JOURNAL OF CELLULAR PHYSIOLOGY 146:379-385( 1991I
Immunolocalization of Basic Fibroblast Growth Factor During Chicken Cardiac Development SETH A. CONSlGLl AND JACQUELYNJOSEPH-SILVERSTEIN*
Department of Biological Sciences, St. john’s University, jamaica, New York 17439 Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFCF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.
Cardiac development involves a complex series of survival and neurite outgrowth (Hatten et al., 1988; coordinated events that ultimately lead to the forma- Vaca et al., 1989; Unsicker et al., 1987). Elucidation of tion of a highly vascularized, innervated, contractile the temporal and spatial expression of bFGF protein organ. Although cardiac morphogenesis has been well throughout cardiac development, as well as correlation described, little is known concerning the molecular of the pattern of bFGF expression with particular signals involved in re ulating its development. Recent developmental events, should greatly aid in identifying immunohistochemica studies performed in our labora- its role(s) during cardiogenesis. tory have identified the heparin-binding growth factor, Our initial immunohistochemical studies (Josephbasic fibroblast growth factor (bFGF) in myocardial Silverstein et al., 1989) suggested that myocyte bFGF cells from 2- to 5-day embryonic chick hearts (stages distribution began to change at embryonic day 5, with 12-26) (Joseph-Silverstein et al., 1989).The presence of a noticeable decrease in ventricular staining. bFGF bFGF in embryonic heart myocytes suggests that this was not detected in any other tissues in the developing growth factor may play a role in cardiac development. heart, including endothelium, epicardium, and vascuBoth basic and acidic FGF have been identified in lar smooth muscle, at any time at 2-5 days. To deterextracts of cultured cardiac myocytes from adult (Speir mine whether the apparent alteration in the spatial et al., 1988) and neonatal rat (Weiner and Swain, expression of bFGF seen in the myocardium of 5-day 1989), as well as in extracts of myocardial tissue hearts persists or undergoes further change as develprepared from a variety of species, including chicken, opment proceeds, we have now extended our studies to rat, sheep, cow (Kardami and Fandrich, 1989), and embryonic day 20, which is just before hatching. We normal human adults (Casscells et al., 1990). In addi- demonstrate here that both temporal and spatial regtion, Claycomb and Moses (1988) demonstrated that ulation of bFGF expression occurs in the developing FGF stimulates the proliferation of cultured rat adult myocardium at days 5-20 of embryogenesis. We also ventricular myocytes. Taken together, these studies demonstrate here that, although alterations occur in suggest that FGF may play an autocrine role in the myocyte bFGF expression during development, no apparent changes occur in the expression of bFGF in other re lation of myocyte proliferation. B G F has a variety of in vitro biological activities cardiac tissues. Endothelium, epicardium, and vascuthat may be relevant to normal cardiogenesis. These lar smooth muscle remain negative for bFGF immunoinclude stimulation of endothelial cell proliferation (Gospodarowicz et al., 1983, 1986; Moscatelli et al., 1986) and angiogenesis (Shing et al., 1985; Moscatelli Received August 27, 1990; accepted December 5, 1990. et al., 19861, and the enhancement of neuronal cell *To whom reprint requests/correspondence should be addressed.
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reactivity throughout development. The studies described here are the first to provide a detailed temporal and spatial map for bFGF immunoreactivity in the developing heart and have led to the identification of a correlation between myocyte proliferation and bFGF expression. This correlation suggests a possible role for bFGF in the autocrine regulation of myocyte proliferation during cardiac embryogenesis. Thus, the generation of a temporal and spatial map for bFGF expression during cardiac development has proved extremely useful in the generation of directly testable hypotheses for its role in this process.
MATERIALS AND METHODS Fertile eggs from White Leghorn chickens were obtained from SPAFAS Inc. (Norwich, CT) and maintained in a humidified incubator at 37°C. Embryos were staged according to Hamburger and Hamilton before dissection. Preparation of antibodies Polyclonal antihuman bFGF antibodies were raised in rabbits, and amma globulin fractions were prepared as previous y described (Joseph-Silverstein et al., 1988). The same serum, depleted of antihuman bFGF activity by adsorption with recombinant human bFGF, was used as a control in these studies as previously described (Joseph-Silverstein et al., 1989). Immunohistochemistry Hearts were dissected from embryos of appropriate stages and fixed immediately in Bouin’s fixative ( 1 5 5 1 saturated aqueous picric acid-formaldehydeglacial acetic acid) from overnight to 4 days, depending on their size. The fixed hearts were dehydrated through a series of ethanol solutions, cleared in xylene, and then infiltrated and embedded in Paraplast Plus (Monojet Scientific, St. Louis, MO). Sections were cut on a rotary microtome, placed in xylene, rehydrated through a series of ethanol solutions (100-70%), and washed in a saturated lithium chloride-70% ethanol solution. For immunohistochemistry using the ABC method (Hsu et al., 1981) sections were incubated for 30 min at room temperature in 0.3%hydrogen peroxide in methanol, to block endogenous peroxidase activity, prior to blocking of nonspecific antibody binding with phosphate-buffered saline (PBS) containing 5% normal goat serum and 0.1% Triton X-100. Sections were then incubated overnight at 4°C in the appropriate primary antibody diluted in PBS containing 5% normal goat serum and 0.1% NP-40. Antihuman bFGF gamma globulins were diluted 100-fold. Adjacent sections were incubated with bFGF-adsorbed gamma globulins as controls. In some studies, adjacent sections were also incubated with either mouse monoclonal anti-actin antibody HUC1-1, which recognizes both a cardiac striated muscle and a vascular smooth muscle actin (Sawtell and Lessard, 1989), or mouse monoclonal anti-actin antibody C-4, which is a generally reactive antibody (Lessard, 1988), as controls for the specificity of the cellular distribution and overall spatial pattern of staining obtained with the anti-bFGF antibodies. Bound antibody was detected in one of two ways. In
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some studies, the peroxidase-based ABC method (Hsu et, al., 1981) employing reagents obtained from Vector Laboratories, Inc. (Burlingame, CA) was used, as previously described (Joseph-Silverstein et al., 1989). Counterstaining with 0.125% methylene blue was carried out in some cases. We also detected bound antibody using an immunogold immunohistochemical technique in conjunction with silver enhancement (Holgate et al., 1983). Briefly, sections that had been incubated overnight with the appropriate primary antibody, as described above, were washed for 30 min. at room temperature in three changes of PBS and then incubated for 1 hr at room temperature in a 50-fold dilution of biotinylated rabbit antigoat IgG (Vector Laboratories, Inc., Burlingame, CA). Sections were then washed again in PBS and incubated with 5 nm colloidal gold particles conjugated to streptavidin (Auroprobe EM, Janssen Biotech, Olen, Belgium) for 1 hr at room temperature, Following extensive washing with nanopure H20 for 1-3 hr, the signal obtained was intensified by incubation of the sections for 2-18 min with reagents in the IntenSE M Silver Enhancement Kit (Janssen Biotech, Olen, Belgium). No counterstain was used with these sections. No differences were seen in the anti-bFGF antibody staining patterns obtained when a comparison of the ABC and immuno old detection methods was done. Photographs were ta en with a Nikon automatic camera using Panatomic X black and white film (Eastman Kodak Co., Rochester, NY). RESULTS A comparison of 4-,7-, 9-, 14-, and 19-day embryonic chick hearts incubated with polyclonal antihuman bFGF immunoglobulins demonstrated a decrease in the amount of myocardial bFGF immunoreactivity with increasing developmental age (Fig. lA,C,E-I). Control slides in which adjacent sections were stained with bFGF-adsorbed antibodies showed no immunoreactivity (Fig. lB,D). As shown in Figure 1,the intensity of anti-bFGF staining was greater in the nontrabeculated outer regions of the ventricles than in the myocardial tissue closest to the cardiac chambers in 7- to 19-day embryos. In addition, with progressive development, the area of the ventricles that stain intensely with anti-bFGF decreased, so that by day 19, only a thin rim of bFGF immunoreactivity, directly underlying the epicardium, remained. Anti-bFGF antibody staining of the atrium, however, remains intense throughout development. The specificity of this staining pattern was demonstrated by incubating adjacent sections with a mouse monoclonal actin antibody, HUC1-1, previously shown to stain embryonic rat hearts in a uniform manner throughout development (Sawtell and Lessard, 1989). The pattern of staining obtained with HUC1-1 was uniform throughout the development of the chick heart, as well (data not shown). As we previously reported for day 4 and 5 embryonic chick hearts, in day 7 to day 20 developing hearts, bFGF immunoreactivity remained confined to the myocardium. In addition, through day 20, bFGF appeared to remain localized mainly in the myocyte cytoplasm (Fig. 2). No nuclear staining was apparent. Neither epicardial nor endocardia1 (Fig. 2) cells were stained with polyclonal anti-human bFGF at any developmen-
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bFGF IN THE DEVELOPING CHICK HEART
Fig. 1. Immunohistochemical identification of bFGF in developing hearts from 4- to 19-day chick embryos. Sections through 4-day (stage 24; A,B), 7-day (C,D), 9-day (E), 14-day (F,I),and 19-day (G,H) chick embryo hearts were incubated with either antihuman bFGF gamma globulins (A,C,E-I) or bFGF-adsorbed gamma globulins (B,D). The detection system used in A and B was immunogold with silver
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enhancement, while the ABC system was used in C-I. Ventricular expression of bFGF is decreased in 7-day through 19-day (A,C,E,H,I) chick embryos. The atria remain darkly stained with anti-bFGF gamma globulins throughout development (C,E-G). Arrows point to atrial myocardium. Magnification bar: 0.33 mm.
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Fig. 2. Immunolocalization of bFGF to myocytes in the developing and chick heart. Sections through 11-day (A),14-day (B), 16-day (C), 20-day (D) chick embryo hearts were incubated with anti-human bFGF gamma globulins. Antibody binding was detected using the ABC method, and sections were counterstained with methylene blue.
Neither epicardium (ep) nor endocardium (arrow) was stained with the anti-bFGF gamma globulins at any of the stages studied. No nuclear (double arrowhead) staining was resent. No endothelial staining was seen (asterisk). Magnification gar: 0.01 mm.
tal stage studied. In addition, no staining of endothelial cells or smooth muscle cells of developing major blood vessels was seen in the hearts (Fig. 3A-C). By contrast, monoclonal anti-actin antibody, (2-4, which is a generally reactive antibody (Lessard, 1988) stained both endothelium and smooth muscle (Fig. 3D). DISCUSSION Recent studies have demonstrated the presence of bFGF in extracts of cardiac tissue (Kardami and Fandrich, 1989; Casscells et al., 1990) and in cultures of neonatal myocytes (Weiner and Swain, 1989). bFGF is capable of stimulating mitogenesis (Claycomb and Moses, 19881, as well as altering the expression of cardiac actin and myosin isozymes (Parker et al., 1990) in cultured cardiac myocytes, su gesting a possible role for bFGF in the autocrine regu ation of myocyte proliferation and differentiation during myocardial development and cardiac hypertrophy. Until recently, however, it was not known whether bFGF is present in embryonic hearts. Studies carried out in our laboratory, using immunohistochemical methods, demonstrated that bFGF is present in 2- to 5-day embryonic chick cardiac myocytes (Joseph-Silverstein et al., 1989). In addition, using a receptor binding assay, Olwin and Hauschka (1990) demonstrated that detectable levels of FGF receptor were present in day-17 chick embryo hearts, but absent in 19-day hearts. Taken together, these studies further suggest a role for bFGF in cardiac development. Several additional hypotheses concerning the role of bFGF in cardiac em-
bryogenesis can be developed on the basis of its in vitro properties. bFGF may be primarily involved in regulating myocyte proliferation and differentiation. However, bFGF is also a potent angiogenic agent (Shing et al., 1985; Moscatelli et al., 1986) and neurotrophic agent (Hatten et al., 1988; Unsicker et al., 1987; Walicke, 1988; Vaca et al., 19851. Thus myocyte-derived bFGF may regulate vascularization and innervation of the developing heart. In this paper we also looked for bFGF expression in cardiac tissues other than myocardium, since the appearance of bFGF at later developmental stages in these tissues may suggest further hypotheses for its role in cardiac development. We have also extended our bFGF immunohistochemical studies to later stages of chick development to determine whether alterations in the spatial and temporal expression of bFGF occur in cardiac tissues during heart development, which correlate with the onset of specific events involved in cardiogenesis. Our studies demonstrate that the intensity of ventricular anti-bFGF expression progressively decreased in day 5-20 embryos. By contrast, atrial expression remained constant. This study is consistent with studies of Kardami and Fandrich (19891, who recently reported that atrial extracts from adult chicken hearts contain increased amounts of bFGF as compared with ventricular extracts. The dramatic pattern of ventricular bFGF expression (see Fig. 11,that is the progressive decrease in expression in the interior, highly trabeculated region of the ventricle, relative to the myocardium adjacent to the
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bFGF IN THE DEVELOPING CHICK HEART
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Fig. 3. Absence of bFGF staining in vascular tissues. Sections through 9-day (A,B) and 15-day (C) chick embryo hearts were incubatedwith antihuman bFGF gamma globulins. Antibody binding was detected using the ABC method, and sections were counterstained with methylene blue. Neither vascular smooth muscle, nor endothe-
lium was stained with anti-bFGF gamma globulins at any of the stages studied. When adjacent sections were stained with monoclonal anti-actin antibody C-4 (D), intense staining was observed. E, epicardium; double arrowhead, coronary artery; arrow, arteriole. Magnification bar: 0.05 mm.
epicardium with development, correlates well with the spatial and temporal attern of myocyte proliferation seen by Jeter and ameron (1971). These workers reported that a progressive increase in the proliferative index of embryonic chick cardiac myocytes occurs from stages 20-23 (approximately days 3-41, followed by a gradual decrease to almost 0 at stage 44 (day 18). In addition, a specific spatial pattern of proliferation, as reflected by 13H]thymidine incorporation, was observed, in which by stage 29 (day 6) proliferative activity was limited to the periphery of the myocardium (Jeter and Cameron, 1971). In our studies, we begin to see a decrease in bFGF immunoreactivity at day 5 in the inner, highly trabeculated region of the ventricles, while by day 19, only a thin rim of bFGF immunoreactivity is present in the ventricle, immediately underlying the epicardium (see Fig. 1). The correlation between the temporal and s atial pattern of bFGF immunoreactivity with that o [3H]thymidine incorporation in the developing heart suggests a role for bFGF in the autocrine regulation of myocyte proliferation. The spatial pattern of bFGF expression in the developing chick heart also parallels the pattern of expression of a-vascular actin isoform seen in the developing rat heart by Sawtell and Lessard (1989). These investigators reported a decrease in a-vascular actin immunoreactivity in the inner layers of the developing ventricle and suggested a correlation between its expression and the level of organization of the sarcomere. a-Vascular actin is the first in a sequence of a-actin isoform genes to be expressed during avian cardiogen-
esis (Ruzicka and Schwartz, 1988), sug esting that the expression of this gene product may re ect the state of myocyte differentiation. Thus, the similarity of the spatial pattern of expression between bFGF and avascular actin may sug est a role for bFGF in the regulation of myocyte di erentiation as well as proliferation. This hypothesis is consistent with the work of Parker et al. (19901, who demonstrated that bFGF alters the expression of myosin and actin isoforms in cultured myocytes. Correlation of the spatial and temporal pattern of bFGF expression with other developmental events key to cardiac morphogenesis is less clear. Electron microscopic studies of the embryonic chick heart by Manasek (1970) demonstrated that by day 4 of development, nonmuscle cells appear in the myocardium that are probably fibroblasts (Manasek, 1970), followed by the appearance of presumptive endothelial cells by day 7 (Manasek, 1971). Tokuyasu (1985)reported the a pearance of typical capillaries in 5- to 8-day chick em ryos. In addition, the appearance of nervous tissue in the heart occurs at approximately stage 22 (day 3.5) in the developing chick embryo (Ruckman, 1985). Thus, the initiation of these events occur somewhat before the loss of large amounts of bFGF from the myocardium, suggesting that bFGF could be involved in both myocardial vascularization and/or innervation. In vitro models of cardiac development in which bFGF activity can be inhibited must be carried out to elucidate this issue. These studies are in progress in our laboratory. bFGF has been localized to cardiac endothelium as
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well as vascular smooth muscle in adult heart, by Kardami and Fandrich (1989) and by Casscells et al. (1990). In our studies, bFGF was not seen in either tissue in the developing chick heart at any time during embryogenesis, although monoclonal actin antibody C-4 stained these tissues in adjacent sections. This difference could reflect a difference in the expression of bFGF in embryonic vs. adult heart. Recent preliminary studies in our laboratory demonstrate immunoreactivity of our antihuman bFGF antibodies with chick embryonic limb vascular endothelium and vascular smooth muscle at some of the same developmental stages studies here (unpublished results), suggesting that the lack of expression in cardiac endothelium and smooth muscle is not a result of epitope unavailability. Thus, bFGF may be differentially expressed in developing endothelium and smooth muscle from different tissues. In our earlier studies, bFGF was localized to the cytoplasm of develo ing myocytes. No change in the distribution of bFG within cells was seen with increasing developmental age. Neither nuclei nor extracellular matrices from 7- to 20-da hearts were stained with the antihuman bFGF anti odies. Renko et al. (1990)recently reported that the 18.6-kD form of bFGF is localized to the cytosol of cultured cells, while larger forms can be found in the nucleus. The antibodies used in the studies described here recognize both the 18.6-kD and larger forms of bFGF (Moscatelli et al., 1987), suggesting that the lack of nuclear staining seen in our studies cannot be accounted for by the inability of these antibodies to recognize nuclear bFGF. In fact, our earlier studies (Joseph-Silverstein et al., 1989) demonstrated that the same pattern of staining was obtained in embryonic striated muscle cells and their precursors regardless of whether affinity-purified antibodies recognizing only the 18.6-kD form of bFGF, or a' gamma lobulin fraction recognizing both the 18.6-kD and a farger form of bFGF was used. bFGF was detectable only in the cytoplasm of the myocardial cells. Weiner and Swain (1989) re orted that although aFGF can be detected in extrace lular matrix from neonatal rat myocytes, no bFGF is detectable in their preparations, consistent with our results. By contrast, Kardami and Fandrich (1989) reported immunolocalization of bFGF to intercalated discs, nuclei, and the pericellular region of adult cardiac myocytes of several species. Several possibilities exist that could account for the differences seen in these two studies, including differences in fixation and the use of different polyclonal antibodies. However, it is also possible that the differences reflect biological differences in bFGF distribution in embryonic vs. adult tissues. We were unable to obtain good preservation of cellular structure in embryonic tissues using the fixation procedure of Kardami and Fandrich, making it impossible to shed further light on the reason for the differences in our results. The studies presented here suggest a role for myocyte-derived bFGF in the autocrine regulation of embryonic cardiac myocyte proliferation and differentiation but do not rule out a role for bFGF in either myocardial innervation or vascularization. Further elucidation of the role for bFGF in cardiac morphogen-
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esis require studies involving the inhibition of bFGF activity in in vitro models.
ACKNOWLEDGMENTS This work was partially supported by a grant-in-aid from the American Heart Association, New York City Affiliate to J.J-S. We would like to thank Dr. J.L. Lessard for the generous gift of his monoclonal anti-actin antibodies and helpful comments. We would also like to thank Dr. L. Trombetta for his helpful technical advice.
LITERATURE CITED Casscells, W., Speir, E., Sasse, J., Klagsbrun, M., Allen, P., Lee, M., Calvo, B., Chiba, M., Haggroth, L., Folkman, J., and Epstein, S.E. (1990) Isolation, characterization and localization of heparinbinding growth factors in the heart. J. Clin. Invest., 85:433441. Claycomb, W.C., and Moses, R.L. (1988) Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells. Dev. Biol., 127:257-265. Gospodarowicz, D., Cheng, J., and Lirette, M. (1983) Bovine brain and pituitary fibroblast growth factors. Comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells. J. Cell Biol., 97:1677-1685. Gospodarowicz, D., Neufeld, G., and Schweigerer, L. (1986) Molecular and biological characterization of FGF, an angiogenic factor which also controls the proliferation and differentiation of mesoderm and neurectoderm derived cells. Cell Diff. 91 :1-17. Hatten, M.E., Lynch, M., Rydel, R.E., Sanchez, J. Joseph-Silverstein, J., Moscatelli, D., and Rifkin. D.B. (1988) In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev. Biol., 125:28&289. Holgate, C.S., Jackson P.. Cowen, P.N.. and Bird, C.C. (1983) Immunogold-silver staining: A new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 31:938-944. Hsu, S.M., Raine, L., and Fanger, H. (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577-580. Jeter, J.R. Jr., and Cameron, I.L. (1971) Cell proliferation patterns during cytodifferentiation In embryonic chick tissues: liver, heart and erythrocytes. J. Embryol. Exp. Morih. 25(3):405-422. Joseph-Silverstein, J., Moscatelli, D. an Rifiin, D B. (1988) The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF: the identification of bFGF in adherent elicited murine peritoneal macrophages. J. Immunol. Methods. 110:183192. Joseph-Silverstein, J., Consigli, S.A., Lyser, K.M., and Ver Pault, C. (1989) Basic fibroblast growth factor in the chick embryo: Immunolocalization to striated muscle cells and their precursors. J. Cell Biol. 108:2459-2466. Kardami. E.. and Fandrich. R.R. (1989)Basic fibroblast growth factor in atria and ventricles' of the vertebrate heart. 3. Cell Biol. 109:1865-1875. Lessard, J.L. (1988) Two monoclonal antibodies to actin: One muscle selective and one generally reactive. Cell Motil. Cytoskel., 10:349362. Manasek, F.J. (1970) Histogenesis of the embryonic myocardium. Am. J. Cardiol., 25:149-168. Manasek. F.J. (1971) The ultrastructure of embrvonic mvocardial blood vessels. Dev. Biol., 26r42-54. Moscatelli, D., Presta, M., and Rifkin, D.B. (1986) Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. Proc. Natl. Acad. Sci. USA, 83:2091-2095. Moscatelli, D., Joseph-Silverstein, J., Monejias, R., and Rifkin, D.B. (1987) Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc. Natl. Acad. Sci. (USA), 84:577%5782. Olwin, B.B., and Hauschka, S.D. (1990) Fibroblast growth factor receptor levels decrease during chick embryogenesis. J. Cell Biol., 110:503-509.
bFGF IN THE DEVELOPING CHICK HEART Parker, T.G., Packer, S.E., Schneider, M.D. (1990) Peptide growth factors can provoke “fetal” contractile protein gene expression in rat cardiac myocytes. J. Clin. Invest., 85.507414. Renko, M., Quarto, N., Morimoto, T., and Rifkin, D.B. (1990) Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J. Cell. Physiol., 244:lOS-114. Ruckman, R.N. (1985) Cardiac morphogenesis: Hemodynamic effects. In: Cardiac Morphogenesis. V.J. Ferrans, G. Rosen uist, and C. Weinstein, eds. Elsevier Science Publishing Co., Inc., J e w York, pp. 146-156. Ruzicka, D.L., and Schwartz, R.J. (1988) Sequential activation of a-actin genes during avian cardiogenesis: Vascular smooth muscle a-actin gene transcripts mark the onset of cardiomyocyte differentiation. J. Cell Biol., 107:2575-2586. Sawtell, N.M., and Lessard, J.L. (1989)Cellular distribution of smooth muscle actins during mammalian embryogenesis: Expression of the a-vascular but not the y-enteric isoform in differentiating striated myocytes. J. Cell Biol., 109:2929-2937. Shing, Y., Folkman, J., Haudenschild, C., Lund, D., Crum, R., and Klagsbrun, M. (1985) Angiogenesis is stimulated by a tumorderived endothelial cell growth factor. J . Cell Biochem., 29:275-287. Speir, E., Yi-Fu, Z., Lee, M., Shrivastav, and Casscells, W. (1988)
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Fibroblast growth factors are present in adult cardiac myocytes, in uiuo. Biochem. Biophys. Res. Commun., 157:1336-1340. Tokuyasu, K.T. (1985) Development of myocardial circulation. In: Cardiac Morphogenesis. V.J. Ferrans, G . Rosenquist, and C. Weinstein, eds. Elsevier Science Publishing Co., Inc., New York, pp. 226-237. Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G., and Sensenbrenner, M. (1987) Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc. Natl. Acad. Sci. USA, 845459-5463. Vaca, K., Stewart, S.S., and Appel, S.H. (1989) Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle. J. Neurosci. Res., 2355-63. Walicke, P.A. (1988) Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci., 8:2618-2627. Weiner, H.L., and Swain, J.L. (1989)Acidic fibroblast growth factor mRNA is expressed by cardiac myocytes in culture and the protein is localized to the extracellular matrix. Proc. Natl. Acad. Sci. USA, 86:2683-2687.
Immunolocalization of Basic Fibroblast Growth Factor During Chicken Cardiac Development SETH A. CONSlGLl AND JACQUELYNJOSEPH-SILVERSTEIN*
Department of Biological Sciences, St. john’s University, jamaica, New York 17439 Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neuronal cells also suggest that myocyte-derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immunohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti-bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFCF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.
Cardiac development involves a complex series of survival and neurite outgrowth (Hatten et al., 1988; coordinated events that ultimately lead to the forma- Vaca et al., 1989; Unsicker et al., 1987). Elucidation of tion of a highly vascularized, innervated, contractile the temporal and spatial expression of bFGF protein organ. Although cardiac morphogenesis has been well throughout cardiac development, as well as correlation described, little is known concerning the molecular of the pattern of bFGF expression with particular signals involved in re ulating its development. Recent developmental events, should greatly aid in identifying immunohistochemica studies performed in our labora- its role(s) during cardiogenesis. tory have identified the heparin-binding growth factor, Our initial immunohistochemical studies (Josephbasic fibroblast growth factor (bFGF) in myocardial Silverstein et al., 1989) suggested that myocyte bFGF cells from 2- to 5-day embryonic chick hearts (stages distribution began to change at embryonic day 5, with 12-26) (Joseph-Silverstein et al., 1989).The presence of a noticeable decrease in ventricular staining. bFGF bFGF in embryonic heart myocytes suggests that this was not detected in any other tissues in the developing growth factor may play a role in cardiac development. heart, including endothelium, epicardium, and vascuBoth basic and acidic FGF have been identified in lar smooth muscle, at any time at 2-5 days. To deterextracts of cultured cardiac myocytes from adult (Speir mine whether the apparent alteration in the spatial et al., 1988) and neonatal rat (Weiner and Swain, expression of bFGF seen in the myocardium of 5-day 1989), as well as in extracts of myocardial tissue hearts persists or undergoes further change as develprepared from a variety of species, including chicken, opment proceeds, we have now extended our studies to rat, sheep, cow (Kardami and Fandrich, 1989), and embryonic day 20, which is just before hatching. We normal human adults (Casscells et al., 1990). In addi- demonstrate here that both temporal and spatial regtion, Claycomb and Moses (1988) demonstrated that ulation of bFGF expression occurs in the developing FGF stimulates the proliferation of cultured rat adult myocardium at days 5-20 of embryogenesis. We also ventricular myocytes. Taken together, these studies demonstrate here that, although alterations occur in suggest that FGF may play an autocrine role in the myocyte bFGF expression during development, no apparent changes occur in the expression of bFGF in other re lation of myocyte proliferation. B G F has a variety of in vitro biological activities cardiac tissues. Endothelium, epicardium, and vascuthat may be relevant to normal cardiogenesis. These lar smooth muscle remain negative for bFGF immunoinclude stimulation of endothelial cell proliferation (Gospodarowicz et al., 1983, 1986; Moscatelli et al., 1986) and angiogenesis (Shing et al., 1985; Moscatelli Received August 27, 1990; accepted December 5, 1990. et al., 19861, and the enhancement of neuronal cell *To whom reprint requests/correspondence should be addressed.
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reactivity throughout development. The studies described here are the first to provide a detailed temporal and spatial map for bFGF immunoreactivity in the developing heart and have led to the identification of a correlation between myocyte proliferation and bFGF expression. This correlation suggests a possible role for bFGF in the autocrine regulation of myocyte proliferation during cardiac embryogenesis. Thus, the generation of a temporal and spatial map for bFGF expression during cardiac development has proved extremely useful in the generation of directly testable hypotheses for its role in this process.
MATERIALS AND METHODS Fertile eggs from White Leghorn chickens were obtained from SPAFAS Inc. (Norwich, CT) and maintained in a humidified incubator at 37°C. Embryos were staged according to Hamburger and Hamilton before dissection. Preparation of antibodies Polyclonal antihuman bFGF antibodies were raised in rabbits, and amma globulin fractions were prepared as previous y described (Joseph-Silverstein et al., 1988). The same serum, depleted of antihuman bFGF activity by adsorption with recombinant human bFGF, was used as a control in these studies as previously described (Joseph-Silverstein et al., 1989). Immunohistochemistry Hearts were dissected from embryos of appropriate stages and fixed immediately in Bouin’s fixative ( 1 5 5 1 saturated aqueous picric acid-formaldehydeglacial acetic acid) from overnight to 4 days, depending on their size. The fixed hearts were dehydrated through a series of ethanol solutions, cleared in xylene, and then infiltrated and embedded in Paraplast Plus (Monojet Scientific, St. Louis, MO). Sections were cut on a rotary microtome, placed in xylene, rehydrated through a series of ethanol solutions (100-70%), and washed in a saturated lithium chloride-70% ethanol solution. For immunohistochemistry using the ABC method (Hsu et al., 1981) sections were incubated for 30 min at room temperature in 0.3%hydrogen peroxide in methanol, to block endogenous peroxidase activity, prior to blocking of nonspecific antibody binding with phosphate-buffered saline (PBS) containing 5% normal goat serum and 0.1% Triton X-100. Sections were then incubated overnight at 4°C in the appropriate primary antibody diluted in PBS containing 5% normal goat serum and 0.1% NP-40. Antihuman bFGF gamma globulins were diluted 100-fold. Adjacent sections were incubated with bFGF-adsorbed gamma globulins as controls. In some studies, adjacent sections were also incubated with either mouse monoclonal anti-actin antibody HUC1-1, which recognizes both a cardiac striated muscle and a vascular smooth muscle actin (Sawtell and Lessard, 1989), or mouse monoclonal anti-actin antibody C-4, which is a generally reactive antibody (Lessard, 1988), as controls for the specificity of the cellular distribution and overall spatial pattern of staining obtained with the anti-bFGF antibodies. Bound antibody was detected in one of two ways. In
y
some studies, the peroxidase-based ABC method (Hsu et, al., 1981) employing reagents obtained from Vector Laboratories, Inc. (Burlingame, CA) was used, as previously described (Joseph-Silverstein et al., 1989). Counterstaining with 0.125% methylene blue was carried out in some cases. We also detected bound antibody using an immunogold immunohistochemical technique in conjunction with silver enhancement (Holgate et al., 1983). Briefly, sections that had been incubated overnight with the appropriate primary antibody, as described above, were washed for 30 min. at room temperature in three changes of PBS and then incubated for 1 hr at room temperature in a 50-fold dilution of biotinylated rabbit antigoat IgG (Vector Laboratories, Inc., Burlingame, CA). Sections were then washed again in PBS and incubated with 5 nm colloidal gold particles conjugated to streptavidin (Auroprobe EM, Janssen Biotech, Olen, Belgium) for 1 hr at room temperature, Following extensive washing with nanopure H20 for 1-3 hr, the signal obtained was intensified by incubation of the sections for 2-18 min with reagents in the IntenSE M Silver Enhancement Kit (Janssen Biotech, Olen, Belgium). No counterstain was used with these sections. No differences were seen in the anti-bFGF antibody staining patterns obtained when a comparison of the ABC and immuno old detection methods was done. Photographs were ta en with a Nikon automatic camera using Panatomic X black and white film (Eastman Kodak Co., Rochester, NY). RESULTS A comparison of 4-,7-, 9-, 14-, and 19-day embryonic chick hearts incubated with polyclonal antihuman bFGF immunoglobulins demonstrated a decrease in the amount of myocardial bFGF immunoreactivity with increasing developmental age (Fig. lA,C,E-I). Control slides in which adjacent sections were stained with bFGF-adsorbed antibodies showed no immunoreactivity (Fig. lB,D). As shown in Figure 1,the intensity of anti-bFGF staining was greater in the nontrabeculated outer regions of the ventricles than in the myocardial tissue closest to the cardiac chambers in 7- to 19-day embryos. In addition, with progressive development, the area of the ventricles that stain intensely with anti-bFGF decreased, so that by day 19, only a thin rim of bFGF immunoreactivity, directly underlying the epicardium, remained. Anti-bFGF antibody staining of the atrium, however, remains intense throughout development. The specificity of this staining pattern was demonstrated by incubating adjacent sections with a mouse monoclonal actin antibody, HUC1-1, previously shown to stain embryonic rat hearts in a uniform manner throughout development (Sawtell and Lessard, 1989). The pattern of staining obtained with HUC1-1 was uniform throughout the development of the chick heart, as well (data not shown). As we previously reported for day 4 and 5 embryonic chick hearts, in day 7 to day 20 developing hearts, bFGF immunoreactivity remained confined to the myocardium. In addition, through day 20, bFGF appeared to remain localized mainly in the myocyte cytoplasm (Fig. 2). No nuclear staining was apparent. Neither epicardial nor endocardia1 (Fig. 2) cells were stained with polyclonal anti-human bFGF at any developmen-
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bFGF IN THE DEVELOPING CHICK HEART
Fig. 1. Immunohistochemical identification of bFGF in developing hearts from 4- to 19-day chick embryos. Sections through 4-day (stage 24; A,B), 7-day (C,D), 9-day (E), 14-day (F,I),and 19-day (G,H) chick embryo hearts were incubated with either antihuman bFGF gamma globulins (A,C,E-I) or bFGF-adsorbed gamma globulins (B,D). The detection system used in A and B was immunogold with silver
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enhancement, while the ABC system was used in C-I. Ventricular expression of bFGF is decreased in 7-day through 19-day (A,C,E,H,I) chick embryos. The atria remain darkly stained with anti-bFGF gamma globulins throughout development (C,E-G). Arrows point to atrial myocardium. Magnification bar: 0.33 mm.
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Fig. 2. Immunolocalization of bFGF to myocytes in the developing and chick heart. Sections through 11-day (A),14-day (B), 16-day (C), 20-day (D) chick embryo hearts were incubated with anti-human bFGF gamma globulins. Antibody binding was detected using the ABC method, and sections were counterstained with methylene blue.
Neither epicardium (ep) nor endocardium (arrow) was stained with the anti-bFGF gamma globulins at any of the stages studied. No nuclear (double arrowhead) staining was resent. No endothelial staining was seen (asterisk). Magnification gar: 0.01 mm.
tal stage studied. In addition, no staining of endothelial cells or smooth muscle cells of developing major blood vessels was seen in the hearts (Fig. 3A-C). By contrast, monoclonal anti-actin antibody, (2-4, which is a generally reactive antibody (Lessard, 1988) stained both endothelium and smooth muscle (Fig. 3D). DISCUSSION Recent studies have demonstrated the presence of bFGF in extracts of cardiac tissue (Kardami and Fandrich, 1989; Casscells et al., 1990) and in cultures of neonatal myocytes (Weiner and Swain, 1989). bFGF is capable of stimulating mitogenesis (Claycomb and Moses, 19881, as well as altering the expression of cardiac actin and myosin isozymes (Parker et al., 1990) in cultured cardiac myocytes, su gesting a possible role for bFGF in the autocrine regu ation of myocyte proliferation and differentiation during myocardial development and cardiac hypertrophy. Until recently, however, it was not known whether bFGF is present in embryonic hearts. Studies carried out in our laboratory, using immunohistochemical methods, demonstrated that bFGF is present in 2- to 5-day embryonic chick cardiac myocytes (Joseph-Silverstein et al., 1989). In addition, using a receptor binding assay, Olwin and Hauschka (1990) demonstrated that detectable levels of FGF receptor were present in day-17 chick embryo hearts, but absent in 19-day hearts. Taken together, these studies further suggest a role for bFGF in cardiac development. Several additional hypotheses concerning the role of bFGF in cardiac em-
bryogenesis can be developed on the basis of its in vitro properties. bFGF may be primarily involved in regulating myocyte proliferation and differentiation. However, bFGF is also a potent angiogenic agent (Shing et al., 1985; Moscatelli et al., 1986) and neurotrophic agent (Hatten et al., 1988; Unsicker et al., 1987; Walicke, 1988; Vaca et al., 19851. Thus myocyte-derived bFGF may regulate vascularization and innervation of the developing heart. In this paper we also looked for bFGF expression in cardiac tissues other than myocardium, since the appearance of bFGF at later developmental stages in these tissues may suggest further hypotheses for its role in cardiac development. We have also extended our bFGF immunohistochemical studies to later stages of chick development to determine whether alterations in the spatial and temporal expression of bFGF occur in cardiac tissues during heart development, which correlate with the onset of specific events involved in cardiogenesis. Our studies demonstrate that the intensity of ventricular anti-bFGF expression progressively decreased in day 5-20 embryos. By contrast, atrial expression remained constant. This study is consistent with studies of Kardami and Fandrich (19891, who recently reported that atrial extracts from adult chicken hearts contain increased amounts of bFGF as compared with ventricular extracts. The dramatic pattern of ventricular bFGF expression (see Fig. 11,that is the progressive decrease in expression in the interior, highly trabeculated region of the ventricle, relative to the myocardium adjacent to the
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bFGF IN THE DEVELOPING CHICK HEART
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Fig. 3. Absence of bFGF staining in vascular tissues. Sections through 9-day (A,B) and 15-day (C) chick embryo hearts were incubatedwith antihuman bFGF gamma globulins. Antibody binding was detected using the ABC method, and sections were counterstained with methylene blue. Neither vascular smooth muscle, nor endothe-
lium was stained with anti-bFGF gamma globulins at any of the stages studied. When adjacent sections were stained with monoclonal anti-actin antibody C-4 (D), intense staining was observed. E, epicardium; double arrowhead, coronary artery; arrow, arteriole. Magnification bar: 0.05 mm.
epicardium with development, correlates well with the spatial and temporal attern of myocyte proliferation seen by Jeter and ameron (1971). These workers reported that a progressive increase in the proliferative index of embryonic chick cardiac myocytes occurs from stages 20-23 (approximately days 3-41, followed by a gradual decrease to almost 0 at stage 44 (day 18). In addition, a specific spatial pattern of proliferation, as reflected by 13H]thymidine incorporation, was observed, in which by stage 29 (day 6) proliferative activity was limited to the periphery of the myocardium (Jeter and Cameron, 1971). In our studies, we begin to see a decrease in bFGF immunoreactivity at day 5 in the inner, highly trabeculated region of the ventricles, while by day 19, only a thin rim of bFGF immunoreactivity is present in the ventricle, immediately underlying the epicardium (see Fig. 1). The correlation between the temporal and s atial pattern of bFGF immunoreactivity with that o [3H]thymidine incorporation in the developing heart suggests a role for bFGF in the autocrine regulation of myocyte proliferation. The spatial pattern of bFGF expression in the developing chick heart also parallels the pattern of expression of a-vascular actin isoform seen in the developing rat heart by Sawtell and Lessard (1989). These investigators reported a decrease in a-vascular actin immunoreactivity in the inner layers of the developing ventricle and suggested a correlation between its expression and the level of organization of the sarcomere. a-Vascular actin is the first in a sequence of a-actin isoform genes to be expressed during avian cardiogen-
esis (Ruzicka and Schwartz, 1988), sug esting that the expression of this gene product may re ect the state of myocyte differentiation. Thus, the similarity of the spatial pattern of expression between bFGF and avascular actin may sug est a role for bFGF in the regulation of myocyte di erentiation as well as proliferation. This hypothesis is consistent with the work of Parker et al. (19901, who demonstrated that bFGF alters the expression of myosin and actin isoforms in cultured myocytes. Correlation of the spatial and temporal pattern of bFGF expression with other developmental events key to cardiac morphogenesis is less clear. Electron microscopic studies of the embryonic chick heart by Manasek (1970) demonstrated that by day 4 of development, nonmuscle cells appear in the myocardium that are probably fibroblasts (Manasek, 1970), followed by the appearance of presumptive endothelial cells by day 7 (Manasek, 1971). Tokuyasu (1985)reported the a pearance of typical capillaries in 5- to 8-day chick em ryos. In addition, the appearance of nervous tissue in the heart occurs at approximately stage 22 (day 3.5) in the developing chick embryo (Ruckman, 1985). Thus, the initiation of these events occur somewhat before the loss of large amounts of bFGF from the myocardium, suggesting that bFGF could be involved in both myocardial vascularization and/or innervation. In vitro models of cardiac development in which bFGF activity can be inhibited must be carried out to elucidate this issue. These studies are in progress in our laboratory. bFGF has been localized to cardiac endothelium as
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CONSIGLI AND JOSEPH-SILVERSTEIN
well as vascular smooth muscle in adult heart, by Kardami and Fandrich (1989) and by Casscells et al. (1990). In our studies, bFGF was not seen in either tissue in the developing chick heart at any time during embryogenesis, although monoclonal actin antibody C-4 stained these tissues in adjacent sections. This difference could reflect a difference in the expression of bFGF in embryonic vs. adult heart. Recent preliminary studies in our laboratory demonstrate immunoreactivity of our antihuman bFGF antibodies with chick embryonic limb vascular endothelium and vascular smooth muscle at some of the same developmental stages studies here (unpublished results), suggesting that the lack of expression in cardiac endothelium and smooth muscle is not a result of epitope unavailability. Thus, bFGF may be differentially expressed in developing endothelium and smooth muscle from different tissues. In our earlier studies, bFGF was localized to the cytoplasm of develo ing myocytes. No change in the distribution of bFG within cells was seen with increasing developmental age. Neither nuclei nor extracellular matrices from 7- to 20-da hearts were stained with the antihuman bFGF anti odies. Renko et al. (1990)recently reported that the 18.6-kD form of bFGF is localized to the cytosol of cultured cells, while larger forms can be found in the nucleus. The antibodies used in the studies described here recognize both the 18.6-kD and larger forms of bFGF (Moscatelli et al., 1987), suggesting that the lack of nuclear staining seen in our studies cannot be accounted for by the inability of these antibodies to recognize nuclear bFGF. In fact, our earlier studies (Joseph-Silverstein et al., 1989) demonstrated that the same pattern of staining was obtained in embryonic striated muscle cells and their precursors regardless of whether affinity-purified antibodies recognizing only the 18.6-kD form of bFGF, or a' gamma lobulin fraction recognizing both the 18.6-kD and a farger form of bFGF was used. bFGF was detectable only in the cytoplasm of the myocardial cells. Weiner and Swain (1989) re orted that although aFGF can be detected in extrace lular matrix from neonatal rat myocytes, no bFGF is detectable in their preparations, consistent with our results. By contrast, Kardami and Fandrich (1989) reported immunolocalization of bFGF to intercalated discs, nuclei, and the pericellular region of adult cardiac myocytes of several species. Several possibilities exist that could account for the differences seen in these two studies, including differences in fixation and the use of different polyclonal antibodies. However, it is also possible that the differences reflect biological differences in bFGF distribution in embryonic vs. adult tissues. We were unable to obtain good preservation of cellular structure in embryonic tissues using the fixation procedure of Kardami and Fandrich, making it impossible to shed further light on the reason for the differences in our results. The studies presented here suggest a role for myocyte-derived bFGF in the autocrine regulation of embryonic cardiac myocyte proliferation and differentiation but do not rule out a role for bFGF in either myocardial innervation or vascularization. Further elucidation of the role for bFGF in cardiac morphogen-
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esis require studies involving the inhibition of bFGF activity in in vitro models.
ACKNOWLEDGMENTS This work was partially supported by a grant-in-aid from the American Heart Association, New York City Affiliate to J.J-S. We would like to thank Dr. J.L. Lessard for the generous gift of his monoclonal anti-actin antibodies and helpful comments. We would also like to thank Dr. L. Trombetta for his helpful technical advice.
LITERATURE CITED Casscells, W., Speir, E., Sasse, J., Klagsbrun, M., Allen, P., Lee, M., Calvo, B., Chiba, M., Haggroth, L., Folkman, J., and Epstein, S.E. (1990) Isolation, characterization and localization of heparinbinding growth factors in the heart. J. Clin. Invest., 85:433441. Claycomb, W.C., and Moses, R.L. (1988) Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells. Dev. Biol., 127:257-265. Gospodarowicz, D., Cheng, J., and Lirette, M. (1983) Bovine brain and pituitary fibroblast growth factors. Comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells. J. Cell Biol., 97:1677-1685. Gospodarowicz, D., Neufeld, G., and Schweigerer, L. (1986) Molecular and biological characterization of FGF, an angiogenic factor which also controls the proliferation and differentiation of mesoderm and neurectoderm derived cells. Cell Diff. 91 :1-17. Hatten, M.E., Lynch, M., Rydel, R.E., Sanchez, J. Joseph-Silverstein, J., Moscatelli, D., and Rifkin. D.B. (1988) In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev. Biol., 125:28&289. Holgate, C.S., Jackson P.. Cowen, P.N.. and Bird, C.C. (1983) Immunogold-silver staining: A new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem. 31:938-944. Hsu, S.M., Raine, L., and Fanger, H. (1981) Use of avidin-biotinperoxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577-580. Jeter, J.R. Jr., and Cameron, I.L. (1971) Cell proliferation patterns during cytodifferentiation In embryonic chick tissues: liver, heart and erythrocytes. J. Embryol. Exp. Morih. 25(3):405-422. Joseph-Silverstein, J., Moscatelli, D. an Rifiin, D B. (1988) The development of a quantitative RIA for basic fibroblast growth factor using polyclonal antibodies against the 157 amino acid form of human bFGF: the identification of bFGF in adherent elicited murine peritoneal macrophages. J. Immunol. Methods. 110:183192. Joseph-Silverstein, J., Consigli, S.A., Lyser, K.M., and Ver Pault, C. (1989) Basic fibroblast growth factor in the chick embryo: Immunolocalization to striated muscle cells and their precursors. J. Cell Biol. 108:2459-2466. Kardami. E.. and Fandrich. R.R. (1989)Basic fibroblast growth factor in atria and ventricles' of the vertebrate heart. 3. Cell Biol. 109:1865-1875. Lessard, J.L. (1988) Two monoclonal antibodies to actin: One muscle selective and one generally reactive. Cell Motil. Cytoskel., 10:349362. Manasek, F.J. (1970) Histogenesis of the embryonic myocardium. Am. J. Cardiol., 25:149-168. Manasek. F.J. (1971) The ultrastructure of embrvonic mvocardial blood vessels. Dev. Biol., 26r42-54. Moscatelli, D., Presta, M., and Rifkin, D.B. (1986) Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. Proc. Natl. Acad. Sci. USA, 83:2091-2095. Moscatelli, D., Joseph-Silverstein, J., Monejias, R., and Rifkin, D.B. (1987) Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor. Proc. Natl. Acad. Sci. (USA), 84:577%5782. Olwin, B.B., and Hauschka, S.D. (1990) Fibroblast growth factor receptor levels decrease during chick embryogenesis. J. Cell Biol., 110:503-509.
bFGF IN THE DEVELOPING CHICK HEART Parker, T.G., Packer, S.E., Schneider, M.D. (1990) Peptide growth factors can provoke “fetal” contractile protein gene expression in rat cardiac myocytes. J. Clin. Invest., 85.507414. Renko, M., Quarto, N., Morimoto, T., and Rifkin, D.B. (1990) Nuclear and cytoplasmic localization of different basic fibroblast growth factor species. J. Cell. Physiol., 244:lOS-114. Ruckman, R.N. (1985) Cardiac morphogenesis: Hemodynamic effects. In: Cardiac Morphogenesis. V.J. Ferrans, G. Rosen uist, and C. Weinstein, eds. Elsevier Science Publishing Co., Inc., J e w York, pp. 146-156. Ruzicka, D.L., and Schwartz, R.J. (1988) Sequential activation of a-actin genes during avian cardiogenesis: Vascular smooth muscle a-actin gene transcripts mark the onset of cardiomyocyte differentiation. J. Cell Biol., 107:2575-2586. Sawtell, N.M., and Lessard, J.L. (1989)Cellular distribution of smooth muscle actins during mammalian embryogenesis: Expression of the a-vascular but not the y-enteric isoform in differentiating striated myocytes. J. Cell Biol., 109:2929-2937. Shing, Y., Folkman, J., Haudenschild, C., Lund, D., Crum, R., and Klagsbrun, M. (1985) Angiogenesis is stimulated by a tumorderived endothelial cell growth factor. J . Cell Biochem., 29:275-287. Speir, E., Yi-Fu, Z., Lee, M., Shrivastav, and Casscells, W. (1988)
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Fibroblast growth factors are present in adult cardiac myocytes, in uiuo. Biochem. Biophys. Res. Commun., 157:1336-1340. Tokuyasu, K.T. (1985) Development of myocardial circulation. In: Cardiac Morphogenesis. V.J. Ferrans, G . Rosenquist, and C. Weinstein, eds. Elsevier Science Publishing Co., Inc., New York, pp. 226-237. Unsicker, K., Reichert-Preibsch, H., Schmidt, R., Pettmann, B., Labourdette, G., and Sensenbrenner, M. (1987) Astroglial and fibroblast growth factors have neurotrophic functions for cultured peripheral and central nervous system neurons. Proc. Natl. Acad. Sci. USA, 845459-5463. Vaca, K., Stewart, S.S., and Appel, S.H. (1989) Identification of basic fibroblast growth factor as a cholinergic growth factor from human muscle. J. Neurosci. Res., 2355-63. Walicke, P.A. (1988) Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J. Neurosci., 8:2618-2627. Weiner, H.L., and Swain, J.L. (1989)Acidic fibroblast growth factor mRNA is expressed by cardiac myocytes in culture and the protein is localized to the extracellular matrix. Proc. Natl. Acad. Sci. USA, 86:2683-2687.
