The development of tubular heart in RNA-treated post-nodal … · RNA-treated chick blastoderm 487...

/ . Embryol. exp. Morph. Vol. 29, 2, pp. 485-501, 1973 4 8 5

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

By M. C. NIU1 AND A. K. DESHPANDE1

From the Department of Biology, Temple University, Philadelphia

SUMMARYPost-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 hthe explants were fed daily with fresh egg-extract medium for 4 days. On the 4th day pulsatingheart was found in the RNA-treated but not in the untreated series. Histological examinationrevealed that cell differentiation other than of erythrocytes and epithelial tissue seldomoccurred in the control and, in contrast, pulsating heart and cardiac myoblasts were presentin 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 anterolateralfragments of chick blastoderm at the definitive streak stage. Both RNA-treatedand untreated explants were cultured in vitro. Only heart RNA was found topromote the development of pulsating tissue. The heart-forming capacity ofheart RNA was sensitive to pancreatic ribonuclease. The point of interest is,however, that no organizing action into pulsating tube resembling normalheart took place. Three possible explanations have been seriously considered,namely (1) the number of cells used was too small to go through the process oforganogenesis, (2) the length of cultivation was too short to achieve organo-genesis and (3) the heart RNA used might contain very low amounts of informa-tional RNA. To overcome these disadvantages, we have now used larger post-nodal pieces (PNP), longer periods of cultivation and nuclear RNA.Furthermore, special precaution was undertaken to excise PNP, avoiding theinclusion of presumptive heart-forming tissue. This communication summarizesthe data, showing that PNP by itself did not differentiate into cardiac tissueand that nuclear (nRNA) and cytoplasmic (cRNA) fractions of heart RNAinitiated the differentiation of pulsating tube and non-beating cardiac tissue aswell. Furthermore, electron-microscopic examination of the beating tissuerevealed 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 thetransection (C C).

MATERIALS AND METHODS

Fertilized White Leghorn eggs were obtained from Shaw's Hatchery, WestChester, Pa., and incubated at 38 °C until they reached the definitive primitivestreak stage of development (Hamburger & Hamilton, 1951, stage 4). Theblastoderms were explanted and the area opaca was removed. The area pellucidawas cut transversely 0-5-0-6 mm posterior to the Hensen's node (Fig. 1), thusmaking 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 onvitelline membrane, previously mounted around a glass ring. Outside the ring,1 ml of nutritive medium was added. This method of cultivation is a modificationof the procedures of New (1955) and Chauhan & Rao (1970). The nutritivemedium used was egg extract which was prepared by mixing a whole unfertilizedegg with 50 ml of Ringer solution. The homogenate was centrifuged at 2000rev/min for 30 min at 4 °C. The supernatant was mixed with an equal volume ofPannett-Compton (1924) solution (PC).

Preparation of heart RNA

Chicken heart was excised immediately after slaughtering and pooled inice-cold isotonic sucrose solution (0-25 M +0-001 M-MgCl2). Unless specifiedotherwise, 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 werechopped up with scissors into fine pieces and blended in a Waring Blender with4 vols. of sucrose solution (0-32 M, +0-003 M-MgCl2 and 0-5 % diethyl pyrocar-bonate). While blending at high speed for 30 sec, 2 more vols. of sucrose solutionwere added and the process continued for 90 sec. The homogenate was filteredthrough 1, 2 and 4 layers of cheese cloth and then 2 layers of flannel. The filtratewas centrifuged using the International PR 2 centrifuge at 4 °C, 2000 rev/minfor 30 min. The supernatant fluid was suctioned off" and used for isolation ofcRNA. 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 M-MgCl2) were added and thoroughly mixed. Crude nuclei were packed in arefrigerated International PR 2 centrifuge at 4 °C, 1800 rev/min, 7 min, andwashing was repeated twice. The loose sediment was packed firmly (30 min)for volume determination, and 10 vols. of 2-4M sucrose + 0001 M-MgCl2 wereused to suspend the crude nuclei evenly. Pure nuclei were collected in a SpincoL2-65B ultracentrifuge at 4 °C, 40000 rev/min for 1 h, and washed twice withbuffered 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 sus-pended 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-EDTA) . An equal volume ofwater-saturated phenol (redistilled) was added, and immediately blended in aLourdes mixer at 30 V for 5 min. Another equal volume of chloroform con-taining 1 % isoamyl alcohol was added. This mixture was again blended at 30 Vfor 5 min at room temperature and centrifuged at 2000 rev/min for 10 min. Thebottom phenol chloroform phase was aspirated out. An equal volume of chloro-form-isoamyl alcohol was added to the aqueous and interphase layers andblended at 30 Y for 5 min. The process was repeated 3 times. To the aqueouslayer 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 blendedin a Lourdes mixer at 60 V for 5 min. The mixture was centrifuged at 1800 rev/minfor 30 min, at 10 °C, and the top aqueous layer saved. To the interphase andphenol layers \ vol. of saline (0-9 % NaCl) was added and blended at 35 V for5 min. The supernatant fluid was combined with the previous and once againextracted with £ vol. of phenol as before. The aqueous layer was removed aftercentrifugation and kept at - 2 0 °C after addition of 95 % ethanol (2 vols.) andK-acetate (up to 2 %).

cRNA from the deep-freeze was centrifuged. The precipitate was redissolvedin saline. Glycogen was removed by centrifugation (Sorvall RC-2) at 2 °C,


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 againtreated with DNase. The enzyme was denatured by chloroform containing 1 %isoamyl alcohol. RNA was reprecipitated by 2 vols. of 95 % ethanol and washedwith 67 % ethanol. The sediment was redissolved in saline. U.v. spectrophoto-metric examination of the RNA solution revealed that both preparations weretypical of nucleic acids. Tests with the Lowry procedure showed that nRNAcontained about 2 % and cRNA 1 % of protein. The Dische reaction indicatedthat 1-1-5 % of DNA was present in nRNA, but none could be detected incRNA. The amounts of RNA calculated from the orcinol reaction and fromthe optical density reading at 260 nm differed less than 10 %, thus indicatingthat RNA was essentially the only substance in the solution.

Brain nRNA was prepared in the same way as the heart nRNA.

RNA treatmentPNPs 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. Chickenheart RNA isolated from nuclei (nRNA) and cytoplasm (cRNA) were used inthis study. For functional comparison, brain nRNA was also used. Controlseries 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 mediumwas replaced every 24 h by feshly made Qgg extract. Daily observation was madeprior to the change of medium. At the end of the 5th day (120 h) the explantswere fixed in Bouin's fluid. Some of them were stained and mounted in toto forphotography. They were later embedded in the same way as others. Serialsections 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 werewashed 3 times in the same buffer containing isotonic sucrose and then postfixedin cold 1 % OsO4 in cacodylate buffer for 1 h at 0 °C. Dehydration was carriedthrough a graded series of alcohols and the tissue embedded in Araldite. Thinsections for electron microscope were cut with a glass knife on a Porter-BlumUltra Microtome MT-2, mounted on 200-mesh grids coated with a thin layerof formvar and carbon, stained with lead citrate and examined in a PhilipsEM-300 microscope.


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 culturedfor 5 days. The prominent heart tube (H) was pulsating from the 4th to the fixationtime at the end of the 5th day. The dark stained area at top is due to the accumulationof blood. x25.

RESULTS

1. Control series(a) PC solution

A total of 48 PNPs were explanted for this study. Daily observation through-out the period of incubation did not reveal the presence of twitching tissue inthis series (Fig. 2). Red blood cells (RBC) became visible as red patches on thethird day of incubation.

Histological findings. Serial sections of the 48 PNPs were examined. RBC andhemopoietic tissue (bf, Fig. 4 A) were present in all cases. Forty-five of the 48had a thin layer of ectoderm and 1-2 layers of endoderm (94 %). Mesodermappeared as undifferentiated condensation (me, Fig. 4B) or a loose network ofmesenchyme. Three of the 48 PNPs (6 %) differentiated further. One formedneural 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, ormyoblasts. Three explants had neuroid tissue and notochord, one each hadneuroid tissue or tubules and four had notochord only. The frequency of differen-tiation was 69 %.


Fig. 4. Photomicrographic sections of two control PNPs (A and B). b, Red bloodcells; bf, blood forming tissue; ec, ectoderm; en, endoderm; and me, condensedmesenchyme. 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). Nine-teen of them were either beating or twitching in some localized area. Nine ofthe explants had well-defined pulsating tubes (Fig. 3) and ten beating patches.Pulsation was noted first on the 4th day of incubation and invariably persistedthroughout the period of cultivation. The rate of beating averaged 20-25/minand gradually slowed down during observation in room temperature.

For a comparison, heart nRNA was also dissolved in HR and used to treat12 PNPs. Five of the 12 developed pulsating tissue, 2 with non-beating cardiactissue, 7 with neuroid tissue, 6 with tubule and 4 with notochord (Table 3).

Histologicalfindings. Examination of serial sections of the 51 explants revealedthat differentiation had occurred in 40 (78 %). The distribution of various organsin nRNA-treated explants is portrayed by Table 2. It can be seen that 19 explantshad beating hearts (48 %) and 11 had non-beating heart tissues (27 %). Twelveof the 40 differentiated explants contained beating heart only and 5 containedneuroid only. When explants containing beating and non-beating hearts onlyare combined, there are 20 out of 40, i.e. 50 % with heart only. The frequencyof tubule and neuroid formation was 23 % and 30 % respectively (Table 1). Across-section of a pulsating heart tube is shown in Fig. 5.

The beating tissues observed in this study resembled those obtained fromthe heart-forming graft in chorio-allantoic membrane (Rawles, 1943). They are


V

Fig. 5. Section of a chicken-heart nRNA-treated PNP passing through bent hearttube. E, Endocardium; EM, epimyocardium; H, tubular heart, x 130.Fig. 6. Section of the PNP treated with chicken heart cRNA, showing looselyarranged beating tissue, m, Cardiac myoblasts. x 130.Fig. 7. Section of the PNP treated with chicken heart nRNA, with compact beatingtissue, m, Cardiac myoblasts. x 130.Fig. 8. Section of the PNP treated with chicken brain nRNA. Note the predominantneural differentiation. NT, Neural tube; P, neural plate; me, mesenchyme. x 130.


either loosely arranged (Fig. 6) or compact (Fig. 7). In early stages of heartdevelopment, myofibrils and cross-striation of myocardiac cells can hardly bediscerned by light microscopy (Goss, 1938, 1940; Copenhaver, 1939; Manasek,1970). Our identification of cardiac tissue was based primarily upon rhythmicbeating and accompanied by (1) histological observation of interlacing strandsand distinctive nuclei and (2) study of fine structure by electron microscopy.Through the latter, we learned that the heart-RNA-induced beating tissue hadproperties typical of cardiac muscle. They are (1) large numbers of individualglycogen 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 cyto-plasm; and (3) cell to cell attachments occur at specialized zones called inter-calated discs (/) and desmosomes. Intercalated discs are cell to cell junctionalcomplexes. Desmosomes represent that portion of the junction where the twoopposed plasma membranes lie parallel and have dense plaques, separated by agap 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 bythree electron micrographs (Figs. 10-12). Fig. 10 shows the tight junctionalcomplexes between adjacent cells. Both intercalated discs (/) and Z-bands (Z) arerecognizable 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, myofibrilsoften converge to them from different directions (Fig. 11). Extension of myofibrilis 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 beatingwas similar to that obtained from nRNA-induced heart. The types of organsformed in the RNA-treated PNP explants are criss-cross classified in Table 2.Fifteen of the 23 differentiated explants (65 %) contained heart tissue only. Ifexplants 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 frequentlythan 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 (Tables1,2). No explants had either beating tissue or tissue resembling myocardiac cells.Histological examination disclosed that 9 of the 20 differentiated explants hadneuroid only (45 % - Fig. 8). The frequency of neural formation, was 85 %.


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 atright angle. F, Myofibril; g, Golgi body; G, glycogen; /, intercalated disc; is, inter-cellular space; L, lipids; M, mitochondria; N, nucleus; Z, Z-band. x 65000.

E M B 29


Table 1. Developmental potentiality of chick blastoderm (PNPs) inpresence and absence of RNA, cultured in vitro for 5 days (120 h)

No. of No. of differentiated PNPs showing development of various organsPNPs , * >

differen- Cardiac tissuetiated , A v

Series and no. Beating Non-beating Tubule Somite Notochord Neuroidcultured

Control (PC) 3/48 0 0 0 0 2 2(6%)

Heart nRNA 40/51 19(48%) 11(27%) 9(23%) 3(8%) 2(5%) 12(30%)(78 %) 30 (75 %)

Heart cRNA 23/37 12 (52 %) 6 (26 %) 0 0 5 (22 %) 5 (22 %)(62%) 18(78%)

Brain nRNA 20/23 0 0 7 (35 %) 1 (5 %) 6 (30 %) 17 (85 %)(87%)

DISCUSSION

Cellular components of PNP, transected at the level 0-4 mm posterior toHensen's node, consist of epiblasts, hyoblasts and migrating cells (presumptivemesoderm). In the culture medium of plasma and embryonic extracts, theydeveloped into epithelium, connective tissue, and red blood cells (Murray, 1932).When they were grown in freshly prepared egg extract, some neuroid tissueand/or notochord developed (Chauhan & Rao, 1970). In the experiments ofChauhan & Rao the frequency was 3 % and in ours 6 %. These frequencies aretoo low to differ statistically from the results of Murray. The PNPs used in thepresent report, transected 0-6 mm posterior to the node, contained some cellscapable of synthesizing myosin (Ebert, 1953) or thymidine labeled cells thatcontribute to heart formation in normal development (Rosenquist, 1970), butwere unable to undergo differentiation. Should the transection be made 0-2 mmposterior 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 explantsfrom anterolateral blastoderm could be due to the inclusion of some heart-forming cells (Niu & Mulherkar, 1970). The increase of heart formation in theheart-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-producingcells 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 andothers same as Fig. 9. x 41000.

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Table 2. Distribution of the differentiated organs in PNP explantstreated with RNAs

Kinds of organs differentiated

Beating heart onlyBeating heart with neuroidBeating heart with tubuleBeating heart with neuroid and tubule

Total

Non-beating cardiac tissue onlyNon-beating cardiac tube with notochordNon-beating cardiac tissue with tubule

and/or somiteNon-beating cardiac tissue with notochord

Total

Tubule onlyTubule and somiteNotochord and tubuleNotochord and somiteNotochord only

Total

Neuroid and tubuleNeuroid and notochordNeuroid, notochord and tubuleNeuroid

Total

Heart nRNA(40 of 51

PNP explantsdifferentiated-

78 %)

12 (6-tube)3 (3-tube)22

19(48%)

8 (8-tube)0

30

11(27%)

120003 (8 %)

0205

7(18%)

Heart cRNA(23 of 37

PNP explantsdifferentiated -

62 %)

11 (5-tube)001

12(52%)

4 (3-tube)1

01

6(26%)

000011(4%)

0202

4(17%)

Brain nRNA(20 of 23

PNP explantsdifferentiated -

87 %)

0000

0

00

00

0

101103 (15 %)

431917 (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 organsNo. of PNPs , "differentiated Cardiac tissue

and no. , A »Experimental series cultured Beating Non-beating Tubule Notochord Neuroid

Without nRNA

Chick heart nRNA

9/13(69%)10/12

(83 %)

0 0 1(8%) 7(51%) 4(30%)

5(40%) 2(16%) 6(48%) 4(32%) 7(56%)7(56%)


PC solution was replaced by HR. When the medium thus obtained wasemployed to grow PNPs, no beating tissue has been observed. However, thefrequency of differentiation was high (69 %, Table 3). The difference in effectbetween PC- and HR-made egg extract on PNP is very striking. Experimentalanalysis of the responsible factor(s) is under way. From the viewpoint offunctional analysis of RNA or other molecules, it appears that the use of PC ispreferable.

The second reason for using PNP in the study of specific differentiation is itsclearly defined boundary, about one-quarter of the area pellucida, in which thereare sufficient numbers of cells under inductive influence to undergo morpho-genesis. When small numbers of cells are used, tissue differentiation (histo-genesis) instead of organogenesis prevails (Grobstein & Zwilling, 1953). Thishas been well illustrated by the formation of beating tissue instead of tubularheart in chorio-allantoic grafts of small pieces of the heart-forming areas(Rawles, 1943). The RNA-induced formation of beating tissue but not beatingtube (Butros, 1965; Niu & Mulherkar, 1970) can similarly be explained.

The function of isolated RNAs from adult chickens was investigated byapplying heart and brain RNAs separately onto excised PNPs. They respondedto brain RNA by forming mostly neural tissue and to heart RNA by developingpredominantly into beating and non-beating hearts, thus establishing the organ-specificity of RNAs. Furthermore, both kidney (Deshpande & Niu, 1971) andthymus RNAs (unpublished) were incapable of inducing the formation oftubular heart and/or beating tissue.

In a recent paper, Jacobson & Duncan (1968) emphasized the lequirement ofspecific heart inducer in the development of heart primordium in the newt,Tarica trosa. According to these authors, this heart inducer came from anteriorendoderm and other embryonic tissue surrounding the heart primordium.Apparently the specific heart inducer they were discussing differs from the heart-forming RNA used in our experiments. Besides, the hypoblasts in our PNPseem to contribute to the formation of posterior endoderm and can hardly playany influence on the RNA-induced heart formation.

Preliminary experiments using Poly A-attached mRNA suggest that theinduced formation of beating heart could be as high as 90 %. This implies thatexogenous heart RNA transforms PNP into heart tube. The RNA synthesizedin the developing heart is responsible for the synthesis of heart-specific proteins(enzymes). It appears that the exogenous heart-RNA-treated PNP has acquiredthe capacity to synthesize heart proteins continuously.

Heart RNA was separated into nRNA and cRNA. The organs developed inthe cRNA-treated PNPs are heart, notochord and neuroid. The nRNA serieshas, in addition, somite and tubule (Table 1). The frequency of the beating andnon-beating heart formation is 30 out of the 40 explants (75 %) in the nRNAseries and 18 of 23 (78 %) in the cRNA (Table 1). Fifty per cent of the nRNAtreated explants have heart developed only and 65 % in the cRNA series. These


data would suggest that cRNA is more selective than nRNA in the initiation ofheart formation. Functional selectivity is a measure of the information thatRNA carries. In this sense, the heterogeneous cRNA is likely to contain moremessenger RNA than nRNA. This is supported by recent advances in metho-dology used to isolate messenger (m) RNA, Poly A-attached RNA, from poly-somes of the cytoplasm (Rosenfeld, Comstock, Means & O'Malley, 1972).The RNA thus isolated was the only mRNA in our hands that initiated thedifferentiation of PNPs into beating tissue and/or tubular heart.

This work was supported by research grants from The Population Council and The NationalFoundation. The authors wish to thank Dr Tze Lin for his aid in the isolation of nuclearRNA and Mrs Sharon L. Howard for her volunteer in histological service. Deep appreciationgoes to L. C. Niu for her expert analysis of the fine structure of the induced heart tissue.

REFERENCESBUTROS, J. (1965). Action of heart and liver RNA on the differentiation of segments of chick

blastoderm. / . Embryo!. exp. Morph. 13, 119-128.CHAUHAN, S. P. S. & RAO, K. V. (1970). Chemically stimulated differentiation of post-nodal

pieces of chick blastoderm. /. Embryo!, exp. Morph. 23, 71-78.COPENHAVER, W. M. (1939). Initiation of beat and intrinsic contraction rates in different

parts of the Amblystoma heart. /. exp. Zoo!. 30, 193-224.DEHAAN, R. L. (1968). Emergence of form and function in embryonic heart. Devi Bio!.

Suppl. 2, pp. 208-250.DESHPANDE, A. K. & Niu, M. C. (1971). Specific function of exogenous RNA in differentia-

tion of post-nodal pieces of early chick embryo blastoderm. Abstract no. 140, EleventhAnnual Meeting of Am. Soc. Cell Bio!.

EBERT, J. D. (1953). Some aspects of protein biosynthesis in development. In ThirteenthGrowth Symposium {Aspects of Synthesis and Order in Growth) (ed. Rudnick), pp. 69-112.Princeton University Press.

Goss, C. M. (1938). The first contraction of the heart in rat embryos. Anat. Rec. 70, 505-524. ^Goss, C. M. (1940). First contractions of heart without cytological differentiation. Anat. «

Rec. 76, 19-27. jGROBSTEIN, C. & ZWILLING, E. (1953). Modification of growth and differentiation of chorio-

allantoic grafts of chick blastoderm pieces after cultivation at a glass-clot interface. /. exp. *Zoo!. 122, 259-284. t

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development ofthe chick embryo. /. Morph. 88, 49-92.

HOWARD, E. (1953). Some effect of NaCl concentration on the development of early chick *blastoderms in culture. /. cell. comp. Physiol. 41, 237-260. x

JACOBSON, A. G. & DUNCAN, J. T. (1968). Heart induction in salamanders. / . exp. Zool.107, 79-103.

MANASEK, F. J. (1970). Histogenesis of the embryonic myocardium. Am. J. Cardiol. 25, *149-168.

MULHERKAR, L. (1958). Induction by regions lateral to the streak in chick embryo. J. Embryo!.exp. Morph. 6, 1-14.

MURRAY, P. D. A. (1932). The development /// vitro of blood of early chick embryo. Proc. *R. Soc. Lond. B 14, 497-521.

NEW, D. A. T. (1955). A technique for the cultivation of the chick embryo in vitro. J. Embryo!.exp. Morph. 3, 326-331.

Niu, M. C. & MULHERKAR, L. (1970). The role of exogenous heart-RNA in development «of chick embryo in vitro. J. Embryo!, exp. Morph. 24, 33-42.

RNA-treated chick blastoderm 501PANNETT, C. A. & COMPTON, A. (1924). The cultivation of tissue in saline embryonic juice.

Lancet 206, 381-384.RAWLES, M. E. (1943). The heart-forming areas of the early chick blastoderm. Physiol. Zool.

16, 22-44.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, 387-391.

ROSENQUIST, G. C. (1970). Localization and movement of cardiogenic cells in the chickembryo: the heart-forming portion of the primitive streak. Devi Biol. 22, 461-475.

{Received 1 September 1972, revised 31 October 1972)

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