/. Embryo!. exp. Morph. Vol. 24, 1, pp. 33-42, 1970
33
Printed in Great Britain
The role of exogenous heart-RNA in development
of the chick embryo cultivated in vitro
By M. C. NIU 1 AND L. MULHERKAR 2
From the Department of Biology, Temple University
SUMMARY
The physiological effect of fresh calf heart-RNA was studied on the explanted chick
blastoderm at the definitive streak stage. It was found that heart-RNA interferes with normal
development of the central nervous system, especially forebrain, and of the body axis, but
not with normal development of the heart. To analyse this effect further, the untreated and
RNA-treated fragments of the antero-lateral blastoderm were investigated by intrablastodermal transplant and in vitro. Approximately 50 % of the treated grafts transplanted
intrablastodermally developed into heart, but none of the controls. In vitro formation of the
heart-like structure was found in 45 % of the heart-RNA-treated series as opposed to 20 % of
the PC saline controls and none of the liver-RNA series. When theexplants of the presumptive
forebrain were treated with heart-RNA and cultured in isolation in vitro, 11 % developed into
brain vesicle compared with 76 % of the controls. It appears, therefore, that heart-RNA has
somehow collaborated with the macromolecules responsible for heart formation but interfered with those responsible for the development of the central nervous system.
INTRODUCTION
Previous experiments have shown that heart-RNA-treated grafts of the excised
chick blastoderm develop differentially from those treated with brain-, liver- or
kidney-RNA. While heart-RNA stimulates the development of heart-like
structures, it seldom permits neural formation within 24 h of cultivation (Sanyal
& Niu, 1966). If the heart-RNA-treated explants are cultivated in vitro for
5 days, twitching is noted in almost half of them (Butros, 1965). We have lately
extended the functional study of the heart-RNA to chick embryos explanted
in vitro. The purpose of this communication is to show that heart-RNA causes
abnormal formation of the central nervous system, but does not interfere with
normal heart development of the whole chick embryo and that heart-RNA
stimulates the differentiation of the explant from the antero-lateral margin of
the area pellucida into heart but inhibits the development of the presumptive
forebrain.
1
Author's address: Department of Biology, Temple University, Philadelphia, Pa. 19122,
U.S.A.
2
Author's address: Department of Zoology, University of Poona, Poona-7, India.
E M B 24
34
M. C. NIU AND L. MULHERKAR
MATERIALS AND METHODS
Freshly fertilized White Leghorn eggs were obtained from Shaw's Hatchery,
West Chester, Pa., and incubated at 37-5 °C to get the definitive primitive streak
stage of development (Hamburger & Hamilton, stage 4, 1951). They were then
explanted and cultured in vitro by the method of New (1955). RNA from calf
organs was extracted by a modified Kirby procedure already published (Niu,
Cordova & Niu, 1961; Hillman & Niu, 1963). Fresh calf heart and liver,
brought ice-cold in sucrose solution (0-25M plus 000033 M-CaCl2) from the
slaughter house, were used for the preparation. The tissue was homogenized and
deproteinized twice with water-saturated phenol (redistilled); polysaccharides
were removed by high speed contrifugation at 2 °C (Sorvall, 20000 rev./min for
30 min). The RNA thus extracted was subjected to three washes with ether
which was removed under negative pressure. Then it was precipitated by
ethanol and redissolved in saline. UV spectrophotometric examination of the
isolated RNA gave a typical spectrum for nucleic acids with the maximum and
minimum absorption at 258 and 230 m/.i respectively. Diphenylamine test for
the presence of DNA was nil (Burton, 1956). Contamination of protein was
estimated by the procedure given by Lowry, Rosebrough, Farr & Randall,
(1951) and the amount was less than 4 %.
Experiments were done in four groups.
Group 1. 55 embryos at the definitive primitive streak stage, explanted by
New's technique, were treated for 2 h at room temperature (21 °C) by adding
about three drops of calf heart-RNA on the ventral surface at a concentration of
OD260 mfl 80/ml of Pannett-Compton saline (PC saline); during this period the
heart-RNA was changed twice to ensure proper concentration over the entire
embryo and then the embryos were incubated with heart-RNA at 37 °C for
about 20 to 22 h.
27 embryos treated with PC saline instead of heart-RNA solution served as
controls. To show the specific effect of heart-RNA, 10 embryos each were
treated in the same manner with heart-RNA (OD 80/ml) after boiling at 100 °C
for 30 min and liver-RNA (OD 80/ml). Four embryos were treated with
RNase-treated heart-RNA (Worthington Pancreatic RNase, 20/*g/ml at 37 °C
for 30 min). On account of the heterogeneity of the heart-RNA used in this
study, the digestion with pancreatic RNase resulted in a loss of approximately
85 % RNA. RNase activity was neutralized by the rabbit antiserum against
pancreatic RNase.
Group 2. Heart-RNA was fractioned into high (HMW) and low molecular
weight (LMW) fractions by 1 M-NaCl. The LMW fraction only was used as the
HMW fraction gave crystals in the concentration used in this study. A total of
75 grafts about 0-3 mm square were taken 0-6-0-9 mm lateral to the anterior •} of
the primitive streak (see Fig. 1). They were treated with or without RNA in the
following manner. 5 or 6 explants were kept in each small Petri dish containing
Role of heart-RNA in development
35
2 ml of the PC saline with or without LMW fraction of heart- and liver-RNA
(OD 28/ml) and kept in a cold room (2-4 °C) for 12-16 h for proper diffusion
of the RNA (28 explants were treated with heart-RNA, 27 with liver-RNA
and 20 with PC saline). After this treatment they were found to be healthy
and formed spherical balls. The 3 kinds of explants were grafted separately in
between the epiblast and hypoblast at the site from which they were excised.
The grafted embryos were incubated for about 20 h and then fixed in Carnoy's
fluid. The sections were cut at 8 ft and stained with haematoxylin.
Fig. 1. Diagram showing the parts removed from explanted chick blastoderm after
20-22 h of incubation at 37-5 °C. F.B. = presumptive forebrain; L.F. = anterolateral fragments of the area pellucida and also the site for grafting the PC salineand RNA-treated explants.
Group 3. 80 grafts isolated as in group 2 were cultured in isolation on an agar
base which contained medium 199 (Microbiological Associates, Bethesda) and
foetal bovine serum. 33 of these were treated with heart-RNA (OD 80/ml), 27 with
liver-RNA (OD 80/ml) and 20 with PC saline for 12-16 h in the cold room,
after which they were grown in vitro for 48 h on the nutritive medium. The
composition of the medium was as follows:
1.
2.
3.
4.
5.
Medium 199 concentrated 10 x
Foetal bovine serum
Penicillin-streptomycin solution
NaHCO3 (7-5 %)
H?O
5000 ml
5-000 ml
0-500 ml
0125 ml
39-375 ml
50000 ml
The above solution was heated carefully to 52 °C and mixed with 5 % agar
heated to 70 °C. The ratio of medium to agar was 5:1.
Group 4. Among 45 isolates of presumptive forebrain region of the definitive
3-2
36
M. C. NIU AND L. MULHERKAR
primitive streak blastoderm, 28 were treated with heart-RNA (OD 80/ml) in a
manner described under group 3 and then grown on nutritive medium for 48 h.
17 isolated were treated with PC saline only and then cultured in vitro as controls.
RESULTS
Group 1. Out of 55 embryos treated with heart-RNA 38 showed malformation,
mainly of the nervous system. In these embryos the brain did not show its
characteristic divisions such as forebrain, midbrain and hindbrain (microcephaly). The brain region showed remarkable regression to the level of the
heart (Figs. 2, 3). The optic vesicles were directed forward instead of being in
their normal lateral position. The neural tube remained open. Shortening of the
body axis was quite evident in 38 cases. The axial mesoderm appeared as a
Figs. 2 and 3. Photographs of explanted chick embryos showing the effect of calf
heart-RNA on their development, 22 h after RNA application. H = heart and
Op = optic vesicle, x 24.
Fig. 4. Photograph of the control of Figs. 2 and 3. S = somites; H = heart and
Op = optic vesicle, x 24.
Role of heart-RNA in development
37
diffuse mass and no segmentation was noted in 32 cases. The heart on the contrary developed normally with some distortion in 29 cases and was beating in all
the cases before fixation; 9 embryos developed a heart which was relatively
smaller in size. 11 embryos were abnormal and 6 embryos died.
I
0-1 mm
I
Fig. 5. Camera lucida drawing of a cross section of the heart-RNA-treated graft
(beating heart). B = red blood cells; E = endocardium and EM = epimyocardium.
Fig. 6. Photograph showing a cross section of the graft in the explanted chick
embryo. H = heart, x 130.
All 27 embryos without treatment with heart-RNA and kept as controls
showed normal development (Fig. 4). Out of 10 embryos treated with boiled
heart-RNA, 8 showed perfectly normal development. The axis was short in one
and one died. The 10 treated with calf liver-RNA developed in the same manner
38
M. C. NIU AND L. MULHERKAR
as those of the PC saline series. 3 of the 4 embryos treated with RNase-digested
heart-RNA developed normally and one had an abnormal heart. The latter was
apparently due to the presence of 15 % undigested RNA.
Group 2. A total of 28 antero-lateral grafts were treated with heart-RNA and
grafted intrablastodermally. 14 differentiated into heart (7 into heart only and
7 into heart and neural tissue as well), 5 into neural tissue, 4 remained as a mass
of cells and 5 were absorbed into the body of the host. Heart tissue was identified by its contractions (Fig. 5) or by structure (Fig. 6) in 9 cases. 7 of the
27 liver-RNA-treated grafts developed neural tissue; 7 formed a mass of identifiable cells and 13 were incorporated into the tissue of the hosts. 20 untreated
grafts were incorporated into the host.
Figs. 7 and 8. Camera lucida drawings of 2 cross sections from 2 different explants
treated with heart-RNA. B = blood cells; E = endocardium and EM = epimyocardium.
Fig. 9. Camera lucida drawing of the control of Figs. 7 and 8.
Group 3. A total of 33 antero-lateral explants were treated with heart-RNA
and grown in isolation. 10 developed into heart tissue and of these 5 developed
heart plus red blood cells (Figs. 7, 8); 4 explants developed into red blood cells
only, 5 into cells arranged in a palisade and 14 appeared as a mass of cells
without any definitive patterns. 3 of the 10 explants with heart tissue were beating
at the time of fixation. Out of the 27 explants treated with liver-RNA, 6 showed
a definite pattern of cells surrounding small cavities or sinuses, 1 underwent
Role of heart-RNA in development
39
neutralization and 20 remained as a mass of cells. The PC saline treated controls
showed 4 with heart-like cavities and 16 appeared as a mass of cells (Fig. 9). No
palisade arrangement of cells was evident.
Group 4. Out of 28 explants of the presumptive forebrain treated with heartRNA and grown in isolation only 3 showed the formation of brain vesicle
(Fig. 11) (forebrain?), 13 formed small vesicles whose neurocoel had filled with
cells (Fig. 10), and 12 remained as a solid mass of unidentifiable cells. 13 of the
17 controls differentiated into brain vesicle and 4 remained as a mass of cells.
Figs. 10 and 11. Camera lucida drawings of one section each from the heart-RNAtreated and control explants of the presumptive forebrain cultivated in nutritive
medium for 48 h. Note the presence of cellular elements in the smaller brain vesicle.
DISCUSSION
All of the untreated chick embryos explanted in vitro developed as normally as
those in vivo. In the RNA-treated embryos 1 out of the 10 treated with boiled
heart-RNA and 6 of the 55 treated with heart-RNA died. These few deaths were
probably not due to the RNA treatment per se, but more likely were the result
of the general operational handling. The fact that the embryos treated with
heart-RNA showed abnormalities (78%) while those treated either with PC saline,
boiled heart-RNA, RNase-treated heart-RNA or liver-RNA showed normal
development suggests that the malformations produced in the embryos are due
to the effect of heart-RNA. The next question concerns the specificity of this
effect. Results such as microcephaly, opening of the neural tube or shortening
of the body axis cannot be said to be specific to heart-RNA as many other
substances such as analogues of purine and pyrimidine metabolism have been
shown to cause such abnormalities (Waddington, Feldman & Perry, 1955;
Blackwood, 1962; Billet, Collini & Hamilton, 1965; Rao, 1967). Apparently
both RNA and the analogues are interfering with normal RNA metabolism of
the developing nervous system. It should be said, however, that the heart-RNAinflicted inhibition requires the participation of intact macromolecules and liverRNA does not cause similar inhibition. On the other hand, liver-RNA has been
shown to inhibit tumour growth (Olenov, 1968; Niu, 1969). We believe both of
40
M. C. NIU AND L. MULHERKAR
these macromolecules act upon the target cells directly. A test for the direct
effect was carried out on in vitro cultures of the control and heart-RNAtreated presumptive forebrain. 13 of the 17 control explants developed into brain
vesicle (76 %), 1 developed into a smaller vesicle with the neurocoel filled with
cells (6 %) and 3 remained as a solid mass of cells. In contrast, 3 of the 28 heartRNA-treated explants developed into brain vesicle (11 %) and 13 into smaller
vesicles with cells (46 %). These data would indicate that heart-RNA acts upon
the cells of the presumptive forebrain and thus interferes with the normal
development of forebrain.
The formation of distorted heart was considered to be the secondary consequence of the axis shortening. Of all the embryos treated with heart-RNA
approximately 78 % had developed a normal sized beating heart at the time of
fixation. Even in extreme cases of microcephaly and axis shortening the distorted hearts were beating regularly. A more convincing picture of the specific
action of the heart-RNA could be seen from the results of intrablastodermal
grafting. About 50 % of the grafts developed into heart and none of the controls.
Similar results were obtained from the posterior third of the primitive streak
(Sanyal & Niu, 1966). If, on the other hand, the grafts were treated with liverRNA, 25 % became neuralized but none developed into heart tissue. Furthermore, when the antero-lateral fragments were cultured in vitro, formation of
heart-like structure with endo- and epimyocardium and/or blood cells occurred
in 15 of the 33 explants treated with heart-RNA (45 %) as opposed to 4 out of
20 in the controls (20%). The development of heart-like tissue in the control
series is explained on the grounds that the explants contain some heart-forming
cells (Rawles, 1936, 1943; Mulherkar, 1958), as the tendency for heart formation at the stage used is extended laterally over a wide area (Ebert, 1953). The
RNA-induced increase from 20 to 45 % in heart formation is specific because
liver-RNA has never induced heart development.
It seems paradoxical to note that the control explants develop, in vitro, into
heart-like tissue in 4 of 20 cases, but none in the 20 when each was grafted into the
explanted chick blastoderm. This disparity was caused by the difficulty of locating
the graft at the site of implantation. Disappearance of the graft might be due
either to cell death or to emigration. Examination of the graft at intervals did
not reveal evidence of cell death, but a gradual reduction in the size of the
grafts did occur. Since the operation was done at such an early stage when the
morphogenetic movements were active, it is highly possible that the disappearance of the grafts is associated with the morphogenetic movements of the
host cells.
The report that freshly isolated RNA was incapable of initiating differentiation in the excised chick blastoderm (Hillman & Hillman, 1967) is not contradictory to the data presented here and those previously described (Niu et al.
1961; Butros, 1965; Sanyal & Niu, 1966; Niu & Leikola, 1968) because {a) they
cultured the explants in Tyrode solution and this balanced salt solution is
Role of heart-RNA in development
4]
insufficient for support of growth or differentiation of the chick tissue and (b) their
results were obtained from experiments using explants without pre-treatment
with RNA. Our pre-treating the explants with RNA is, in some ways, similar to
the procedure of infecting cells with virus or viral RNA first and studying the
viral-RNA induced transformation in the cells. Furthermore, the procedure for
RNA isolation used by Hillman & Hillman (1967) has already been reported
(Niu et al. 1961; Hillman & Niu, 1963) and is routinely used in our laboratory.
RESUME
Le role du RNA exogene de coeur dans le developpement de
Vembryon de Poulet cultive in vitro
On a etudie 1'effet du RNA extrait a partir du coeur de veau frais sur le blastoderme de
Poulet explante au stade de la ligne primitive.
On a observe que le RNA de coeur interfere avec le developpement normal du systeme
nerveux central, plus particulierement avec le developpement du cerveau anterieur et du
systeme nerveux troncal tandis que le developpement du coeur n'est pas affecte. Pour continuer Panalyse, des fragments antero-lateraux du blastoderme, traites ou non par de TARN
furent etudies apres transplantation intrablastodermique et in vitro.
Environ 50 % des greffons transplanted dans des blastodermes et traites a I'ARN ont
forme des coeurs; aucun n'a ete trouve dans les temoins.
In vitro, des structures de type cardiaque ont ete trouvees dans 45 % des series, traitees par
I'ARN de coeur, par opposition a 20 % dans les temoins dans la solution saline PC et aucune
structure cardiaque dans les series traitees au RNA de foie.
Quand des explants de cerveau anterieur sont traites par du RNA de coeur et cultives in
vitro, 1.1 % developpent des vesicules cervicales contre 76 % chez les temoins.
II semble done, que le RNA de coeur ait quelque peu collabore avec les macromolecules
responsables de la formation du coeur mais ait interfere avec celles responsables du developpement du systeme nerveux central.
This work was supported in part by a grant from The Population Council, New York City.
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