Effects of nucleotides on neurulation in amphibian embryos

DEVELOPMENTAL

BIOLOGY,

Effects

of

ELISABETH Deportments

5, 452-467

( 1962)

Nucleotides Amphibian

on Neurulation Embryos

AMBELLAN~

of Biochemistry

AND

GEORGE

und Zoology, Columbus.

Accepted

September

The

in

WEBSTER~ Ohio

State

University,

Ohio II,

1962

INTRODUCTION

Various nucleotides specifically accelerate neural tube closure in Rana pipiens embryos (Ambellan, 1955, 1958). The acceleration is obtained whether the nucleotides are applied at the g-cell stage, at any time during early growth, or at the flat neural plate stage. Of all the nucleotides examined, ATP” was found to be the most effective, but it produced certain abnormalities. A-3-P was least effective. Other nucleoside di- and triphosphates also appeared to promote neural tube closure. Whether the nucleotides accelerate neural tube closure by promoting nucleic acid synthesis (and thereby protein synthesis) or whether they have a different mode of action is not known. However, it is evident that the specific effects of the nucleotides provide another means for gaining insight into some of the chemical events that occur during the process of neurulation. These effects have, therefore, been examined in more detail, in order to establish some of their parameters, prior to the initiation of experiments on the manner in which the nucleotides affect neural tube closure. ’ Present address: Institute of Animal Morphology, Free University of Brussels, Brussels 16, Belgium. ‘Present address: Institute for Enzyme Research, University of Wisconsin, Madison 6, Wisconsin. 3 Abbreviations used in the text: ATP, adenosine-5’-triphosphate; ADP, adenosine-5’-diphosphate; A-5-P, adenosine-5’-monophosphate; A-3-P, adenosine-3’monophosphate; DPN, diphosphopyridine nucleotide or (new name) nicotinamide adenine dinucleotide, and DPNH, the reduced form of this material. 452

EFFECTS

OF NUCLEOTIDES

MATERIALS

ON

AND

NEURULATION

453

METHODS

Frogs (Rana pipiens) were obtained from J. M. Hazen CO., Alberg, Vermont. Ovulation was induced by injection of 2-6 frog pituitary glands in l-2 ml of 10% Ringer’s solution into the peritoneal cavity of healthy females. Experimental methods for morphological studies were essentially those described previously (Ambellan, 1955). Ten to twenty handdejellied eggs per stender dish in about 15 ml of freshly made solutions were kept at a temperature that was constant for different dishes within any one experiment, but different experiments were performed at different temperatures. Solution volumes and number of eggs were constant within any one experiment. All samples were at least in duplicate, often in triplicate. Selection of an apparently homogeneous starting population by eliminating any abnormal eggs and any that were ahead of or behind the average stage of development for the clutch resulted in 90-100% of the eggs within dishes maintaining the same rate of development throughout most of the experiments. In studies using C’” -nucleotides large numbers of eggs were required, often over 1000 in 50 ml or less of solution at the start of an experiment. Despite selection of a homogeneous population, under such overcrowded conditions, development was slow and there was large variability between embryos. However, since the primary purpose of these later studies was no longer to determine morphological effects, the results are reported as “hours of treatment,” and the corresponding morphological stages observed applied to only about 60-70% of the embryos. For these studies embryos were enzymatically dejellied with cysteine-activated papain by a modification of the method of Spiegel (1951). Embryos were shaken gently for about 1 hour at room temperature in 5-20% papain in 10% Ringer’s solution (pH 6.6-6.S) containing 0.12%cysteine. Dejellied embryos fall to the bottom of the dish, cluster in the center with swirling, and can be removed with an embryo lifter; the remaining embryos with jelly can be floated off the top. The “hours advance” in neurulation in experimental animals was the time taken for control embryos grown in 10% Ringer’s solution to reach the same stage of neural tube closure as the experimental embryos. In early studies (Ambellan, 1955) extent of closure was determined by measuring the width of the open anterior folds with an

454

AMBELLAN

AND

WEBSTER

ocular micrometer, and precise timing was determined by fixing experimental animals in Bouin’s solution and measuring the time for controls to reach the same stage. Later experiments followed the sample principle, but determinations were made by visual observations only, and finer determinations were made by arbitrary substaging of the Shumway developmental stages as pictured by Rugh (1948). Nucleotides were added to 10% Ringer’s solution and pH adjustments were made with 0.1 N HCl or with “Ringer’s hydroxides,” a combination of sodium, potassium, and calcium hydroxides having the same cation concentration as Ringer’s solution. There was no evidence of a significant degradation of unlabeled ATP in solution in these experiments. An ascending paper chromatogram (methanol-formic, acidwater, 80: 15: 5) of 0.002 M ATP kept in 10% Ringer’s solution, pH 5.6, 14” C, at zero time and at 24 and 48 hours showed no detectable spots for ADP or A-5-P. The triphosphate breakdown is presumably small at the pH and within the temperature ranges of 10-18” C used in most of the experiments. In experiments using C” -labeled materials, radioactivity of the incubation solutions was determined after solutions were added to the embryos in order to avoid dilution errors. After removal from solutions, embryos were washed until no further radioactivity was detectable in a total of 3-4 ml of wash medium; they were then given an additional rinse. The washed eggs were homogenized in a glass homogenizer in a small volume of Ringer’s solution, and aliquots were assayed for radioactivity. Perchloric acid was added to the remaining homogenate to a final concentration of 7%. The mixtures were left in the cold for 1 hour with occasional stirring and then centrifuged for 15 minutes at 10,000 g. The sediment was washed twice with small volumes of 5% perchloric acid. The combined supernatant and rinses represent the “acid-soluble fraction.” Samples were neutralized with KOH, and the precipitate was discarded. Supernatants were assayed for radioactivity by plating samples at infinite thinness (less than 0.1 mg/cm?) on glass planchets. The self-absorption of less than 2% was neglected. Radioactivity was determined on a Nuclear-Chicago model D-47 gas flow counter. The counting errors were never more than 3%. ATP, ADP, UTP, GDP, and CDP were all obtained as sodium salts from the Sigma Chemical Co. The purity of these compounds was determined by checking their spectra and molar extinction coefficients. Correspondence with published values was obtained in all cases ex-

EFFECTS

OF

NUCLEOTIDES

ON

NEURULATIOS

455

cept for ADP, which appeared to contain a 20% excess of water of hydration. The nucleotides all migrated as single substances when subjected to ascending paper chromatography in methanol-formic acidwater (80: 15: 5). A-3-P was obtained from Boehringer and Co. (Germany). Radioactive adenine nucleotides, all labeled at C-8, were obtained from Schwartz Laboratories; sodium ATP (2.8 &mg); sodium A-5-P (0.7 +/mg); sodium A-3-P ( 1.1 &mg) ; and lithium ADP (1.8 &mg). The lithium ADP was converted to sodium ADP via the barium salt. Chromatography of these materials showed that Cl”-ATP contained about 10% ADP, and that Cl’-ADP contained about 15% ATP. The monophosphates showed no contamination by other nucleotides. The fixation of embryos for histological and radioautography studies was performed in collaboration with Dr. Ruth Kleinfeld of the Department of Pathology, The Ohio State University. Embryos were fixed for 1 hour in 5% trichloroacetic acid containing 1.37% lanthanum acetate. After being rinsed with water, the embryos were kept in tertiary butyl alcohol. Embryos were embedded in paraffin, sectioned, and fixed for autography. Radioautograms were prepared by Dr. Robert Painter, Battelle Memorial Institute, Columbus, Ohio, using a 30-day exposure with Kodak AR-10 stripping film. The studies were then developed and stained with Azur B. RESULTS

Time Course of A&nine

Nucleotide

Effects

The morphological effects of adenylates on neurulation over a period of time are illustrated in Fig. 1, which records a typical experiment showing the effects of 1 mM concentrations of ATP, ADP, A-3-P, and A-5-P. It can be seen that neural tube closure begins earliest with ATP treatment, next earliest with ADP, and latest with A-3-P. Neural tubes of embryos treated with A-5-P failed at first to close and at one time were about 4 hours behind controls. With this low concentration and short duration of treatment (14 hours), all animals were apparently normal by tailbud stage. This is indicated by the dashed line in Fig. 1.

Concentration

Effects of Adenine Nucleotides

Many experiments indicated that the neurulation advance depended upon the concentration of the nucleotide solutions used. Results from one 48-hour experiment are shown in Fig. 2. At 0.125 mM both ATP

456

AMBELLAN

AND

WEBSTER

16 14 12 IO 8 6 6 B 4 Ia

2 controls

0 -2 -4 0

2

Observation

4

6 time

8

IO I2 during

FIG. 1. Time course of different adenine closure. Treatment was started at late gastrula, tion, 10” C. Solutions were 1 mM; 12 embryos

20 neurulation

”

(hours

nucleotide 14 hours per dish.

4 )

effects on neural tube before onset of neurulaSamples in duplicate.

0 A-5-P I 0.125

I 0.500

2.000

1 8.000

Concentration

FIG.

2. Effects of varying concentrations of adeninc tube closure. Treatments were started at early gastrula, of neurulation, 120” C. Solution concentrations of 1 mM terminations; 15 embryos per dish. Samples in duplicate.

nucleotides 24 hours confirmed

on neural before onset by UV de-

EFFECTS

OF

NUCLEOTIDES

ON

457

NEURULATION

and ADP were effective, but the monophosphates were not. ATP treatment was lethal at 8 mM, and at 2 mM the embryos were abnormal. ADP and A-3-P at 8 mM produced slightly abnormal embryos. The most rapid tube closure was found with ATP at both 2 mM and 8 mM; the next most effective was ADP, which showed the same hours advance over controls at all four of the concentrations used here. A-3-P has the least effect, and even at 8 mM was only about half as effective as ADP. Again the treatment with A-5-P blocked neural tube closure for several hours at all four concentrations, although these animals later recovered.

8

ADP

6

2

4

Observation

time

6 during

0 neurulation

IO (hours)

FIG. 3. Comparison of nucleoside di- and triphosphate effects on neural tube closure. Abscissa shows observation times during neurulation, which it took about 14 hours for controls to complete (14” C). Treatments were started during gastrulation, 24 hours prior to neurulation. Solutions were 1 mM at pH 5.6; 12 embryos per dish. Samples in duplicate. One separate experiment using the diphosphates and another separate experiment using triphosphates, gave results similar to the above combined experiment.

358 Comparison

AMBELLAN

of Different

AND

WEBSTER

Nucleotides

The effects of replacing ATP with UTP, CTP, or GTP, or of replacing ADP with UDP, CDP, or GDP are shown in Fig. 3. Among the triphosphates, the effective order for advancing neurulation was ATP > UTP > CTP > GTP. In the case of the diphosphates, it was ADP > UDP > GDP > CDP. The adenine nucleotides were clearly the most effective and the uridine nucleotides the next most effective. Differences between guanosine and cytidine nucleotides do not appear to be significant. The triphosphates were relatively more effective than the diphosphates, ADP being slightly more effective than UTP. Efects of Other Materials The effect on neurulation of numerous other compounds related to the nucleoside phosphates was investigated. Conditions of concentration and pH which are effective with nucleotides were used, and in TABLE EFFECTS Materials~

ADP (standard) Nicotinamide* DPN* Yeast RN.1 Adenine* Adenosine* Pi Ribose Ribose-5-P* Glucose* DPNH* DPN* ADP Bromoacetate Iodoacetate A-5-P

OF OTHER Concentration (m.+f)

2 2-8 2 mg/ml* l-8 l-8 l-8 1-8 2 l-8 2 l-4 0.54 9 l-3 2-8

I-4

MATERIALS

1 0~

NEIJRULATION Iteuults

PH

5.6 5.7 5.7 5.7 and 5.7 and 5.7 and 5.7 and 5 .7 and 5.7 5.7 and 5.7 and 7.2 7.2 G 5.6 and 5.6 and

7.2 7.2 7.2 7.2 7 .2 7.2 7.2

7.2 7.2

4 Hr advance 2 Hr advance 2 Hr advance 2 Hr advance No effect No effect No effect No effect No effect, No effect No effect, No effect No effect Neurulation block Neurulation block Neurulation block

a Materials were in 10% Ringer’s solutions, 18-24 hours, 20 embryos samples in duplicate. * Not expressed in molarity. Single experiments are indicated by asterisk. Several concentrations within the limits indicated. Other materials, not starred, were repeated one experiment.

per dish,

were in more

all

used than

EFFECTS

OF NUCLEOTIDES

ON

NEURULATION

459

some cases the effect at pH 7.2 was also examined. Table 1 reports those that are particularly interesting. The only materials other than nucleotides so far found to produce any positive effects, however slight, are nicotinamide or DPN (NAD) at pH 5.6, and yeast RNA at both pH 5.6 and 7.2. DPN (NAD) had no effect at pH 7.2 nor did DPNH (NADH) at either pH. Adenine, adenosine, and the other components of the adenine nucleotide molecule, separately and in various combinations, were tested at both pH values without any effects. For the nucleotides to advance neurulation under these in vivo conditions seems to require the minimum structural integrity of the base-ribose-phosphate bonds. A number of analogs of purines and pyrimidines that have been reported to block morphogenesis, such as 5-bromouracil, were examined, but all produced nonspecific cytolysis. In contrast, A-5-P specifically blocks neurulation while all other parts of the embryos apparently develop normally. Embryos were seen as late as tailbud stage with neural tubes still open. Two other compounds, bromoacetate and iodoacetate, not related to nucleotides also specifically blocked neural tube closure, The effects of chloropicrin and iodoacetate on neural tube blockage in the European species Rana fusca has been previously reported (Rapkine and Brachet, 1951). The mode of action of these substances is not known but may be related to their ability to combine with sulfhydryl groups. Effects

of pH

It had been previously found (Ambellan, 1958) that the effective pH for advancing neurulation in R. pipiens was about 5.6, and that ATP was highly toxic at pH 7.5. More detailed studies of the effects of pH with ADP and ATP are summarized in Fig. 4. The effect on neurulation (Fig. 4A) is limited to a range of about 5.9-6.5. Above this pH, ADP-treated embryos generally resemble controls, and the viability curve ( Fig. 4B ) , clearly shows the toxicity of ATP beginning at about pH 7-7.5. The viability curve for ADP on the alkaline side, and for both ADP and ATP in acid, follows that for untreated controls at the same pH values (not shown on the figure). Within the pH range 6.5-7.5 there is wide variability among different clutches of eggs (three similar experiments) and among embryos in a dish that was not observed at other pH values. This is indicated by the broken lines in Fig. 4. Maintenance of a constant pH is often difficult because cytolyzing eggs tend to acidify solutions and growing eggs

460

AMBELLAN

AND

WEBSTER

100 80 60 40 20 0

100 80

0

4.0

5.0

6.0

7.0 8.0

9.0

PH Effects of ATP and ADP on viability and on neurulation with varying pH. (A) Effect on neurulation. (B) Effect on viability. In two experiments using 1 mM and one experiment using 2 mM solutions (all samples in duplicate), the curves were similar. Variable effects on neurulation with ADP and variable effects on viability with ATP within the pH range 6.5-7.5 are indicated by dashed lines. The viability range for untreated controls, not shown here, is about 4.5-9.0, some individual variation occurring among embryos near the upper and lower pH limits. With the methods employed, variations between embryos within any one experimental dish, or between duplicate sample dishes, were rarely observed (except as specially indicated). FIG.

4.

secrete ammonia and urea, which tend to make solutions basic. These activities can produce a change of 2 pH units during an experiment, so the pH of solutions containing large numbers of embryos must be constantly adjusted.4 4 The specific were later found

conditions to vary

for advancing widely among

neurulation different

with amphibian

ADP or with ATP species, and for

EFFECTS

OF NUCLEOTIDES

ON

461

NEURULATION

An experiment comparing the effects of A-3-P and A-5-P at two different pH values is reported in Table 2. Advanced neurulation TBBLE

2

A-3-P A-3-P A-3-P

4 2 1

Advanced Advanced No effect

5.6

A-5-P A-5-P A-5-P

4 2 1

Delayed neurulation Delayed neurulation No effect

7.2

A-3-P” A-3-P A -3-P

4 2 1

Cytolysis Delayed Delayed

closure, closure,

then then

A-5-P A-5-Y A-5-P

4 2 1

Delayed Delayed Delayed

closure, closure, closure,

then cytolysis then cytolysis then cytolysis

5.6

7.2

neurulation neurulation

(later

= Treatment was for 30 hours at 14” C, starting at early gastrula. contained 12 embryos each, all in duplicate. b Results with A-Z-I’, at pH 7.2 vary widely between experiments. were no effects. c Exhibited slight variability between experiments.

recovery)

cytolysis cytolysis

Sample Often

dishes there

with A-3-P at pH 5.6 is consistent and repeatable, as is delayed neural tube closure with A-5-P at pH 7.2. The effect of the latter is slightly less constant at pH 5.6. The effects of A-3-P at pH 7.2 were, however, variable from one experiment to another. further biochemical analyses of the morphological phenomenon, the optimum conditions must be determined in each case. With the South African Xenopns, the pH optimum is at 5.6 and lethal over pH 7, but the tolerable concentrations are about l/10 that found for Rana pipiens. The European pleurodele requires the same weak concentrations as Xenopus, but the pH optimum is about 7, and below that the nucleotides are toxic. With the European R. tempomaria or R. ftrsca there was no difference in pH effects with ADP or ATP between pH 5 and 7.6 within the effective concentration ranges used, which were as wide as with the R. pipiens reported here.

462 Penetration

.&fBELLAN

of Nucleotides

AND

WEBSTER

into Embryos

It is difficult to judge the relative effectiveness of various substances toward some cellular process unless one knows something about their relative efliciencies of penetration into the cells. Likewise, the effect of pH on certain cellular processes has been found to be related to the increased penetration of some substance at a particular pH. Measurements were made, therefore, to determine whether W-labeled nucleotides are taken up by the embryos prior to neurulation. The uptake of C4-ATP and Cl”-ADP during the periods before neurulation is shown in Fig. 5. In experiment 1, embryos were treated with C”-ATP for 48 hours before neurulation. The uptake reached a

20,000 2 :$

16,000

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12,000

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4.000

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8

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pd

I

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I

16 Hours

24

32

40

of treatment

FIG. 5. Uptake of CJ-adenine nucleotides as a function of developmental stage of embryos. Experiment 1: Treatment was started at early gastrula, 48 hours before neurulation, with 0.5 mM ATP. Initial radioactivity was 3.6 X 10” cpm in 50 ml (7.2 x lo* cpm/ml). Samples of 250 eggs each, in duplicate, were removed after 8, 24, and 48 hours of treatment. Experiment 2: Treatment was started at late gastrula, 12 hours before neurulation, with 0.5 mM ADP. Initial radioactivity was 1.8 x lo6 cpm in 25 ml (7.2 x lo4 cpm/ml). Samples of 240 eggs removed after 1, 3, 6, and 12 hours of treatment. Experiment 3: Treatment was started 1 hour prior to neurulation with 0.5 mM ADP. Initial radioactivity was 1.1 x 10’ cpm in 35 ml solution (3.0 X 10” cpm/ml). Samples of 250 eggs each removed after 15, 30, and 60 minutes treatment. The counts expressed on the ordinate are x 10 for this experiment.

EFFECTS

OF

NUCLEOTIDES

ON

463

NEURULATION

low stationary level between the 8-hour and 24-hour periods, i.e., during gastrula stages. The sharp increase in uptake occurred at some undetermined time before neurulation and is indicated by dashed lines. Experiment 2 using Cl”-ADP for 12 hours before neurulation showed no change in uptake between the 3-hour and 6-hour periods measured-again during gastrula stages in this experiment. The level reached here approximately equaled that found in experiment 1, and the sharp increase again occurred at an undetermined time between end of gastrula and the mid-neurula stages. It might appear at first glance that the ATP used in experiment 1 penetrates more slowly than the ADP used in experiment 2. However, other experiments (one reported in Table 3) had demonstrated that there is no difference in penetration between these two materials, and ATP is actually more effective in promoting neurulation. In contrast, in experiment 3, embryos were treated with Cl’-ADP at the flat neural plate stage just prior to neural tube closure. Assay after I5-, 3O-, and 60-minute periods showed that there was an immediate, linear uptake that far exceeded that found in experiments 1 and 2, even when adjustment is made for higher starting counts. This finding is consistent with the morphological observations previously reported, that a l-hour treatment at this period can promote neurulation, whereas, when applied at earlier stages, higher concentrations of materials or longer durations of treatments are required. Two experiments comparing the uptake of different adenine nucleotides are shown in Table 3. With nonlabeled materials, the increased TABLE PENETRATION

OF

ADELUXE

Experiment la: ~&b&d acid-soluble

rxtraets

Rlaterials

ATP ADP A-3-P A-5-P Controls

(abwrptiotl at 260 m,,)

0.485 0.480 1.780 -

3

NUXEOTIDES

(total

INTO

WHOLE

EMBRYOS

Experiment 2” Cl”-labeled nucleotides radioactivity abnorbrd.

cpm)

Whole homogumtrr

!)50. 1140. 12,600. 1110.

350. 350. 1505. 455.

0.140

a Treatment for 21 hours at 18” C, 0.004 M solutions. Samples b Treatment for 20 hours at 14” C, 0.001 M solutions. Total about 1.3 X 106. Samples, 0.1 ml, plated in duplicate.

in duplicate. starting counts

all

464

AMBELLAN

AND

WEBSTER

UV absorption of the acid-soluble fraction is taken as an approximate measure of penetration into the embryos. With C14-nucleotides, total counts in whole homogenates as well as acid-soluble fractions are reported. The slight differences in uptake between ATP, ADP, and A-5-P are apparently not significant, but there is a striking difference between the uptake of these substances and that of A-3-P. The latter exhibits a tenfold greater uptake in the whole homogenate and about a fourfold greater uptake in the acid-soluble fractions in both experiments. This indicates more rapid penetration of A-3-P than of ADP or ATP, which are actually more effective in promoting neurulation. Experiments using C ‘*-ADP to test whether the difference in morphological results between pH 5.6 and pH 7.5 could be due to differences in penetration are summarized in Table 4. There is

PENETRATION

OF

ADI’

IN

ACII)IC

AND

IN

1tadioactivit.y

HI,IC:HTI,Y

BASIC

SOI,~:TIOKS~~

(rpm)

1

5.6-5.8 7.2-7.5

1 .06 1 o:<

1!420 1824

0.18 0.17

2

5.6-5.8 7.2-7.5

I .57 1.58

!K<75 7200

0.5!) 0.45

5.G5.8 7.2-7.5

I .04 1.14

5500 4400

0.53 0.40

3

a Solutions were 0.5 mM ADP. Treatments were for 30 hours at 14” C, starting at early gastrula. In experiment 1, samples were 260 embryos each. In experiments 2 and 3, samples were 380 embryos each.

slightly better penetration in acid, averaging about 25%. That this difference is not significant to explain the lack of morphological effects at pH 7.5 can be seen by referring to the effects of concentration reported above (Fig. 2); these results show that it requires manyfold differences in concentrations before clear morphological distinctions can be observed. In the present experiment, ADP concentration was 0.5 mM at both pH values, which was about four times the amount required to produce visible advanced neurulation at pH 5.6. (Thus a decrease of 25% in penetration of 0.5 mM ADP at pH 7.2 still leaves

AFFECTS

OFNU~LEOTIDES

ON NEURULATION

465

a relative effective penetration of 0.375 mM, or 3 times the concentration shown in Fig. 2 required to advance neurulation.) Radioautograph,y

Studies

When embryos treated with C?-ADP or Cl”-ATP were observed histologically at the time of precocious neurulation, the radioactive grains were seen to be densely concentrated throughout the neural and chordamesodermal tissues. The concentration was greatest in the neural ectoderm and decreased downward to the roof of the archenteron. A control series of slides was treated with cold 10% trichloroacetic acid for 1 hour to remove acid-soluble material that had not already been removed by the same treatment during the initial fixation. Over half of the grains remained, and those remaining showed the same distribution as on the untreated slides. DISCUSSION

The experiments reported above leave little doubt that the nucleotides have specific effects on neural tube closure. Depending upon the nucleotide employed, one can obtain either promotion or inhibition of neurulation. The biochemical reality underlying the visible morphological effects was clearly demonstrated by the dependence upon nucleotide concentration, pH, and time of treatments. There are some obvious possibilities to explain why these effects are obtained: (1) the nucleotides produce some superficial reaction corresponding to the “chaotic neurulation” effects induced in explants under various conditions such as pH or temperature shock; (2) the nucleotides could penetrate into the embryos and promote one or more processes(RNA synthesis is one possibility) in which nucleotides are limiting; or (3) the apparent specificity of the effect on neurulation is due to the fact that only at this time, after the invagination of the ectoderm, does the internal effective concentration of the nucleotides become sufficient to affect tube closure. However, these latter two possibilities are not mutually exclusive, one being an explanation at the biochemical level, the other at the morphological level. Both imply a requirement for a certain internal level of nucleotides to affect neural tube formation. Regarding the first possibility, the controls showed that the pH and temperature conditions used in these experiments have by themselves no effect on neurulation in whole embryos. Nor indeed, is there any necessary reason to extrapolate from the bc-

466

AMBELLAN

AND

WEBSTER

havior of explants which are quite delicate, or transplant or injection experiments where attendant cellular cytolysis is suspected and often visible, to the behavior of entire, intact embryos to which the present investigation is limited. The possibility of a sublethal cytolysis cannot be eliminated without thorough cytological studies. If this were shown to exist un’der optimum conditions of nucleotide treatment when all development is apparently enhanced, then it is possible that this kind of “sublethal cytolysis” is a part of normal development at this time, as Brachet has suggested. The present studies have demonstrated that the nucleotides do indeed penetrate into the embryos. Although there is no direct evidence for the penetration of the phosphate group attached to the C’“nucleotides, a dephosphorylation at the cell surface is not compatible with the observed differential effects of mono-, di-, and triphosphorylated compounds, nor with the lack of any visible effects of adenine or adenosine. It is not possible as yet to say exactly how the nucleotides promote neurulation. The effectiveness of nucleoside di- and triphosphates is compatible with the idea that their effects might be on the synthesis of RNA. However, the effectiveness of adenosine-3’-phosphate and the inhibitory action of adenosine-5’-phosphate is difficult to explain only in terms of RNA synthesis. It is evident that further work on the fate of the nucleotides, once they have penetrated into the cells, and on the effects they have on synthetic processes, will be necessary for a clearer understanding of the manner in which the nucleotides act to promote neural tube closure. SUMMARY

Conditions have been found under which treatments of frog embryos with solutions of nucleotides promote closure of neural tubes many hours ahead of controls. Under optimum conditions, growth of the whole embryo is apparently enhanced. Adenine nucleotides were more effective than other nucleotides in promoting neurulation, and the nucleotide triphosphates were more effective than the diphosphates. Adenosine-3’-monophosphate is much less effective, and adenosine-S-monophosphate is inhibitory. The effects were dependent upon the concentrations of nucleotides, duration of treatment, time of application, and pH. The most effective time of treatment was at the start of neural tube formation.

EFFECTS

OF NUCLEOTIDES

ON

NEURULATION

467

The neurulation effect was not related to the extent of penetration of a nucleotide into the cells. Adenosine-3’-monophosphate, the least effective promoting material, penetrates ten times as fast as ADP or ATP. The pH dependence of morphological effects was not related to penetration. Cl”-ADP at pH 7.2 does not promote neurulation, but penetrates to about the same extent as at pH 5.6, which does promote neurulation. Slight positive effects on neurulation were found with nicotinamide or with DPN (NAD) at pH 5.6 and with yeast RNA at both pH 5.6 and 7.2. Adenine and adenosine had no effect. Radioautography studies showed that the W-labeled material was limited principally to the neural and chordamesodermal tissues. This investigation was supported by a Research Grant (C-3725) from the National Cancer Institute, Public Health Service, and by a Fellowship (GF-9374) to one of us (E.A.) from the Division of General Medical Sciences, United States Public Health Service. The cooperation of Professors John Price and Henry Plaine and of the Zoology Department of the Ohio State University is gratefully acknowledged. REFERENCES AMBELLAN, E. ( 1955). Effect of adenine mononucleotides on neural tube formation of frog embryo. PTOC. Natl. Acad. Sci. U. S. 41, 428432. AMBELLAN, E. (1958). Comparative effects of mono-, di-, and triphosphorylated nucleosides on amphibian morphogenesis. J. Embryol. Exptl. Morphol. 6, 86-93. BRACHET, J. Personal communication. RAPKINE, L., and BHACHET, J. (1951). A n investigation of the role of sulfhydryl groups in morphogenesis. Bull. sot. chim. biol. 33, 427-438. RUGH, R. ( 1948). “Experimental Embryology.” Burgess, Minneapolis, Minnesota. SPIEGEL, M. (1951). A method for removal of the jelly and vitelline membranes of the egg of Rana pipiens. Anat. Record 111, 544.