1. RNA was prepared from chick embryo hearts and livers respectively by a modified procedure of Kirby.

  2. Post-nodal fragments, obtained by transecting definite streak or heard process blastoderms at 0·2 mm. posterior to the primitive pit and trimmed to 1 × 1 mm.2, were cultured for 8 days on a basic medium containing TC 199, whole egg suspension, embryo extract, agar and penicillin.

  3. Half of the cultured pieces received one drop of either RNA twice daily; the other pieces were given a drop of the solvent.

  4. Heart RNA promoted twitching in localized areas. Differentiation of myoblasts and nerve fibres was enhanced.

  5. Liver RNA stimulated the folding of the entoderm layer and modification of the cells to columnar, goblet form.

  6. The results are discussed in the fight of current literature on transformation in cells of higher organisms.

The posterior segment isolated from definitive streak or head process blasto-derms by a transverse cut 0·5–0·2 mm. posterior to Hensen’s node seems to be a suitable experimental object for the study of differentiation. Its limited pro-spective significance and potency in chorioallantoic transplantation or in vitro compared with the versatile node has been the subject of several studies (Hunt, 1931; Willier & Rawles, 1931; Rawles, 1936; Rudnick, 1938a, b; Waddington 1933; Spratt, 1952). The present author has shown that rooster testis DNA and 5-day chick embryo DNA (0·25–0·50 mg./ml.) induce neural tubes and somite-like aggregations of the mesoderm in post-nodal explants cultured on albumin-agar (Butros, 1960). When axial structures were grafted on the explants, the latter were induced to develop nephrotomes, ducts, neural folds and notochords (Butros, 1962). Improved procedures for culturing post-nodal explants, which circumvent the tendency of these pieces to form hollow vesicles, were reported recently (Butros, 1963a). A programme was then initiated for the study of the action of nucleic acids extracted from chick embryo organs on these fragments.

As a first approach to the problem, the fragments were cultuted in a protein-deficient medium containing the RNA for 2 days and were then grafted on to the chorioallantoic membrane for a second period of about 9 days. Embryonic brain RNA produced in the treated tissues papillomatous epidermis with giant epidermal cysts that were keratinized and highly fibrillar. Adult heart RNA caused hyperplasia of the epidermis with some keratinized, papillary projections. In the control fragments there was no keratinization nor papillary formation of the epidermis, the cysts were very small and devoid of fibrillar organizations. Adult pancreatic RNA enhanced the development of the entodermal layer (Butros, 19636). It is the purpose of this report to describe the action of embryonic liver RNA and embryonic heart RNA on isolates obtained by transection somewhat more anteriorly than in the previous reports (0-2 mm. posterior to the primitive pit) and cultured in vitro throughout the experimental period with no further transplantation on the chorioallantoic membrane.

Preparation of the RNA

The following procedure, based on Kirby’s phenol method (Kirby, 1956), was used to extract heart RNA and liver RNA respectively from over a hundred, 8–12-day chick embryos. The organs were frozen on dry CO2 and all subsequent operations were done in the cold. Homogenization was performed in 3 vol. of water in Servall omni-mixer for 60 sec. after which the material was stirred slowly while receiving an equal volume of 90 per cent, liquified phenol. Moderate stirring continued for 1 hr. followed by centrifugation at 12,000 rev./min. for 20 min. The aqueous layer was removed by suction and the residue washed with a little water and re-centrifuged three more times repeatedly, each time collecting the aqueous layers. The combined aqueous layers were made 2 per cent, with potassium acetate and the RNA was precipitated by adding 2 vol. of ethanol and collected by centrifugation at 5000 rev./min. It was then washed with ethanol water (3:1), absolute ethanol and ether several times, followed by further washing with ethanol and re-dissolving in water for dialysis. Insoluble material appearing after dialysis was centrifuged down and final precipitation of the RNA was accomplished with ethanol. The product was stored in sterile vessels in ethanol until used, upon which samples were dried aseptically in a desiccator and dissolved in Dulbecco’s solution for application. Both heart and liver RNA thus extracted were readily soluble in water and gave a typical ultra-violet absorption curve with a maximum at 259 mμ. Tests were positive (green) with orcinol but were negative with diphenylamine and Biuret.

Procedure

Eggs from white Leghorns were obtained from the University Farm. Blasto-derms of the definite streak or head process stages were transected at 0·2 mm. posterior to the primitive pit and the anterior parts discarded. The post-nodal pieces were then trimmed laterally, through the area pellucida, and posteriorly to give 1×1 mm.2 pieces which were pipetted to the watch-glass, containing 0·5 ml. of the same basic medium for both experimental and control groups, supported on cotton rings in petri-dishes (Fell & Robison, 1929).

Application of the RNA solution (1·0 mg./ml. in Dulbecco’s solution) began 24 hr. following explanation. This was dome once every 12 hr. with a micro-pipette either directly on the post-nodals or on pieces of lens paper affixed to the upper side of the latter. Control explants received plain Dulbecco’s solution. The fragments were transferred every other day to fresh vessels and at the end of the experimental period (8–9 days) were fixed in Bonin’s solution, embedded in paraffin, serially sectioned at 7 μ and stained with Hematoxylin-eosin.

The basic medium

To discourage migration of the mesodermal core, serum was avoided (Butros, 1963a), but 10-day chick embryo extract (EE50) and whole egg suspension (shaken with equal volume of Ringer’s solution and centrifuged) supplemented the synthetic medium. The stock penicillin solution contained 450 units/ml. and the stock agar was 5 per cent, in Ringer’s solution. The composition of the basic medium was as follows :

Predominance of neural tissue at the end of culture period

Although the fate of explants isolated 0·2 mm. posterior to the primitive pit has been described (Hunt, 1931; Rawles, 1936; Rudnick, 1938; Spratt, 1952), the relatively prolonged culture technique and the lateral and posterior trimming of the area pellucida of the pieces in the present work create a new situation that needs exploration. Sixty-five explants isolated at the 0·2 mm. level were compared with a similar number isolated at 0·4 mm. after 8 days of culture on the basic medium. Of tissues recognizable histologically, the neural was maintained in good condition in about one-eighth of the pieces (Plate, Fig. A). A small number had cardiogenic myoblasts but none had notochords (Table 1).

TABLE 1.

Tissues surviving in post-nodal explants trimmed down to 1 x 1 mm.2 cultured on the basic medium for 8 days

Tissues surviving in post-nodal explants trimmed down to 1 x 1 mm.2 cultured on the basic medium for 8 days
Tissues surviving in post-nodal explants trimmed down to 1 x 1 mm.2 cultured on the basic medium for 8 days

Furthermore, the neuroblasts had extensively proliferated and spread over a large surface of the explant, showing in very few cases differentiated nerve fibres. The shape of the neural tube was often distorted by flattening. Undifferentiated, primitive mesodermal proliferation was noted in the majority of the pieces.

Heart RNA applied on lens paper laid on the post-nodals

(In this and remaining experiments transection is at 0·2 mm.)

Heart RNA was applied drop-wise on lens paper attached to the upper surface of thirty-five cultured explants. A second set of thirty-five fragments similarly received Dulbecco’s solution. In all pieces, there was extensive migration and peripheral expansion along the lens paper resulting in thinning out. Serial sections showed further that the ectoderm cells were extremely elongate, and arrayed in concentric layers parallel to the paper. This was partially true for the mesoderm layer. Cells of both germ layers were primitive, undifferentiated and the development of neural tissue was suppressed in both controls and experimentals.

Heart RNA applied drop-wise directly on the post-nodals

A total of seventy explants were cultured in this series, thirty-five of which were used as controls. Routine observations during the first 2 days showed that all seventy fragments were developing in accordance with expectation at this level of transection: i.e., neural axis in the centre, a few somites on the flanks and isolated twitching heart primordia (Spratt, 1952, 1955; Rawles, 1943). With culture beyond the early morphogenetic stages, less structures became discernible on gross examination and a smaller number of twitching, cardiogenic areas were noted. However, a significant difference was noted in the number of explants showing twitching between experimentals and controls. By the 5th day, fifteen of the thirty-five experimentals had twitching areas as against one in the thirty-five controls. These numbers declined to four experimentals and no controls by the 8th day.

Histological findings

In about half the fragments of both treated and control groups the epidermis had periderm and basal layers but it was not discernible in the remaining pieces. Neural tubes of variable sizes and shapes persisted to the end of the culture period and were the predominant, differentiated tissue. Flattening was a common distortion, and some multiple tubes were noted in both groups. Experimental fragments showed, however, a superiority in the number of well-differentiated tubes having nerve fibres (Table 2 and Plate, Figs. B & C). Regions of cardiogenic tissue showed syncytial myoblasts whose nuclei were large and round displaying coarse chromatin granules. Myofibrils were discernible in the cytoplasm (Plate, Figs. D & E). More pieces showed myoblasts than twitching during the culture period in both experimental and control groups (Table 2).

TABLE 2.

Gross observations and differentiated tissues appearing in post-nodal explants at the end of 8 days in culture

Gross observations and differentiated tissues appearing in post-nodal explants at the end of 8 days in culture
Gross observations and differentiated tissues appearing in post-nodal explants at the end of 8 days in culture

Liver RNA

Ninety explants were cultured, half of which received liver RNA, drop-wise, as in the case of heart RNA. Two fragments from the treated group showed twitching in limited areas on the 5th day; none were seen in the controls.

Histological findings

Unlike those treated with heart RNA, there was no marked improvement in the differentiation of nerve fibres in this experimental group over their controls (Table 3). Myoblasts with fibrils were noted, as in the heart RNA groups, insignificantly more in the experimentals (Table 3). Here, again, a large number of pieces showed myoblasts upon histological examination than those observed twitching during the culture period. A striking finding was the enhancement of the entodermal layer by liver RNA. In some pieces cells were highly columnar with large vacuoles assuming the characteristics goblet cells (Plate, Fig. F). Deep folds were noted in other treated pieces.

TABLE 3.

Gross and histological observations on post-nodal explants treated with liver RNA

Gross and histological observations on post-nodal explants treated with liver RNA
Gross and histological observations on post-nodal explants treated with liver RNA

Enzyme treated RNA

Liver RNA and heart RNA were treated with 25 μg./ml. RNA-ase (Sigma) for 30 min. at 37°C., after which the solutions were deproteinized with phenol and the latter extracted with ether. Explants treated during culture with these enzyme-treated solutions did not differ appreciably from the controls described in the preceding two sections.

During the culture period of explants by the present procedure there was apparently a competition among the main presumptive tissues initially included in the 1 × 1 mm.2 sector. Some tissues noted at this level (Spratt, 1952, 1955; Rawles, 1943) in the first 2 days, such as somites and presumptive cardiac tissue, suffered gradual displacement by neural tissue which seemed to survive best under the present conditions. Twitching declined during the culture period though myoblasts survived in some pieces that did not exhibit twitching any more. Differential preference of early tissues to various nutritive substances used singly has been described by Spratt (1950) and may be the explanation for this overall effect of neural dominance.

Heart RNA had apparently a beneficial action on the small cardiogenic areas as there were many more twitches in the experimental group than in controls, especially in the first half of the culture period. Perhaps neural dominance masked the myoblast stimulation. Stimulation of nerve fibres by heart RNA may be an indication of some kind of versatility of embryonic cells that can make use of heart RNA to build neurofibrils. It could be that the applied RNA was being depolymerized and resynthesized in the neural tissue in accordance with their type and need. In the previous report a similar versatility was noted in the explants transected at 0·4 mm., at which level no neural tube can appear. Brain RNA, there, supposed to support neurofibril formation, was used by epidermal cells to induce keratinization (Butros, 1963).

In this connexion it should be recalled that a possible relation may exist between some neural protein or its derivative (neurokeratin) and epidermal keratin (Block, 1938; Low, 1953). Liver RNA also showed an enhancing action in inducing goblet cells and folds in the entodermal layer which was not noted in the previous report, perhaps due to the lower dosage used then. The effect noted presently resembles the action of adult pancreatic RNA noted in the previous work (Butros, 1963). That liver RNA did not stimulate myoblast nor nerve fibre development is not surprising owing to the lack of similarities between the proteins of liver and these tissues.

The action of a nucleic acid may be very specific as to act like a transformation agent even in higher organisms. Szybalska & Szybalski (1962) reported a DNA-mediated change in a mutant strain of mammalian cells that cannot utilize hypoxanthine, due to lack of inosinic acid pyrophosphorylase, to a positive strain. Kraus (1961) described alterations in the type of hemoglobin synthesized in tissue culture of human bone marrow treated with human DNA. Weisberger (1962) reported a similar alteration by incubating sickle cell marrow cells with nucleo-protein isolated from normal marrow, or other such combination. Niu (1963) found that liver RNA can induce tryptophan pyrrolase and glucose-6-phosphatase enzymes in ascites cells, whereas kidney RNA induced the biosynthesis of greater amounts of 1-amino acid oxidase, peculiar to kidney tissue. A similarly specific action was recently described in chick organogenesis by Hillman & Niu (1963).

Chick blastoderms of stage 5 were extirpated of their notochord and cultured by New’s method for about 1 day to show early morphogenesis. These extirpates usually develop abnormalities in the mid-brains and hind-brains, but when notochord and brain RNA’s were added drop-wise, normal brains developed. Only notochord RNA initiated the development of notochord beyond the extirpated level (Hillman & Niu, 1963). In the present work the RNA action was not as specific as in the above examples. Our procedures, however, deviate basically from those used by the above authors in two aspects.

First, the systems used for demonstrating transformation were dissociated, individual cells rather than intact, complex tissue fragments like our post-nodals. Second, exposure to the nucleic acid in the transformation experiments cited was brief (as in enzyme assay), and the observations were made soon after the entrance of the agent. For example, in the blastoderms studied by Hillman & Niu, the effects were noted within 1 day. The results might have been different had the blastoderms been cultured long enough to observe histogenesis. Incidentally, such culture is not possible by New’s procedure which they use as development does not continue beyond 2–3 days (New, 1955).

Evidently two diverse effects may be elicited with nucleic acids using embryonic reacting systems: (1) a rapidly acting morphogenetic effect exemplified by the finding of Hillman & Niu. Our explants manifested such an action in the early culture days, i.e. enhancement of twitching by heart RNA. (2) A slow histogenetic effect. The latter was possibly obscured by competition with neural tissue. If one chooses to eliminate this variable, one should trim the explants in such a manner as to leave only one type of presumptive tissue on which the action of various RNA’s could be studied.

Action d’acides ribonucléiques d’origine cardiaque et hépatique sur la différenciation de segments de blastoderme de poulet

  1. On a préparé de l’ARN à partir, respectivement, de coeurs et de foies d’embryons de poulet à l’aide d’un procédé de Kirby, modifié.

  2. Des fragments post-nodaux, obtenus en sectionnant des blastodermes aux stades de la ligne primitive ou du processus céphalique, à 0,2 mm. en arrière de la fossette primitive, et taillés à 1 × 1 mm., ont été cultivés pendant 8 jours sur un milieu contenant du TC 199, une suspension d’oeuf complet, de l’extrait embryonnaire, de l’agar et de la pénicilline.

  3. La moitié des fragments cultivés ont reçu, deux fois par jour, une goutte de l’un ou l’autre ARN; les autres fragments ont reçu une goutte de solvant.

  4. L’ARN cardiaque a provoqué l’apparition de contractions dans des zones localisées. La différenciation de myoblastes et de fibres nerveuses a été accrue.

  5. L’ARN hépatique a stimulé le plissement de la couche entodermique et la modification des cellules qui ont pris une forme columnaire, en gobelet.

  6. On discute les résultats obtenus, à la lumière des travaux en cours sur la transformation des cellules chez les organismes supérieurs.

This work was supported by grant No. G-17841 from the National Science Foundation, Washington, U.S.A., to whom the author is deeply indebted.

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Plate 1

FIG. A. Post-nodal explant isolated by a cut 0 · 2 mm. posterior to the primitive pit, trimmed to 1 x 1 mm.2, and cultured for 8 days on the basic medium, showing a convoluted neural tube lacking fibrillar elements. x430. 1 = lumen of neural tube;m = mitotic neuroblasts.

FIG. B. Nerve fibres differentiated around neural tube in an explant receiving embryonic heart RNA during the culture period. x430. n.f. = nerve fibre; ns. = neuroblasts migrating out of neural tube; n.t. = neural tube.

FIG. C. Another specimen showing the effect of embryonic heart RNA in promoting nerve fibre differentiation. x430. n.f. = nerve fibres; p.n. = proliferating neuroblast away from neural tubes.

FIG. D. Lateral region of an explant treated with embryonic heart RNA showing a syncytium of myoblasts and the beginning of myofibril formation. x430. f = myofibrils; m = myoblasts.

FIG. E. Dense proliferation of myoblasts noted in another explant treated with embryonic heart RNA. x430. f = myofibrils;m = myoblasts.

FIG. F. Goblet cells noticed in the entodermal layer of an explant treated with embryonic liver RNA. x 430. c = mesodermal core of the post-nodal; e = entodermal layer; g = goblet cells with conspicuous vacuoles.

Plate 1

FIG. A. Post-nodal explant isolated by a cut 0 · 2 mm. posterior to the primitive pit, trimmed to 1 x 1 mm.2, and cultured for 8 days on the basic medium, showing a convoluted neural tube lacking fibrillar elements. x430. 1 = lumen of neural tube;m = mitotic neuroblasts.

FIG. B. Nerve fibres differentiated around neural tube in an explant receiving embryonic heart RNA during the culture period. x430. n.f. = nerve fibre; ns. = neuroblasts migrating out of neural tube; n.t. = neural tube.

FIG. C. Another specimen showing the effect of embryonic heart RNA in promoting nerve fibre differentiation. x430. n.f. = nerve fibres; p.n. = proliferating neuroblast away from neural tubes.

FIG. D. Lateral region of an explant treated with embryonic heart RNA showing a syncytium of myoblasts and the beginning of myofibril formation. x430. f = myofibrils; m = myoblasts.

FIG. E. Dense proliferation of myoblasts noted in another explant treated with embryonic heart RNA. x430. f = myofibrils;m = myoblasts.

FIG. F. Goblet cells noticed in the entodermal layer of an explant treated with embryonic liver RNA. x 430. c = mesodermal core of the post-nodal; e = entodermal layer; g = goblet cells with conspicuous vacuoles.