/ . Embryol. exp. Morph. Vol. 29, 2, pp. 485-501, 1973
Printed in Great Britain
485
The development of tubular heart in RNA-treated
post-nodal pieces of chick blastoderm
By M. C. NIU 1 AND A. K. DESHPANDE 1
From the Department of Biology, Temple University, Philadelphia
SUMMARY
Post-nodal pieces of the stage-4 chick blastoderms were transected 0-6 mm posterior to
Hensen's node and cultured in vitro with and without chicken-heart RNA. After 24 h
the explants were fed daily with fresh egg-extract medium for 4 days. On the 4th day pulsating
heart was found in the RNA-treated but not in the untreated series. Histological examination
revealed that cell differentiation other than of erythrocytes and epithelial tissue seldom
occurred in the control and, in contrast, pulsating heart and cardiac myoblasts were present
in most of the RNA-treated explants that had differentiated.
INTRODUCTION
In previous experiments (Niu & Mulherkar, 1970), ribonucleic acids (RNA)
were isolated from heart and liver and used separately to treat the anterolateral
fragments of chick blastoderm at the definitive streak stage. Both RNA-treated
and untreated explants were cultured in vitro. Only heart RNA was found to
promote the development of pulsating tissue. The heart-forming capacity of
heart RNA was sensitive to pancreatic ribonuclease. The point of interest is,
however, that no organizing action into pulsating tube resembling normal
heart took place. Three possible explanations have been seriously considered,
namely (1) the number of cells used was too small to go through the process of
organogenesis, (2) the length of cultivation was too short to achieve organogenesis and (3) the heart RNA used might contain very low amounts of informational RNA. To overcome these disadvantages, we have now used larger postnodal pieces (PNP), longer periods of cultivation and nuclear RNA.
Furthermore, special precaution was undertaken to excise PNP, avoiding the
inclusion of presumptive heart-forming tissue. This communication summarizes
the data, showing that PNP by itself did not differentiate into cardiac tissue
and that nuclear (nRNA) and cytoplasmic (cRNA) fractions of heart RNA
initiated the differentiation of pulsating tube and non-beating cardiac tissue as
well. Furthermore, electron-microscopic examination of the beating tissue
revealed fine structure typical of cardiac muscle cells.
1
Authors' address: Department of Biology, Temple University, Philadelphia, Pa. 19122,
U.S.A.
486
M. C. NIU AND A. K. DESHPANDE
PNP
Fig. 1. Diagram of the stage-4 chick blastoderm indicating the level of the
transection (C C).
MATERIALS AND METHODS
Fertilized White Leghorn eggs were obtained from Shaw's Hatchery, West
Chester, Pa., and incubated at 38 °C until they reached the definitive primitive
streak stage of development (Hamburger & Hamilton, 1951, stage 4). The
blastoderms were explanted and the area opaca was removed. The area pellucida
was cut transversely 0-5-0-6 mm posterior to the Hensen's node (Fig. 1), thus
making sure to exclude the presumptive heart-forming areas (Rawles, 1943;
Mulherkar, 1958; DeHaan, 1968; Rosenquist, 1970). The post-nodal piece
(PNP) thus obtained was transferred to, and flattened with, epiblast resting on
vitelline membrane, previously mounted around a glass ring. Outside the ring,
1 ml of nutritive medium was added. This method of cultivation is a modification
of the procedures of New (1955) and Chauhan & Rao (1970). The nutritive
medium used was egg extract which was prepared by mixing a whole unfertilized
egg with 50 ml of Ringer solution. The homogenate was centrifuged at 2000
rev/min for 30 min at 4 °C. The supernatant was mixed with an equal volume of
Pannett-Compton (1924) solution (PC).
Preparation of heart RNA
Chicken heart was excised immediately after slaughtering and pooled in
ice-cold isotonic sucrose solution (0-25 M +0-001 M-MgCl2). Unless specified
otherwise, preparation of RNA was carried out in the cold room at 4 °C.
RNA-treated chick blastoderm
487
Connective tissue and fat were removed from the hearts. Batches of 70 g were
chopped up with scissors into fine pieces and blended in a Waring Blender with
4 vols. of sucrose solution (0-32 M, +0-003 M-MgCl2 and 0-5 % diethyl pyrocarbonate). While blending at high speed for 30 sec, 2 more vols. of sucrose solution
were added and the process continued for 90 sec. The homogenate was filtered
through 1, 2 and 4 layers of cheese cloth and then 2 layers of flannel. The filtrate
was centrifuged using the International PR 2 centrifuge at 4 °C, 2000 rev/min
for 30 min. The supernatant fluid was suctioned off" and used for isolation of
cRNA. The sediment was saved for preparation of nRNA.
Isolation of nRNA
To the above sediment 50-100 vols. of sucrose solution (0-32 M +0-003 MMgCl2) were added and thoroughly mixed. Crude nuclei were packed in a
refrigerated International PR 2 centrifuge at 4 °C, 1800 rev/min, 7 min, and
washing was repeated twice. The loose sediment was packed firmly (30 min)
for volume determination, and 10 vols. of 2-4M sucrose + 0001 M-MgCl2 were
used to suspend the crude nuclei evenly. Pure nuclei were collected in a Spinco
L2-65B ultracentrifuge at 4 °C, 40000 rev/min for 1 h, and washed twice with
buffered saline (0-14M NaCl and 001 M Tris, pH 7-1). The insoluble material,
sedimented in a Sorvall RC-2 centrifuge at 5000 rev/min for 20 min, was suspended in 10 vols. of SDS (sodium dodecyl sulphate) buffer (0-5% SDS,
0-1 M-NaCl, 0-01 M acetate buffer, pH 5-2, 0-001 M - E D T A ) . An equal volume of
water-saturated phenol (redistilled) was added, and immediately blended in a
Lourdes mixer at 30 V for 5 min. Another equal volume of chloroform containing 1 % isoamyl alcohol was added. This mixture was again blended at 30 V
for 5 min at room temperature and centrifuged at 2000 rev/min for 10 min. The
bottom phenol chloroform phase was aspirated out. An equal volume of chloroform-isoamyl alcohol was added to the aqueous and interphase layers and
blended at 30 Y for 5 min. The process was repeated 3 times. To the aqueous
layer from the last extraction were added 2 vols. of 95 % ethanol and K-acetate
(up to 2 %) and the fraction was stored at - 20 °C.
Isolation of cRNA
To the above supernatant an equal volume of phenol was added and blended
in a Lourdes mixer at 60 V for 5 min. The mixture was centrifuged at 1800 rev/min
for 30 min, at 10 °C, and the top aqueous layer saved. To the interphase and
phenol layers \ vol. of saline (0-9 % NaCl) was added and blended at 35 V for
5 min. The supernatant fluid was combined with the previous and once again
extracted with £ vol. of phenol as before. The aqueous layer was removed after
centrifugation and kept at - 2 0 °C after addition of 95 % ethanol (2 vols.) and
K-acetate (up to 2 %).
cRNA from the deep-freeze was centrifuged. The precipitate was redissolved
in saline. Glycogen was removed by centrifugation (Sorvall RC-2) at 2 °C,
488
M. C. NIU AND A. K. DESHPANDE
18000 rev/min for 20 min. The supernatant was washed with ether 3-5 times.
Ether was expelled under negative pressure. Both nRNA and cRNA were again
treated with DNase. The enzyme was denatured by chloroform containing 1 %
isoamyl alcohol. RNA was reprecipitated by 2 vols. of 95 % ethanol and washed
with 67 % ethanol. The sediment was redissolved in saline. U.v. spectrophotometric examination of the RNA solution revealed that both preparations were
typical of nucleic acids. Tests with the Lowry procedure showed that nRNA
contained about 2 % and cRNA 1 % of protein. The Dische reaction indicated
that 1-1-5 % of DNA was present in nRNA, but none could be detected in
cRNA. The amounts of RNA calculated from the orcinol reaction and from
the optical density reading at 260 nm differed less than 10 %, thus indicating
that RNA was essentially the only substance in the solution.
Brain nRNA was prepared in the same way as the heart nRNA.
RNA treatment
PNPs were prepared as described above with 1 ml of nutritive medium
added outside the vitelline membrane. The RNA was dissolved in PC solution
(80 o.D./ml) and applied 0-1 ml inside and 0-1 ml outside the glass ring. Chicken
heart RNA isolated from nuclei (nRNA) and cytoplasm (cRNA) were used in
this study. For functional comparison, brain nRNA was also used. Control
series of PNPs received 0-2 ml of PC or HR (Howard Ringer; Howard, 1953)
instead of RNA solution. All cultures were incubated at 37-5 °C. The medium
was replaced every 24 h by feshly made Qgg extract. Daily observation was made
prior to the change of medium. At the end of the 5th day (120 h) the explants
were fixed in Bouin's fluid. Some of them were stained and mounted in toto for
photography. They were later embedded in the same way as others. Serial
sections were cut at 6 /tin and stained with hematoxylin and eosin.
Fine structure of the RNA-induced beating tissue
Six explants of the pulsating tissue were fixed in 2-5 % buffered glutaraldehyde
(0-1 M-Na cacodylate, pH 7-4) for 1 h at 0 °C. At intervals of 30 min they were
washed 3 times in the same buffer containing isotonic sucrose and then postfixed
in cold 1 % OsO4 in cacodylate buffer for 1 h at 0 °C. Dehydration was carried
through a graded series of alcohols and the tissue embedded in Araldite. Thin
sections for electron microscope were cut with a glass knife on a Porter-Blum
Ultra Microtome MT-2, mounted on 200-mesh grids coated with a thin layer
of formvar and carbon, stained with lead citrate and examined in a Philips
EM-300 microscope.
RNA-treated chick blastoderm
489
H
\
Fig. 2
Fig. 3
Fig. 2. Whole mount of a PNP cultured in the egg-extract medium for 5 days.
None of the axial structures were seen, x 25.
Fig. 3. Whole mount of a PNP treated with chicken-heart nRNA and cultured
for 5 days. The prominent heart tube (H) was pulsating from the 4th to the fixation
time at the end of the 5th day. The dark stained area at top is due to the accumulation
of blood. x25.
RESULTS
1. Control series
(a) PC solution
A total of 48 PNPs were explanted for this study. Daily observation throughout the period of incubation did not reveal the presence of twitching tissue in
this series (Fig. 2). Red blood cells (RBC) became visible as red patches on the
third day of incubation.
Histologicalfindings. Serial sections of the 48 PNPs were examined. RBC and
hemopoietic tissue (bf, Fig. 4 A) were present in all cases. Forty-five of the 48
had a thin layer of ectoderm and 1-2 layers of endoderm (94 %). Mesoderm
appeared as undifferentiated condensation (me, Fig. 4B) or a loose network of
mesenchyme. Three of the 48 PNPs (6 %) differentiated further. One formed
neural tube, one notochord, and one both neural and chordal tissue.
(b) HR solution
A total of 13 PNPs were examined. None of these had beating tissue, or
myoblasts. Three explants had neuroid tissue and notochord, one each had
neuroid tissue or tubules and four had notochord only. The frequency of differentiation was 69 %.
490
M. C. NIU AND A. K. DESHPANDE
Fig. 4. Photomicrographic sections of two control PNPs (A and B). b, Red blood
cells; bf, blood forming tissue; ec, ectoderm; en, endoderm; and me, condensed
mesenchyme. x 120.
2. Experimental series
(a) Differentiation of the PNPs treated with chick-heart tiRNA
Fifty-one PNPs were treated with nRNA (Tables 1, 2 pages 494 and 498). Nineteen of them were either beating or twitching in some localized area. Nine of
the explants had well-defined pulsating tubes (Fig. 3) and ten beating patches.
Pulsation was noted first on the 4th day of incubation and invariably persisted
throughout the period of cultivation. The rate of beating averaged 20-25/min
and gradually slowed down during observation in room temperature.
For a comparison, heart nRNA was also dissolved in HR and used to treat
12 PNPs. Five of the 12 developed pulsating tissue, 2 with non-beating cardiac
tissue, 7 with neuroid tissue, 6 with tubule and 4 with notochord (Table 3).
Histologicalfindings. Examination of serial sections of the 51 explants revealed
that differentiation had occurred in 40 (78 %). The distribution of various organs
in nRNA-treated explants is portrayed by Table 2. It can be seen that 19 explants
had beating hearts (48 %) and 11 had non-beating heart tissues (27 %). Twelve
of the 40 differentiated explants contained beating heart only and 5 contained
neuroid only. When explants containing beating and non-beating hearts only
are combined, there are 20 out of 40, i.e. 50 % with heart only. The frequency
of tubule and neuroid formation was 23 % and 30 % respectively (Table 1). A
cross-section of a pulsating heart tube is shown in Fig. 5.
The beating tissues observed in this study resembled those obtained from
the heart-forming graft in chorio-allantoic membrane (Rawles, 1943). They are
RNA-treated chick blastoderm
491
V
Fig. 5. Section of a chicken-heart nRNA-treated PNP passing through bent heart
tube. E, Endocardium; EM, epimyocardium; H, tubular heart, x 130.
Fig. 6. Section of the PNP treated with chicken heart cRNA, showing loosely
arranged beating tissue, m, Cardiac myoblasts. x 130.
Fig. 7. Section of the PNP treated with chicken heart nRNA, with compact beating
tissue, m, Cardiac myoblasts. x 130.
Fig. 8. Section of the PNP treated with chicken brain nRNA. Note the predominant
neural differentiation. NT, Neural tube; P, neural plate; me, mesenchyme. x 130.
492
M. C. NIU AND A. K. DESHPANDE
either loosely arranged (Fig. 6) or compact (Fig. 7). In early stages of heart
development, myofibrils and cross-striation of myocardiac cells can hardly be
discerned by light microscopy (Goss, 1938, 1940; Copenhaver, 1939; Manasek,
1970). Our identification of cardiac tissue was based primarily upon rhythmic
beating and accompanied by (1) histological observation of interlacing strands
and distinctive nuclei and (2) study of fine structure by electron microscopy.
Through the latter, we learned that the heart-RNA-induced beating tissue had
properties typical of cardiac muscle. They are (1) large numbers of individual
glycogen granules, both dispersed and accumulated in large masses or pools
(G, Fig. 9). These accumulations are frequently associated with lipid droplets
(L); (2) both orderly and randomly arranged myofibrils (F) are present in cytoplasm; and (3) cell to cell attachments occur at specialized zones called intercalated discs (/) and desmosomes. Intercalated discs are cell to cell junctional
complexes. Desmosomes represent that portion of the junction where the two
opposed plasma membranes lie parallel and have dense plaques, separated by a
gap of extracellular space which is filled with fibrillar proteinaceous material.
Myofibrils insert into the intercalated disc at right angles (see inset, Fig. 9).
Histogenesis of myofibrils in the developing myocardiac cells is depicted by
three electron micrographs (Figs. 10-12). Fig. 10 shows the tight junctional
complexes between adjacent cells. Both intercalated discs (/) and Z-bands (Z) are
recognizable here. Myofibrils appear in unorganized, random fashion. It seems,
however, that they tend to be attaching to / a t the right top corner of the picture.
As fibrogenesis progresses and Z bands become readily discernible, myofibrils
often converge to them from different directions (Fig. 11). Extension of myofibril
is accomplished through crystallization of amorphous proteinaceous material
(Am, Fig. 12).
(b) Differentiation of the PNPs treated with chick-heart cRNA
A total of 37 PNPs were treated with cRNA and 12 of them were beating.
Five of the 12 were tubular. Pulsation started on the 4th day. The rate of beating
was similar to that obtained from nRNA-induced heart. The types of organs
formed in the RNA-treated PNP explants are criss-cross classified in Table 2.
Fifteen of the 23 differentiated explants (65 %) contained heart tissue only. If
explants with cardiac tissue, notochord and neuroid are separately combined,
the frequencies of the three tissues were 78 %, 22 % and 22 % respectively
(Table 1). In other words, heart formation occurred 3-5 times more frequently
than the development of either notochord or neuroid.
(c) Differentiation of PNPs treated with chicken-brain nRNA
Twenty of the 23 brain-nRNA-treated PNPs underwent differentiation (Tables
1,2). No explants had either beating tissue or tissue resembling myocardiac cells.
Histological examination disclosed that 9 of the 20 differentiated explants had
neuroid only (45 % - Fig. 8). The frequency of neural formation, was 85 %.
RNA-treated chick blastoderm
493
Fig. 9. Electron micrograph of cells from the heart-RNA induced beating tissue,
x 16000. The inset shows an enlarged intercalated disc with myofibril attached at
right angle. F, Myofibril; g, Golgi body; G, glycogen; /, intercalated disc; is, intercellular space; L, lipids; M, mitochondria; N, nucleus; Z, Z-band. x 65000.
E M B 29
494
M. C. NIU AND A. K. DESHPANDE
Table 1. Developmental potentiality of chick blastoderm (PNPs) in
presence and absence of RNA, cultured in vitro for 5 days (120 h)
Series
No. of No. of differentiated PNPs showing development of various organs
PNPs ,
*
>
differenCardiac tissue
A
v
tiated ,
and no. Beating Non-beating Tubule
Somite Notochord Neuroid
cultured
Control (PC)
3/48
0
0
0
(6%)
Heart nRNA 40/51 19(48%) 11(27%) 9(23%)
(78 %)
30 (75 %)
Heart cRNA 23/37 12 (52 %) 6 (26 %)
0
(62%)
18(78%)
Brain nRNA 20/23
0
0
7 (35 %)
(87%)
0
3(8%)
2
2
2(5%)
12(30%)
0
5 (22 %)
5 (22 %)
1 (5 %)
6 (30 %)
17 (85 %)
DISCUSSION
Cellular components of PNP, transected at the level 0-4 mm posterior to
Hensen's node, consist of epiblasts, hyoblasts and migrating cells (presumptive
mesoderm). In the culture medium of plasma and embryonic extracts, they
developed into epithelium, connective tissue, and red blood cells (Murray, 1932).
When they were grown in freshly prepared egg extract, some neuroid tissue
and/or notochord developed (Chauhan & Rao, 1970). In the experiments of
Chauhan & Rao the frequency was 3 % and in ours 6 %. These frequencies are
too low to differ statistically from the results of Murray. The PNPs used in the
present report, transected 0-6 mm posterior to the node, contained some cells
capable of synthesizing myosin (Ebert, 1953) or thymidine labeled cells that
contribute to heart formation in normal development (Rosenquist, 1970), but
were unable to undergo differentiation. Should the transection be made 0-2 mm
posterior to the node, thus allowing the inclusion of various organ-forming cells,
there would be axial organs and some pulsating tissue developed (Butros, 1965,
and others). Similarly, the formation of beating tissue in the control explants
from anterolateral blastoderm could be due to the inclusion of some heartforming cells (Niu & Mulherkar, 1970). The increase of heart formation in the
heart-RNA-treated explants should result from the action of exogenous RNA.
In the latter case it may act on the recipient cells, reinforce the myosin-producing
cells and/or inhibit others with different potentiality.
FIGURES
10-12
Electron micrographs of cells from the heart-RNA induced beating tissue.
Am, Amorphous proteinaceous material; D, desmosome; Ri, ribosomes and
others same as Fig. 9. x 41000.
Fig. 10. For legend see p. 494.
Fig. 11. For legend see p. 494.
o
m
o
>
2
o
4
Fig. 12. For legend see p. 494.
I
o
498
M. C. NIU AND A. K. DESHPANDE
Table 2. Distribution of the differentiated organs in PNP explants
treated with RNAs
Heart nRNA Heart cRNA Brain nRNA
(40 of 51
(23 of 37
(20 of 23
PNP explants PNP explants PNP explants
differentiated- differentiated - differentiated 78 %)
62 %)
87 %)
Kinds of organs differentiated
Beating heart
Beating heart
Beating heart
Beating heart
Total
only
with neuroid
with tubule
with neuroid and tubule
Non-beating cardiac
Non-beating cardiac
Non-beating cardiac
and/or somite
Non-beating cardiac
Total
12 (6-tube)
3 (3-tube)
2
2
19(48%)
tissue only
tube with notochord
tissue with tubule
tissue with notochord
12(52%)
0
0
0
0
0
8 (8-tube)
4 (3-tube)
1
0
0
3
0
0
1
0
0
6(26%)
0
1
0
1
1
0
0
0
0
0
0
1
1(4%)
0
2
0
5
2
0
2
1
9
7(18%)
4(17%)
17 (85 %)
1
2
0
0
0
3 (8 %)
Neuroid and tubule
Neuroid and notochord
Neuroid, notochord and tubule
Neuroid
Total
0
0
1
0
11(27%)
Tubule only
Tubule and somite
Notochord and tubule
Notochord and somite
Notochord only
Total
11 (5-tube)
3 (15 %)
4
3
Table 3. Developmental potentiality of chick blastoderm (PNPs) in
Howard Ringer with and without nRNA cultured for 5 days
No. showing development of various organs
No. of PNPs ,
"
differentiated
Cardiac tissue
A
,
»
and no.
Experimental series
cultured
Beating Non-beating Tubule Notochord Neuroid
Without nRNA
Chick heart nRNA
9/13
(69%)
10/12
(83 %)
0
0
5(40%) 2(16%)
7(56%)
1(8%)
7(51%)
4(30%)
6(48%)
4(32%)
7(56%)
RNA-treated
chick blastoderm
499
PC solution was replaced by HR. When the medium thus obtained was
employed to grow PNPs, no beating tissue has been observed. However, the
frequency of differentiation was high (69 %, Table 3). The difference in effect
between PC- and HR-made egg extract on PNP is very striking. Experimental
analysis of the responsible factor(s) is under way. From the viewpoint of
functional analysis of RNA or other molecules, it appears that the use of PC is
preferable.
The second reason for using PNP in the study of specific differentiation is its
clearly defined boundary, about one-quarter of the area pellucida, in which there
are sufficient numbers of cells under inductive influence to undergo morphogenesis. When small numbers of cells are used, tissue differentiation (histogenesis) instead of organogenesis prevails (Grobstein & Zwilling, 1953). This
has been well illustrated by the formation of beating tissue instead of tubular
heart in chorio-allantoic grafts of small pieces of the heart-forming areas
(Rawles, 1943). The RNA-induced formation of beating tissue but not beating
tube (Butros, 1965; Niu & Mulherkar, 1970) can similarly be explained.
The function of isolated RNAs from adult chickens was investigated by
applying heart and brain RNAs separately onto excised PNPs. They responded
to brain RNA by forming mostly neural tissue and to heart RNA by developing
predominantly into beating and non-beating hearts, thus establishing the organspecificity of RNAs. Furthermore, both kidney (Deshpande & Niu, 1971) and
thymus RNAs (unpublished) were incapable of inducing the formation of
tubular heart and/or beating tissue.
In a recent paper, Jacobson & Duncan (1968) emphasized the lequirement of
specific heart inducer in the development of heart primordium in the newt,
Tarica trosa. According to these authors, this heart inducer came from anterior
endoderm and other embryonic tissue surrounding the heart primordium.
Apparently the specific heart inducer they were discussing differs from the heartforming RNA used in our experiments. Besides, the hypoblasts in our PNP
seem to contribute to the formation of posterior endoderm and can hardly play
any influence on the RNA-induced heart formation.
Preliminary experiments using Poly A-attached mRNA suggest that the
induced formation of beating heart could be as high as 90 %. This implies that
exogenous heart RNA transforms PNP into heart tube. The RNA synthesized
in the developing heart is responsible for the synthesis of heart-specific proteins
(enzymes). It appears that the exogenous heart-RNA-treated PNP has acquired
the capacity to synthesize heart proteins continuously.
Heart RNA was separated into nRNA and cRNA. The organs developed in
the cRNA-treated PNPs are heart, notochord and neuroid. The nRNA series
has, in addition, somite and tubule (Table 1). The frequency of the beating and
non-beating heart formation is 30 out of the 40 explants (75 %) in the nRNA
series and 18 of 23 (78 %) in the cRNA (Table 1). Fifty per cent of the nRNA
treated explants have heart developed only and 65 % in the cRNA series. These
500
M. C. NIU AND A. K. DESHPANDE
data would suggest that cRNA is more selective than nRNA in the initiation of
heart formation. Functional selectivity is a measure of the information that
RNA carries. In this sense, the heterogeneous cRNA is likely to contain more
messenger RNA than nRNA. This is supported by recent advances in methodology used to isolate messenger (m) RNA, Poly A-attached RNA, from polysomes of the cytoplasm (Rosenfeld, Comstock, Means & O'Malley, 1972).
The RNA thus isolated was the only mRNA in our hands that initiated the
differentiation of PNPs into beating tissue and/or tubular heart.
This work was supported by research grants from The Population Council and The National
Foundation. The authors wish to thank Dr Tze Lin for his aid in the isolation of nuclear
RNA and Mrs Sharon L. Howard for her volunteer in histological service. Deep appreciation
goes to L. C. Niu for her expert analysis of the fine structure of the induced heart tissue.
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^
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*
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{Received 1 September 1972, revised 31 October 1972)