MICROSCOPY RESEARCH AND TECHNIQUE 40:446–454 (1998) Cytochemical Localization of Adenylate Cyclase in the Limb Buds of Bufo bufo SIMONETTA TEI, DANIELA VAGNETTI, MONICA DI GIACOMO, AND ROSALBA M. FARNESI* Institute of Comparative Anatomy, University of Perugia, v. A. Pascoli, Italy KEY WORDS cAMP; cytochemical and ultrastructural analysis; limb bud; Amphibia ABSTRACT The importance of cyclic nucleotides in the regulation of the processes of differentiation and embryonic development is known. The possible role that cyclic adenosine monophosphate (cAMP) plays during the development of the posterior limb of Bufo bufo is studied by the cytochemical localization of adenylate cyclase (AC), an enzyme that catalyzes the synthesis of the cyclic nucleotide. The method is based on the reaction between the enzyme AC and its specific substrate AMP-PNP (58-adenylylimidodiphosphate) in the presence of lead. The lead precipitates that form as secondary reaction products are evidence of enzymatic activity. Reaction products are present only at the epithelial level in the limb bud; initially, such products are visible only at the base of the bud, particularly on the epithelial fascia located at the boundary with the body. During successive elongation and toe formation, AC activity is only present on the cells of the proximal portion of each new segment. Enzymatic activity is never present in correspondence to the ectodermal apical crest. cAMP is probably not involved in the processes of cellular proliferation but, rather, in the processes of inducing differentiation of the internal mesenchymal cells. Microsc. Res. Tech. 40:446–454, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION In embryonic vertebrate limb development, the complex interactions between the ectoderm and the underlying mesenchyme are essential for the normal morphogenesis of this structure. These relationships, present during the entire period of limb development, are represented by the formation of the apical ectodermal ridge (AER) (Laufer, 1993), with the induction by AER of the expression of some HodX complex genes in the mesenchymal cells (Vogel et al., 1995), and with the regulation by the ectoderm of the extracellular matrix composition in the subepidermal mesoderm (Knudson et al., 1995). The formation of the AER, upon the induction of the apical mesenchyme of the limb bud, occurs very early and the structure looks like a thickened ectodermal hood covering the bud apex (Balinsky, 1972). The mesoderm not only induces the formation of the AER but also maintains it; in fact, when non-limb mesoderm is transplanted under the AER, the crest shrinks and limb development ceases (Gilbert, 1988). The AER interacts with the mesenchyme and is essential for limb growth (Zwilling, 1955; Saunders et al., 1957; Saunders, 1972). It permits the polarized development of the limb along the proximal–distal axis (Bodemer, 1968; Kosher and Savage, 1980) stimulating the proliferation of the underlying mesenchymal cells (Bodemer, 1968; Laufer, 1993). The control of cellular proliferation should occur as the result of the antagonistic action of two proteins, FGF-4 and BMP-2, the genes of which are expressed in the AER (Niswander and Martin, 1993). In addition, the AER keeps the immediately underlying mesenchymal cells in an undifferentiated state (Kosher et al., 1979; Kosher and Savage, 1980) by a gradient-like diffusion of particular inhibir 1998 WILEY-LISS, INC. tory molecules; the concentration of these molecules is, however, greater in the subridge mesenchymal cells. When these subridge cells move away from this zone, as a result of polarized proximal to distal outgrowth, they respond less to the action of these molecules, lose the labile state, and differentiate into chondrogenic cells. Kosher and Savage (1980) also observed that in the presence of cyclic adenosine monophosphate (cAMP) derivatives, limb explants, formed from subcrest mesoderm covered by AER and surrounded by dorsal-ventral ectoderm, failed to undergo polarized outgrowth and characteristic contour changes. The cessation of morphogenesis is accompanied by the precocious chondrogenic differentiation of the mesenchymal cells. These results, according to the authors, indicate that a greater cAMP content in the subcrestal mesenchymal cells enables them to overcome the negative influences of cytodifferentiation and the positive influences of morphogenesis being imposed upon them by the AER. It therefore seems that cAMP plays a fundamental role in morphogenesis and limb differentiation (Kosher and Savage, 1980). In order to understand more about cAMP action in the processes of embryonic development, a cytochemical study was carried out to localize adenylate cyclase (AC), an enzyme directly involved in cAMP synthesis, in the epithelium of the hind limb of Bufo bufo during some of the earliest, most significant stages of development. The results of the cytochemical test suggest that cAMP may be involved in the differentiation processes and limb morphogenesis. *Correspondence to: Rosalba M. Farnesi, Istituto di Anatomia Comparata via A. Pascoli 06100 Italy. Received 22 July 1996; accepted in revised form 15 November 1996. ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO MATERIALS AND METHODS Bufo bufo embryos were kept in aquaria (10°C, pH 6.5, natural light) until the desired stages were reached (embryonic stages 23 and 25; larval stages I and V (Rossi, 1959)) (Fig. 1). Thirty embryos in each stage were prefixed for 30 minutes in 1% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.4) with 4% sucrose. For the cytochemical assay, the samples were incubated according to the method of Howell and Whitfield (1972), modified by Cutler and Christian (1980), in 80 mM Tris-maleate buffer (pH 7.4), 8% glucose, 2 mM theophylline, 2 mM magnesium sulphate, 4 mM lead citrate, 10 mM sodium fluoride, and 0.5 mM 58adenylyl-imidodiphophate (AMP-PNP) (Boehringer Mannheim, Mannheim, Germany). The control samples of corresponding stages were randomly selected and incubated in a substrate-free medium. All specimens were incubated at 30°C for 45 minutes, with gentle shaking. After the incubation, all samples were rinsed in 80 mM Tris-maleate buffer (pH 7.4) and then in 0.1 M cacodylate buffer (pH 7.4). The material was then postfixed for 1 hour at 4°C in 1% osmium tetroxide and processed for electron microscopy following standard procedures (Mollenhauer, 1964). After a brief staining in a saturated solution of uranyl acetate in 50% ethanol, thin sections were examined with a Philips EM 300 at 60 Kv. RESULTS At embryonic stage 23, the hind limb bud is a small subspherical lateral bump located at the boundary between the body and the tail. Ultrastructural examination showed that it has an external two-layered epithelium and mesenchymal cells to the inside. The epithelial cells are flattened with numerous electron-dense pigments throughout the cytoplasm and abundant mucous secretions at the cell tip. The intercellular spaces between the contiguous and underlying epithelial cells are enlarged (Fig. 2A). At the level of the fold which marks the border between the body and the limb, the body epithelium is cuboidal and ciliated, while that of the limb bud is non-ciliated and rich in secretions (Fig. 2A); the epithelium is multilayered in the fold region. The mesenchyme is made up of undifferentiated cells with a large nucleus and nucleolus; they have less cytoplasm and have scarce organelles. There is a noticeable distance between the cells, which have irregular borders and long cytoplasmic strands that sometimes connect them. At the bud base, the cells are more abundant, closer together, and have the long shape typical of movement cells. The extracellular matrix is very transparent (Fig. 2B). In this precocious stage of limb formation, only some of the superficial epithelial cells located at the border between the body and limb are positive for the AC reaction (Figs. 2A,C). The reaction products are only found along the lateral and basal membranes. At embryonic stage 25, the epithelium of the bud apex becomes multilayered with cube-like elements separated by large intercellular spaces (Fig. 3A). Based on this characteristic appearance and its location, this epithelial region is regarded as the presumptive AER. In the mesenchymal portion, the cells are more numer- 447 Fig. 1. (a) Embryo of Bufo bufo at stage 23, in which the limb bud is visible at the base of the tail. (b–e) Limb bud at the embryonic and larval stages considered. ous and closer together than in the preceding stage. A large blood vessel is present in the subcrestal mesenchyme (Fig. 3B). Also at this stage, the enzymatic reaction is observed only on lateral-basal membranes of the superficial cells located near the fold at the boundary with the body (Figs. 3C,D). At larval stage I, the limb bud is longer and has a subcylindrical shape. More layers of epithelial cells can be seen at the fold; the innermost cells represent the germinative layer and show slightly differentiated electron-dense cytoplasm with mitotic patterns (Figs. 4A,B). The cells of the upper layers are well differentiated, with abundant pigment-rich cytoplasm containing numerous organelles and lipid drops (Fig. 4A). The epithelium again becomes two-layered along the lateral surfaces of the limb bud (Fig. 4C). The enzymatic reaction product is present at the fold of the border with the body, along the lateral and basal membranes of the outermost cell layer (Fig. 4A). The reaction is lacking in a short region that follows (Fig. 4C) and then reappears, more distally on the basallateral membranes of the epithelial cells of the outer layer (Fig. 4D). Reaction products were not observed on the AER, which was still present on the bud apex. The subcrestal mesenchymal cells are still undifferentiated, while the more internal ones show signs of differentiation; among these elements it is possible to see clusters of nervous layers and blood vessels. At larval stage V the limb bud is flattened, apically enlarged with two barely visible curvatures: one between the stylopodium and zeugopodium and the other between the zeugopodium and the autopodium. In the bud apical zone, the lateral protuberances of the 3rd, 5th, and 4th digits can be seen; the latter is more accentuated and centrally located. The epithelium is multilayered at the two-curvatures level, and twolayered along the rest of the surface. The AER is no longer visible. Within the limb, with the exception of the nervous and vascular components (Fig. 5A), definite tissues are still not visible, even though aggregations of cellular Fig. 2. Hind limb bud of Bufo bufo at embryonic stage 23. (A) Separation fold between ciliated epithelium of the body and that of the limb; the latter has flattened cells with mucous secretions (m) and pigments (p). The AC reaction products (arrows) are present along the lateral-basal membranes of some superficial cells; (c) cilia. 5,300x. (B) Elongated mesenchymal cells (mc) that seem to penetrate to the interior of the limb; the cells are distant from each other and the extracellular matrix is transparent; (e) epithelium of limb bud. 3,200x. (C) Localization of reaction products (arrows) in the epithelial cells near the fold between the body and limb. 8,400x. ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO Fig. 3. Hind limb bud of Bufo bufo at embryonic stage 25. (A) Multilayered apical ectodermic ridge (aer); mesenchmyal cells (mc) can be seen below. 2,550x. (B) Subridge blood vessel (v) surrounded by 449 as yet undifferentiated mesenchymal cells. 2,700x. (C, D) Localization of enzymatic reaction products in the multilayer epithelium in the region of the limb fold. 4,500x; 6,800x. Fig. 4. Hind limb bud of Bufo bufo at larval stage I. (A) Multilayered epithelium in the fold region: the outermost cells are differentiated, the deeper cells, which have a high nucleus/cytoplasm ratio, make up the germinative layer. The reaction products (arrows) are localized only on the basal-lateral membranes of a few superficial cells. 4,500x. (B) Particular of the germinative layer of the epithelium showing a cell in mitosis. 3,600x. (C) Two-layered epithelium of the proximal region of the limb bud; no reaction products can be seen. 5,700x. (D) Two-layered epithelium of the intermediate and distal regions of the limb. The reaction products (arrows) are visible along the cell membranes in the superficial layer. 3,600x. Fig. 5. Hind limb bud of Bufo bufo at larval stage V. (A) Nervous processes (arrows) and blood vessel (v) between differentiating mesenchymal cells. 2,000x. (B) Clustering of mesenchymal cells around the chondrogenic centers; (v) blood vessel. 2,100x. (C) Differentiating mesenchymal cells; in the cytoplasm myofibrils in formation can be seen (arrows). 5,400x. (D) Particular of intercellular substance between the differentiating mesenchymal cells; numerous collagen fibrils can be seen. 18,500x. Fig. 6. Hind limb bud of Bufo bufo at larval stage V. (A, B) Multilayered epithelium at the level of the curvature between stylopodium and zeugopodium (A) and the curvature between the zeugopodium and autopodium (B). Abundant reaction products (arrows) can be seen along the cellular membrane. 4,500x; 2,800x. (C) Two-layered epithelium located at the boundary between the basal zone in which reaction products are present and the apical zone of the 4th digit, in which the reaction is lacking. 2,200x. (D) Epithelium of the apical region of the 4th digit; reaction products are lacking. 2,800x. (E) Epithelial region of the limb bud in which the cells clearly show signs of degeneration: the cytoplasm is very transparent and the mitochondria and rough reticulum are fragmented. 6,500x. ADENYLATE CYCLASE IN LIMB BUD OF BUFO BUFO elements can be seen that delimit centers of tissue condensation (Fig. 5B) and some cellular elements have myofibrils being formed (Fig. 5C). Noticeable accumulations of collagen fibrils are present in the matrix, which is now denser than before (Fig. 5D). The reaction products can be seen in the epithelium of the curvatures (Figs. 6A,B) and at the base of the 4th (middle) digit (Fig. 6C), while they are lacking at the tip of the 4th digit (Figs. 6C,D). At this stage some welldefined epithelial areas with some degenerating cells can be seen (Fig.6E). DISCUSSION The morphologic and cytochemical data obtained enable us to make some observations. In the first stages of hind limb development in Bufo bufo, there are a few undifferentiated mesenchymal elements; those located at the boundary between the body and limb seem to migrate from inside the body, as reported in the literature (Balinsky, 1972). Mitosis is rare, if not completely lacking, in both the epithelium and in the mesenchyme, even though the bud is increasing in size. This can probably be explained for the mesenchyme by the continual migration of cellular elements from within the body, while for the epithelium, by the flattening and spreading of the cells. In the mid-apical portion of the limb bud the AER is present. This structure has been described in the limb bud of vertebrates but its presence has been a matter of discussion in amphibians (Tschumi, 1957; Bodemer, 1968; Dober and Tschumi, 1971; Tarin and Sturdee, 1969; Balinsky, 1972). In the more advanced developmental stages (larval stages I and V), the external morphology of the limb bud begins to be delineated, while there is still no internal tissue differentiation. The presence of areas of cell degeneration observed at larval stage V could be related precisely to the modelling of the limb shape. The results of the cytochemical tests indicate that AC activity is constant during limb development at the epithelial level. The reaction products are localized only on the lateral and basal membranes of the cells in the outermost layer or in the most external layers in the multilayered epithelial zones. The reaction is never present in the basal germinative layer, which confirms that cAMP is not associated with cellular proliferation events (Otte et al., 1989, 1990; Hadden et al., 1972; Kram and Tomkins, 1973; Rudland et al., 1974a,b). The enzymatic activity of stage 23, at the epithelial cell level, is localized along the original fold of the limb and could be correlated with the dynamic activity of this epithelial zone at this particular moment of development. During the successive stages, the lead deposits are not uniformly distributed along the entire epithelium; rather, they are always localized at the base of each new segment. The reaction products are never present in the AER cells and this suggests a possible role of cAMP during limb development. The fact that the apical ridge, which may have the function of maintaining the immediately underlying mesenchymal cells in an undifferentiated state (Kosher et al., 1979; Kosher and Savage, 1980), lacked enzymatic activity suggests that cAMP is not involved in the functions of the AER. 453 Kosher and Savage (1980) observed that in the presence of cAMP derivatives, explants of subcrestal mesenchymal cells overcome the negative influences on cytodifferentiation and the positive influences on their morphogenesis imposed by the ridge. These authors suggested that the increase of cAMP may either block the mesenchymal cell response to the influence of the AER or reduce the function of the ridge itself. In the epithelial zone of the limb bud in which enzymatic activity can be found, the cAMP may have, on the contrary, a positive effect on cytodifferentiation. 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