Well-defined growth factors promote cardiac development in axolotl
mesodermal explants
ANTHONY J. MUSLIN and LEWIS T. WILLIAMS*
Howard Hughes Medical Institute, Program of Excellence in Molecular Biology, Cardiovascular Research Institute, University of California,San Francisco, San Francisco, CA 94143-0724, USA
* Corresponding author
Summary
The effect of growth factors on the formation of cardiacmesoderm in the urodele, Ambystoma mexicanum(axolotl), has been examined using an in vitro explantsystem. It has previously been shown that cardiacmesoderm is induced by pharyngeal endoderm duringneurula stages in urodeles. In this study, explants ofprospective cardiac mesoderm from early neurula stageembryos rarely formed beating cardiac tissue in culture.When transforming growth factor beta-1 (TGF-/5i) orplatelet-derived growth factor BB (PDGF) was added tosuch explants, the frequency of heart tissue formationincreased markedly. The addition of other growth
factors to these explants did not enhance cardiacmesoderm formation. The addition of basic fibroblastgrowth factor (bFGF) to prospective heart mesodermderived from later stage embryos resulted in a decreasedtendency to form cardiac tissue. These results suggestthat growth factors analogous to TGF-ft, PDGF, andbFGF may regulate the initial stages of vertebratecardiac development in vivo.
The vertebrate heart develops from embryonic meso-derm. The process of cardiac development has beenstudied most extensively in amphibian species. Fatemap and embyonic explant studies have identified thelocation of cardiac precursor cells in several amphibianspecies. These studies have established that duringearly neurula stages amphibian cardiac primordia arepaired dorsoanterior structures which migrate ventrallyand ultimately fuse in the ventral midline (Chuang andTseng, 1957; Jacobson, 1960; Jacobson, 1961; Jacobsonand Duncan, 1968; reviewed by Jacobson and Sater,1988). The results of explant experiments have alsoshown that amphibian cardiac development requires aninductive interaction. The timing of cardiac inductionvaries among amphibian species: it is completed by lategastrula stages in the anuran Xenopus laevis (Sater andJacobson, 1989), yet in the urodele Taricha torosa,induction is not completed until late neurula stages(Jacobson and Duncan, 1968). The likely source of acardiac inducing substance in urodeles, as determinedby explant experiments, is the pharyngeal endoderm(Fullilove, 1970). This inducing substance has not yetbeen identified.
Cardiac differentiation is also regulated by thepresence in vivo of inhibitory signals. A variety of
explant experiments have suggested that certain tissues(especially neural cells) release an inhibitory signalwhich prevents cardiac differentiation (reviewed byJacobson and Sater, 1988; Smith and Armstrong, 1990).This inhibitory signal has not been identified.
Polypeptide growth factors are likely candidates asinitiators of inductive interactions in embryonic devel-opment. The presence of a variety of growth factors invertebrate embryos have been demonstrated, includ-ing: platelet-derived growth factor (PDGF), fibroblastgrowth factor (FGF), epidermal growth factor (EGF),insulin-like growth factor, nerve growth factor, andmembers of the transforming growth factor beta (TGF-P) family of molecules (Nexo et al. 1980; Adamson andMeek, 1984; Wilcox and Derynck, 1988; Miller et al.1989; Lyons etal. 1989; Ruiz i Altaba and Melton, 1989;Akhurst et al. 1990; Thomsen et al. 1990; reviewed byWhitman and Melton, 1989). A variety of experimentshave suggested that FGF and a TGF-/3-like molecule(possibly Activin B) are the natural substances thatinduce the formation of mesoderm (Slack and Forman,1980; Slack etal. 1987; Kimelman etal. 1988; Kimelmanand Kirschner, 1987; Rosa etal. 1988; Green etal. 1990;Thomsen et al. 1990; reviewed by Smith, 1989).Whether these factors or others regulate cardiacdevelopment is not known. The finding that mRNA for
?! is expressed within or adjacent to the cardiac
1096 A. J. Muslin and L. T. Williams
plate of murine embryos has suggested that this factorplays a role in cardiac development (Akhurst et al.1990).
In this study, embryos from the species Ambystomamexicanum (axolotl) were used to examine the poten-tial role of growth factors in cardiac induction. Axolotlembryos are advantageous compared to other amphib-ian embryos because of their large size, easy avail-ability, and slow pace of development (Armstrong andMalacinski, 1989). Early neurula stage axolotl pre-cardiac mesoderm rarely formed beating heart tissue inculture. When certain growth factors were added tosuch mesodermal explants, beating heart tissue forma-tion was dramatically enhanced.
Materials and methods
Embryonic explant preparationAxolotl embryos were obtained from the Indiana UniversityAxolotl Colony. Embryos were stored at 18°C in 20%modified Steinberg's Solution (MSS), pH7.4 (Armstrong andMalacinski, 1989), and were staged according to the systemdescribed by Schreckenberg and Jacobson (1975). Embryoswere de-jellied manually. Watchmaker's forceps were used toremove the vitelline membrane from embryos. Pre-cardiacmesoderm with underlying ectoderm was isolated fromneurula stage embryos using glass needles, eyebrow hairknives, and hair loops. All microsurgery was performed inagar-lined plastic culture dishes in 100% MSS with addedpenicillin G 100 units ml"1, streptomycin sulfate 100 ^gml"1,and fungizone 1.25 ^gml"1. Embryonic explants were incu-bated for thirty minutes in 100 % MSS with added antibioticsin culture dishes at 18°C. (Sater and Jacobson, 1989). Duringthis incubation period, the underlying ectoderm formed anepithelial vesicle surrounding the explanted mesoderm.Epithelial vesicles were found to promote the survival ofenclosed embryonic tissues. In some cases, growth factorswere added to incubation solutions. Explants were thentransferred in incubation solutions to cover slips to makehanging-drop cultures. Explants were maintained at 18°C andwere observed daily for 30 days using a compound micro-scope.
Growth factorsGrowth factors were added to incubation solutions thatbathed explants of mesoderm. Human recombinant TGF-/3!(Genentech, Inc.), human recombinant PDGF BB (ChironCorp.), human recombinant basic FGF (Chiron Corp.),human recombinant EGF (Genzyme), all-trans retinoic acid(Sigma), and porcine insulin (Collaborative Research) wereadded at the concentration indicated in the text and figures.Bovine serum albumin (final concentration O.lmgml ) wasadded to the incubation solutions containing TGF-/?!, bFGF,PDGF, and EGF. Each explant that was exposed to growthfactor was matched with a control explant obtained from pre-cardiac mesoderm derived from the contralateral side of theembryo. Control explants were incubated in 100 % MSS withadded antibiotics and the appropriate diluents.
HistologyExplants for histological analysis were fixed in 4 % parafor-maldehyde for l-2h, washed in 50% ethanol, dehydrated to100% ethanol, placed in xylenes, then impregnated withparaplast. Eight micron sections were cut and dried onto
gelatin-subbed glass slides. Sections were stained withtoluidine blue.
Statistical AnalysisAs noted above, mesodermal explants were observed for 30days for the presence of beating cardiac tissue. Explants whichexhibited cell autolysis within the 30 day observational periodwere excluded from statistical analysis. Paired experimentaland control explants, obtained from contralateral pre-cardiacmesoderm, were compared using Chi-square analysis orFisher's Exact Test (when Chi-square analysis was inappro-priate).
Results
Timing of cardiac induction in axolotlsThe timing of cardiac induction in axolotls wasdetermined by examining anterolateral mesodermalexplants for the formation of beating heart tissue.Mesodermal explants were isolated from embryos atvarious developmental stages within vesicles of ectoder-mal epithelium and were maintained in hanging-dropcultures (Fig. 1). Explants were initially darkly pig-
Stage 14 *
Ectoderm
y~~-f— Precardiacmesoderm
Add growth factor,30 min. incubation
Vesicle forms•i spontaneously
Hanging dropculture, 18°Cfor 30 days
Fig. 1. Schematic representation of the experimentalmethod. Anterolateral mesoderm and overlaying ectodermwas isolated from early neurula stage axolotl embryos andexposed to various reagents in agar-lined culture dishes.After an incubation period explants spontaneously formedvesicles and were then placed in hanging drop cultures (seetext for details).
GFs promote axolotl heart development 1097
merited but became translucent after 5 to 6 days inculture. Beating cardiac tissue was easily recognized asa small clump of cells that contracted synchronously at arate of 20 to 30 beats per minute (Fig. 2). This rate ofcontraction is consistent with amphibian myocardium(Jones, 1967; Jones and Shelton, 1963). Early neurula(stage 14) mesodermal explants rarely formed beatingcardiac tissue, while late neurula (stage 18) explantsoften formed such beating tissue (Fig. 3).
When pharyngeal endoderm was included in meso-dermal explants from early neurula stage embryos,heart tissue formed frequently (Fig. 3). In pairedexplant experiments, stage 14 mesodermal explantswith added endoderm formed beating heart tissue in63% of cases (12/18), compared with 0% (0/19) ofcontrol explants without added endoderm. The effect ofadded endoderm on the rate of heart tissue formationwas significant by Chi-square analysis (Chi-square withcontinuity correction 14.7, P=0.0001). The mean timebefore beating was observed in endoderm-containingexplants was 9.1 ±1.1 (S.E.) days. In addition, explantsat all neurula stages developed larger and morestructured hearts when endoderm was included.
Addition of growth factors to early neurula-stagemesodermal explantsThe effect of growth factors on cardiac induction wasexamined using pre-cardiac mesodermal explants fromearly neurula stage axolotl embryos. Stage 14 mesoder-mal explants, which rarely formed beating heart tissuein culture, were used to assay the cardiac inducingpotentials of several growth factors. Explants werebathed in solutions containing growth factors and wereobserved daily for the formation of heart tissue. Theconcentrations of growth factors used were based inpart on experiments in which blastula stage animal capexplants were used as a model of mesodermal inductionin Xenopus (Ruiz i Altaba and Melton, 1989; Green etal. 1990). The growth factors used in this study have allbeen localized in the embryonic tissues of vertebrates.In general, the amino acid sequences of the growthfactors tested here are highly conserved among ver-tebrate species, allowing for the use of mammalianfactors with amphibian tissues.
When stage 14 mesodermal explants were bathed inincubation solution containing 30ngmPJ of TGF-ft,59% (20/34) formed beating heart tissue comparedwith 0% (0/34) of paired controls (Figs 2 and 4). Thisresult was highly significant by Chi-square analysis(Chi-square with continuity correction 25.6,P=0.0001). The mean time before beating was ob-served in mesodermal explants exposed to TGF-ft was11.3±1.2 (S.E.) days. When SOngml"1 PDGF BB wasincluded in the incubation solution, 41% (12/29) ofexplants formed beating tissue compared with 0%(0/29) of paired controls (Chi-square with continuitycorrection 12.7, F=0.0004). The mean time beforebeating was observed in explants exposed to PDGF BBwas 12.8±2.1 days. Incubation solutions containingSOngml"1 of EGF increased heart formation to 22%(7/32) compared with 6% (2/32) of paired controls.
Fig. 2. Early neurula stage axolotl mesodermal explants inepithelial vesicles after 2 weeks in culture. A, Controlexplant without beating cardiac tissue formation.B, Explant exposed to TGF-ft (30-ngmr1). Beatingcardiac tissue formation was observed at the site indicatedby the arrow. C, Explant containing pharyngeal endoderm.The beating cardiac tissue in the endoderm-containingexplant (arrow, panel C) was larger and more developedmorphologically than the comparable beating tissue in theexplant exposed to growth factor (arrow, panel B). Scalebar, 250 fim.
1098 A. J. Muslin and L. T. Williams
100 I
c80-
(0a>
CD 60
v>c(0
XUJ
20-
+Endoderm
Mesoderm
4 E
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nts
Sta
ge
1
+Endo i1 ^
+TGF-B1 •
+PDGF H
46GF H
+TGF/FGF H
+RA
+WGF
+lnsulin
0 %
0 %
Meso H
C
• 7% (2/27)
3% (1/29)
(0/23)
(0/25)
3% (6/218)
20
^ ^ ^ • 4 1 % (11/19)
(7/32)
40 60% Beating
3% (12/19)
(20/34)
80 U
14/15 16/17 18/19
Embryonic Stage
Fig. 3. Effect of endoderm on formation of beating cardiactissue in mesodermal explants. The percentage ofembryonic explants that formed beating cardiac tissue isplotted as a function of the embryonic stage used as asource of the explants. All explants were observed for 30days and the presence of beating cardiac tissue formationwas assessed by microscopic observation. The hollowsquares represent pre-cardiac mesodermal explants inepithelial vesicles, while the solid diamonds representmesodermal explants with pharyngeal endoderm includedin the epithelial vesicle. Embryos were staged using themethod described by Schreckenberg and Jacobson (1975).
The effect of EGF on beating tissue formation inmesodermal explants was not significant by statisticalanalysis (Fisher's Exact Test, 2-tailed, P>0.05). Themean time to beating for EGF-exposed explants was13.0±3.5 days.
Stage 14 mesodermal explants incubated in saltsolution containing 50ngml~x of basic FGF did notform beating heart tissue in any of 23 explants (0/23)(Fig. 4). Insulin, at a concentration of 600ngml~1 hadno effect on heart formation in this assay (0/25).Retinoic acid was also ineffective at promoting heartdevelopment at a concentration of 1/ZM (3%, 1/29).
Fate map and embryonic explant studies havedemonstrated that posterior mesoderm from neurulastage urodele embryos does not usually form cardiactissue (reviewed by Jacobson and Sater, 1988). Toassess whether TGF-& could respecify posterior meso-derm to form heart, Stage 14 posterior mesodermalexplants were examined for their ability to form beatingheart tissue in hanging-drop culture. In no cases didposterior mesodermal explants cultured in salt solution(0/23) or in TGF-fr SOngmT1 (0/23) form beatingheart tissue in culture.
Inhibition of cardiac induction by bFGFFGF inhibits skeletal muscle development in certaincell lines (Vaidya et al. 1989). In this study, FGF did notpromote cardiac development in early neurula stagemesodermal explants (Fig. 4). To assess the possibility
Fig. 4. The effect of added growth factors on beating heartformation in stage 14 mesodermal explants. Humanrecombinant basic FGF (SOngml"1), porcine insulin(600ngml~1), retinoic acid all-trans (1/iM), humanrecombinant EGF (SOngmT1), human recombinant PDGFBB (SOngml"1), or human recombinant TGF-ft(SOngmT1) were added to explants at the time tissue wasremoved from embryos. All explants were observed for 30days for the presence of beating cardiac tissue. The lowestbar represents pooled mesodermal explants incubated incontrol solutions. The uppermost bar representsmesodermal explants with anterior endoderm included.Beating heart formation in explants was usually detectedwithin 14 days of culture.
that FGF has an inhibitory effect on cardiac develop-ment, early neurula mesodermal explants were incu-bated in solutions containing both 50 ng ml"1 bFGF and30ngmrx TGF-ft. Explants bathed in both growthfactors formed beating heart tissue in 7% (2/27) ofcases, which was similar to the rate of heart formationin control explants (4%, 1/27), and was considerablyless marked than the effect of TGF-/3! alone onmesodermal explants (57%, 17/30).
The inhibitory potential of FGF was also tested onmesodermal explants from midneurula stage embryosin which heart induction is often completed. Inmesodermal explants from Stage 15-17 embryos,beating heart formation occurred in 37 % of cases(14/38). However, when SOngml"1 of bFGF was addedto incubation solutions, heart formation was decreasedto 13% (Chi-square with continuity correction'4.5,P=0.03).
Histologic appearance of explantsEarly neurula mesodermal explants were maintained inculture for 2 weeks then were fixed and stained as notedabove. Mesodermal explants incubated in salt solutionwere composed largely of thin cells immediately withinthe epithelial vesicle, and loose collections of fibroblast-like cells (Fig. 5). These cell types have been referred toas mesothelium and mesenchyme, respectively, byGreen et al. (1990). Mesodermal explants exposed toTGF-/3j sometimes exhibited tightly-packed sphericalcollections of cardiomyocytes contained within a thin
GFs promote axolotl heart development 1099
5A
BFig. 5. The histologic appearance of early neurula stagemesodermal explants fixed after 2 weeks in culture.A, control mesodermal explant. Note the thin sheet ofmesothelium-like cells (me) adjacent to the epithelialvesicle. B, Mesodermal explant exposed to TGF-^. Notethe circular collection of tightly packed cardiomyocytes(cm), contained within a thin-walled sac. Scale bar, 180fan.
sac which, in turn, was contained within the thick-walled epithelial vesicle. Other mesodermal explantsexposed to TGF-^ were composed of small collectionsof cardiomyocytes within the epithelial vesicle.
Discussion
We have examined the effect of growth factors onaxolotl cardiac induction using a mesodermal explantsystem. Previous studies have suggested that cardiacinduction in amphibians results from the release of asignal from endoderm which influences mesoderm todifferentiate into myocardium (reviewed by Jacobsonand Sater, 1988). The timing of this inductive interac-tion appears to vary between amphibian species,occurring earlier in anurans than in urodeles (Sater andJacobson, 1989). Recently, Smith and Armstrong(1990) reported that cardiac specification in axolotlsoccurs during early neurula stages, using isolated
mesodermal explants without the addition of inducingsubstances. In their study, more stage 14 mesodermalexplants formed beating heart tissue (20 %) than in ourwork, however, this may be explained by their use of adifferent staging system in which stage 14 is defined byslightly more mature embryos.
Taking advantage of the fact that cardiac induction isnot completed in early neurula stage axolotl embryos,we tested the ability of various growth factors toenhance heart formation in mesodermal explants. Twowell defined growth factors enhanced heart formationin this in vitro system. Undifferentiated anteriormesoderm, when exposed to TGF-ft or PDGF BB,formed beating heart tissue. Although our results donot prove that these factors induce cardiac tissue invivo, both TGF-y^ and PDGF have been found inneurulating vertebrate embryos. One interpretation ofour results is that TGF-& and PDGF replicate theeffects of a natural inducer molecule, stimulating apattern of gene expression in undifferentiated meso-derm that culminates in the formation of heart.However, addition of TGF-/?X or PDGF to mesodermalexplants did not completely compensate for theremoval of pharyngeal endoderm: cardiac tissue thatdeveloped in growth factor-exposed mesodermalexplants was smaller, simpler in structure, and haddelayed onset of beating compared with endoderm-containing explants.
The assay system used in this study, the observationof rhythmically beating heart cells, examines a latestage in cardiac development. A large variety ofstructural changes are required to transform anundifferentiated mesodermal cell into a beating heartcell. In our observation system, 10 to 14 days wererequired to observe the effects of growth factors. Theprolonged nature of this system affects the interpret-ation of the data presented in several ways. First, it ispossible that some of the ineffective growth factorstested may stimulate the synthesis of some but not all ofthe proteins required for spontaneous beating. Second,it is not clear that TGF-ft and PDGF stimulation ofmesoderm establishes a pattern of gene expressionwhich culminates in heart formation. These factorsmight simply enhance a pre-established pattern ofcardiac gene expression which results in sufficientprotein synthesis for spontaneous beating.
The fact that more than one growth factor waseffective in promoting beating heart formation raisesfurther questions about cardiac induction in amphib-ians. Each growth factor may cause intracellularmetabolic alterations that reproduce the effects of thenatural inducer, such as activation of an intracellularkinase. Alternatively, one of the growth factors testedmay be highly homologous to the native inducer and theother effective factor may simply increase production ofthe in vivo inducer. For example, PDGF may stimulateproduction in mesoderm of TGF-/?! which, in turn, isthe natural inducing substance.
In this study, only anterolateral mesoderm from earlyneurula stage embryos was observed to form beatingheart tissue in culture. When posterior mesoderm was
1100 A. J. Muslin and L. T. Williams
cultured in the presence of TGF-&, beating tissueformation did not occur. This suggests that TGF-ft, atthe concentation tested, was unable to respecifyposterior mesodermal cell fate.
We found that bFGF inhibited myocardial develop-ment in axolotl mesodermal explants. During normalembryogenesis, pre-cardiac mesoderm is located adjac-ent to the neural plate during early neurula stages. Asneurulation proceeds, pre-cardiac mesoderm migratesventrally, away from the forming neural tube. Jacobsonand Duncan (1968) noted that amphibian neural tissuereleased some inhibitory substance which inhibitsbeating heart tissue formation in mesodermal explants.Several studies have demonstrated the presence of FGFin developing vertebrate neural tissue (Gonzalez et al.1990; Mascarelli etal. 1987; Risau, 1986). It is thereforepossible that FGF may be a natural inhibitor of cardiacinduction.
The ability of bFGF to inhibit cardiac differentiationsuggests that although cardiac specification is com-pleted in many embryos by mid-neurulation, cardiacdetermination has not yet occurred. In fact, Smith andArmstrong (1990) concluded that axolotl cardiacinduction required 2 signalling interactions: an initialsignal that causes cells to begin differentiating intomyocardium, and a second signal that leads to theorganization of functional sarcomeric myofibrils. De-velopment of organized myofibrils, which is necessaryfor the onset of rhythmic beating, may occur spon-taneously after the initial inductive interaction ifinhibitory signals are absent (such as in mesodermalexplants). Given this two-step model of cardiacinduction, our findings suggest that: (1) TGF-^ orPDGF can replicate the effect of the natural first-stepinducer and stimulate the development of functionalmyocardium in the absence of inhibitors (in explants),and (2) bFGF simulates the effect of the in vivoinhibitor and prevents the completion of myocardialcell differentiation in tissue that has already receivedthe initial inductive signal. Exposure of stage 14mesodermal explants to both TGF-/}j and bFGF did notresult in an increased frequency of beating heartformation suggesting that TGF-^j was unable tocompensate for bFGF inhibition of cardiac differen-tiation.
The identification of highly specific early marker geneproducts of cardiac induction will be helpful to furtherexamine the interactions that lead to heart formation.Unfortunately, many cardiac sarcomeric protein geneproducts are not uniquely expressed in embryoniccardiac tissue. No regulatory genes of cardiac develop-ment have been identified at this time. This axolotlmesodermal explant system, however, offers an ap-proach to identifying early marker and regulatory geneproducts of cardiac induction.
We thank L. S. Cousens (Chiron Corp.) for providingPDGFBB and bFGF, R. Ebner and R. Derynck (Genentech,Inc.) for providing TGF-ft, and E. Amaya, J. Escobedo, W.J. Fantl, M. W. Kirschner and A. K. Sater for helpfuldiscussions. This work was supported by N.I.H. Program of
Excellence in Molecular Biology grant HL43821 (L.T.W.) andNational Research Service Award 1F32HL08035-01(A.J.M.).
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