The role of exogenous heart-RNA in development of the chick embryo cultivated in vitro

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.

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 (0·25 M plus 0·00033 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 μ 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 OD_260mμ 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 μ 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 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 μ and stained with haematoxylin.

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:

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 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.

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 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.

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 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.

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 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 heart-RNA 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.

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-RNA-inflicted inhibition requires the participation of intact macromolecules and liver-RNA 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 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-RNA-treated 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 heart-RNA-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 liver-RNA, 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 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.

On a étudié l’effet du RNA extrait à partir du coeur de veau frais sur le blastoderme de Poulet explanté au stade de la ligne primitive.

On a observé que le RNA de coeur interfère avec le développement normal du système nerveux central, plus particulièrement avec le développement du cerveau antérieur et du système nerveux troncal tandis que le développement du coeur n’est pas affecté. Pour continuer l’analyse, des fragments antéro-latéraux du blastoderme, traités ou non par de l’ARN furent étudiés après transplantation intrablastodermique et in vitro.

Environ 50% des greffons transplantés dans des blastodermes et traités à l’ARN ont formé des coeurs; aucun n’a été trouvé dans les témoins.

Jn vitro, des structures de type cardiaque ont été trouvées dans 45% des séries, traitées par l’ARN de coeur, par opposition à 20% dans les témoins dans la solution saline PC et aucune structure cardiaque dans les séries traitées au RNA de foie.

Quand des explants de cerveau antérieur sont traités par du RNA de coeur et cultivés in vitro, 11 °/₀ développent des vésicules cervicales contre 76% chez les témoins.

Il semble donc, que le RNA de coeur ait quelque peu collaboré avec les macromolécules responsables de la formation du coeur mais ait interféré avec celles responsables du développement du système nerveux central.

This work was supported in part by a grant from The Population Council, New York City.