Differentiation (1992) 51 : 177-186

Differentiation Ontogeny, Neoplasia and Differentiation Therapy

0 Springer-Verlag 1992

The mammalian anti-a-smooth muscle actin monoclonal antibody recognizes an a-actin-like protein in planaria (Dugesia lugubris s.1.) Rita Pascolini"", Ines Di Rosa', Anna Fagottil, Fausto Panara', and Giulio Gabbiani

' Istituto di Anatomia Cornparata, Facolta di Scienie MM. FF. NN., Universiti di Perugia, Via Pascoh, 1-06100 Perugia, Italy Departement de Pathologie, CMU, Faculte de Medicine, Universite de Geneve, 1, rue Michel-Servet, CH-1211 Genkve 4, Switzerland Accepted in revised form June 17, 1992

Abstract. The presence of an a-smooth muscle (a-sm) actin-like protein in planaria (Dugesia lugubris s.1.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti a-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the a-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of a-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the a-sm actin can be considered to be a marker for myoid differentiation. The suggestion that a-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.

Introduction

The expression of actin isotypes has been used to study cell differentiation under normal and pathological conditions [3, 16, 17, 28, 361. Actin, a globular 42 kDa molecule, is one of the most conserved eukariotic proteins. Using two-dimensional polyacrylamide gel electrophoresis and amino acid sequence analysis, six isotypes have ~~~

Correspondence to: R. Pascolini

been found in muscle tissues and two in all other cell types [12, 44, 451. The isoactins differ mainly in their NH,-terminal amino acid sequences, but are strongly homologous in the other domains [46]. Invertebrate muscle actins are not as well characterized as those of vertebrates [20, 21, 461. However, a detailed study on the NH,-terminal domain indicated that most invertebrate muscle actins are homologous to each other and to the isoforms of vertebrate cytoplasmic actins [46]. We used flatworm planarians - commonly thought to hold a key position in the metazoan phylogeny [9, 321 - to study cell migration and cell differentiation in a living, low evolutive level organism. Adult fresh-water planarians are characterized by continuous cell turnover in which the substitution of terminally differentiated cells is dependent upon a proliferating [2] and migrating [31] stem cell population (these cells can also be designated as replacement cells or neoblasts). Planarians grow and degrow continuously depending upon various natural (e.g. food availability) and experimental (e.g. starvation) conditions [4, 51. The regenerative power of planarians is widely known. Although head regeneration requires 3-4 weeks, the major molecular and cellular events which are at the basis of this phenomenon occur in the first week of the regenerative process [2, 5 , 11, 141. Cell migration and differentiation are difficult to study in planarians under normal physiological conditions because, at any given time, only a limited number of cells are implicated in the process. However, when a planarian is induced to regenerate, cell migration and differentiation increase greatly and can be observed more easily. Using a monoclonal antibody which reacts with all vertebrate actin isoforms, we demonstrated the presence in planarians of a 42 kDa actin-like molecule that was localized in differentiated muscle cells and in migrating cells like the phagocytic- [30] and blastema-forming cells [31]. It has been hypothesized that various actin isoforms are present and are differentially expressed in planarians [31], although direct evidence of this phenomenon has not been reported. The anti a-smooth mus-

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cle actin monoclonal antibody (a-sml mAb) is the only well-characterized monoclonal antibody specific for the mammalian a-sm actin isotype [35]. This antibody recognizes a very restricted epitope in the NH,-terminal region of a-sm actin and does not cross-react with the other actin isoforms [35]. In this work we present evidence that the mammalian anti a-sml mAb recognizes a 42 kDa a-actin-like protein in specific regions of the freshwater planarian Dugesia lugubris s.1. and that this protein is expressed during cell differentiation. Recently a new yeast actin-like gene (ACT2) has been isolated and sequenced and the defined putative actinlike protein seems to be involved in a regulatory mechanism in the cell cycle [34]. It is of interest that by lowstringency Southern hybridization the authors detected ACT2-related sequences in genomic DNA of several lower eukaryotes indicating that one or more classes of actin-like proteins are universally present in eukaryotes [34]. In order to investigate the possible relationships between planarian and yeast actins we assayed the specificity of anti a-sml mAb on Saccharomyces cerevisiae by Western-blotting analysis carried out during cell growth.

Methods Planarian tissue preparation. We used worms at various regenerative stages but the results reported here concern only non-injured worms (0 hours of regeneration) and 4-day-regenerants in which blastema was growing and the beginning of cell differentiation was the major event [2, 5, 11, 141. Head regeneration was induced by decapitating chloretone-anaesthetized specimens as previously described [31]. SDS-PAGE and Western blotting. The planarian tissues extract for actin immunocharacterization, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and Western blotting was prepared as previously described [31]. One-dimensional SDS-PAGE on 10% acrylamide slab gel was performed according to [23]. After blotting [42], the nitrocellulose sheets were immunodecorated with the primary antibody and revealed with an appropriate secondary antibody coupled with alkaline phosphatase (Bio-Rad, USA). The monoclonal antibody to a-sm actin (anti a-sml) has been characterized [35] and used for immunoblotting experiments at 1 : 100 dilution in TRIS buffered saline (TBS: 0.02 M TRIS, 0.9% NaC1, pH 7.2) containing 1 YOgelatin. The monoclonal antibody to a-sarcomeric actin (anti a-sr) [38] was purchased from Sigma (USA). The specificity was assayed (1 : 100 dilution in TBS-gelatin) by SDS-PAGE and Western blotting as reported above using purified bovine skeletal and cardiac actin [40]. Monoclonal antibodies against various actin isoforms (anti-total actin mAb) were obtained from Amersham (UK). The specificity was assayed by twodimensional electrophoresis 1291 and Western blotting

(1 : 500 dilution in TBS-gelatin) using bovine aorta and chicken gizzard extracts [35] or purified a-sr actin. The extract from yeast was prepared as follows : Saccharomyces cerevisiae (strain DBVPG 6173) was kindly supplied by the Industrial Yeast Collection from the Dept of Biologia Vegetale (University of Perugia) and seeded on plates of YEPG containing yeast extract (1 %), peptone YO), glucose (2%) and agar (2%) at 25" C for 24 h. The cell suspension, made in liquid YEPG, was used for inoculating several 250 ml bottles containing 2.5 x lo8 cells/100 ml of culture medium. Cells were grown at 25" C with shaking (200 rpm). Under these conditions the early log phase occurred at about 4 h, the middle at about 9 h and the late at about 18 h. At the indicated times and under constant conditions, a sufficient number of bottles were inoculated to obtain 1-2 g of cells (fresh weight). The cells were pelleted and washed three times by centrifugation in phosphate buffered saline (PBS) and 1 g was homogenized in PBS containing 20 m M EGTA, 20 pg/ml leupeptin, 156 pg/ml benzamidine, 80 pg/ml aprotinin, and 1 m M phenyl methyl sulfonyl fluoride (PMSF) by a Bead Beater device (Biospec, Bathersville, USA) using glass spheres (diameter 0.5 mm) for 15 s. The homogenate was solubilized by adding an equal volume of Laemmli sample quenching [23] and boiling it for 3 min at 100" C. SDSPAGE and Western blotting analyses were carried out as described above and the nitrocellulose replicas were immunostained with anti a-sm 1 mAb. Proteins were determined by dye-binding assay [6] using bovine serum albumin (BSA) as the standard. Imrnunocytochemistry. For immunoperoxidase staining, the specimens were fixed in 100% ethanol and embedded in paraffin according to [l].After rehydration in phosphate buffered saline (PBS : 10 m M sodium phosphate, 0.9% NaCI, pH 7.5) deparaffinized sections were treated with normal horse serum and then incubated with anti a-sml mAb (1:500 dilution in PBS containing 0.1 M EDTA) [l] for 45 min at 20" C. In some experiments, specimens were incubated overnight at 4" C but no differences were observed with respect to a 45 min incubation. The antigen was localized using avidin-biotinylperoxidase complex (ABC-P, Vector Laboratories). Peroxidase activity was demonstrated with 3-3'-diaminobenzidine tetrahydrochloride (0.1 YO)and H,O, (0.02%) solution in 0.1 M TRIS buffer, pH 7.2. For immunoelectron microscopy samples were fixed, embedded in Lowicryl4 KM and processed as previously described [31]. The ultrathin sections were rehydrated in 20 m M TBS+0.1% BSA, pH 8.2, labelled with the primary antibody (anti a-sml mAb diluted 1 : 100; anti a-sr mAb diluted 1:lOO or 1:500; and anti-total actin mAb diluted 1 : 1000 in TBS containing 0.1 YOBSA) and stained with goat anti-mouse IgG or IgM coupled to 10 nm colloidal gold (Janssen, Beerse, Belgium). Grids were examined with a Philips 400 T electron microscope. Controls with pre-immune serum were carried out in all experiments. Some specimens were processed for conventional electron microscopy [31].

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anti a-sml anti a - s r anti-total actin Fig. 1. A Coomassie blue stain of SDS-PAGE analysis of purified bovine skeletal muscle actin (lane I ) , bovine aorta extract (lane 2) and whole planarian extract (lane 3). The molecular weights of protein markers (M) are indicated in kDa. B Immunoblots of the same samples as in A immunodecorated with anti a smooth muscle actin (I-sml) mAb. C Immunoblots of the same samples as in A immunodecorated with anti a-sarcomeric actin (a-sr) mAb. D Immunoblots of the same samples as in A immunodecorated with anti-total actin mAb

Results Electrophoresis and Western blotting analysis

As a first step, the actin-like molecules present in planarian extract were characterized after SDS-PAGE and Western blotting using the following mAbs: anti a-sml mAb (Fig. 1 B); anti a-sr mAb (Fig. 1C) and anti-total actin mAb (Fig. 1 D). In these experiments we used purified bovine skeletal muscle actin and an extract of tunica media from bovine aorta as reference standards. In planarian extract, the anti a-sml mAb specifically bound a 42 kDa protein on nitrocellulose replica (Fig. 1 B,

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Fig. 2. A Schematic representation of the head amputation in planaria. The body stump is divided into three separate zones (i, ii and iii) and each treated for SDS-PAGE and Western blotting. B Western blotting decorated with anti a-sml mAb at 0 h (lane I) and 4-day regenerants (lane2) of the body portions i, ii and iii prepared as described above

lane 3). This protein band had the same electrophoretic mobility as actin isolated from bovine skeletal muscle (Fig. 1A, lane 1 ) and the 42 kDa band immunolabelled in the bovine aorta extract (Fig. 1 B, lane 2). The mAb did not react with bovine skeletal actin (Fig. 1 B, lane 1). The anti cesr mAb recognized the bovine a-skeletal actin (Fig. 1C, lane 1) but did not react with any protein molecule in the bovine aorta and planarian extracts (Fig. 1 C, lanes 2 , 3 ) . When the nitrocellulose replicas were immunodecorated with anti-total actin mAb, a 42 kDa protein band was clearly stained in both the bovine aorta (Fig. 1 D, lane 2) and planarian extracts (Fig. 1 D, lane 3) and comigrated with the purified bovine skeletal actin (Fig. 1 D, lane 1). The Western blotting detection of a-sm actin-Iike molecule in planarian was carried out at various regenerative stages. The data were obtained in four different experiments each with at least ten specimens for each regeneration point. The results indicated that the de-

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growth. Western-blotting analysis of the same samples immunodecorated with anti a-sml mAb revealed the presence of the labelling only on bovine aorta extract used as standard (Fig. 3B). Identical results were obtained by stratifying excess protein extract on gel lanes or by delaying the staining times.

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anti a - s m l Fig. 3. A Coomassie blue stained SDS-PAGE of proteins (about 60 pg) from yeast extracts at 4 h (lane I ) , 9 h (lane 2) and 18 h (lane 3) of cell growth. Purified bovine skeletal muscle actin was used as molecular weight marker (lane 4). B Western blotting analysis of the same samples as in A with the exception that fane4 was stratified with bovine aorta extract. The immunodecoration was accomplished with anti a-sml mAb followed by a secondary appropriate antibody conjugated with alkaline phosphatase

tected antigen increased, with the maximum occurring between 70-120 hours after head amputation. From the sixth to twenty-first day the actin immunostaining progressively decreased to the same level as that in noninjured animals (not shown). Previous experiments showed that the antigen immunodetection was proportional to the amount of protein extract charged on the gel. To determine whether the increase in the detection of a-sm actin-like protein in planarian extract was related to the regeneration process and in particular, to blastema development, we divided the worm stump into three separate zones, as indicated, in both intact and 4-day-regenerant planarians (Fig. 2 A). The SDS-PAGE of each extract and Western-blotting of proteins immunodecorated with anti a-sml mAb clearly demonstrated that the increase of cI-sm actin-like protein immunodetection was observed only in the regenerative zone (Fig. 2B, i). In the other two portions, no differences were revealed in antigen stain at the two times (Fig. 2B, ii, iii). The low antigen content found in the pharynxcontaining portion (Fig. 2B, ii) was not surprising and confirmed the low level observed in the area of presumptive blastema development in non-injured worms. The more intense labelling observed in the caudal piece (Fig. 2B, iii) is of interest and is currently being investigated. On the basis of recently published results on yeast actins [34], we tested the reactivity on yeast preparations of the anti a-sm 1 mAb by Western-blotting experiments. Figure 3A shows the Coomassie blue stained SDSPAGE of proteins extracted from yeast at the initial (4 h), middle (9 h) and late (18 h) log phase of cell

Figure 4 a shows the micrograph of the epidermis and underlying tissues in non-injured planarians. The epidermis is monostratified and the well-differentiated epithelial cells show the so-called “foot” anchored to a basal lamina by hemidesmosomes. The underlying zone is occupied by a muscle cell layer and below that is the parenchyma in which various types of gland cell, phagocytic and replacement cells are observed. In the 4-day-regenerating planarians, the structural organization of epidermal and subepidermal tissues is strongly modified (Fig. 4 b d ) . The epithelial cells of the newly formed epidermis are still differentiating. The socalled basement membrane is disorganized and contains some cell debris and phagocytic cells (Fig. 4b, c). The blastema area is mainly characterized by a random mosaic of blastema-forming cells with morphological features that are similar to migrating mesenchyme-like cells (Fig. 4c, d). They are spindle-shaped or unipolar and have pseudopodia and typical filopodia at the leading edge (Fig. 4d). The cytoplasm is rich in mitochondria, free polysomes and rough endoplasmic reticulum vesicles. The euchromatic nucleus contains one or more prominent nucleoli and occupies a large portion of the cell cytoplasm (Fig. 4c, d). Microfilament bundles are also often observed at the level of the thin cytoplasm extensions (Fig. 4d). At this stage of regeneration no apparent sign of phenotypical differentiation can be detected in blastema-forming cells which appear as a homogeneous cell population. However, the myoblasts have a recognizable phenotype (Fig. 4b) since they appear to be the first committed cell type. The developing myoblasts are clearly seen together with a few differentiated myofibers in the subepidermal tissues adjacent to the wound area (Fig. 4b). The muscle layer is almost completely destroyed due to degeneration and apoptosis. The differentiation of myoblasts into myofibers seems to be a very important first step for correct tissue patterning and regeneration. Immunocytochemistry Figure 5 shows the light microscopy micrographs of ABC-P-stained 4-day-regenerating planarians treated with anti a-sml mAb. A strongly positive reaction was detectable mainly at the basal portion of the epidermis and also in blastema-forming cells that were apparently moving in clusters which arose from various foci in the underlying tissues (Fig. 5 a). When the experiment was carried out with non-injured planarians, the immunostaining was detectable in the epidermis and occasionally in some sparse parenchymal cells (not shown). Deparaf-

Fig. 4a-d. Conventional electron microscopy. a Epidermis and a portion of underlying tissues of intact planarian. b-d: 4-day regenerants. Blastema underling the epidermis ajdacent to the wound (b). Blastema under wound epidermis (c). Blastema forming cells (d). BM, basement membrane; Ep, epidermis; ML, muscle layer;

P,parenchyma; BC blastenia forming celis; GCP, gland ceil process; M , myofiber; M B , myoblast; n, nucleus; PhCP, phagocytic cell process; PhC, phagocytic cell; Rh, rhabdyte; RhC, rhabdytic cell. Arrows indicate microfilaments. Burs, 5 pm (a); 2.5 pm (b, c, and d)

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Fig. 5. ABC-P staining of 4-day-regenerating planarians treated with anti a-sml mAb (a). Control specimens treated with non immune serum instead of the first antibody. Nonspecific reaction is detectable in rhabdites (b). Arrowheads indicate the staining of the basal portion of the epidermis; arrows indicate clusters of blastema-forming cells. B, blastema; Ep, epidermis. Bar, 10 pin (a, b)

finized control sections, treated with non-immune serum instead of the first antibody, were predominantly free of positive ABC-P reaction (Fig. 5b). Although the rhabdites appeared to be stained in both treated and control sections, suggesting an unspecific reaction in these cell types, the epidermis basal portion was immunostained only in treated specimens (Fig. 5 b). At the ultrastructural level, the immunogold labelling of intact planarians was mainly localized in the so-called “foot” of epidermal cells (Fig. 6a). In these structures intermediate size filaments and microfilaments are seen in the area close to the hemidesmosomes (Fig. 6b); it is of interest that a-sm actin-like protein is associated in the same cytoplasmic domain. Muscle and gland cell processes were devoid of positive immunostaining (Fig. 6 a). When the immunolocalization was carried out using the anti-total actin mAb, the muscle cells were strongly marked (Fig. 6c). No label was observed with the anti a-sr mAb (not shown). On 4-day-regenerants, the gold labelling in epidermal cells was more marked than in non-injured worms (Fig. 6 d). Undifferentiated blastema-forming cells were also stained by the anti a-sml mAb (Fig. 7a-c). In these cells the immunogold particles were localized at the level of the cell surface mainly along the pseudopodia and filopodia (Fig. 7a, b). It is interesting to note that a migrating phagocytic-like cell was not labelled by the anti a-sml mAb (Fig. 7a). Myoblasts were also positively labelled with anti a-sm-1 mAb, but labelling was not detected at the level of the differentiating myofiber (Fig. 7c); rather, it was stained with anti-total actin mAb (Fig. 7d). Discussion Vandekerckhove and Weber [46] demonstrated that invertebrate muscle actins have a cluster of only three

NH,-terminal acidic residues whereas vertebrate muscle actins generally contain four acidic residues. They concluded that invertebrate muscle actins display structural homology with vertebrate non-muscle actins rather than muscle actins. The existence of multiple isoactins in invertebrates has been demonstrated in insect muscles [18, 191, in body wall muscle in the mollusc Aplysia californicu [25] and in adductor muscle of mollusc Pecten maximus [20, 211. A characteristic feature of these actin molecules is their isoelectric point which is appreciably more basic than vertebrate skeletal muscle actin 118, 19, 411. This is due to one less negative net charge at the NH,-terminal sequence [46]. With the exception of the permanent Schneider L-2 cell line, derived from Drosophilu embryos [41], few data are reported concerning the structural characterization and distribution of non-muscle cell actin in invertebrates. Our data suggest that in planarian tissues at least two different isoactins are present. One isoform is expressed mainly in differentiated muscle cells and is immunologically different from vertebrate a-sarcomeric and a-smooth isotypes. Another non-muscle form is related to cell differentiation and has a molecular weight and immunological properties which are similar to the warm-blooded vertebrate a-smooth muscle actin. We have also reported evidence that the a-sm actin-like protein detected in planarians does not correspond to any yeast actin-like molecule [34]. Moreover the amino acid sequence of the a-sm NH,-terminal decapeptide used for monoclonal antibody preparation is completely different from that of yeast ACT2 protein as deduced from ACT2 gene nucleotide sequence [34, 35,461. Although planarian muscle cells do not express isoactins recognized by the antibody anti-vertebrate a-sr actins, it is interesting to note that the transient expression of the a-sm actin-like molecule in differentiating planarian muscle cells resembles that observed during myogenesis of rat and mouse embryos [l, 8, 22, 471. Like vertebrates, the planarian myoblasts expressed an a-sm actinlike antigen during the first step of development but when they acquired the differentiated phenotype, the antigen was no longer detected. Our biochemical data suggest that the changes in the 42 kDa actin-like protein observed during regeneration may be related to the increased number of blastemaforming cells which apparently move into this region and which undergo differentiation. It should be pointed out that the expression of two homeobox genes, recently cloned and sequenced in planaria, showed a maximum at the same regenerative stage [l 11. Epidermal cells were labelled by the anti a-sm 1 mAb at the level of the adhesion areas. These cells are probably contractile, even if a true microfilament meshwork was more easily detectable in newly differentiating epidermal cells [31]. Moreover, the use of a-sm actin as a marker for cells with contractile functions such as myofibroblast-like cells [24, 26, 371 and microvascular pericytes [27, 391 has been demonstrated. Another consideration is that planarian epidermal cells may be functionally similar to myoepithelial cells that have intermediate-

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Fig. 6. Intact planarians (a-c). a Immunocytochemical decoration with anti a-sml mAb; 10 nm gold particle labelling of the epidermal cell foot. b Conventional electron microscopy showing microfilamen1 bundles (arrows) and some intermediate-sized filaments (arrowheads) in feet of an epidermal cell. c Immunocytochemical decoration with anti-total actin mAb, 10 nm gold particle labelling

of myofibers. d Immunocytochemical decoration with anti a-sml mAb of 4-day-regenerating animals, 10 nm gold particle labelling of the basal portion of the epidermis. *, reticular material (this structure has been described in [43]); other symbols are as in Fig. 3. Bars, 0.5 pm (a); 1 pm (b, c and d)

Fig. 7a-d. Immunoelectron micrographs of 4-day-old regenerants. a, b Undifferentiated blastema-forming cells stained with anti a-sml mAb labelled with 10 nm colloidal gold. Myoblasts decorated with anti cl-sml mAb (c) and with anti-total actin mAb (d). rer, rough endoplasmic reticulum; arrowheads indicate organizing myofibers. Other symbols are as in Fig. 3. Bars, 0.5 pm (a, c and d); I pm (b)

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sized filaments associated with actin molecules [ 101 and are specifically stained by the anti a-sml mAb [15, 351. In these systems, as well as during planarian regeneration, these cells may help to contract the wound edges in order to create the first protective epidermal covering. Blastema-forming cells are also positively stained by the anti a-sml mAb. Although the origin of these cells in planaria is an open question, their functional and morphological characteristics are typical of migrating mesenchyme-like cells [ 131. The hypothesis that a-sm actin expression could be considered a marker for mesenchyme-like cells was recently discussed by Schmitt-Graff et al. [33] in their work concerning the appearance of a-sm actin in human lens cells of anterior capsular cataract and in cultured bovine lens-forming cells. These cell types, despite their embryonic ectodermal origin, under some pathological or experimental conditions, expressed a marker of myoid differentiation or they seemed to be transformed into mesenchyme-like cells [ 13, 331. Moreover it has recently been shown that during experimental wound healing in the rat, fibroblastic stromal cells which normally do not contain a-sm actin modulate into myofibroblasts and express temporarily a-sm actin, which disappears when the wound closes [7]. To date, it is not known whether the immunological properties of the 42 kDa a-sm actin-like protein detected in planarians also reflects structural similarity with the corresponding protein found in warm-blooded vertebrate smooth-muscle cells. However, the following considerations are worth mentioning: first, the anti a-sml mAb recognizes a specific and restricted amino acid sequence which constitutes the epitope at the NH,-terminal domain of vertebrate a-sm actin and the sequence is different than that of other sarcomeric and non-sarcomeric actin isoforms [35]. Second, the immunohistochemical experiments indicate that the antigen is localized at the sites of the cytoplasmic domains in which a positive reaction would be expected for non-muscle actin. Third, like vertebrate muscle cells, the planarian a-sm actin-like protein is expressed during the myoblast differentiation process. In conclusion we report, for the first time in an invertebrate, the presence of an actin-like protein that is recognized by the anti a-sm 1 mAb elicited against the mammalian vascular a-sm actin isotype. This molecule is expressed in mesenchyme-like cells during formation and growth of regenerative blastema and at early stages of myoid cell differentiation. Acknowledgements. We thank Dr. L. Lanfaloni for yeast cultures and Sister Nancy Hutchinson for critical reading of the manuscript. This work was in part supported by Swiss National Science Faindation Grant No 31/30796.91 and in part by Italian CNR grant No 90.01522.CT04.

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