie et Vextent des syntheses pendant laformation de la region hypostomale.

1. Des techniques biochimiques ont ete utilisees pour etudier (a) l'existenceeventuelle de gradients axiaux de synthese de DNA, de RNA ou de proteines,(b) l'hypothese selon laquelle la formation de la region hypostomale s'accom-pagne de modifications profondes de l'activite metabolique de cette region. Unenouvelle technique d'administration de precurseurs radioactifs a ete mise aupoint pour repondre a la seconde question.

2. L'incorporation de la 3H-thymidine est quasi constante dans la regiongastrique, tres similaire dans la region digestive et legerement moindre dans laregion de l'hypostome et des tentacules, ainsi que dans celle du pedoncule et dudisque basal.

3. La topographie de l'incorporation de la 3H-uridine revele un gradientdistoproximal; l'incorporation la plus elevee s'effectue au niveau de l'hypostomeet des tentacules.

4. L'incorporation d'un hydrolysat de proteines-14C est maximale au niveau


de l'hypostome et des tentacules; dans les autres regions, on observe uneincorporation moindre et relativement uniforme.

5. On n'observe pas d'augmentation de la synthese du DNA au moment de laformation d'un nouvel hypostome; par contre, la synthese du RNA est fortementaugmentee et la synthese proteique legerement accrue. Cette stimulation de lasynthese du RNA affecte non seulement la region hypostomale presomptive,mais aussi toutes les autres parties de l'hydre, pendant les 190 premieresminutes de la regeneration.

6. La signification de ces resultats est discutee en fonction des hypothesesactuelles concernant la regulation de la morphogenese chez l'hydre.

I am deeply indebted to Professor Lewis Wolpert for his advice and encouragement. Wewish to thank the Agricultural Research Council for a scintillation counter, and the NuffieldFoundation for their support of this work.

REFERENCES

BONTING, S. L. & JONES, M. (1957). Determination of microgram quantities of deoxy-ribonucleic acid and protein in tissues grown in vitro. Archs Biochem. Biophys. 66, 340-53.

BURNETT, A. L. (1961). The growth process in hydra. / . exp. Zool. 146, 21-84.BURNETT, A. L. (1966). A model of growth and cell differentiation in hydra. Am. Nat. 100,

165-90.BURNETT, A. L., BALRD, R. & DIEHL, F. (1962). Method of introducing tritiated thymidine

into the tissues of hydra. Science, N. Y. 138, 825-6.CAMPBELL, R. D. (1965). Cell proliferation in hydra: An autoradiographic approach.

Science, N. Y. 148, 1231-2.CAMPBELL, R. D. (1967a). Tissue dynamics of steady state growth in Hydra Httoralis. I.

Patterns of cell division. Devi Biol. 15, 487-502.CAMPBELL, R. D. (19676). Growth patterns of Hydra: Distribution of mitotic cells in H.

pseudoligactis. Trans. Am. microsc. Soc. 86, 169-73.CERiorn, G. (1955). Determination of nucleic acids in animal tissues. / . biol. Chem. 214,

59-70.CHILD, C. M. (1941). Patterns and Problems of Development. University of Chicago Press.CLARKSON, S. G. (1969). Nucleic acid and protein synthesis and pattern regulation in hydra.

II. Effect of inhibition of nucleic acid and protein synthesis on hypostome formation./ . Embryol. exp. Morph. 21, 55-70.

CLARKSON, S. G. & WOLPERT, L. (1967). Bud morphogenesis in hydra. Nature, Lond. 214,780-3.

CROWELL, S., WYTTENBACH, C. R. & SUDDITH, R. L. (1965). Evidence against the concept ofgrowth zones in hydroids. Biol. Bull. mar. biol. Lab., Woods Hole 129, 403. (Abstr.)

HALE, L. J. (1964). Cell movements, cell division, and growth in the hydroid Clytiajohnstoni.J. Embryol. exp. Morphol. 12, 517-38.

HAM, R. G. & EAKIN, R. E. (1958). Time sequence of certain physiological events duringregeneration in hydra. / . exp. Zool. 139, 33-54.

Li, Y. F. & LENHOFF, H. M. (1961). Nucleic acid and protein changes in budding HydraHttoralis. In The Biology of Hydra, ed. W. F. Loomis and H. M. Lenhoff. Coral Gables,Florida: University of Miami Press.

LOOMIS, W. F. (1955). Glutathione control of the specific feeding reaction of hydra. Ann. N.Y.Acad. Sci. 62, 209-28.

LOOMIS, W. F. & LENHOFF, H. M. (1956). Growth and sexual differentiation of hydra in massculture. / . exp. Zool. 132, 555-74.

LOWRY, O. H., ROSEBROUGH, N. J., FARR, H. L. & RANDALL, R. J. (1951). Protein measure-ment with the Folin phenol reagent. / . biol. Chem. 193, 265-76.

54 S. G. CLARKSON

MUSCATINE, L. (1961). Symbiosis in marine and freshwater coelenterates. In The Biology ofHydra, ed. W. F. Loomis and H. M. Lenhoff. Coral Gables, Florida: University of MiamiPress.

PARK, H. D., ORTMEYER, A. & BLANKENBAKER, D. (1967). Mitotic activity in Hydra pseudo-ligactis during regeneration of apical structures. Am. Zoo]. 7, 750-1. (Abstr.)

SHOSTAK, S., PATEL, N. G. & BURNETT, A. L. (1965). The role of mesoglea in mass cellmovements in Hydra. Devi Biol. 12, 434-50.

TARDENT, P. (1963). Regeneration in Hydrozoa. Biol. Rev. 38, 293-333.WEBSTER, G. (1964). A study of pattern regulation in hydra. Ph.D. Thesis, University of

London.WEBSTER, G. (1966a). Studies on pattern regulation in hydra. II. Factors controlling hypo-

stome formation. / . Embryo!, exp. Morph. 16, 105-122.WEBSTER, G. (1966/?). Studies on pattern regulation in hydra. III. Dynamic aspects of

factors controlling hypostome formation. / . Embryol. exp. Morph. 16, 123-141.WEBSTER, G. & WOLPERT, L. (1966). Studies on pattern regulation in hydra. I. Regional

differences in time required for hypostome formation. J. Embryol. exp. Morph. 16,91-104.

{Manuscript received 30 April 1968)

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