Comparative Effects of Moni-, Di-5 and
Triphosphorylated Nucleosides on Amphibian
Morphogenesis
by ELISABETH AMBELLAN1
From the Institute of Cancer Research, Columbia University, College of Physicians & Surgeons,
New York
INTRODUCTION
previously reported (Ambellan, 1955) established the fact that
short-term treatment of Rana pipiens blastulae with solutions of mononucleotides under specific conditions resulted in accelerated formation of neural tubes:
at 18° C. they were about 4 hours ahead of controls. As briefly mentioned in
the previous paper, slight overdoses or excessive periods of treatment resulted
in visible side-reactions affecting other aspects of development in conjunction
with the precocious neural tube formation. A comparison of the different effects
that result from overdoses of adenosinemonophosphate (AMP-3), adenosinediphosphate (ADP), adenosinetriphosphate (ATP), and other compounds that
differ from each other only in the number of phosphate bonds may throw some
light on the biochemical nature of the morphogenetic processes differentially
EXPERIMENTS
affected by these substances and is the subject of this paper.
MATERIALS AND METHODS
A homogeneous population of dejellied embryos was selected and groups
raised each in about 15 ml. of solution in covered Stender dishes in a constant
temperature water-bath at about 18° C. Compounds under investigation were
added to 10 per cent. Ringer's solution, then brought to pH 5-6-5-8 with Ringer's
hydroxides (a combination of K, Na, and Ca hydroxides with the same cation
concentrations as in Ringer's solution). Controls were at the same pH.
'Optimum conditions' were arbitrarily defined as the concentration of substance and duration of treatment at which there were no apparent effects on the
embryo other than on neurulation; the embryos developed normally to hatching.
These generally were: between 12-24 hours of treatment with ATP 1 mM. or
with AMP-3 1 mM., and up to 8 mM. of ADP or IDP (inosinediphosphate),
although there was some clutch-to-clutch variability in concentrations tolerated.
1
Author's address: Institute of Cancer Research, Columbia University, College of Physicians &
Surgeons, 630 West 168th Street, New York 32, N.Y., U.S.A.
[J. Embryol. exp. Morph. Vol. 6, Part 1, pp. 86-93, March 1958]
PHOSPHORYLATED NUCLEOSIDES
87
RESULTS
Treatment of R. pipiens embryos with solutions of ATP, AMP-3, ADP, IDP,
or mixtures of AMP-3 + ATP and mixtures of other diphosphorylated nucleosides accelerated neural-tube formation under 'optimum conditions'. The morphogenetic acceleration was of the order of 6 hours at 16° C , 4 hours at 18° C ,
or 3 hours at 23 ° C. With excessive but sub-lethal concentrations of materials or
TEXT-FIG. 1. Effect of mononucleotides on neuraltube closure. All animals from same clutch raised
under constant temperature conditions. Top:
control, open neural plate (stage 14). Middle:
ATP-treated embryo, closed neural tube (stage
15), defective sense plates. Bottom: ADPtreated embryo, closed neural tube, sense plates
normal.
duration of treatment specific morphogenetic effects of the several nucleotides
were observed in addition to early neural-tube closure (see Text-fig. 1). The
development of head and gill structures (Shumway stages 16 and 17) immediately following neurulation in normal morphogenesis was blocked with overdoses of ATP, but not with overdoses of any other substance used (see Textfig. 2). With overdoses of AMP-3 the first visible defect in morphogenesis was
a block in the growth of the tail-bud (Shumway stage 18). At similar concentrations ADP or IDP had no adverse effects, and development following precocious
neurulation was apparently further stimulated, although the advance over
controls was difficult to measure objectively. A typical experiment in which
treatment was continuous, beginning during the blastula stage, is reported in
Table 1.
88
E. A M B E L L A N — D E V E L O P M E N T A L E F F E C T S OF
The effects of the mononucleotides were morphologically specific for the process of neural-tube closure in that regardless of the time of treatment there were
no visible differences between control and experimental animals during blastula,
TEXT-FIG 2. Effect of mononucleotides on headstructure development. Both animals are the
same chronological age. Top: ATP-treated
embryo showing microcephaly. Bottom: control embryo at stage 16. ADP- and AMP-3
treated embryos resemble controls.
TABLE 1
Comparative effects of AMP-3, ADP, and ATP on frog morphogenesis
Observation times after fertilization at 18° C
Substance in solution (mM.)
60 hours
10% Ringer (controls) .
Open neural plate*
ADP 40
AMP-3 4-0
ATP 4 0
Closed neural tube§
Closed neural tube
Closed neural tube
72 hours
Head structures
developf
+
+
Microcephalic
96 hours
Tail-bud growth^
+
Stunted
Stunted
+ Apparent advance over controls; head and gill structures often enlarged at 72 hours, tailbuds often longer and general improved viability compared to controls at 90 hours.
* Shumway stage 14.
t Shumway stage 16.
% Shumway stage 19.
§ Morphological resemblance to Shumway stage 15.
gastrula, or early neural-plate stages. However, while controls then required
about 8 hours (18 ° C.) to completely close the neural tubes, experimental animals
formed completed neural tubes in 4 hours. Eggs treated at the 2-cell stage with
ATP (as suggested by Dr. L. G. Barth) or with ADP, left overnight at 8° C. and
washed thoroughly before being returned to Ringer's solution, showed no differences from controls during blastula or gastrula stages, but exhibited precocious neurulation, which occurred 2-3 days after removal of the eggs from the
PHOSPHORYLATED NUCLEOSIDES
89
treatment solutions. In most of the experiments treatment was started during
blastula stages, but it was also effective when applied as late as stage 14 when
embryos have flat, open neural plates.
Some differences were noted in the rate of neural-tube closure between the
effective substances. When stage-14 embryos were treated at 23° C , the neural
tubes closed in about 45 minutes with ATP solutions, in about 1 hour with ADP,
and in about 1 | hours with AMP-3. Controls took about 3 hours to completely
close the neural tubes at this temperature. In experiments in which substances
had been applied 2 days prior to neurulation and in which the precise onset of
tube closure happened to be observed, the process started first in the ATP group,
about 20 minutes later in the ADP group, and about 30 minutes after that in the
AMP-3-treated embryos.
Specific effects of ATP treatment
Side effects observed with overdoses of ATP, occurring with an intensity
proportional to concentration, were: delayed closing of the blastopore, microcephaly following precocious neurulation, and, finally, lack of development of
neural ridges, gill plates, and sense plates at the time of tube closure (Text-figs. 1
and 2). Some or all of these abnormalities were noted in over forty experiments
using ATP solutions and were never observed with overdoses of any other
materials used. These specific results of ATP treatment did not generally appear
when equimolar AMP-3 was added to the ATP solutions. The ATP experimental animals were bloated and stunted by stage 18, but these later defects
were also observed with AMP-3 and ADP treatment.
Specific effects of AMPS treatment
With precocious neurulation resulting from AMP-3 the development of neural
ridges, sense, and gill plates was apparently normal. Following neurulation
embryos often showed grossly exaggerated head and gill structures, but never
microcephaly. The first block in development with overdose treatment did not
appear until the tail-bud stage when embryos were stunted as with ATP treatment; but they were less severely so, and recovery upon return to Ringer's solution was more frequent. These after-effects from AMP-3 treatment were more
variable than with other substances used and occasionally did not appear at all.
It had previously been found that AMP-5 did not consistently advance neurulation under these same conditions and it was not used in these investigations.
Effects of ADP, IDP, and other diphosphorylated nucleosides
There were no visible side effects or after effects from ADP or IDP when used
at the same concentrations that had proved toxic with ATP or AMP-3. The
advanced developmental rate observed during neurulation apparently continued
through the following stages when head structures and tail-buds develop, and
embryos often showed improved viability compared to controls. An ADP concentration about four times that of ATP or AMP-3 was required to produce any
90
E. AMBELLAN—DEVELOPMENTAL EFFECTS OF
abnormal effects. These abnormalities then resembled the AMP effects described
above. Head structures were normal or exaggerated, and stunted tail-buds were
the first defects to appear.
Embryos treated with equimolar solutions of AMP-3 + ATP generally
resembled those treated with ADP alone, although there was some individual
TABLE 2
Comparative and concentration effects of di- and triphosphorylated nucleosides
on frog morphogenesis
Observation times after fertilization at 20° C
Substance in solution (mM.)
10% Ringer (controls) .
ADP: 0-25, 0-5, 10, 4 0 , and 8 0 (5
separate dishes)
.
.
.
.
ADP 0-75+IDP 0-25 .
ADP, CDP, GDP, U D P : 0-25 each
ATP 4 0
„
10
.
.
.
„ 0-5
.
.
.
ATP 1 0 + A M P - 3 1 0 .
„
0-5+ „
0-5 .
48 hours
Open neural plate
60 hours
Head structures
develop
Closed neural tubes
+
Microcephalic
Like controls*
Like controls
+
72 hours
Tail-bud growth
+
+
+
Stunted
Stunted
Stunted
Like controls
Like controls**
4- Apparent advance over controls, head and gills often larger.
* Two embryos in this group were microcephalic.
** One-half of the ten embryos in this group were microcephalic.
No apparent variability occurred between ten embryos in each of the other experimental groups
or four different groups of controls.
variability within experimental groups not found when ADP alone was used.
Although treatment with 4 mM. ATP resulted in typical microcephaly, treatment with a mixture of ATP + AMP-3, 4 0 mM. each, resulted in normal head
development with no apparent abnormalities until tail-bud stage. Embryos
treated with this mixture sometimes cytolysed at about stage 19, which did not
happen when ADP alone was used.
IDP can effectively replace ADP in this system. In an experiment using this
material (kindly supplied by Dr. S. Ochoa) at 8,4, and 1 mM., both continuously
and for 12-hour treatments started at blastula stages all embryos in all the experimental groups showed A\ hours advance in neural-tube closure. By the tail-bud
stages embryos treated at the highest concentration (8 mM. continuously) had
stunted bodies, and those treated at the lowest concentration (1 mM. for 12
hours) resembled controls. Embryos in the other four experimental groups (1 and
4 mM. continuous, and 4 and 8 mM. 12-hour treatments) lived for 8 days while
three different groups of controls died in 6 days under the slightly overcrowded
conditions of this experiment. Embryos from this same clutch of eggs treated
continuously with ATP at 1 and 4 mM. cytolysed immediately after the precocious neurulation.
PHOSPHORYLATED NUCLEOSIDES
91
Combinations of ADP with uridinediphosphate (UDP), guanosinediphosphate
(GDP), and cytosinediphosphate (CDP) were effective in advancing neurulation,
but no more effective—at the lower ranges of concentrations used—than was
ADP alone. Results of experiments carried out with Dr. Herman Kalckar at the
National Institutes of Health (Bethesda, Md.) are reported in Table 2. Treatments were continuous starting at blastula stages. Solutions were at pH 5-6-5-8.
DISCUSSION
The original hypothesis that ATP in solutions might serve as an energy source
in morphogenesis had to be modified when it was found that formation of the
neural tube—the first structure formed in morphological differentiation in amphibia as in all vertebrates—was accelerated by all of the mononucleotides,
including AMP-3 which does not have the labile, 'high energy' phosphate bond.
This part of the results generally supports Brachet's hypothesis (1947) on the
role of mononucleotides in morphogenesis, although in these experiments the
visible effects were on neural-tube and not on neural-plate formation. While
precocious neurulation was produced by any of the mononucleotides, the rate
of this acceleration was directly proportional to the number of phosphate
groups on the nucleotides used: fastest with ATP, next with ADP, and slowest
with AMP-3.
The formation of head and gill structures follows neural-tube formation in
normal development. Horstadius (1950) has shown that neural-crest cells that
appear at the time of neurulation are concerned in the formation of these structures. This developmental system is apparently affected by treatment with solutions of ATP. Even mild overdoses result in microcephaly by stage 16, and with
larger overdoses this microcephaly is foreshadowed by the lack of visible neural
ridges, sense, and gill plates during the time of rapid neural-tube formation.
Treatment with AMP-3, ADP, IDP, or other diphosphorylated nucleosides,
never blocked neural-crest or head-structure development at any concentrations
used. Embryos, in fact, often showed exaggerated formation of these structures,
and it may be that this next morphogenetic process, as well as neurulation, can
be enhanced with overdoses of these substances.
The diphosphorylated nucleosides were the most innocuous materials used to
advance neurulation inasmuch as other developmental processes were least
disturbed. This suggests that the mechanism by which these materials are utilized
to advance neurulation may more closely resemble normal synthetic mechanisms
of the embryo than utilization of ATP or AMP-3. The details of the dose effectiveness reported in Table 2 also suggest that utilization of the mono- and triphosphorylated nucleosides may be by way of a kinase catalysing the reaction
2 ADP ^
ATP+AMP,
92
E. AMBELLAN —DEVELOPMENTAL EFFECTS OF
since the abnormal effects of ATP were largely reversible by the addition of
equimolar AMP-3 to solutions.
While there is no direct evidence in these experiments for penetration of the
effective materials, three aspects of the results make this a likely assumption:
(a) the pH dependence of the results, (b) the reversibility of the specific morphogenetic effects of ATP treatment by addition of AMP-3 to treatment solutions,
and (c) the clear separation in time between application and effect of treatment,
especially in experiments in which the visible effects appeared 2-3 days after
removal of embryos from treatment solutions and in which ultra-violet spectrophotometric readings confirmed that all free, unbound nucleotides had been
removed.
The possibility that the mononucleotides may be utilized metabolically, either
directly or indirectly, in the synthesis of key materials is now under active
investigation.
SUMMARY
1. Treatment of Rana pipiens embryos with adenosinetriphosphate (ATP),
adenosinemonophosphate (AMP-3), adenosinediphosphate (ADP), and other
diphosphorylated nucleosides, either under 'optimum conditions' or with an
overdose, resulted in formation of neural tubes about 4 hours (at 18° C.) ahead
of untreated controls. With overdoses specific side-reactions appeared that
differed with the substance applied.
2. The rate of neurulation was directly related to the number of phosphate
groups of the nucleotides used; it was fastest with ATP, intermediate with ADP,
and slowest with AMP-3. The other side-reactions varied specifically and consistently with the number of phosphate groups, but the intensity of these reactions was not directly proportional to the number of phosphate groups present.
3. Overdose treatment with ATP blocked development of head and gill structures immediately following precocious neurulation. This effect was largely
reversible by addition of equimolar AMP-3 to solutions.
4. Overdose treatment with AMP-3 apparently enhanced formation of head
and gill structures. Development was first blocked at the tail-bud stage.
5. ADP, IDP, and other diphosphorylated nucleosides at similar concentrations had no adverse effects following precocious neurulation and apparently
stimulated growth of the entire embryo.
6. All of the mononucleotides specifically accelerated neural-tube formation
whether applied at the 2-cell stage, during the blastula stage, or at the stage of
the open neural plate.
ACKNOWLEDGEMENTS
In addition to the assistance acknowledged in the text, I would like to express
thanks to Drs. Z. Dische and Alfred Gellhorn for laboratory hospitality during
PHOSPHORYLATED NUCLEOSIDES
93
these past two years; to Dr. France Baker for the attached drawings; to Drs. Eric
Hirschberg, Alan Wachtel, and S. Ochoa for manuscript suggestions; and to Dr.
Herman Kalckar whose active interest in this work was indispensable.
REFERENCES
AMBELLAN, E. (1955). Effect of adenine mononucleotides on neural tube formation of frog
embryos. Proc. nat. Acad. Sci., Wash. 41, 428-32.
BRACHET, J. (1947). Metabolism of nucleic acids during embryonic development. Cold Spr. Harb.
Symp. quant. Biol. 12,18-25.
HORSTADIUS, S. (1950). The Neural Crest. New York: Oxford University Press.
(Manuscript received 23: v: 57)