Transforming growth factor-ill localized within the heart of the chick embryo ..... TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of ...
Anat Embryol(1991) 183:345-352
Anatomy and Embryology 9 Springer-Verlag1991
Transforming growth factor-ill localized within the heart of the chick embryo Michael Choy 1, Margaret T. Armstrong 2, and Peter B. Armstrong 2 1 Divisionof Pediatric Cardiology,Department of Pediatrics, Universityof California Davis MedicalCenter, Sacramento, CA 95817, USA 2 Department of Zoology,Universityof Californiaat Davis, Davis, CA 95616, USA Accepted December 21, 1990
Summary. Transforming growth factor-ill is a pleiotropic peptide mediator of growth, differentiation, and extracellular matrix synthesis. In the embryonic chick heart prior to the formation of the endocardial cushions, evidence from in vitro experiments suggests that transforming growth factor-ill may be an inducer of the differentiation of atrioventricular endothelial cells into endocardial cushion mesenchyme. Further in vitro evidence suggests that the factor stimulates mesenchymal cell proliferation, and, thus, growth of the cushions. Using an antibody made against a peptide duplicating the aminoterminal 30 amino acid sequence of transforming growth factor-ill, we stained sections of stage 11, 18, 23, 26, and 36 chick hearts by an in situ immunofluorescence technique. Transforming growth factor-/~l staining localized to the endocardial surface and epicardial surface of the stage 11 heart, but it decreased from these locations in later stages. The cardiac jelly (stage 11), endocardial cushions (stage 18, 23, and 26), and, subsequently, the heart valve leaflets (stage 36) stained intensely for the growth factor. Key words: Heart morphogenesis - Transforming growth factor-ill - Endocardial cushions - Atrioventricular valves
Introduction Transforming growth factor-fl (TGF-/~) type 1 is a pleiotropic peptide mediator of growth, differentiation, and extracellular matrix synthesis. Our previous work and that of other investigators suggest at least two functions of the growth factor in the development of the atrioventricular endocardial cushions in the heart of the chick embryo. At 50 to 55 h of age (stage 15, Hamburger and Hamilton 1951), a distinct pattern of fibronectin, apparOffprint requests to .' M. Choy
ently produced by the myocytes, appears in the matrix of the atrioventricular and outflow regions of the embryonic chick heart (Mjaatvedt et al. 1987). The fibronectin may bind or co-localize with a substance that induces the endothelial cells to differentiate into mesenchymal cells which migrate into the extracellular matrix. TGF-/~ type i or 2, in combination with ventricular myocardial tissue, will induce transformation of organ-cultured cardiac endothelial cells into mesenchyme (Potts and Runyan 1989). Thus, TGF-fll is a candidate for the co-localizing factor in the extracellular matrix that induces the endocardium to mesenchyme transformation. A potential function of TGF-fll in later development of the endocardial cushions is the stimulation of cell proliferation. We have demonstrated that TGF-fll, in the absence of other mitogens, is a potent stimulator of proliferation of cushion mesenchyme maintained as 3-dimensional tissue aggregates in organ culture (Choy et al. 1990). The stimulation appears to be a two-step process, the initial event being the deposition of an adhesive interstitial matrix rich in fibronectin in the cultured aggregate. It is suggested that this matrix is a sufficient condition for subsequent proliferation. In this study we show that TGF-fll is present in the atrioventricular cardiac jelly of the early heart prior to the formation of the endocardial cushions (stage 11), in the endocardial cushions during growth (stages 18, 23, and 26), and in the atrioventricular valves when they are still remodeling (stage 36). Thus, it is present at times and locations consistent with an inductive function at early stages and a function as regulator of proliferation at later stages.
Materials and methods Antibodies. The anti-A1/30 antibody used for TGF-/~I staining
(also calledanti-CC1/30 antibody)was obtained from CeltrixLaboratories (A subsidiaryof CollagenCorporation, 2500 Faber Place, Palo Alto, Calif., USA). The polyclonal antibody is produced in the rabbit against a syntheticpolypeptide that duplicates the 30 amino acid sequence of the amino-terminalend of human TGF-fll
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cranial
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0.5mm Fig. 1. Section of a stage 11 chick heart stained for TGF-~I by an immunofluorescence technique described in the text. The stage 11 cardiac tube has looped forming a primitive atrium (a) and ventricle. Prior to the formation of the atrioventricular endocardial cushions, extracellular TGF-fil staining is seen in the cardi-
(Ellingsworth et al. 1986). The human TGF-fil is identical to the chicken TGF-/?I (Jakowlew et al. 1988a), and it differs by one amino acid in the mouse, but the 30 amino acid amino-terminal ends in all three species are the same (Derynck et al. 1986). The anti-A1/30 antibody in previous investigations (Heine et al. 1987; Flanders et al. 1989; Thompson et al. 1989) has been well characterized and stained TGF-/?I in extracellular sites. Flanders et al. (1989) suggested that secretion of the latent TGF-~I complex (Wakefield et al. 1988; Miyazono et al. 1988) from the cells may change the spatial arrangement of the TGF-fil molecule in the complex, exposing the epitope recognized by this antibody. Thus, the antibody may recognize the latent form as well as the active form of TGF-~I (Thompson et al. 1989). It does not recognize intact TGF-p2 on Western blots or by ELISA (Flanders et al. 1989). The goat anti-chicken plasma fibronectin antibody used for fibronectin staining was obtained from Dr. E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, Calif., USA). The second antibody for the TGF-fll staining was a fluoresceinconjugated sheep anti-rabbit IgG (Organon Teknika-Cappel, 1 Technology Court, Malvern, Pa., USA). For the fibronectin staining, a fluorescein-conjugated rabbit anti-goat IgG was obtained from ICN ImmunoBiologicals (P.O. Box 1200, Lisle, Ill., USA).
Histology. White Leghorn chicken eggs were incubated at 37.5~ C in humidified air for 2, 3, 4, 5, and 10 days. This produced embryos of stage 11, 18, 23, 26, and 36 (Hamburger and Hamilton 1951). The stage 11, 18, 23, and 26 embryos, and the isolated hearts at stage 36, destined for TGF-fil staining, were placed in Bouin's fixative (75.0 ml saturated picric acid, 25.0 ml concentrated formalin, 5.0 ml glacial acetic acid). Similarly staged embryos destined for fibronectin staining were fixed in Carnoy's solution (6.0 ml ethyl alcohol, 3.0 ml chloroform, and 1.0 ml glacial acetic acid). The embryos or isolated hearts were embedded in paraffin and sectioned in the frontal plane at a thickness of 6 gm. Serial sections were mounted on gelatin-coated slides and stained as described below. For the TGF-fll staining, 14-day-old Swiss Webster embry-
i
caudal
ac jelly (j) adjacent to the endothelial cells (e), and along the pericardia1 surface of the epimyocardium (epm). In addition, TGF-~I extends through the cardiac jelly at least in the atrioventricular region (av). The line drawing demonstrates the section of heart with the more caudal structures
onic mouse hearts were fixed, sectioned, and mounted onto glass slides in order to obtain positive controls (Heine et al. 1987).
Immunohistochemical staining. For TGF-fil staining the appropriate sections were deparaffinated with toluene and treated with 1.0 mg/ml testicular hyaluronidase (Sigma Chemical Company, P.O. Box 14508, St. Louis, Mo., USA) in 0.1 M sodium acetate (pH 5.5) in saline (30 rain, 37~ C). Nonspecific protein-binding sites were blocked with 1.0 mg/ml bovine serum albumin in saline, and the sections were incubated in 0.1 mg/ml anti-A1/30 antibody (60 min). After washing in phosphate-buffered saline, the sections were exposed to fluorescein-conjugated sheep anti-rabbit antibody (30 min). Negative control sections were incubated in saline containing bovine serum albumin, or nonimmune rabbit serum, and stained with the second antibody. A standard immunofluorescence procedure was performed for fibronectin staining. The tissue was not exposed to hyaluronidase, and negative control sections were incubated in saline containing bovine serum albumin or nonimmune goat serum. The sections were viewed and photographed using either a Zeiss WL or Leitz Dialux microscope, both equipped with epifluorescence. Results
Controls I n b o t h negative c o n t r o l sections, T G F - / ? I a n d fibronectin s t a i n i n g are n o t seen in the chick tissue. I n the positive c o n t r o l sections, TGF-/~I staining is clearly seen in the e n d o c a r d i a l cushions of the e m b r y o n i c m o u s e heart, b u t n o n e is present in the m y o c a r d i u m . This localization is the same as t h a t described by H e i n e et al. (1987) in the mouse.
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Fig. 2A, B. Section of stage 18 or i9 chick heart stained for either TGF-fil (A) or fibronectin (B). A By stage 18, the endocardial cushions (ec) in the atrioventricular region contain an abundant extracellular matrix with some mesenchymal cells. TGF-/~I staining is more prominent in the cushion tissue than in the cardiac jeIly of the atrioventricular region of the stage 11 heart. The correspond-
ing line drawing demonstrates the entire section of heart, identifying the primitive atrium (a) and ventricle (v). B The endocardial cushion of a heart a little more advanced reveals a higher density of cells. The extracellular matrix contains a significant amount of fibronectin. The line drawing shows the orientation of the primitive atrium and ventricle
Stage 11
a t r i u m a n d ventricle. T h e e n d o c a r d i a l cushions o f the a t r i o v e n t r i c u l a r r e g i o n h a v e n o t f o r m e d a t this stage. E x t r a c e l l u l a r T G F - / ~ I staining is intense a d j a c e n t to the e n d o t h e l i a l cells o n the side o f the c a r d i a c jelly b o t h
In the stage 11 chick e m b r y o the c a r d i a c t u b e has a l r e a d y m a d e a significant r i g h t w a r d b e n d , f o r m i n g the p r i m i t i v e
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in the atrium and ventricle (Fig. 1). At least in the atrioventricular region, the TGF-/~I appears to extend into the cardiac jelly all the way to the epimyocardium. In addition, the epimyocardium reveals some TGF-/?I staining along the pericardial side.
Stage 18, 23, and 26 The endocardial cushions of the atrioventricular region, which are established by these stages, demonstrate abundant TGF-/?I. In stage 18 (Fig. 2), the cushion has few cells, and TGF-fll extends through the cardiac jelly as in stage 11. However, by stage 23 (not shown) and 26 (Fig. 3), the cushion has become more densely populated with mesenchymal cells, and TGF-/~I is present throughout the matrix. The extracellular matrix contains fibronectin throughout these stages (Fig. 2). In stage 18 (Fig. 2), the TGF-/?I staining is still seen at the base of the endothelium, but it decreases at later stages. The epicardium continues to stain. In stage 26, the epicardium is beginning to establish two layers the outer epicardial epithelium and the inner lacey network of mesenchymal cells with a voluminous fibronectin-rich extracellular matrix (Fig. 4). The staining for TGF-fil at this stage is found in the extracellular matrix (Fig. 3).
Stage 36
0.5mm
~
By stage 36, the two layers of the epicardium are well defined, but TGF-fll is much less prominent (Fig. 5). The cardiac valves have formed, although limited remodeling will occur in later stages. Intense TGF-fll staining is found in the extracellular matrix of all the valves, and, interestingly, very little staining is seen in the endothelium of the valve leaflets. The extracellular matrix of both atrioventricular valves stains for fibronectin in a pattern similar to the TGF-/?I staining (Fig. 6). However, unlike the TGF-fll staining pattern, the extracellular matrix in the epicardium remains fibronectin-rich at stage 36 (Armstrong 1985).
