DEVELOPMENTALBIOLOGY

Cardiac

148,243-248(1991)

Differentiation Induced by Dopamine in Undifferentiated Cells of Early Chick Embryo MANUEL

SARASA AND SALVADOR CLIMENT

Accepted

July

23. 1.991

Evidence suggests that neurotransmitters can act as possible chemical signals involved in cell division and morphogenetic movements long before neurons appear in the embryo. However, whether they are playing a role in differentiation is now unknown. 11 was recently observed (M. Sarasa and S. Clirnent, 1987, J. Eq Zool. 241,181-190) lhat Lhe neurotransmitter dopamine exerted a stimulating effect on cardiac differentiation in the chick ill 0110. We show here that dopamine acts as a specific inducer of heart muscle differentiation i?~ vitro. When cells of the gastrula of embryos treated with dopamine were dissociated and reaggregated, the aggregates obtained almost entirely underwent cardiac muscle different.iation. Also, when small postnodal pieces obtained from the most posterior region of the gastrula were cultivated in the presence of dopamine, they differentiated into myocardic tissue instead of following their fate map. Therefore, dopamine can trigger a process that both causes undifferentiated cells to differentiate into heart muscle and compels cells already determined to another way of differentiation to become myocardic tissue. ‘?I 1991 Academic Press, Inc.

ence of large hearts, suggesting that dopamine exerted a stimulating effect on cardiac differentiation (Sarasa and Climent, 1987). The present study was carried out to ascertain whether such an effect was exerted on undifferentiated cells or whether, on the contrary, it was exerted on cells already differentiated or in the process of differentiating into heart. It was approached through two separate types of experiments. First, the cells from the entire area pellucida of embryos at stage 4 treated with dopamine before incubation were dissociated to obtain a suspension of single cells and resuspended for reaggregation in a liquid medium. Then the aggregates were cultivated for differentiation on a semisolid medium. In such experiments, most cells of the aggregates differentiated into myocardie tissue. On the other hand, studies have revealed that explants prepared from regions more than 0.4-0.6 mm posterior to the Hensen’s node of stage 4 chick embryos do not develop into any histologically identifiable structure, except for blood, when grown in unsupplemented medium. This inability to undergo differentiation is not the consequence of a deficiency in nutrients or the physical properties of the substratum because nearly identical results can be obtained with different culture techniques. From there these explants, which are called postnodal pieces (PNPs), have been widely used as a model system to study mechanisms of induction (Butros, 1965; Chauhan and Rao, 1970; Niu and Deshpande, 1973; Lee and Kalmus, 1975; Deshpande and Siddiqui, 1976,1977; Ranzi and De Bernardi, 1983; Lee et ab, 1985).

INTRODUCTION

Heart differentiation is a developmental process that has attracted the attention of many workers. In both amphibians and chicks cardiac morphogenesis has been studied almost step by step (reviewed by DeHaan, 1965) and seems to be an inductive process mediated by the endoderm subjacent to the precardiac splanchnopleure (Orts-Llorca, 1964; Orts-Llorca and Ruano, 1965; Jacobson and Duncan, 1968). Recent molecular biology evidence has revealed that RNA could act as a possible inducer of cardiac differentiation both in salamanders (Davis and Lemanski, 1987) and in chicks (Niu and Deshpande, 1973; Deshpande and Siddiqui, 1977, 1978; Siddiqui, 1983). However, the precise inductive mechanism of action of the RNA or the possible existence of simpler molecules that could trigger the stimulation of genes responsible for the transcription of RNAs specific for cardiac differentiation remains unknown. Catecholamines, known as neurotransmitters and hormones, are broadly distributed in the animal kingdom and occur during early stages of development in all species studied (reviewed by Buznikov, 1981). In the chick, Ignarro and Shideman (1968) detected in the yolk the presence of large quantities of catecholamines, which may enter the embryo. Burack and Badger (1964) observed that the activity of dopa decarboxylase, the enzyme that converts dopa into dopamine, increases from stage 4 (Hamburger and Hamilton, 1951) onward. Recently it was observed that the most outstanding feature of 48-hr chick embryos treated with dopamine, either alone or combined with glucose or ethylenediaminetetraacetic acid, before incubation was the pres243

0012-1606/91 Copyright All rights

$3.00

0 1991 hy Academic Press, Inc. of reproduction in any form reserved.

244

DEVELOPMENTAL

BIOLOGY

In our second type of experiments, PNPs small enough to be sure that they were devoid of precardiogenie cells were used to test the ability of dopamine to induce heart differentiation. At stage 4 it has been demonstrated (Niu and Deshpande, 19’73; Deshpande and Siddiqui, 1976,197’7) that PNPs obtained by transverse cuts 0.6 mm posterior to Hensen’s node were unable to undergo cardiac differentiation in vitro. However, based on tritiated thymidine-labeled grafts obtained from various stages, it was observed that, at this stage, the precardiogenic region extends almost halfway between the anterior and the posterior ends of the primitive streak (Rosenquist, 1970). Thus, to be completely sure that every possible heart-forming cell was excluded, PNPs obtained by transecting the area pellucida of stage 4 embryos halfway between the anterior and the posterior ends of the primitive streak or smaller were used. In these experiments, the presence of dopamine in the culture medium induced cardiac differentiation in a dose-dependent manner. MATERIAL

AND

METHODS

Injection procedures. Fertile White Leghorn eggs were obtained from a local supplier. Before incubation, 0.65 mmole dopamine (Sigma) dissolved in 100 ~1 Ringer’s saline was injected into the subblastodermic cavity as previously described (Sarasa and Climent, 1987). Controls received 100 ~1 Ringer’s saline. Dissociation, reaggregation, and cultivation procedures. Blastoderms were removed after 20 hr of incuba-

tion at 38°C identified for staging, and thoroughly washed in calcium-magnesium-free saline (Moscona, 1961). The pellucid areas were precisely trimmed, cut into small pieces, and incubated for 10 min at 37°C in a solution of 0.5% trypsin (Difco) and 0.3% carboxymethyl cellulose (Schuchardt) in Moscona’s saline. The pieces were profusely washed in fresh Moscona’s saline, placed in glass tubes, and disaggregated into single cells using the free vibrating arm of a shaker (Auerbach and Grobstein, 1958; Murillo et al, 1975). For reaggregation, cells were resuspended in a liquid medium, which consisted of Eagle’s MEM (GIBCO) supplemented with 10% horse serum (Difco), at a concentration of 0.5-0.6 X lo6 viable cells per milliliter. Cell suspension was distributed in 15-ml tubes at a rate of 1 ml per tube and incubated at 37°C in a gyratory shaker at 70 rpm and up to obtain aggregates. After 24 hr, aggregates were transferred to culture dishes containing modified Wolff & Haffen’s semisolid medium (Wolff and Haffen, 1952) consisting of a mixture (vol/vol) of 7 parts of 1% bactoagar (Difco) in Gey’s saline, 3 parts of Tyrode’s saline, and 3 parts of horse serum. The dishes were sealed and incubated for 5 days at 37°C. At regular intervals they were examined to observe the appearance of pulsating tissues.

VOLUME

148,199l

lmm

FIG. 1. Camera lucida drawing chick embryo. PNPs were obtained line) halfway between the anterior tive streak. They were cultivated supplemented with dopamine to from 6.5 to 0.0065 mM

of the area pellucida of a stage 4 by a precise transection (dashed and the posterior ends of the primifor 5 days on a semisolid medium obtain final concentrations ranging

Obtention and cultivation of the PNPs. Because the size of the area pellucida and the length of the primitive streak at stage 4 vary broadly, only embryos with streaks longer than 1.8 mm were used. PNPs were obtained by transecting the area pellucida halfway between the anterior and the posterior ends of the primitive streak (Fig. 1). In addition, to rule out the possibility of a gradient of precardiogenic cells, subregions of the PNP were also used. Explants were cultivated in the semisolid medium described for aggregates but supplemented with diverse concentrations of dopamine. As controls, PNPs obtained in the same way and cultivated in unsupplemented semisolid medium were used. Morphological study. After in vitro culture, all pieces were fixed in 2.5% glutaraldehyde in Sorensen’s buffer (0.1 Mphosphate, pH 7.3) at 4°C for 30 min, postfixed in 2% osmium tetroxide in the same buffer (4°C 30 min), dehydrated in an ethanol series, stained with uranyl acetate, and embedded in Epon. Semithin sections were stained with basic toluidine blue and examined under the light microscope. Ultrathin sections were stained with lead citrate and examined under a Philips EM 301. Determination of acetylcholinesterase activity. The increase in acetylcholinesterase activity has been used as a biochemical marker of cardiac myocyte differentiation (Deshpande and Siddiqui, 1977,1978). Although cardiac muscle differentiation of a population of undifferentiated cells may be ascertained by functional rhythmical beating and/or morphological appearance of myofibrils in its mononuclear cells, in contrast to the

SARASA AND CLIMENT

Dopamine

nonrhythmical contraction and the presence of several nuclei in the differentiation of skeletal muscle cells, we have also employed the acetylcholinesterase enzyme assay as a biochemical marker of the cardiac myocyte differentiation. Both the 16-day-old and the 5-day-old chick embryonic hearts, the cardiac tubes obtained after the differentiation of the cardiogenic areas (these cultured in the same semisolid medium described above), the aggregates, and the PNPs used for the assays were homogenized in cold 50 mM sodium-potassium phosphate buffer, pH 7.0,l mMEDTA-Na,. The homogenates were then centrifuged for 15 min at 10,000 rpm and the supernatant was used. Acetylcholinesterase activity was spectrophotometrically assayed according to the method of Ellman et al. (1961), which consists of the appearance of the dinitrothiobenzoic acid derivative of thiocholine at 412 nm. Thus, the reaction mixture was composed of 0.1 ml of the supernatant plus 0.4 ml of 0.1 M sodium phosphate buffer, pH 8.0, plus 60 ~1 of 10 mMdinitrothiobenzene and 12 ~1 of 75 mM acetylthiocholine. The addition of 2 ~1 of the inhibitor eserine completely stopped the reaction. Protein determination was made according to the method of Lowry et al. (1951). The assays were performed in duplicate for each experiment. RESULTS

AND

DISCUSSION

Dopamine Aggregates

In all cases, including those in which spontaneous pulsations were not observed, almost the entire aggregate differentiated into a well-defined heart muscle tissue (Fig. 2; Table 1). The first contractions were observed after 48 hr of culture in the semisolid medium and became rhythmic by 60 hr. The entire aggregate beat at the same time and the beats lasted until the moment of fixation. Three aggregates, including those that did not show pulsations, consisted mainly of myocardic tissue, although a central mass of cells containing enormous amounts of bundles of intermediate filaments could also be seen. Unlike those that beat, myocardic tissue of nonbeating aggregates was compacted and did not display spaces filled with cardiac jelly-like material. It is noteworthy that dopamine aggregates, obtained after 5 days of culture, showed more acetylcholinesterase activity than the 5-day-old embryonic hearts or the cardiac tubes differentiated from cardiac areas at stage 5 cultured in the same semisolid medium during the same time (Table 2). Control

Aggregates

Control aggregates, obtained from cells of the area pellucida of 20-hr (stage 4) saline-treated embryos, followed their typical pattern of sorting and further dif-

Induces

Heart

Muscle

245

ferentiation (Sanders and Zalik, 1976; Murillo et al., 1978). They exhibited deep epithelial cords, surrounded by mesenchyme, and hypoblastic-like cells occupied the most peripheral position. Most aggregates displayed large cavities containing blood. Unlike the dopamine aggregates, only a small nodule of pulsating tissue could be seen in control aggregates (Fig. 3; Table 1). Moreover, the control aggregates clearly showed an acetylcholinesterase activity lower than that of the dopamine aggregates (Table 2). These results seemed to indicate that dopamine was inducing heart muscle differentiation. However, the question of whether dopamine controlled the state of determination or whether it stimulated the expression of heart muscle phenotype in cells that were already determined remained. For example, the results might be explained if dopamine somehow selectively killed precursors to those cell types that did not subsequently appear in aggregates or if it had only stimulated multiplication and/or differentiation of only the cells of the area pellucida already determined to differentiate into heart. Therefore, to test whether dopamine was really able to induce cells to differentiate into heart muscle, an undifferentiated region of the early embryo, devoid of precardiogenic cells, was selected to be exposed to the action of dopamine. Table 3 shows that dopamine induced PNPs to differentiate into pulsating heart muscle in a dose-dependent manner. The highest inductive effect was recorded at 0.65 mM. At this dose most explants showed rhythmic and spontaneous pulsations, and as occurred in the dopamine aggregates, heart muscle differentiation predominated in all explants. No blood, cavities, or other structures resembling any primary organ were present. Contractions began by the fourth day of culture. At 0.065 mM dopamine, explants did not beat, but when analyzed histologically the greatest part of every explant had differentiated into heart muscle. Nevertheless, the degree of myofibrillar organization was lower at this dose than at 0.65 mM. Heart muscle tissue differentiated in nonbeating explants constantly lacked cardiac jelly-like material. Cells of PNPs cultivated at 0.0065 mM dopamine and those cultivated on the basic medium (controls) did not differentiate into heart muscle tissue, but rather into blood, following their fate map (Settle, 1954; Wilt, 1967; Bellairs, 1971). At 6.5 mMdopamine, PNPs did not develop. Smaller explants, obtained by cutting caudally halfway between both ends of the primitive streak, also differentiated into myocardic tissue in the presence of 0.65 mM dopamine. Explants consisting of very small regions of the PNP did not grow or develop under our experimental conditions. PNPs obtained from stage 5 embryos, when they are completely devoid of precardiogenic cells because cardiogenic areas are well defined, cranially located, and already commit-

246

DEVELOPMENTALBIOLOGY

vOLUME148,1991

FIG. 2. Semithin (a) and ultrathin (b) sections of an aggregate obtained from cells of the area pellucida of embryos treated with dopamine before incubation and cultivated for 5 days on a semisolid medium. (a) The entire core differentiated into pulsating heart muscle tissue consisting of irregular strands separated by spaces filled with a cardiac jelly-like material. The periphery is formed by hypoblastic-like cells. Bar = 0.1 mm. (b) Myocardic tissue consisted of highly differentiated myocytes similar to normal embryonic myocardial cells. Bar = 1 pm. FIG. 3. Semithin section of an aggregate obtained from cells of the area pellucida of control (saline-treated) embryos and cultivated for 5 days on a semisolid medium. Blood cells inside blood lacunae (small arrows), epithelial cords, and a region of contractile tissue (large arrow) can be observed. Bar = 0.1 mm.

ted to differentiating into heart, also differentiated into pulsating tissue in some cases if 0.65 mM dopamine was present in the culture medium. The latter results clearly showed that dopamine behaves as a specific inducer of heart muscle differentiation. In PNPs at stage 4 obtained by transecting the area pellucida 0.6 mm behind Hensen’s node, and therefore larger than ours, heart muscle differentiation was obtained with sulfhydryl-containing amino acids (Lee and Kalmus, 1975), cyclic AMP (Deshpande and Siddiqui, 1976), and total RNA from embryonic chick heart (Niu

and Deshpande, 1973). However, unlike dopamine, all these substances induced, in addition to heart muscle tissue, several other mesoblastic differentiations such as notochord, nephric tubules, somites, and blood, as well as neural tissue. Also, such PNPs could contain precardiogenic cells according to studies using tritiated thymidine-labeled grafts (Rosenquist, 1970). Heart muscle differentiation has also been obtained with a distinct low-molecular-weight RNA from chick embryonic heart (7 S CEH-RNA; Siddiqui, 1983). This RNA appears to play a crucial role in embryonic chick

SARASA AND CLIMENT

Dopamine

TABLE 1 DIFFERENTIATION IN VITRO OF AGGREGATES OF DISSOCIATED CELLS FROM THE AREA PELLUCIDA OF CONTROL AND DOPAMINE-TREATED STAGE 4 CHICK EMBRYOS

Induces

Heart

247

Muscle

TABLE 3 DIFFERENTIATION IN VITRO OF PNPs OF STAGE 4 CHICK CULTIVATED IN SEMISOLID MEDIUM CONTAINING DIFFERENT TRATIONS OF DOPAMINE Heart

Heart muscle tissue (% ) Number of aggregates Control Dopamine

8

15

Total 6 (75) 15 (100)

Beating 6 (75) 13 (87)

Blood

(%)

Others

(W)

8 (100) 0 (0)

7 (87) 3 (20)

Concentration of dopamine (mM) 6.5 0.65

12

11

0.065 0.0065

None

heart formation because when added to PNPs it promotes specific changes that are similar to the embryonic myogenic process. In a critical analysis of the heart muscle differentiation induced by 7 S CEH-RNA in PNPs, Siddiqui (1983) noted that only precardiogenic cells seem to respond to the 7 S CEH-RNA. When PNPs were isolated by sequential cuts, at intervals of 0.1 mm, beginning with 0.4 mm posterior to Hensen’s node, those PNPs obtained by cutting below 0.8 mm posterior to Hensen’s node failed to develop heart-like tissue. Unlike 7 S CEH-RNA, dopamine is capable of inducing myocardic differentiation in PNPs obtained by cutting below 0.9 mm posterior to Hensen’s node. Altogether these results underline the specific determinant character of dopamine to induce heart muscle differentiation in PNPs. Recently it was observed (Wiens et al., 1984) that collagen synthesis inhibitors prevented the functional differentiation of precardiac mesoderm obtained from embryos at stages 6-8. Because the synthesis of actin was not affected, whereas myofibrillogenesis was depressed, it was suggested that the extracellular protein collagen was necessary for either the synthesis of nonactin cardiac contractile proteins or the assembly of cardiac contractile proteins into myofibrils. In both aggregates and PNPs, a feature of beating myocardic tissue was the presence of cardiac jelly-like material surrounding the contractile tissue while nonbeating myocardic tissue displayed cell compaction and lacked spaces filled with cardiac jelly-like material. Results suggest that (i) given

TABLE 2 ACETYLCHOLINESTERASE ACTIVITY IN 16-DAY-OLD AND ~-DAY-OLD CHICK EMBRYONIC HEARTS, CARDIAC TUBES DIFFERENTIATED FROM CULTURED CARDIAC AREAS, DOPAMINE AGGREGATES, AND CONTROL AGGREGATES 16-day-old heart Sday-old heart Cultured cardiac area Dopamine aggregate Control aggregate

Note.

Standard

5.079 1.011 0.550 2.695 1.050

deviation

(+0.1X9) (kO.037) (eO.106) (t0.129) (kO.011)

~mole/min/mg flmole/min/mg ~mole/min/mg ~mole/min/mg flmole/min/mg

is in parentheses.

protein protein protein protein protein

(l/1000) (l/1000) (l/1000) (l/1000) (l/1000)

Number of PNPs

8 8

(control)

27

Total 0 11 8

0 0

EMBRYOS CONCEN-

muscle tissue Beating 0 8 0 0 0

Blood 0 0 0 8

27

Others 0 0 0 0 0

the great amount of highly organized sarcomeric myofibrils displayed by the nonbeating myocardic tissue, factors other than collagen must also be taken into account to explain the sarcomeric organization of myofibrils, and (ii) extracellular matrix materials seem to be necessary for the functional differentiation or beating of myocardic tissue. We do not yet know if dopamine is responsible for these effects or if they are the result of a by-product. Our studies also do not reveal whether dopamine behaves as a morphogenetic hormone, inducing cells to differentiate into heart muscle tissue. For example, we do not know whether it binds cytosolic or membrane receptors and whether it is working through some messenger(s). Note that preliminary results indicate that dopamine could act through its binding to some receptor because its equiagonist, epinine, also induces heart muscle differentiation in PNPs. This action seems to be specific to dopamine receptors because other neurotransmitters, such as serotonin or acetylcholine, did not induce myocardic differentiation. Likewise, earlier studies showed that cyclic AMP, both a presumed morphogen and an intracytoplasmic messenger in some dopamine effects, can produce a number of differentiations in PNPs, including heart muscle tissue (Deshpande and Siddiqui, 1976). Both 7 S CEH-RNA (Siddiqui, 1983) and dopamine (this report) specifically induce heart muscle differentiation. Together these results suggest that dopamine can be used as an efficient tool to elucidate how cells commit to heart muscle and how they then differentiate. Alternatively, results also suggest that dopamine, or any other functionally related molecule, may be able to trigger the cascade of events leading to the acquisition of heart muscle phenotype. Several years ago, it was suggested (McMahon, 1974) that neurotransmitters were directly involved in the biochemistry of induction. To our knowledge, this is the first evidence that a neurotransmitter can induce the acquisition of a particular state of determination. Studies are currently in progress to ascertain whether dopa-

248

DEVELOPMENTAL BIOLOGY

mine is playing the same role in normal that it seems to be playing in vitro.

development

We thank Dr. Patricia F. Silva for his aid in the determination of acetylcholinesterase activity and our colleagues for the critical reading of the manuscript. REFERENCES AUERBACH, R., and GROBSTEIN, C. (1958). Inductive interaction of embryonic tissues after dissociation and reaggregation. Exp. Cell Res. 15,384-397. BELLAIRS, R. (1971). “Developmental Processes in Higher Vertebrates.” Logos Press, London. BURACK, W. R., and BADGER, A. (1964). Sequential appearance of dopa decarboxylase, dopamine P-oxidase and norepinephrine N-methyl transferase activities in the embryonic chick. Fed Proc. 23, 561. BUTROS, J. (1965). Action of heart and liver-RNA on the differentiation of segments of chick blastoderm. J. Embryo1 Exp. Morphol. 13, 119-128. BUZNIKOV, G. A. (1981). Neurotransmitters in early development. In “Problems in Developmental Biology” (N. Khrushchev, Ed.), pp. 76104. MIR, Moscow. CHAUHAN, S. P. S., and RAO, K. V. (1970). Chemically stimulated differentiation of post-nodal pieces of chick blastoderms. J. Embryol. Exp. Morphol. 23, 71-78. DAVIS, L. A., and LEMANSKI, L. F. (1987). Induction of myofibrillogenesis in cardiac lethal mutant axolotl hearts rescued by RNA derived from normal endoderm. Development 99,145-154. DEHAAN, R. L. (1965). Morphogenesis of the vertebrate heart. In “Organogenesis” (R. L. DeHaan and H. Ursprung, Eds.), pp. 377-419. Holt, Rinehart & Winston, New York. DESHPANDE, A. K., and SIDDIQUI, M. A. Q. (1976). Differentiation induced by cyclic AMP in undifferentiated cells of early chick embryo in vitro. Nature 263, 588-591. DESHPANDE, A. K., and SIDDIQUI, M. A. Q. (1977). A reexamination of heart muscle differentiation in the postnodal piece of chick blastoderm mediated by exogenous RNA. Dev. Biol. 58,230-247. DESHPANDE, A. K., and SIDDIQUI, M. A. Q. (1978). Acetylcholinesterase differentiation during myogenesis in early chick embryonic cells caused by an inducer RNA. Dzferentiation 10,133-137. ELLMAN, G. L., COURTNEY, K. D., ANDRES, V. J. R., and FEATHERSTONE, R. M. (1961). A new and rapid calorimetric determination of acetylcholinesterase activity. B&hem. Pharmacol. 7,88-95. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49-92. IGNARRO, L. J., and SHIDEMAN, F. E. (1968). Appearance and concentrations of catecholamines and their biosynthesis in the embryonic and developing chick. J. Pharmacol. Exp. Ther. 159,38-48. JACOBSON, A. G., and DUNCAN, J. T. (1968). Heart induction in salamanders. J. Exp. 2001. 167,79-103. LEE, H., and KALMUS, G. W. (1975). Studies on cell differentiation: Inducing capacity of sulfhydryl-containing amino acids on postnodal pieces of chick blastoderms. J. Exp. Zoo1 193,37-48. LEE, H., NAGELE, R. G., and ROISEN, F. J. (1985). Nerve growth factor induces neural differentiation in undifferentiated cells of early chick embryos. J. Exp. Zool. 233,83-91.

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LEMANSKI, L. F., PAULSON, D. J., and HILL, C. S. (1979). Normal anterior endoderm corrects the heart defect in cardiac mutant salamanders (Ambyostoma mexicanurn). Science 204,860-862. LINASK, K. K., and LASH, J. W. (1986). Precardiac cell migration: Fibronectin localization at mesoderm-endoderm interface during directional movement. Dew. Biol. 114,87-101. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. BioL Chem.

193,265-275.

MCMAHON, D. (1974). Chemical messengers in development: A hypothesis. Science 185,1012-1021. MOSCONA, A. (1961). Rotation-mediated histogenetic aggregation of dissociated cells. Exp. Cell Res. 22,455-475. MURILLO, N. L., CLIMENT, S., and GOTZENS, V. (1978). Cell affinity and determination during early development. Ann. Fuc. Vet. Zuragoza 12,89-97. [In Spanish] MURILLO, N. L., CLIMENT, S., and VILLAR, J. M. (1975). Differentiation neurale a partir de cellules t&s jeunes dissociees. Bull. Assoc. Anat. 59,689-705.

NIU, M. C., and DESHPANDE, A. K. (1973). The development of tubular heart in RNA-treated postnodal pieces of chick blastoderm. J. Embryol

Exp.

Morphol.

29,485-501.

NIU, M. C., and MULHERKAR, L. (1970). The role of exogenous heartRNA in development of the chick embryo cultivated in vitro. J. Embryol.

Exp.

Morphol.

24, 33-42.

ORTS-LLORCA, F. (1964). Les facteurs determinants de la morphogenese et de la differentiation cardiaque. Bull Assoc. Anat. 123, 3-126.

ORTS-LLORCA, F., and RUANO, G. D. (1965). Influence of the endoderm on heart differentiation. Wilhem Roux’ Arch. Entwicklungsmech. Org. 156,368-370.

RANZI, S., and DE BERNARDI, F. (1983). Nucleic acids and proteins in the first stages of embryonic development. In “Control of Embryonic Gene Expression” (M. A. Q. Siddiqui, Ed.), pp. 115-166. CRC Press, Boca Raton, FL. ROSENQUIST, G. C. (1970). Location and movements of cardiogenic cells in the chick embryo: The heart-forming portion of the primitive streak. Deu. Biol. 22, 461-475. SANDERS, E. J., and ZALIK, S. E. (1976). Aggregation of cells from early chick blastoderms. Dzflerentiaticm 6, l-11. SARASA, M., and CLIMENT, S. (1987). Effects of catecholamines on early development of the chick embryo: Relationship to effects of calcium and CAMP. J. Exp. Zool. 241,181-190. SE~LE, G. W. (1954). Localization of erythrocyte-forming areas in the early chick blastoderm cultivated in vitro. Contrib. Embryol. 35,221; 237. SIDDIQUI, M. A. Q. (1983). Control of gene activity in early cardiac muscle development. In “Control of Embryonic Gene Expression” (M. A. Q. Siddiqui, Ed.), pp. 255-279. CRC Press, Boca Raton, FL. WIENS, D., SULLINS, M., and SPOONER, B. S. (1984). Precardiac mesoderm differentiation in vitro: Actin-isotype synthetic transitions, myofibrillogenesis, initiation of heartbeat, and the possible involvement of collagen. Diflzrentiation 28,62-72. WILT, F. H. (1967). The control of embryonic hemoglobin synthesis. A&.

Morphog.

6,89-125.

WOLFF, E., and HAFFEN, K. (1952). Sur une methode de culture d’organes embryonnaires in vitro. Tex. Rep. Biol Med. 10,463-472.