Smooth muscle actin expression during rat gut development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium

Differentiation (1990) 43: 87-97

Differentiation Ontogeny and Neoplasia 0 Springer-Verlag 1990

Smooth muscle actin expression during rat gut development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium Michkle Kedinger *, Patricia Simon-Assmann, Franqoise Bouziges, Christiane Arnold, Eliane Alexandre, and Katy Haffen INSERM Unite 61, Biologie Cellulaire et Physiopathologie Digestives, 3 avenue Moliere F-67200 Strasbourg, France Accepted in revised form March 3, 1990

Abstract. Cytodifferentiation of smooth muscle cells has been analyzed immunocytochemically during rat intestinal development and in chimaeric intestines by using monoclonal antibodies reacting specifically with smooth muscle actin species (CGA7 [lo] and anti-a SM-1 [40]). As development proceeds, the various intestinal muscle layers differentiate in the following order: (1) cells expressing smooth muscle actin appear within the mesenchyme of the 15-day fetal rat intestine, in the circular muscle-forming area, the differentiation of cells in the presumptive longitudinal muscle layer starting with a 48-h delay; (2) smooth muscle fibers appear within the connective tissue core of the villi shortly after birth, in parallel with a progressive formation of the muscularis mucosae, which becomes clear-cut only in the course of the 2nd week after birth; (3) a distinct cell layer in the innermost part of the circular muscle layer arises during the perinatal period. Thereafter, the fluorescence pattern remains unchanged until the adult stage. Chimaeric intestines were constructed by the association of 14-day fetal intestinal epithelium and cultured fetal rat or human skin fibroblasts. These fibroblastic cells did not express actin at the time at which they were associated. The immunocytochemical analysis of smooth muscle actin in the hybrid intestines, which had developed as intracoelomic grafts for 12 days, revealed that the skin fibroblastic cells had been induced by the intestinal epithelial cells to differentiate into smooth muscle cells. Such a result was also obtained with allantoic endoderm. It was not obvious in cocultures of intestinal epithelium with skin fibroblastic cells. However, when intestinal epithelial cells were cocultured with intestinal mesenchymal cells, actin expression was stimulated in the latter cell population.

Introduction The small intestinal wall is surrounded by a smooth muscle coat composed of two distinct layers: The inner circu-

* To whom offprint requests should be sent

lar muscle layer is thick and continuous, extending from the upper oesophagus to the anal canal, and the external longitudinal layer is less developed and less regular along the whole digestive tract. Intestinal muscle cells are actively involved in transit processes and indirectly in absorption. Indeed, coordinated contractions of these two layers bring about mixing and oral- aboral movements of the luminal content. In addition to this muscle coat, the small intestinal wall comprises a continuous sheet of smooth muscle two to five cells thick - the muscularis mucosae- separating the mucosa from the submucosa [8]. Finally, the connective tissue core of the villi - the lamina propria - contains smooth muscle cells oriented more or less along the longitudinal axis of the villi. These cells - as well as the muscularis mucosae may also influence intestinal function by contributing to the movements of villi rather than to the overall motility WI. The small intestinal musculature as well as the connective tissue differentiate from the gut embryonic mesenchyme, while the intestinal epithelium derives from the inner embryonic intestinal enlage, the endoderm. Although ontogenic studies have been increasingly concerned with the structural and biochemical aspects of epithelial differentiation (for reviews see [5, 18, 19, 431) little is known about the developmental pattern of the gastroenteric smooth muscle [8, 23, 291. In previous experiments using recombinants between various embryonic tissue anlagen we have demonstrated that interactions between the endoderm and its surrounding mesenchyme are instrumental in intestinal morphogenesis and cellular differentiation (for reviews see [15, 191). Epithelial differentiation does not take place in the absence of a mesenchymal support, and viceversa. Moreover, we have shown that quite-normal morphogenesis of the endoderm occurs when mesenchymal cell types foreign to the intestine - such as skin fibroblasts - are substituted for gut embryonic mesenchyme [22]. In turn, endoderm induces the associated fibroblastic cells to organize themselves into smooth muscle-like layers and into the connective tissue axis of the villi. Survival, proliferation and differentiation of intestinal ~

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endodermal cells have also been obtained in vitro provided that the epithelial cells were seeded on top of confluent fibroblastic cells of intestinal or nonintestinal origin [21]. These previous studies were mainly directed towards the cytodifferentiation of the epithelial cell layer and especially of the absorptive cells, which was followed via the expression of specific markers, the brush border digestive hydrolases. In the present work we focus our attention on the cytodifferentiation of smooth muscle cells using smooth-muscle-specific monoclonal antibodies (CGA7,[lo] and a SM-1 [40]) that have been useful for studies relevant to cell differentiation and to tumor characterization [2, 10, 40-421. These antibodies bind specifically to smooth muscle cells and myoepithelial cells, but fail to react with actin of skeletal muscle, epithelium or fibroblasts; immunoblot experiments have revealed that CGA7 recognizes ci and y smooth muscle actin species in extracts of rat colon [lo]; anti-a-SM-1 reacts only with the a-smooth muscle isoform of human and animal actin [40]. The present investigation was conducted to analyze the expression of smooth muscle actin in the developing rat small intestine and to explore the inductive effect of epithelial cells on smooth muscle differentiation. The latter aspect has been approached experimentally by : (1) the construction of chimaeric intestines made up of fetal intestinal or allantoic endoderm, surrounded by a sheet of cultured fetal intestinal mesenchymal cells or skin fibroblasts and developed as intracoelomic grafts ; (2) the examination of in vitro cocultures of intestinal endodermal cells and fetal gut mesenchymal cells or skin fibroblasts.

Methods Materials. Syntheiic culture media Dulbecco's modified Eagle's (DMEM), Ham's F10, F12 and C M R L 1066 and also fetal calf serum and trypsin were purchased from Gibco. Type IV collagenase ( N _ 150 Ujmg) came from the Worthington Biochemical Corporation. Anirnals. Wistar rat fetuses wcrc obtained in our laboratory and litters were taken daily between 14 days of gestation and birth, the existence of a vaginal plug determining day 0. Newborn rats on day 3, 10 and 15 after birth and adult rats were also used. Chick embryos were used at 3 or 4 days counted as days of incubation at 38" C.

Cell cultures. Intestinal mesenchymal cell cultures were derived from 14-day fetal rat intestinal mesenchyme that had bccn separated from the endoderm after a l - h incubation in collagenase (0.03% in C M R L 1066 a t 37" C) [21]. Fetal rat skin fibroblastic cell cultures were derived from enzymatically dissociated 20-day fetal rat aponeurosis (treated with 0.01% collagcnasc and 0.01 YOtrypsin in C a Z + ,Mgz+-free Ham's F10 medium for 1 h a t 37" C) [14]. Fetal human skin fibroblasts were derived from skin fragments assigned for karyotypic examination and kindly provided by Dr. Ruch (Laboratoire de cytogtnktique, Centre hospitalo-universitaire, Strasbourg, France). Subcultures were performcd after trypsinization of confluent cultures (0.25% trypsin solution and 0.02% EDTA in CaZ+-, Mg2+-free PBS for 10-15 min at 37" C). The isolated cells were seeded at a plating density of 5 x lo4 cells/cmz. The culture medium

consisted of a 131 mixture of DMEM and Ham's F12 supplemented with 15% fetal calf serum and gentamycin (0.2 mgiml). The skin fibroblastic cell cultures were used at confluency between the first and the third passage. In the case of cocultures, microexplants of pure intestinal endoderm (scparated from the surrounding mesenchyme of 14-day fetal rat intestines), were seeded on top of confluent monolayers of intestinal mesenchymal cells or of skin fibroblastic cells as previously described [21]. They have been examined after a 6- to 12-day epithelial-fibroblastic cell contact. Chimueric intestines. Segments of 14-day fetal rat intestinal endoderm (separated from the mesenchyme as described in the preceding paragraph) as well as of collagenase-dissociated allantoic endoderm of 4-day chick embryos [13] were enveloped in a sheet of cultured intestinal mesenchymal cells, or of rat or human cultured skin fibroblastic cells. After overnight culture on agar-gelified medium to allow their cohesion, the recombinants were grafted into the coelomic cavity of 3-day chick embryos. The resulting chimaeric intestines were recovered 12 days later. Immunojhorescence study. Proximal rat intestinal segments taken a t various developmental stages as well as the chimaeric intestines or cocultures were embedded in Tissue Tek, snap-frozen in Freon, cooled in liquid nitrogen and stored a t -70" C until use. Fivemicrometer frozen sections were prepared, air-dried and kept frozen a t -20" C for subsequent immunocytochemical studies. The monoclonal antibody CGA7 [lo] used in the present study was kindly provided by Dr. Gown, University of Washington, Seattle, USA. The sections were treated with CGA7 at a 1jlOO dilution in phosphate-buffered saline (PBS) for 2 h in a humified chamber, washed and stained with fluorescein-isothiocyanate-conjugated goat antibodies to mouse immunoglobulins (Nordic) diluted ( I / 200) in PBS. The preparations were mounted in glycerol/PBS/phenylenediamine under a coverslip, observed and photographed with an Orthoplan Leitz microscope. The specificity of the immunofluorescent reaction was ascertained by preabsorption of the antibodies with pure rabbit muscle actin (Sigma) in the presence of Tween. In parallel, immunocytochemistry was also performed using another monoclonal antibody that reacts only against a-smooth muscle actin (anti-a SM-1, 1/20 in PBS, [40]; a generous gift from Dr. Gabbiani, University of Geneva, Switzerland).

Results Developmental pattern of intestinal smooth muscle cell dcferentiation assessed by actin expression

In the early gut anlage of 14-day rat fetuses, no specific labelling of actin with CGA7 antibodies could be detected (Fig. 1 A, B). At 15 days of gestation, the stage at which the intestinal tube is still composed of morphologically undifferentiated endodermal and mesenchymal tissue anlagen, CGA 7 antibodies decorated a circular zone of three to four subperipheral cell layers within the mesenchyme of the proximal intestine, corresponding to the presumptive inner muscle layer (Fig. l C , E and F). Although a few positive cells could be visualized at 14 days in this latter area with the anti-ci SM-1 monoclonal, at 15 days the first clear-cut expression of actin was confirmed by a strong fluorescence signal with that antibody (Fig. 1 D). The fact that anti-a SM-1 decorated cells in the same area indicates that of the ci and y actin isoforms reacting with CGA7 at least the former one is expressed. At 17 days of gestation, the stage at which

Fig. 1A-J. Immunocytochemical localization of actin with monoclonal antibodies to smooth muscle specific isoactins, CGA7 (A, C, G H ) or anti-aSM-1 (D-I) in fetal rat intestine a t 14 (A), I 5 (C, D), 17 ( G ) and 20 (H, I) days of gestation. B, E, F and J illustrate 5 p (B, E), 0.5-p (F) histological sections and 5-p (J) cryosections stained with periodic-acid Schiff and hematoxylin (B, E,J) or toluidin blue (F) of fetal intestine at respectively, 14, 15,

151/2 and 20 days. e , endoderm; m, mesenchyme; E, villus epithelium; cml, circular muscle layer; Iml, longitudinal muscle layer; L p , connective tissue or lamina propria; *, negative triangular areas presumably corresponding to nerve complexes; 0. presumptive circular muscle layer. The broken line outlines the epithelial-mesenchymal or-lamina propria interface. Bur, 30 p. A, B x 80; C, D, I x 315; E x 225; F x 385; G x 160; H x 190; J x400

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villus primordia begin to protrude, faint labelling became obvious within the longitudinal muscle-forming area (Fig. 1 G, CGA7 antibodies). From this stage until birth, the staining pattern of smooth muscle actin remained unchanged : bright labelling within the well-defined circular muscle layer, the longitudinal one remaining very thin during this period (Fig. l H , CGA7 antibody; 11, anti-a SM-1; and 15). The fluorescent signal was apparent within the whole cytoplasm of the smooth muscle cells. Between the two muscle layers, triangular negative areas could be distinguished ; they presumably correspond to the location of intramuscular nerve bundles [S]. Until birth, the connective tissue underlying the villi was devoid of labelling; vascular smooth muscle cells were unstained although the vessels were outlined by basement membrane molecules [37]. From birth onwards, the following changes were obvious. Firstly, there was progressive thickening of the outer muscle layer as development proceeded (Fig. 2 A, C and D). Secondly, in the 3-day rat intestine, positively labelled smooth muscle cells appeared within the villus core, concomittant with the individualization of an irregular layer of labelled cells - the muscularis mucosae separating the submucosa from the inucosal connective tissue, visualized at this stage mainly with anti-a SM-1 antibodies (Fig. 2B). The intensity of the reaction and the thickness of the latter cell layer were progressively accentuated from day 3 to day 15 (Fig. 2B-D). Thirdly, during this postnatal period, the staining pattern also revealed a thin layer of brightly labelled cells in the innermost part of the circular muscle (Fig. 2C, D). In addition, from birth onwards, smooth muscle actin was expressed by the vascular smooth muscle cells (Fig. 2B, C). When compared to the postnatal stages, the adult intestinal muscle layers exhibited less-intense labelling (Fig. 2 E) ; however, bright staining remained obvious in the muscularis mucosae and in the innermost cell layer of the muscle coat. Interestingly, in addition to the positive fibers that populated the villus core, elongated decorated cells surrounding the crypts, already irregularly visible at 10-1 5 days after birth, were found consistently in the adult intestine (Fig. 2F). It must be pointed out that CGA7 antibodies also react with human tissue. Indeed, an overall similar labelling pattern was observed in adult human small intestine, the pericryptal fibers being particularly obvious (Fig. 2 G, H).

Induction of smooth muscle actin expression in skin.fibroblastic cells by intestinal epithelial cells In previous studies we showed that fetal human or rat skin fibroblastic cells can replace the intestinal mesenchyme allowing intestinal endodermal cells to achieve cytodifferentiation and villus morphogenesis [22]. Whether skin fibroblastic cells in turn differentiate into muscle layers has been examined by the analysis of smooth muscle actin expression. Neither CGA7 nor anti- aSM-1 antibodies stained cultured fetal human (Fig. 3A) or rat skin fibroblastic

cells (not illustrated) when they were associated with endoderm. Recombinants composed of skin fibroblastic cells, whatever the species of origin, associated with 14day fetal rat intestinal endoderm and grafted into the coelomic cavity of 3-day chick embryos for 12 days, developed autonomously into cylindrical organs that were loosely attached to the thoracic or abdominal wall of the host by a vascular peduncle providing the blood supply of the transplants (Fig. 4 B). Histologically, these grafts were composed of small villi covered by an epithelial cell layer, the intermediate connective tissue expanding into the centers of the villi and the presumptive peripheral muscle layers (Fig. 3 C). The ultrastructural features of the latter cell layer (Fig. 5) were closely similar to those of the muscle coat of a 3-day rat intestine developed in situ (not illustrated). The cells were densely packed into groups separated by spaces containing connective tissue elements (Fig. 5A). In addition, abundant extracellular material lining the intercellular spaces was obvious (Fig. 5 B). Parallel-oriented smooth muscle cells formed sheet-like layers (Figs. 5 D and E). The most obvious observation was the presence of numerous bundles of actin microfilaments within the cytoplasm of the cells (Fig. 5 C, E and F). Immunocytochemical examination of the chimaeric intestines, the developmental stage of which corresponded approximately to that of the rat near birth, revealed smooth muscle actin within the smooth muscle layer (Fig. 3D). No labelled muscularis mucosae, nor positive fibers within the core of the villi, were obvious. Similar data were obtained with recombinants composed of intestinal endoderm associated with cultured 14-day intestinal mesenchymal cells (Fig. 3 G, H) or with intact mesenchyme (not illustrated). In a parallel set of experiments, intestinal endoderma1 cells were cocultured in vitro with human or rat skin fibroblasts. Under these conditions, endodermal cells attached to the fibroblastic cell layer, proliferated, and formed a monolayer of polarized cells linked together with characteristic tight junctions that delineate apical brush borders endowed with digestive hydrolases [21]. Basement membrane molecules were progressively deposited at the epithelial-fibroblastic interface [38] mimicking an in vivo organization. Despite these differentiation characteristics, the fibroblastic compartment composed of several piled cell layers failed to react with smooth muscle actin antibodies even after 11-12 days in coculture (Fig. 3 B). In cocultures composed of intestinal mesenchymal cells instead of skin fibroblasts, although the overall behavior of the cocultures was strictly similar to that described above, clear-cut labelling was already obvious after 4 days, within the whole mesenchymal cell population (Fig. 3F). It should be noted that in contrast to the skin fibroblastic cells which were devoid of positive signal - intestinal mesenchymal cells cultured in isolation expressed actin sporadically, the positive cells being randomly distributed (Fig. 3 E), suggesting very limited self-differentiation potential. Related to these data, an interesting observation was made using 4-day chick allantoic endoderm. Allantoic endoderm is known to differentiate into intestinal epithelium when recombined with heterologous mesenchyme -

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Fig. 2A-H. Localization of actin-expressing cells in postnatal (A, B 3 days; C 10 days; D 15 days) and adult (E, F) rat intestines and in adult human ileum (G, H) with CGA7 (A, C-E, G , H) or anti- OL SM-1 (B, F) antibodies. F Transverse section in the crypt region. E, epithelium; Lp, lamina propria; mm, muscularis mucosae; cml, circular muscle layer; I ml, longitudinal muscle layer;

(a)actin-expressing fibers within the villus core; (3)positively labelled blood vessels; (D) brightly labelled cell layer at the innermost part of the circular muscle layer; (=)positive elongated cells underlying the crypt epithelium. Bur, 30 kt (A-D, F, H) or 200 p (E, C). A, D, H x315; B x470; C x 190; E x4 0; F x 125; G x 60

Fig. 3A-H. Immunodetection of actin with antibody anti-a SM-1 in fetal human skin fibroblasts (A) and in fetal rat intestinal mesenchymal cells (E), cultured in isolation for 7 days, in cocultures of human skin fibroblasts (B) or intestinal mesenchymal cells (F) with rat intestinal endodermal cells after respectively 11 and 4 days. Morphological (C, G) and immunocytochcmical (D, H ; antibody CGA7) features of chimaeric intestines developed as intracoelomic grafts for 12 days. They are composed of rat intestinal endoderm

and of human skin fibroblasts (C, D) or rat intestinal mesenchymal cells (G, H). C, G Same as D and H, respectively, but stained with periodic-acid Schiff and hematoxylin after examination with the fluorescence microscope. e, intestinal endodermal cells; ( 2 ) epithelial cell layer; ,f. skin fibroblasts; m, intestinal mesenchymal cells; E, villus epithelium; ml, muscle layers; Lp, lamina propria. Bar, 30 p. A, B, E, F x 315; C, G x 170; D, H x 160

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Fig. 4A-C. Gross morphology of recombinants composed of human skin fibroblastic cells and of chick allantoic endoderm (A, C) or 14-day fetal rat intestinal endoderm (B) photographed under the dissecting microscope at the time of intracoelomic grafting (A)

and 12 days later (B, C). The views (A, C) taken at the same magnification illustrate the growth of an hybrid intestine. (H) host-derived vascularization of the graft. Bur, 500 p. A-C x 12

[13, 451. In the present work, we showed that allantoic endoderm associated with skin fibroblastic cells and grafted in ovo for 12 days (Fig. 4A, C) developed into subnormal intestinal structures (Fig. 6A). Interestingly, also in this case, fibroblastic cells were induced to differentiate into smooth muscle cells expressing actin and were organized in at least one smooth muscle layer (Fig. 6B).

sand [29] : 15-day rat embryos display low-to-undetectable levels of mRNA, which are dramatically increased by birth and finally decrease near weaning. Other interesting features of smooth muscle cell differentiation in the postnatal rat intestine are the progressive differentiation of the muscularis mucosae after birth, between 3 and 15 days, the individualization around 10 days of actin-expressing cells in the innermost part of the circular muscle coat, and the staining of pericryptal fibers in the adult intestine. The so called “special layer” of the innermost circular muscle coat in the mouse intestine derives from fibroblast-like precursor cells [7]. According to Gabella [8] these small electron-dense muscle cells are separated from the bulk of the circular layer by a large number of nerve fibers, some of these being sensory fibers working in conjunction with smooth muscle cells in the manner of a stretch receptor. The existence of a fibroblastic cell population that surrounds the crypts, and whose members proliferate and presumably migrate along with the epithelial cells, has been described in the large and small intestine [27, 30, 321. Our immunocytochemical observations indicate that these pericrypt cells contain smooth muscle actin isoforms, corroborating the findings reported recently in human colon [33,35]. In the developing human colon, this cell type first appears a t 21 weeks of gestation [35], while in the rat small intestine sparse immunoreactive cells, which could correspond to this subset of specialized cells, are visible only around 10 days after birth. Despite the apparent functional similarity of the subepithelial fibroblastic cell populations at various developmental stages, our data indicate that those involved in inductive epithelial-mesenchymal cell interactions during the fetal and perinatal phases of morphogenesis in the rat intestine [14, 20, 281 do not express smooth muscle actin. Only the cells surrounding morphologically mature crypts exhibit smooth muscle differentiation, indicating that the function of these cells may include contractile activities that could be involved in epithelial-cell migration along the crypt-villus axis. Expression of actin

Discussion In the present study, an immunocytochemical approach was used to analyse first the developmental pattern of smooth muscle phenotypes arising from the mesenchyma1 anlage in the rat intestine and second the epithelial control of smooth muscle cell differentiation. The use of specific antibodies allowed us to follow the stepwise segregation of mesenchymal cells expressing smooth muscle actin as a differentiation marker from fetal life to the adult stage. Individualization of actin-containing cells starts in the presumptive circular muscle layer and parallels the previously reported segregation of extracellular matrix proteins in the peripheral mesenchyme at 15-1 6 days of gestation [37]. Similarly, using a monoclonal antibody that reacts with skeletal and smooth muscle cells, Rong et al. [34] showed that in the chick embryonic intestine muscle cells arise proximo-distally at developmental stages that correspond morphologically to that of the 14- to 15-day fetal rat. Differentiation of the longitudinal smooth muscle layer is delayed, the first cells expressing actin at the periphery of the intestinal wall appearing at 17 days of gestation. After birth, the progressive thickening of the muscle layers is accompanied by intensification of the smooth muscle actin staining, the latter decreasing in the adult intestine. The developmental pattern of intestinal smooth muscle actin described here is corroborated by an overall similar pattern of y enteric actin mRNA, as recently reported by McHugh and Les-

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Fig. 5A-F. Ultrastructural features of the smooth muscle coat in recombinants made up of human skin fibroblastic cells and rat intestinal endoderm developed as intracoelomic grafts for 12 days. Transverse (A, B) and longitudinal (C-F) sections of the smooth muscle cell layers. Thc muscle cells are assembled into groups (A) and an abundant extracellular material (H), presumably collagen

fibrils, is obvious between individual cells (B). Note the presence

or numerous cytoplasmic bundles of actin microfilaments ( *). CT, connective tissue elements separating groups of cells. Bar, 2 p (A-E); 0.3 p (F). A, I) x 2900; B x 7100; C x 5900; E x 3900; F x 28700

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Fig. 6A, B. Chimaeric intestine developed as intracoelomic graft for 12 days from the association of human skin fibroblasts and chick allantoic endoderm. Morphological appearance (A) and immunocytochemical detection of smooth muscle actin with CGA7

antibody (B). The section has been stained with periodic-acid Schiff and hematoxylin after observation with the fluorescence microscope. E, villus epithelium; ml, muscle layers. Bar, 30 p. A x 170; B x 160

in the pericrypt cells seems to be independent from the individualization of the muscularis mucosae and the appearance of smooth muscle fibers within the villus core, which both occur perinatally. Thus, although the exact origin of myofibroblasts in the intestine is still undefined, they could derive, as suggested by Oda et al. [31] from modified fibroblasts that have undergone differentiation in response to a specific extracellular microenvironment. It must be noted that whatever the developmental stage studied, CGA7 antibodies never reacted with the intestinal epithelium, as already shown by Gown et al. [ 101, while polyclonal antibodies strongly stained the cytoskeletal core of the microvilli [4, 6, 161. This observation is in accordance with the finding that isoactins in the brush border of rat intestinal epithelial cells contain a set of epitopes that distinguishes them from the muscle isoforms [36]. The data outlined in the second part of the present study clearly demonstrate that muscle differentiation of the fetal intestinal mesenchyme is triggered by the epithelial cells. Indeed, whereas mesenchymal cells cultured alone were almost devoid of actin, its expression was strongly stimulated by intestinal endodermal cells in cocultures, and mainly in grafted associations. Such a triggering property of epithelial cells is also carried by human colonic carcinoma cells (HT-29 and Caco2 lines), which allow avian embryonic gut mesenchyme to differ-

entiate into morphologically well-defined muscle layers [ 121. In relation to these observations, epithelial cells have been shown to enable intestinal embryonic mesenchyme to express tenascin, an extracellular matrix molecule [l]. Another interesting finding arising from the present recombination experiments using a reliable marker protein for distinguishing between smooth muscle and fibroblastic cells, is that under the influence of intestinal epithelial cells there is a reprogramming of the fibroblastic nuclei that were not destined to be muscle, which are thereby induced to express smooth muscle genes. This heterodifferentiation, clearly visible under grafting conditions, was not induced in coculture conditions, suggesting that either systemic factors and/or the assembly of the induced cells into three-dimensional aggregates are prerequisite for completion of subsequent differentiation. Several points that can be raised exclude the possibilities, on one hand, that the muscle coat of the graft may have developed from mesenchymal cells that remained attached to the endoderm, and on the other hand, that nonvascular cells from the host invaded the graft. Firstly, use of the suitable nucleolar marker of quail cells [25] in chimaeric quail/rat or quail/chick epithelial-mesenchymal constructions grafted either into the coelomic cavity [I 1, 241 or on the chorio-allantois of chick embryos [46], has underlined the absence of recip-

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rocal cellular infiltration between the enzymatically dissociated endodermal and mesenchymal cell compartments. Secondly, intestinal endoderm or fibroblastic cell sheets grafted alone involuted or formed empty vascularized vesicles, respectively (data not illustrated). Thirdly, recent experiments involving chick/rat tissue recombinants, to which species-specific antibodies directed against extracellular matrix molecules were applied, from their appropriate specificity of labelling, persuasively indicated the phenotype of host-derived cells corresponding to blood vessels only [38, 391. Finally, recombinants composed of intestinal endoderm and of human colonic cancer cells (cell line HT-29) instead of fibroblastic cells, developed into small vascularized structures composed of tumoral cells without external smooth muscle actinpositive cells (data not illustrated). With respect to functional smooth muscle differentiation, it must be noted that the typical peristaltic movements or coiling displayed by muscle derived from fetal gut mesenchyme are missing in the fibroblast-endodermal recombinants. This lack might be linked to the absence of intrinsic innervation, which has been shown to derive from neural crest cells that colonize the intestinal mesenchyme in situ [9, 261. Future electrophysiological studies could facilitate access to the mechanisms governing functional activity of fibroblasts induced to differentiate into smooth muscle cells. The fact that fibroblastic cells whatever their intestinal or skin origin - were able to differentiate into smooth muscle cells, as well as the data obtained with allantoic endoderm, suggest that two successive steps are involved in intestinal tissue interactions : the fibroblastic cells support epithelial development and differentiation; in turn, the epithelial cells change the genetic program of the fibroblastic cells. Similar changes are obtained in heterocaryons formed by fusion of mouse muscle cells and human fibroblasts, in which human nonmuscle cell nuclei are induced to express muscle genes [17]. The way in which cell interactions control cellular events allowing activation of a gene that was repressed remains unknown. Yet, in the last decade there has been an increasing belief that extracellular matrix molecules can play an important role in the regulation of tissue-specific gene expression via membrane receptors [3]. ~

Acknowledgements. We are grateful to Drs. A.M. Gown (University of Washington, Seattle, USA) and G. Gabbiani (University of Geneva, Switzerland) for their kind gifts of antibodies and Dr. J. Ruch (University of Strasbourg, France) who provided us with human skin fibroblasts. We thank Mrs. C. Leberquier for excellent technical assistance, Mrs. c . Haffen for photographic work and Mrs. Mathern for typing the manuscript. This work was supported by the INSERM and a grant from the Fondation pour la Recherche Mkdicale.

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