Smooth muscle cells of neural crest origin form the aorticopulmonary septum in the avian embryo.код для вставкиСкачать
THE ANATOMICAL RECORD 226:360-366 (1990) Smooth Muscle Cells of Neural Crest Origin Form the Aorticopulmonary Septum n the Avian Embryo ARTHUR C. BEALL AND THOMAS H. ROSENQUIST The Heart Development Group, Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000 ABSTRACT Previous studies have shown that the cells of the aorticopulmonary (AP) septum are similar to the smooth muscle cells of the mediae of the great vessels in their common origin from the cardiac neural crest and in their common expression of a n elastic extracellular matrix. The purpose of this study was to test the cells of the AP septum for the presence of certain cytoplasmic proteins, especially smooth muscle alpha-actin (SMAA) whose presence is definitive of smooth muscle. A monoclonal antibody against SMAA was applied to normal chicken embryos a t 3.5-8 days of incubation and to age-matched embryos from which the cardiac neural crest had been ablated surgically. Antibodies against the intermediate filaments desmin, cytokeratin, and vimentin also were applied. The results showed that the AP septal cells expressed SMAA during the process of septation, days 5-8; but when the cardiac neural crest was ablated and septation was defective, no cells in the conotruncal connective tissue expressed SMAA. None of the intermediate filament proteins were detected in the septum. These results indicate that the AP septal cells are smooth muscle and therefore may be hypothesized to have a n active role in septation. The process of aorticopulmonary (AP) septation has been the object of study in numerous laboratories over many years. Excellent recent reviews of the process have been written by Thompson et al. (1984,1987). The AP septum proper in the chicken embryo is formed at the confluence of a dorsal and a smaller ventral condensation of cells, each of which is in contact with the myocardial cuff (also called the myocardial sheath or mantle) of the truncus arteriosus. Each of these two condensations extends downstream or cephalad within the archaic connective tissue of the spiralling conotruncal ridges and joins the other at its cephalic terminus, between the lumina of aortic arches 4 and 6. The conjoint condensation is the AP septum. After it is formed, the AP septum moves relative to the surrounding tissues toward the heart, while its dorsal and ventral tributaries become relatively shortened. In this way the AP septum is transferred upstream to the heart. The mechanism by which the septum becomes transferred upstream was traditionally considered a “zipper-like’’ fusion of the dorsal and ventral condensations (reviewed by Thompson et al., 1984). In 1979 however Thompson and Fitzharris introduced a n attractive alternative mechanism, wherein the retracting myocardium pulled the septum upstream by the conjoint dorsal and vectral condensations. They referred to this a s the “tractor and sling” concept. The myocardial sheath provided the locomotion, while the cellular aggregations and their extracellular matrix provided a sling for passive upstream transfer of the myocardial energy. 0 1990 WILEY-LISS, INC. Later it was shown that the cells which condense to form the AP septum were ectomesenchyme of neural crest origin which migrate into the truncus via aortic arches 3, 4, and 6 (Kirby e t al., 1983). When this “cardiac” neural crest is ablated surgically from chicken embryos at about stage 10 (Hamburger and Hamilton, 1951), the embryos fail to produce the typical whorl-like cellular aggregation typical of the AP septum, and septation is invariably abnormal. The embryos so treated develop congenital anomalies of the heart, especially persistent truncus arteriosus (Kirby et al., 1985; Nishibatake et al., 1987; Kirby, 1988). The biological or molecular basis of this special role of the cardiac ectomesenchyme is not completely understood. However i t has been shown recently that one of the features of the ectomesenchyme in the AP septum that distinguishes i t from the contiguous non-neural crest mesenchyme is the assumption by the aggregating ectomesenchyme of a n elastogenic phenotype (Rosenquist et al., 1988). Indeed the expression of the tropoelastins and the aldehyde-rich protein is a characteristic unique to the cardiac ectomesenchyme in the early chicken embryo, whether that ectomesenchyme is in the AP septum or in the walls of the great Received March 16, 1989; accepted May 16, 1989. Address reprint requests and correspondence to Dr. Thomas Rosenquist, Professor of Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000 AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE Fig. 1. Sham-operated embryo, incubation day 6. All sections, x 740. A Transmitted light, phase-contrast image of a section through the truncus arteriosus flanked by the two atria (A). Dorsal is to the left and ventral to the right. A central condensation of cells forms the AP septum (*) which separates the two great vessels. Arrows = the myocardial cuff. B: Same section as in a, green epifluorescence, antismooth muscle alpha actin (SMAA). All of the cells of the septum (*) are positive but the SMAA-positive region ends abruptly ventrally. There are a few cells in the lumina1 aspect of walls of the great vessels 36 1 that are positive, just dorsal to the septum. The myocardial cuff (arrows) is negative a t this level. c: Next more distal section from that A and B, epifluorescence, polyclonal anti-desmin. All of the cells of the septum (*) are positive. The most intense reaction is found in the myocardial cuff (arrows); there is also binding of the antibody in the flanking atria and in the proximalmost great vessel walls. D. Next more distal section from C, epifluorescence, monoclonal anti-desmin. The septum (*) bound no antibody while the myocardial cuff was faintly positive (arrows). 362 A.C. BEALL AND T.H. ROSENQUIST vessels (Rosenquist et al., 1988), both of which are derived from the cardiac neural crest (LeLievre and LeDouarin, 1975; Kirby et al., 1983). Thus the ectomesenchymal cells in the cardiovascular outflow region appear to be a homogeneous and continuous population that includes both the septum and the great vessels. The distinct histologic similarity between cells of the artery wall and the septum and the clear distinction between those cells and all other mesenchyme in their vicinity was noted over 20 years ago (Jaffee, 1967), before their common origin was established. Because the AP septal cells are so like the medial cells in their origin, general histologic features, and early expression of elastin, we hypothesized that some of the cells of the AP septum also would have the capacity to express a smooth muscle phenotype. (That a significant fraction of the medial cells of the great vessels do not become smooth muscle was reported by Hughes in 1943, and was confirmed recently by Skalli et al., 1986.) To test this hypothesis we applied antibodies against smooth muscle alpha-actin to sections of normal chicken embryos. The results show that smooth muscle alpha-actin is a constant component of essentially all of the cells of the AP septum and suggest the possibility of a n active role for these cells in the process of septation. To be sure that the cells expressing the muscle-specific protein in the septum were in fact of neural crest origin, we compared control embryos with experimental embryos from which the neural crest had been ablated surgically. Smooth muscle alpha-actin was not expressed by any mesenchymal cells in the truncus arteriosus of the experimental embryos during the septation period. An earlier study by Sumida e t al. (1987) had reported that the intermediate filament desmin was also found in the AP septum of the chicken embryo. Therefore the distribution of desmin was compared in the present study with the distribution of smooth muscle alphaactin; however, the presence of desmin in the AP septum was not confirmed. The AP septum also was negative for two other intermediate filaments, vimentin and cytokeratin. MATERIALS AND METHODS which the stain was applied and the vitelline membrane torn but no further manipulation of the embryo was carried out. Shams were collected at the same intervals as the experimental embryos. Some of the shams were collected after 12-14 days when most of the cells had assumed their ultimate phenotype (Schmid et al., 1979). A rooster of unknown breed, weight 2 kg, was purchased from a local poultry dealer and his great vessels were prepared as described below. The late embryonic and adult great vessels were used to check the validity of certain negative reactions that were found in the early embryonic AP septum andlor great vessels (see below). Experimental and sham-operated embryos and the adult great vessels were placed in a solution of methyl alcohol, chloroform, and acetic acid (“methacarn”; Puchtler et al., 1970) at 4°C for 12-24 hr. Then they were processed through alcohols and xylenes at 4°C into lowmelting-point Paraplast at 57°C. We have found the best preservation of epitopes for immunohistochemistry, as well a s preservation of excellent histologic detail with this procedure (Rosenquist et al., 1988). Sections of 10 pm were cut and mounted serially on glass slides. lmmunohistochemistry Sections generally were rehydrated rapidly with the routine, xylene/xylene/lOO% ethano1/100% ethanollwatertwater, each for 3 min. If a particular antibody was found not to bind to the embryo a t any site, the paraffin removal step was lengthened to include 1 hr in the initial xylene to be sure that the lack of reaction was not a n artifact of retained paraffin. After rehydration the sections were placed in phosphate-buffered saline, pH 7.4, with 0.5% bovine serum albumin (PBS/BSA), for 30 min at 37°C. Then they were incubated with 20 pllsection of the diluted primary antibodies detailed below for 1 h r at 37”C, rinsed with PBSIBSA, and incubated with 20 phection of the appropriate secondary antibody labelled in all cases with rhodamine. (Note: the intense yellow-green autofluorescence of some embryonic proteins under blue epif luorescence could be taken in error for the emission of a fluorescein-labelled secondary antibody, e.g., the illustrations in Rosenquist and McCoy, 1987; or Rosenquist et al., Preparation of the Tissues Fertile Arbor Acre chicken eggs (Central Soya of Athens, GA) were incubated for 30 h r in a humidified atmosphere a t 38°C. A window was made in the shell and the embryos (approximately HH stage 9; Hamburger and Hamilton, 1951) were stained lightly with neutral red. The vitelline membrane was torn over the embryos, and all of the neural folds over somites 1-3 and two somite lengths above somite 1 (the “cardiac neural crest”; Kirby et al., 1985) was removed bilaterally with a microcautery needle (Narayanan, 1970). This technique causes minimal damage to surrounding tissues (Bockman and Kirby, 1984). After surgery the eggs were sealed and returned to the incubator for periods of time which, in the control or sham-operated embryos (see below), yielded embryos of HH stages 2135, days 3.5-8. These incubation times bracketed completely the time of septation (Kirby et al., 1983). Sham-operated embryos (“shams”) were prepared in Fig. 2. The same sham-operated embryo shown in Figure 1, same magnifications throughout. A. Transmitted light, phase-contrast image of a section through the truncus arteriosus at a point nearer the heart than in Figure 1.The characteristic “whorl” conformation of the AP septum (*) is obvious dorsally. The undivided lumen of the truncus is traversed by fixed blood (B) which may give the impression of a completed septum by its location in this section. The blood gives a distinctive, intense autofluorescence (see A-C, below). A = atria; arrows = myocardial cuff. B: Same section as in A, epifluorescence, anti-SMAA. The dorsal limb of the septum (*) is well-marked by the antibody. An infolding of the myocardial cuff in direct contact with the septum and just to its left in this section also is positive. On the other hand the rest of the myocardial cuff (arrows) is negative. C: Section immediately proximal to that in A and B, epifluorescence, polyclonal anti-desmin. The cells in the dorsal limb of the septum are positive (*I, while the most intense reaction is in the myocardial cuff (arrows); the atria show a lesser reaction. D: Section immediately proximal to that in C, epifluorescence, monoclonal anti-desmin. The dorsal limb of the septum is negative (*) while the myocardial cuff is positive. AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE Fig. 2. 363 364 A.C. BEALL AND T.H. ROSENQUIST Fig. 3. Sham-operated embryo, incubation day 8. x 1,480. A Transmitted light, phase-contrast image of a section through the proximal truncus just above the heart a t the time of completion of septation, same orientation as Figures 1 and 2. The dense aggregation of cells which forms the septum (S) is obvious. Arrows = myocardial cuff. B: Same section as A, green epifluorescence, anti-SMAA. The cells of the septum (S) react intensely whereas all other areas including the entire myocardial cuff are negative. 1988; therefore it is essential that fluorescein be avoided during epifluorescence studies of embryonic tissues.) Each slide carried four or more sections. One section on each slide received neither the primary nor the secondary antibody; one section received only the secondary antibody; and the two or more remaining sections received both the primary and the secondary antibody. Details of the primary antibodies are given below; the ratios that are given are for our empirically optimum working dilutions of the antibodies in PBSI BSA. The following monoclonal primary antibodies were localized with the secondary antibody, rhodamine-labelled rabbit anti-mouse IgG l:lO, ICN Immunobiochemicals: 1) Anti-smooth muscle alpha-actin clone 1A4 (Skalli et al., 19861, ascites 1:200, Sigma; 2) Antidesmin clone DE-U-10 (Debus et al., 1983), ascites 1: 10, Sigma; 3) Anti-desmin clone DE-B-5 (Debus et al., 19831, Sigma, ascites l:lO, Sigma; 4) Anti-vimentin clone V9 (Osborn et al., 19841, ascites 1:4, Boehringer Mannheim Biochemica; 5) Anti-cytokeratin clone K813 (Gigi et al., 1982), ascites l:lO, Sigma. Polyclonal rabbit anti-desmin primary antibody, whole serum l:lO, was localized with the secondary antibody, rhodamine-labelled goat anti-rabbit IgG 1: 10, 1"and 2" antibodies both Sigma. Finally all sections were rinsed well with PBS/BSA and coverslips were mounted over the sections with Elvanol, Dupont. Sections were observed and photographed in a Zeiss Standard WL or a Zeiss Axioskop microscope under green epifluorescence: exciter filter BP 510-560, dichromatic beam splitter FT 580, barrier filter DP 590. Photographs were taken on Kodak Tri-X film. TABLE 1. Results of antibody binding studies Antigen SMAA (Mab) DES (Mab) DES (PabI3 Vimentin (Mab) Cytoker (Mab) Location during septation Arterial Septal Skeletal Cardiac SM SM muscle muscle muscle muscle - + + - ++- ++ - - - - - - + + f2 'Abbreviations: SMAA = smooth muscle alpha-actin; DES = desmin; Cytoker = cytokeratin; Pab = polyclonal antibody; Mab = monoclonal antibody. 'Only in the myocardial cuff or sheath of the truncus arteriosus. 3Also bound by tracheo-esophageal mesenchyme. AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE RESULTS The results of the antibody binding studies are summarized in Table 1. Smooth Muscle Alpha-Actin (SMAA) Cardiac neural crest cells migrate into the connective tissue space between the myocardial cuff and the endocardium and aggregate in a peculiar whorl-like configuration that is easily identified after about 5 d of incubation (Figs. 1-3). This aggregation becomes the definitive AP septum. Essentially all of the cells that compose the septum are SMAA-positive, incubation d 6-8 (Figs. 1-3). Of the two condensations of ectomesenchyme which converge to form the definitive septum (Thompson et al., 1984) (Fig. l),only the dorsal limb is obviously SMAA-positive throughout its length (Fig. 2). In the experimental embryos of 5-8 d, there were either no cell aggregations typical of the formation of the AP septum, or there was a small atypical dorsal aggregation of cells like that described by Kirby (1988); in neither case did any cells express SMAA in the conotruncal connective tissue (between the myocardial cuff and the endocardium). The distalmost truncus arteriosus was SMAA-positive (Figs. 2, 3). Desmin The monoclonal anti-desmin antibodies were identical and showed no reaction in the AP septum (Figs. 1, 2). On the other hand, the AP septal cells bound the polyclonal antibody against desmin a s soon as they were found in their characteristic dense aggregation (Figs. 1, 2). The medial cells of the embryonic great vessel walls failed to express desmin; however, the cells of the adult great vessels were positive for the monoclonal antibodies (not shown). The somites and the skeletal muscle derived from the somites were reactive for the monoclonal anti-desmin antibodies at all stages. The ventricles and the myocardial cuff of the heart also were reactive at all stages (Figs. 1, 2). Visceral smooth muscle was initially negative but expressed desmin by 7 d (not shown). The following regions all bound the polyclonal antidesmin a t all stages (not shown): the somites and all skeletal muscle; all visceral and vascular smooth muscle cells; and the tracheoesophageal mesenchyme. Vimentin was not detected in any embryonic or adult tissue. Cytokeratin was not detected in the AP septum or any cells of the great vessels but was expressed by several embryonic tissues including the skin, epithelium of the gut, and the pericardium (not shown). DISCUSSION The expression of different muscle-specific proteins and the expression of intermediate filament proteins may vary greatly during development 1) geographically among the various cell types and 2) chronologically, within a given cell type. Furthermore a given cell type in the embryo may express simultaneously more than one isoform of a protein. The regulation and modulation of the expression of genes for the various mus- 365 cle proteins are not well-defined but are the topic of a great deal of contemporary research (e.g., Hayward et al., 1986; Minty et al., 1986; Ordahl, 1986; Carroll et al., 1988; Ruzicka and Schwartz, 1988). The functional meaning and consequence of the presence of a given muscle-specific protein or intermediate filament protein is unclear. However it has been established that smooth muscle alpha-actin (SMAA) is found only in cells which have the capacity to contract (Skalli et al., 1986). Vascular smooth muscle cells (SMC) which have achieved phenotypic maturity and mitotic quiescence express SMAA rather than the beta form of actin (Clowes e t al., 1988). Thus SMAA is the actin isoform characteristic of all vascular smooth muscle cells when they are functional, and its expression in a n embryonic cell is necessary and sufficient for its positive identification as smooth muscle (Gabbiani et al., 1981; Skalli et al., 1987). It may be surmised therefore that the expression of SMAA by the cells of the aorticopulmonary septum indicates their differentiation into smooth muscle and implies their contractile capacity (although contraction per se has neither been observed here nor reported elsewhere). The presence of SMAA in these cells of neural crest origin, taken with their elastogenic capacity, appears to justify fully the conclusion that yielded the title of this study: smooth muscle cells of neural crest origin form the aorticopulmonary septum in the avian embryo. The absence of any cells that express SMAA in the conotruncal connective tissue following ablation of the cardiac neural crest and the failure of septation in the same embryos constitute a bit of circumstantial evidence in favor of a n active role for septal cells. The potential for active participation by the AP septal cells in the process of septation in the chicken embryo has been indicated also by data reported previously by others. Sumida e t al. (1989) have reported that the mushroom-derived toxin phalloidin was bound to the myocardium as well as to the cells of the AP septum in the 5 d chicken embryo; and phalloidin has a specific affinity for F-actin (reviewed by Sumida et al., 1989). A polyclonal antibody against desmin that was bound by cells of the AP septum suggested a contractile potential to Sumida e t al. (1987). However in the present study desmin was not detected in the AP septum with the monoclonal antibodies although the validity of the antibodies was supported by their reaction in the same embryos with other tissues that typically express desmin. The binding in this study and in the study of Sumida et al. (1987) of a polyclonal “anti-desmin” antibody by the AP septum may have been the result of the presence of some epitope other than one of the desmin epitopes, e.g., one of the fibronectin epitopes. Indeed the similarity between the distribution of the polyclonal anti-desmins used by us and by Sumida et al. (1987) and the distribution of the monoclonal antifibronectin antibody used by Icardo (1985) is striking and was remarked upon also by Sumida e t al. (1987, 1989). Whatever the basis for the discrepancy between the results of the polyclonal compared with the monoclonal antibodies, the true functional roles of the intermediate filaments including vimentin, cytokeratin, and desmin are not known, and any essential relationship that they may have to contractility has not been clearly established. The fact that no intermediate-type A.C. BEALL AND T.H. ROSENQUIST 366 filament was detected in the AP septum in the present study cannot therefore be interpreted as evidence against a contractile capacity for the septal cells. A contribution by septal cell smooth muscle contraction to septation may be hypothesized from these data and those of Sumida et al. (1987,1989); however, based upon the work of Thompson and others (cited above), it seems likely that the putative septal contraction would be only one of several simultaneous, coordinated events that result in successful septation. Nevertheless the concept of active septal contraction may have a n impact on teratology, for example in the interpretation of data from studies where beta-stimulating agents have been used to produce cardiovascular malformations that involve improper septation (reviewed by Ishikawa e t al., 1980). In such cases a direct pharmacologic impact upon the septal smooth muscle by the drug in question would have to be given consideration as a potential teratogenic agent, along with hemodynamic change and other factors. In summary, the present study has shown the presence of SMAA in essentially all of the cells of the AP septum during the process of septation in the chicken embryo. On the other hand, no cells which expressed SMAA were observed in the conotruncal connective tissue in chicken embryos which had undergone surgical ablation of the cardiac neural crest. These data support the possibility that the ectomesenchymal cells may contribute actively to the process of AP septation. ACKNOWLEDGMENTS This study was supported by grants HL 36059 and HL 42164 from the National Institutes of Health, National Heart, Lung and Blood Institute, and by a grant from the American Heart Association, Georgia Affiliate. Technical assistants were Harriet Stadt and Donna Kumiski, surgery; Charlotte Fray, histology; and Judy McCoy, photography. LITERATURE CITED Bockman, D.E., and M.L. Kirby 1984 Dependence of thymus development on derivatives of the neural crest. Science, 223t498-500. Cayroll, S.L., D.J. Bergsma, and R.J. Schwartz 1988 A 29-nucleotide DNA segment containing an evolutionarily conserved motif is required in cis for cell-type-restricted repression of the chicken alpha-smooth muscle actin gene core promoter. Mol. Cell. Biol., 8t241-250. Clowes, A.W., M.M. Clowes, 0. Kocher, P. Ropraz, C. Chaponnier, and G. Gabbiani 1988 Arterial smooth muscle cells in uiuot Relationship between actin isoform expression and mitogenesis and their modulation by heparin. J. 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