The development of tubular heart in RNA-treated post-nodal pieces of chick blastoderm

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1 /. Embryol. exp. Morph. Vol. 29, 2, pp , Printed in Great Britain 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 -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, 197), 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 , U.S.A.

2 486 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 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, 197). 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 (197). The nutritive medium used was egg extract which was prepared by mixing a whole unfertilized egg with 5 ml of Ringer solution. The homogenate was centrifuged at 2 rev/min for 3 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 (-25 M +-1 M-MgCl 2 ). Unless specified otherwise, preparation of RNA was carried out in the cold room at 4 C.

3 RNA-treated chick blastoderm 487 Connective tissue and fat were removed from the hearts. Batches of 7 g were chopped up with scissors into fine pieces and blended in a Waring Blender with 4 vols. of sucrose solution (-32 M, +-3 M-MgCl 2 and -5 % diethyl pyrocarbonate). While blending at high speed for 3 sec, 2 more vols. of sucrose solution were added and the process continued for 9 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, 2 rev/min for 3 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 5-1 vols. of sucrose solution (-32 M +-3 M- MgCl 2 ) were added and thoroughly mixed. Crude nuclei were packed in a refrigerated International PR 2 centrifuge at 4 C, 18 rev/min, 7 min, and washing was repeated twice. The loose sediment was packed firmly (3 min) for volume determination, and 1 vols. of 2-4M sucrose + 1 M-MgCl 2 were used to suspend the crude nuclei evenly. Pure nuclei were collected in a Spinco L2-65B ultracentrifuge at 4 C, 4 rev/min for 1 h, and washed twice with buffered saline (-14M NaCl and 1 M Tris, ph 7-1). The insoluble material, sedimented in a Sorvall RC-2 centrifuge at 5 rev/min for 2 min, was suspended in 1 vols. of SDS (sodium dodecyl sulphate) buffer (-5% SDS, -1 M-NaCl, -1 M acetate buffer, ph 5-2, -1 M-EDTA). An equal volume of water-saturated phenol (redistilled) was added, and immediately blended in a Lourdes mixer at 3 V for 5 min. Another equal volume of chloroform containing 1 % isoamyl alcohol was added. This mixture was again blended at 3 V for 5 min at room temperature and centrifuged at 2 rev/min for 1 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 3 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 - 2 C. Isolation of crna To the above supernatant an equal volume of phenol was added and blended in a Lourdes mixer at 6 V for 5 min. The mixture was centrifuged at 18 rev/min for 3 min, at 1 C, and the top aqueous layer saved. To the interphase and phenol layers \ vol. of saline (-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 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,

4 488 M. C. NIU AND A. K. DESHPANDE 18 rev/min for 2 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 % 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 26 nm differed less than 1 %, 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 (8 o.d./ml) and applied -1 ml inside and -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 -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 (12 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 (-1 M-Na cacodylate, ph 7-4) for 1 h at C. At intervals of 3 min they were washed 3 times in the same buffer containing isotonic sucrose and then postfixed in cold 1 % OsO 4 in cacodylate buffer for 1 h at 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 2-mesh grids coated with a thin layer of formvar and carbon, stained with lead citrate and examined in a Philips EM-3 microscope.

5 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. Histological findings. 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 %.

6 49 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 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 2-25/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 4 (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 4 differentiated explants contained beating heart only and 5 contained neuroid only. When explants containing beating and non-beating hearts only are combined, there are 2 out of 4, i.e. 5 % with heart only. The frequency of tubule and neuroid formation was 23 % and 3 % 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

7 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 13. Fig. 6. Section of the PNP treated with chicken heart crna, showing loosely arranged beating tissue, m, Cardiac myoblasts. x 13. Fig. 7. Section of the PNP treated with chicken heart nrna, with compact beating tissue, m, Cardiac myoblasts. x 13. 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 13.

8 492 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, 194; Copenhaver, 1939; Manasek, 197). 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. 1-12). Fig. 1 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 /at 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 2 differentiated explants had neuroid only (45 % - Fig. 8). The frequency of neural formation, was 85 %.

9 RNA-treated chick blastoderm 493 Fig. 9. Electron micrograph of cells from the heart-rna induced beating tissue, x 16. 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 65. E M B 29

10 494 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 (12 h) No. of No. of differentiated PNPs showing development of various organs PNPs, * > differen- Cardiac tissue A tiated, v Series and no. Beating Non-beating Tubule Somite Notochord Neuroid cultured Control (PC) 3/ (6%) Heart nrna 4/51 19(48%) 11(27%) 9(23%) 3(8%) 2(5%) 12(3%) (78 %) 3 (75 %) Heart crna 23/37 12 (52 %) 6 (26 %) 5 (22 %) 5 (22 %) (62%) 18(78%) Brain nrna 2/23 7 (35 %) 1 (5 %) 6 (3 %) 17 (85 %) (87%) DISCUSSION Cellular components of PNP, transected at the level -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, 197). 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 -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, 197), but were unable to undergo differentiation. Should the transection be made -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, 197). 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 1-12 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 41.

11 Fig. 1. For legend see p. 494.

12 Fig. 11. For legend see p o > o m o 4

13 Fig. 12. For legend see p o I

14 498 M. C. NIU AND A. K. DESHPANDE Table 2. Distribution of the differentiated organs in PNP explants treated with RNAs Kinds of organs differentiated Beating heart only Beating heart with neuroid Beating heart with tubule Beating heart with neuroid and tubule Total Non-beating cardiac tissue only Non-beating cardiac tube with notochord Non-beating cardiac tissue with tubule and/or somite Non-beating cardiac tissue with notochord Total Tubule only Tubule and somite Notochord and tubule Notochord and somite Notochord only Total Neuroid and tubule Neuroid and notochord Neuroid, notochord and tubule Neuroid Total Heart nrna (4 of 51 PNP explants differentiated- 78 %) 12 (6-tube) 3 (3-tube) (48%) 8 (8-tube) 3 11(27%) (8 %) 2 5 7(18%) Heart crna (23 of 37 PNP explants differentiated - 62 %) 11 (5-tube) 1 12(52%) 4 (3-tube) 1 1 6(26%) 1 1(4%) 2 2 4(17%) Brain nrna (2 of 23 PNP explants differentiated - 87 %) (15 %) (85 %) 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 9/13 1(8%) 7(51%) 4(3%) Chick heart nrna (69%) 1/12 5(4%) 2(16%) 6(48%) 4(32%) 7(56%) (83 %) 7(56%)

15 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, 197) 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 9 %. 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 3 out of the 4 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

16 5 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. REFERENCES BUTROS, J. (1965). Action of heart and liver RNA on the differentiation of segments of chick blastoderm. /. Embryo!. exp. Morph. 13, CHAUHAN, S. P. S. & RAO, K. V. (197). Chemically stimulated differentiation of post-nodal pieces of chick blastoderm. /. Embryo!, exp. Morph. 23, COPENHAVER, W. M. (1939). Initiation of beat and intrinsic contraction rates in different parts of the Amblystoma heart. /. exp. Zoo!. 3, DEHAAN, R. L. (1968). Emergence of form and function in embryonic heart. Devi Bio!. Suppl. 2, pp DESHPANDE, A. K. & Niu, M. C. (1971). Specific function of exogenous RNA in differentiation of post-nodal pieces of early chick embryo blastoderm. Abstract no. 14, Eleventh Annual Meeting of Am. Soc. Cell Bio!. EBERT, J. D. (1953). Some aspects of protein biosynthesis in development. In Thirteenth Growth Symposium {Aspects of Synthesis and Order in Growth) (ed. Rudnick), pp Princeton University Press. Goss, C. M. (1938). The first contraction of the heart in rat embryos. Anat. Rec. 7, ^ Goss, C. M. (194). First contractions of heart without cytological differentiation. Anat. «Rec. 76, j GROBSTEIN, C. & ZWILLING, E. (1953). Modification of growth and differentiation of chorioallantoic grafts of chick blastoderm pieces after cultivation at a glass-clot interface. /. exp. * Zoo!. 122, t HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, HOWARD, E. (1953). Some effect of NaCl concentration on the development of early chick * blastoderms in culture. /. cell. comp. Physiol. 41, x JACOBSON, A. G. & DUNCAN, J. T. (1968). Heart induction in salamanders. /. exp. Zool. 17, MANASEK, F. J. (197). Histogenesis of the embryonic myocardium. Am. J. Cardiol. 25, * MULHERKAR, L. (1958). Induction by regions lateral to the streak in chick embryo. J. Embryo!. exp. Morph. 6, MURRAY, P. D. A. (1932). The development /// vitro of blood of early chick embryo. Proc. * R. Soc. Lond. B 14, NEW, D. A. T. (1955). A technique for the cultivation of the chick embryo in vitro. J. Embryo!. exp. Morph. 3, Niu, M. C. & MULHERKAR, L. (197). The role of exogenous heart-rna in development «of chick embryo in vitro. J. Embryo!, exp. Morph. 24,

17 RNA-treated chick blastoderm 51 PANNETT, C. A. & COMPTON, A. (1924). The cultivation of tissue in saline embryonic juice. Lancet 26, RAWLES, M. E. (1943). The heart-forming areas of the early chick blastoderm. Physiol. Zool. 16, ROSENFELD, G. C, COMSTOCK, J. P., MEANS, A. R. & O'MALLEY, B. W. (1972). A rapid method for the isolation and partial purification of specific eucaryotic mrna. Biochem. biophys. Res. Comnum. 47, ROSENQUIST, G. C. (197). Localization and movement of cardiogenic cells in the chick embryo: the heart-forming portion of the primitive streak. Devi Biol. 22, {Received 1 September 1972, revised 31 October 1972)

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