/ . Embryol. exp. Morph. Vol. 37, pp. 149-157, 1977
Printed in Great Britain
149
Biosynthetic events of hydrid regeneration
II. Patterns and profiles of RNA synthesis during
distal morphogenesis
By DENNIS C. BROOKS,1 JOSEPH R. VOLAND
AND GEORGIA E. LESH-LAURIE
From the Department of Biology,
Case Western Reserve University, Cleveland
SUMMARY
The pattern of RNA synthesis during the first 48 h of distal regeneration in Hydra
oligactis has been investigated. In addition the RNA synthetic profiles during selected
time periods have been studied.
RNA synthesis was found to increase five-fold during the first 3 h of regeneration and
remained high throughout the rest of the 48 h period. An additional increase was observed
27-30 h following subhypostomal excision, immediately preceding the appearance of the
first tentacle pair. The RNA synthetic level then returned to that found at 3 h of regeneration.
This pattern was similar to that leported previously for DNA synthesis.
Profiles of radioactively labeled RNA remained constant during most of the times studied,
showing the presence of newly synthesized 28S, 18S, 5S, and4S RNA. During two time periods,
28-32 h and 36-40 h, a novel 8S RNA species was observed. The occurrence of this species
coincided with times of increased overall RNA synthesis and immediately preceded tentacle
elaboration.
INTRODUCTION
The freshwater cnidarian Hydra has repeatedly served as an experimental
system for studies of regeneration and morphogenesis. It regenerates rapidly
and consistently after distal amputation and it reforms only two types of
structures, a mouth and several tentacles.
Reports from this and other laboratories have demonstrated a requirement
for RNA synthesis during normal distal regeneration in hydra (Kass-Simon,
1969; Clarkson, 19696; Lesh-Laurie & Hang, 1972; Lesh-Laurie, 1974). In
each of these studies distal regeneration was examined in the presence of an
inhibitor of RNA synthesis, and results ranging from the total inhibition oi
regeneration to an arrest after the emergence of the first tentacle pair have
been obtained. Although each of these investigators concluded that RNA
synthesis was necessary for regeneration, only Clarkson (1969a) attempted
to measure the synthetic events of normal distal regeneration. His study,
1
Author's address: Department of Macromolecular Science, Case Western Reserve
University, Cleveland, Ohio 44106, U.S.A.
150
D. C. BROOKS, J. R. VOLAND AND G. E. LESH-LAURIE
which examined only the first 2-5 h after subhypostomal excision, revealed a
ten-fold increase in RNA synthesis during this time period. No effort was
made to examine synthetic activity beyond 2-5 h of regeneration.
In a previous report (Lesh-Laurie, Brooks & Kaplan, 1976) we described
the pattern of [3H]thymidine incorporation into DNA during the first 48 h
of distal regeneration in Hydra oligactis. Peaks of incorporation occurred
during the intervals 0-3, 24-27, and 33-36 h after excision. These periods
corresponded temporally to wound healing, the emergence of the first tentacle
pair, and the emergence of the third tentacle, respectively.
In this paper we report the results of an investigation of [3H]uridine incorporation into RNA during the first 48 h of hydrid distal regeneration.
Analyses of incorporation into total TCA-insoluble RNA revealed increases
in incorporation rates corresponding to the times of wound healing and tentacle
formation. In addition, fractionation of labeled RNA on polyacrylamide gels
demonstrated the production of a low molecular weight RNA species preceding
tentacle initiation.
MATERIALS AND METHODS
Culture methods
H. oligactis were mass cultured at 18° ± 2 °C in 14 cm finger bowls according
to the methods of Loomis & Lenhoff (1956) with distilled water substituted
for tap water. Cultures were fed to repletion with Artemia salina nauplii every
other day and cleaned 4-6 h later.
Experimental procedures
Non-budding animals, fed one day before subhypostomal excision, were
used for all experiments. For at least 24 h prior to use hydra were cultured
in sterile culture solution containing 0-05 % (v/v) Kantrex (0-33 g/ml kanamycin
sulfate, Bristol Laboratories, Syracuse, New York). This treatment has been
shown autoradiographically to reduce bacterial precursor incorporation in
the mucous coat of the animal to background levels (Lesh-Laurie et ah 1976).
Removal of the hypostome and tentacle ring was accomplished with one
clean cut immediately proximal to the tentacle bases using iridectomy scissors
or a scalpel. Following excision, animals were returned to sterile, kanamycintreated culture solution (SKCS) until used.
Incorporation of[3H]uridine into total RNA. Analyses of uridine incorporation
were carried out during the first 48 h of regeneration. By this time at least
two and often three tentacles were present. Groups of 15 hydra were incubated
for 3 h in 0-5 ml of SKCS containing 15/tCi/ml [3H]uridine (5-[3H]uridine,
29 Ci/mM, Amersham/Searle Corporation). A group of hydra was labeled
during each 3-h interval from 3 h prior to excision until 48 h after excision.
Following the 3-h pulse, they were washed four times over a period of five
min in non-radioactive uridine (3 mg/ml), homogenized, and the resulting
Biosynthetic events of hydrid regeneration. II
151
material precipitated by addition of ice-cold 100 % TCA to a final concentration
of 5 %. Precipitation was allowed to proceed for at least 1 h at 2 °C.
The RNA was extracted by the Schmidt-Thannhauser technique (1945).
After washing twice with ice-cold 5 % TCA, once with 95 % ethanol, and
once with 95% ethanol:ether (3:1), the RNA was hydrolyzed in 2ml of
0-5 N-KOH by heating at 37 °C overnight. To each sample 0-2 ml of 70 %
perchloric acid was added and the macromolecules were allowed to precipitate
on ice for 10 min. This procedure removed the DNA, which was resistant to
alkaline hydrolysis, from the hydrolyzed RNA (Munro & Fleck, 1966).
After centrifugation the supernatant was titrated with KOH to a pH of
approximately 6-8, diluted to 5 ml with distilled water, and chilled on ice
for 20 min. Following another centrifugation, the supernatant was divided
with 1 ml counted in 10 ml of Bray's solution (Bray, 1960) and 1-5 ml analyzed
for RNA content by the Dische modification (Dische, 1954) of the orcinol
reaction. Standards were prepared from yeast RNA (Type XI, Sigma Chemical
Company) and absorbance at 665 nm was read in a Zeiss PMQII Spectrophotometer.
A specific activity (cpm//*g RNA) was then calculated for each labeling
interval. In addition an overall average specific activity was calculated for
each experiment. The specific activities of the individual labeling intervals for
a given experiment were expressed as percentages of the average specific
activity for that experiment. This procedure eliminated differences among
experiments resulting from changes in the condition of the organisms and
from errors in the preparation of labeled precursor solutions.
Each labeling interval is designated as a single time, that of homogenization
at the end of the labeling interval. For example, animals labeled from 24 to
27 h after excision are referred to as 27-h regenerates.
Fractionation of labeled RNA. To study the changes in specific RNA fractions
during regeneration, groups of 50 animals were incubated for 4 h in 4 ml of
SKCS containing 20/^Ci/ml [3H]uridine. The longer labeling period and higher
radioactive concentration were required to insure a significant level of radioactivity in the fractionated RNA. A group of animals was labeled during each
4-h interval from 4 h prior to excision until 48 h after excision. Immediately
after labeling, RNA was extracted with phenol-chloroform and electrophoresed
on 2-4 % polyacrylamide gels as previously described (Voland, 1975).
Gels were analyzed in a Gilford Recording Spectrophotometer equipped
with a linear transporter. Scanning was carried out at 260 nm at a scanning
rate of 2 cm/min. Once A260 patterns were obtained, gels were consecutively
sliced into 1 mm sections, placed in glass vials and solubilized with 0-1 ml
concentrated NH 4 OH. The vials were capped and allowed to sit overnight at
room temperature. The vials were then filled with Bray's solution and counted
in a liquid scintillation counter.
Results from the polyacrylamide gels are expressed as cpm/^g 18S RNA.
152
D. C. BROOKS, J. R. VOLAND AND G. E. LESH-LAURIE
To compute the amount of 18S RNA present, A26c profiles were photocopied,
the 18S peak cut out and weighed. This weight was normalized to a weight at
a full scale deflexion of 1-5 on the Gilford Recording Spectrophotometer and
the micrograms of 18S RNA determined from a standard curve (courtesy of
Dr S. Macintyre) of the weight of the 18S peak at a full scale deflexion of
1-5 versus micrograms of 18S RNA.
Representative radioactive patterns are presented for selected regeneration
times. Each represents an average profile for the time selected (i.e. each experiment was repeated a minimum of three times, and the pattern selected is neither
the high nor the low extreme).
RESULTS
Preliminary experiments revealed that exposure of 15 hydra to 15/^Ci/ml
[3H]uridine for 3 h resulted in sufficient labeling levels (> 1000 cpm/ml) and
RNA concentrations (> 8 /*g/ml) after TCA extraction for analysis by scintillation counting and the orcinol procedure. Using these conditions, radioactive
specific activities were determined at 3-h intervals during regeneration and
are shown graphically in Fig. 1.
A five-fold increase in incorporation occurred in the first 3-h interval after
excision, with incorporation levels remaining high throughout the 48-h period
studied. Specific activity levels began to increase again at 24 h, climbing to
a peak at 30 h of regeneration. The 30-h specific activity level represented a
20-fold increase over the zero hour level.
After this peak of incorporation, the specific activity level fell to that of the
0-3-h interval, then climbed to approximately twice this value and remained
nearly constant for the remainder of the period studied.
The observed specific activities from 3 to 48 h of regeneration were significantly
higher (P < 0-01) than the zero-hour level when analyzed by a two-way analysis
of variance with a test of contrasts (Snedecor & Cochran, 1956). By the same
test, the levels at 27 and 30 h were significantly higher (P < 0-005) than those
from 3 to 48 h. Although fluctuations in specific activity were observed in
individual experiments after 30 h, no significant changes occurred in the
average values.
Because consistent patterns of incorporation were found in TCA-insoluble
material, fractionation and analysis of the newly synthesized RNA was carried
out. Due to the higher radioactive concentrations and longer incubations
required for these procedures, the time periods studied do not correspond
exactly to those employed for the previous experiments.
Both absorbance and radioactive profiles were obtained for phenol-chloroform
extracted RNA. The A26o profiles were similar for all intervals studied regardless
of the extent of regeneration. These profiles were also similar to those obtained
for intact hydra (Voland, 1975), a result consistent with the hypothesis that
the majority of hydrid RNA is not altered during the regeneration process.
Biosynthetic events of hydrid regeneration. II
3
6
9
12
15
18
21
24
27
30
33
36
39
42
153
45
48
Hours of regeneration
Fig. 1. Uridine incorporation during distal regeneration. Groups of 15 regenerating
hydra were labeled for 3 h in 15 /tCi/ml[3H]uridine, homogenized, and the homogenates were precipitated with 5 % TCA. After alkaline hydrolysis, the radioactivities and RNA concentrations of the hydrolysates were measured, and specific
activities were calculated for each labeling interval. An average specific activity
was computed for each experiment, and the individual specific activities were
expressed as percentages of this average. For each labeling interval, expressed
as the hours of regeneration at the end of the interval, the percentages from three
experiments are plotted and the mean values for each time period aie connected
by a line.
The levels of uridine incorporation, however, showed considerable variation
with time during regeneration. The qualitative changes were similar to those
found for TCA-insoluble RNA, with peaks of incorporation occurring at
0-4 and 28-32 h of regeneration. An increase over the zero-hour level was
also noted at 36-40 h after excision.
The basic radioactive profile was relatively constant for most times during
regeneration. Representative profiles are presented in Fig. 2 for time intervals
preceding and following the 28-32-h peak of incorporation. Uridine was incorporated into ribosomal RNA (28S, 18S, 5S), transfer RNA (4S), and DNA.
Incorporation into bacterial ribosomal RNA (23S and 16S) was also noted.
The observed profiles showed a lower level of incorporation into the 18S and
16S species than was anticipated. The reason for this result is probably an
instability of these species related to the extraction procedure.
154
D. C. BROOKS, J. R. VOLAND AND G. E. LESH-LAURIE
DNA 18S
DNA I8S
300
280
260
240
220
z
oo
8S
1 5S
Mt4S
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"
200 •
180
160
140
120
100
80
60
40
20
J: 1100
L.
01 1000
2 900
M 800
"f 700
§" 600
500
400
300
200
100
•
-
-
'A
•fJv \
I \
•
JA
t\
v_
0 10 20 30 40 50 60 70 80 90
mm of gel
0 10 20 30 40 50 60 70 80 90
m m of gel
DNA
000
900
8S
|5S
18S
S
I16S
23S * |
-'1
If
DNA 18S
400 . \ 28S
•
800
B
350
700
<
600
-
500
•
300
250
SP 200
150
00
OO
400
300
200
-
100
JI
n \
D.
o
100
50
i
0
8S
l5S
M4S
1800 [28S 116S
1700 . I l23Sh
1600
1500
1400
1300
1200
I
I
10 20 30 40 50 60 70 80 90
mm of gel
- f
UA
0
8S
|5S
14S
1
D
t
10 20 30 40 50 60 70 80 90 100
mm of gel
Fig. 2. Radioactive profiles for RNA from regenerating animals. RNA was
extracted using a modified phenol-chloroform procedure and electrophoresed on
2-4 % polyacrylamide gels for 2-5 h. The gels were sliced into 1 mm sections,
solubilized with NH4OH and counted in Bray's solution. Data are presented as
cpm//tg 18SRNA. (A) - 4 to Oh regenerates. (B) 0-4h regenerates. (C) 16-20 h
regenerates. (D) 44-48 h regenerates.
Biosynthetic events ofhydrid regeneration. II
18S
700 iDNA
' |28Sli6S
650 .I|23S1|
600
550
500
450
400
350
300
250
200
150
100
50
•
8S
I5S
l|fS
1
A
DNA
1000
900
18S
I16S
B
800
700
155
8S
15S
Vs
600
•
500
•
I
•
10 20 30 40 50 60 70 80 90
mm of gel
400
300
200
100
0
10 20 30 40 50 60 70 80 90 100
mm of gel
Fig. 3. Radioactive profiles for RNA from regenerating animals immediately
prior to tentacle formation. RNA was extracted using a modified phenol-chloroform
procedute and electrophoresed on 2-4% polyacrylamide gels for 2-5 h. The gels
were sliced into I mm sections, solubilized with NH 4 OH and counted in Bray's
solution. Data is presented as cpm//tg 18S RNA. Note the presence of an additional
peak of activity labeled 8S, not seen in the profiles in Fig. 2. (A) 28-32 h regenerates.
(B) 36-40 h regenerates.
While the profiles presented in Fig. 2 are representative of most time
intervals, they do not match the profiles obtained during the intervals from
28-32 and 36-40 h of regeneration. These two profiles are shown in Fig. 3.
In addition to the normal incorporation into DNA, ribosomal RNA, and
transfer RNA, there is a new peak of incorporation designated 8S. This low
molecular weight RNA species was seen consistently in six repeats of the
28-32-h interval and in three repeats of the 36-40-h interval. Moreover, it was
never observed at any other time interval.
DISCUSSION
In this investigation, incorporation of uridine into RNA was measured,
not RNA synthesis per se. In order to relate uridine incorporation to RNA
synthesis, two assumptions must be made. First, the cellular precursor pools
must equilibrate rapidly with each other and with the culture solution. That
this is the case in hydra has been shown by Clarkson (1969a), who found
that [3H]uridine incorporation increased linearly with incubation periods of
from 5 min to 8 h.
The second assumption, that the specific activity of the immediate precursor
pool (UTP) is constant over the 48-h period studied, is much more difficult
to verify. Because hydra consists of an asynchronous population of mixed cell
types, measurement of the TCA soluble pool, or even the UTP pool, would
156
D. C. BROOKS, J. R. YOLAND AND G. E. LESH-LAURIE
yield results which could not be interpreted with certainty (see Hauschka,
1973). Indirect evidence that the observed fluctuations in incorporation rates
are not due to changes in pool specific activities can, however, be obtained by
studying incorporation of different precursors into RNA. When the incorporation
of [3H]adenosine or inorganic [32P] was examined, patterns of incorporation
into phenol-chloroform extracted RNA were similar to those obtained with
[3H]uridine (Voland, unpublished results). Considering these results and the
similarity of the uridine incorporation pattern reported here to the pattern
of thymidine incorporation into DNA reported earlier (Lesh-Laurie et al.
1976), we believe that, under the conditions used in this investigation, uridine
incorporation rates accurately reflect RNA synthetic rates.
The results of this investigation, then, show that amputation of distal
structures in hydra results in a rapid increase in RNA synthesis. This synthesis
remains high throughout the first 48 h of regeneration, with a large increase
in synthesis occurring between 24 and 30 h. This peak of activity closely
follows the 24-27-h peak of DNA synthesis in regenerating hydra, and immediately precedes the emergence of the first tentacle pair (Lesh-Laurie et al.
1976). Because actinomycin D is capable of suppressing regeneration completely, it may be presumed that RNA synthesis is necessary for regeneration
and therefore involved in tentacle morphogenesis.
Examination of radioactive profiles of phenol-chloroform-extracted RNA
confirmed the pattern of synthesis found using TCA-insoluble RNA. Although
bacterial contamination was present, increases in hydrid RNA synthesis were
seen to occur during the 0-4 and 28-32 h periods following subhypostomal
excision.
Moreover, this fractionation of hydrid RNA revealed the synthesis of a low
molecular weight RNA species during the 28-32 h period not present at other
times. The correspondence of this novel RNA synthesis, the increase in overall
RNA synthesis, and the emergence of the first tentacle pair strongly indicates
an interrelationship among these events.
The 8S RNA species was present again during the 36-40-h interval, a time
at which the level of RNA synthesis was relatively high, with individual
experiments showing peaks of incorporation between 36 and 42 h of regeneration
(see Fig. 1). This time period precedes the formation of the third tentacle, an
event that shows less synchrony than the emergence of the first pair. Because
of this lack of temporal consistency, the relationship of the biochemical and
morphogenetic events after 30 h of regeneration is unclear.
In conclusion, this investigation has demonstrated a clear pattern of RNA
synthesis following hypostomal amputation. The increases in total RNA
synthesis, as well as the occurrence of the low molecular weight species revealed
by RNA fractionation, preceded events of major morphogenetic significance,
i.e. wound healing and tentacle initiation. The pattern of DNA synthesis in
regenerating hydra, which is remarkably similar to this RNA synthetic pattern,
Biosynthetic events ofhydrid regeneration. II
157
is closely linked to tentacle morphogenesis in the animal (Lesh-Laurie et ah
1976). We believe that the RNA synthesis observed in these experiments is
similarly involved in the morphogenetic process in hydra. Further investigations
of this involvement employing actinomycin D to inhibit RNA synthesis have
been undertaken, as have analyses of protein synthesis and mesogleal collagen
secretion during regeneration. Results of these studies will be presented in
subsequent papers.
The authors express their gratitude to Dr Marlene Samuelson for many helpful discussions.
This work was supported by a Brown-Hazen grant from Research Corporation, an institutional grant from the American Cancer Society, and the Graduate Alumni Fund of
CWRU.
REFERENCES
BRAY, G. A. (I960). A simple efficient liquid scintillator for counting aqueous solutions in
liquid scintillation counteis. Analyt. Biochem. 1, 279-285.
CLARKSON, S. G. (1969o). Nucleic acid and protein synthesis and pattern regulation in
hydra. I. Regional patterns of synthesis and changes in synthesis during hypostome
formation. J. Embryol. exp. Morph. 21, 33-54.
CLARKSON, S. G. (19696). Nucleic acid and protein synthesis and pattern regulation in
hydra. II. Effect of inhibition of nucleic acid and protein synthesis on hypostome formation.
J. Embryol. exp. Morph. 21, 55-70.
DISCHE, Z. (1954). Color reactions of nucleic acid components. In Nucleic Acids, vol. i
(ed. Chaigaff & Davidson), pp. 285-305. New York: Academic Press.
HAUSCHKA, P. (1973). Analysis of nucleotide pools in animal cells. In Methods in Cell
Biology, vol. 7 (ed. Prescott), pp. 361-462. New York: Academic Press.
KASS-SIMON, G. (1969). The regeneration gradients and the effects of budding, feeding,
actinomycin and RNase on reconstitution in Hydra attenuata Pall. Rev. suisse Zool. 76,
565-599.
LESH-LAURIE, G. E. (1974). Tentacle morphogenesis in Hydra: a morphological and biochemical analysis of the effect of actinomycin D. Amer. Zool. 14, 591-602.
LESH-LAURIE, G. E. & HANG, L. (1972). Tentacle morphogenesis in hydra. I. The morphological effect of actinomycin D. Wilhelm Roux Archiv. EntwMech. Org. 169, 314-334.
LESH-LAURIE, G. E., BROOKS, D. C. & KAPLAN, E. R. (1976). Biosynthetic events of hydrid
regeneration. I. The role of DNA synthesis during tentacle elaboration. Wilhelm Roux
Arch. EntwMech. Org. (In the Press.)
LOOMIS, W. F. & LENHOFF, H. M. (1956). Growth and sexual differentiation of hydra in
mass culture. /. exp. Zool. 132, 555-573.
MUNRO, H. N. & FLECK, A. (1966). The determination of nucleic acids. In Methods of
Biochemical Analysis, vol. xiv (ed. Glick), pp. 113-176. New York: Interscience Publishers.
SCHMIDT, G. & THANNHAUSER, S. S. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phcsphoproteins in animal tissues, /. biol. Chem.
161, 83-89.
SNEDECOR, G. W. & COCHRAN, W. G. (1956). Statistical Methods. Ames, Iowa: Iowa State
University Press.
VOLAND, J. R. (1975). Biochemical interactions and morphogenesis in Hydra. Masters thesis,
Case Western Reserve University.
{Received 24 June 1976)
EMB 37