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

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 [10] and anti-α 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.     

Action of general and alpha-smooth muscle-specific actin antibody microinjection on stress fibers of cultured smooth muscle cells

Arterial smooth muscle cells express alpha- and gamma-smooth muscle, as well as beta- and gamma-cytoplasmic actins. Two actin antibodies, one recognizing smooth muscle and cytoplasmic actin isoforms, the other recognizing specifically alpha-smooth muscle actin, were microinjected into cultured aortic smooth muscle cells. The effect of these antibodies on stress fiber organization was examined by staining with rhodamine-labeled phalloidin and by immunofluorescence with the same antibodies. Microinjection of the general actin antibody abolished most of the stress fiber staining with all reagents, but did not significantly affect the shape of the injected cells. This suggests that stress fiber integrity is not absolutely necessary for the maintenance of cell shape within the time of observation. Microinjection of the specific alpha-smooth muscle antibody abolished to various extents the staining of stress fibers with this antibody, but left practically intact their staining with rhodamine-labeled phalloidin and with the general actin antibody. This suggests that the incorporation of alpha-smooth muscle actin is not absolutely necessary for the maintenance of stress fiber integrity in cultured smooth muscle cells.     

Analysis of surface membrane antigens, cytoskeletal proteins and N-myc oncogene in pediatric solid malignant tumors, their diagnostic usefulness and relevant problems]

Neuroblastoma (NB), primitive neuroectodermal tumor (PNET), Ewing's sarcoma and rhabdomyosarcoma (RMS) are solid malignant tumors in childhood. Microscopically these tumors are grouped as small-round-cell tumors, and a different diagnosis is sometimes difficult. Cell surface membrane antigen, cytoskeletal protein and N-myc amplification and over-expression were analyzed in these cell lines and tumor tissues for the accurate diagnosis. NB and PNET could be distinguished from Ewing's sarcoma and RMS by the panel of monoclonal antibodies against cell surface membrane antigens. The cytoskeletal protein analysis is useful for the diagnosis of RMS and leiomyosarcoma. Alpha-smooth muscle actin and/or desmin were demonstrated in the S-type (epithelial-like) cells in 3 NB cell lines, suggesting the differentiation pathway of NB into smooth muscle cells. N-myc amplification and over-expression were observed in NB cell lines as well as one RMS cell line. The occurrence of N-myc amplification and over-expression in the RMS cell line cautions us against using N-myc as a distinguishable marker for NB.     

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha- smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha- smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm- 1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha- smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti- desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin- negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.     

Effect of sympathectomy on the phenotype of smooth muscle cells of middle cerebral and ear arteries of hyperlipidaemic rabbits

This study was carried out to determine whether sympathectomy influences the phenotypic modulation of smooth muscle cells in the peripheral and cerebral arteries of heritable hyperlipidaemic rabbits. Unilateral superior cervical ganglionectomy (common origin of innervation to the middle cerebral artery and the central ear artery) was performed on four Watanabe heritable hyperlipidaemic rabbits. Cross-sections of the ipsi- (sympathectomized) and the contralateral (intact) cerebral and ear arteries were prepared 2 months later and labelled with monoclonal antibodies against vimentin and desmin, two markers of the differentiation of smooth muscle cells, and α-smooth muscle actin, a marker of these cells. Sections from control and sympathectomized arteries were analysed with a confocal laser scanning microscope. Compared with contralateral intact ear arteries, the sympathectomized ear artery developed a thickened intima with dedifferentiated smooth muscle cells, expressing α-smooth muscle actin but no desmin, whereas the middle cerebral artery remained unchanged. These results suggest that sympathectomy may favour the progression of atherosclerosis in peripheral but not in cerebral arteries of Watanabe heritable hyperlipidaemic rabbits     

Distribution of actin and tropomyosin in Cryptosporidium muris

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (×2,000). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various cellular functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites'' firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the chemical coding of each class of ICs.     

Stretch-dependent growth and differentiation in vascular smooth muscle: role of the actin cytoskeleton

The smooth muscle cells in the vascular wall are constantly exposed to distending forces from the intraluminal pressure. A rise in blood pressure triggers growth of the vessel wall, which is characterized primarily by hypertrophy of smooth muscle cells with maintained differentiation in a contractile phenotype. Growth factor stimulation of dissociated smooth muscle cells, on the other hand, causes proliferative growth with loss of contractility. This type of response is also found in neointima development following angioplasty and in atherosclerotic lesions. An intact tissue environment is therefore critical for preserved differentiation. Recent advances point to a role of actin polymerization in the expression of smooth muscle differentiation marker genes, in concert with serum response factor (SRF) and cofactors, such as myocardin. Stretch of intact venous smooth muscle activates Rho and inhibits the actin filament severing factor cofilin, resulting in increased actin polymerization. Concomitantly, the rates of synthesis of SRF-regulated differentiation markers, such as SM22alpha, calponin, and alpha-actin, are increased. This increase in differentiation signals is parallel with activation of the mitogen-activated protein (MAP) kinase pathway. Thus stretch-induced growth in a maintained contractile phenotype occurs by dual activation of signal pathways regulating both growth and differentiation. A current challenge is to identify sites of crosstalk between these pathways in intact smooth muscle tissue.     

Switching of actin isoforms in skeletal muscle differentiation using mouse ES cells

Among six actin isoforms, α-skeletal and α-cardiac actins have similar amino acid components and are highly conserved. Although skeletal muscles essentially express α-skeletal actins in the adult tissue, α-cardiac isoform actin is prominent in the embryonic muscle tissue. Switching of actin isoforms from α-cardiac to α-skeletal actin occurs during skeletal muscle differentiation. The cardiac type α-actin is expressed in the regeneration and patho-physiological states of the skeletal muscles as well. In the present study, we demonstrate the morphological switching of α-type actin isoforms from α-cardiac to α-skeletal actin in vitro using mouse ES cells for the first time. Immunofluorescent double staining with two specific antibodies revealed that α-cardiac actin appeared first in myoblasts. After cell fusion to form myotubes, the cardiac type actin decreased and α-skeletal actin conversely increased. Finally, the α-skeletal isoform remained as a main actin component in the fully mature skeletal muscle fibers. The exchange of isoforms is not directly linked to the sarcomere formation. As a result, ES cells provide a useful in vitro system for exploring skeletal muscle differentiation.     

The monoclonal antibody GB 42 — a useful marker for the differentiation of myofibroblasts

The expression patterns of a variety of cytoskeletal antigens were studied in normal human tissues (placenta, umbilical cord, myometrium, colon, mammary gland, testis, skeletal muscle, myocardium) as well as in abnormal human tissues (palmar fibromatosis, fibrocystic disease of the mammary gland, mammary carcinoma). The immunohistochemical binding patterns of the monoclonal antibody GB 42 were compared to those of commerical antibodies directed against vimentin, desmin, smooth muscle myosin, pan actin, -smooth muscle actin and -smooth muscle actin. Methods applied comprised immunohistochemistry on cryostat sections and paraffin sections. Immunogold immunocytochemistry was performed on Lowicryl sections. The patterns of GB 42-binding were confirmed biochemically by SDS-PAGE and Western-blotting, and quantitative amino acid analysis. Our data suggest that the monoclonal antibody GB 42 recognizes an actin isoform which is identical to, or closely related to, -smooth muscle actin. Unlike the commercially available antibody against -smooth muscle actin, GB 42 does not cross-react with -skeletal or -cardiac actins. The GB 42-antigen is expressed in smooth muscle cells, myoepithelial cells and in later stages of differentiation of myofibroblasts, in all the tissues investigated. Throughout the development of smooth muscle cells and myofibroblasts, the appearance of the GB 42-antigen occurs after the expression of vimentin, desmin and -smooth muscle actin, but prior to the expression of smooth muscle myosin. GB 42 is a reliable marker for higher stages of differentiation of smooth muscle cells and myofibroblasts.     

Renal leiomyoma: an immunohistochemical, ultrastructural and comparative genomic hybridization study

Renal leiomyoma is a rare neoplasm. We report such a case in a 57-year-old Japanese woman who was found to have a mass in the left kidney. The histological examination disclosed the proliferation of spindle cells showing a benign appearance. Entrapped tubular cells were observed in the peripheral area of the tumor. The immunohistochemical examination of spindle neoplastic cells showed a positive reaction for alpha smooth muscle actin, h-caldesmon, l-caldesmon, calponin, muscle actin, myosin and desmin. Additionally, the ultrastructural examination of the tumor showed membrane caveolae and myofilaments in the cytoplasm. This tumor was considered to show a differentiation into smooth muscle cells. The comparative genomic hybridization of the tumor detected the combined losses of chromosomes 4, 6, 12 and 14 which has not been previously described in renal tumors. Finally, the immunohistochemical panel of smooth muscle markers and ultrastructural and genetic study may be useful in diagnosing renal leiomyoma.     

Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-beta 1

Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with beta-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-beta1 secretion, alpha-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-beta1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-beta1 in response to activation by mast cell beta-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of alpha-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma- enteric isoform in differentiating striated myocytes

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha- vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.     

Smooth muscle-specific alpha tropomyosin is a marker of fully differentiated smooth muscle in lung.

Tropomyosin (Tm) is one of the major components of smooth muscle. Currently it is impossible to easily distinguish the two major smooth muscle (sm) forms of Tm at a protein level by immunohistochemistry due to lack of specific antibodies. Alpha-sm Tm contains a unique 2a exon not found in any other Tm. We have produced a polyclonal antibody to this exon that specifically detects alpha-sm Tm. We demonstrate here the utility of this antibody for the study of smooth muscle. The tissue distribution of alpha-sm Tm was shown to be highly specific to smooth muscle. Alpha-sm Tm showed an identical profile and tissue colocalization with alpha-sm actin both by Western blotting and immunohistochemistry. Using lung as a model organ system, we examined the developmental appearance of alpha-sm Tm in comparison to alpha-sm actin in both the mouse and human. Alpha-sm Tm is a late-onset protein, appearing much later than actin in both species. There were some differences in onset of appearance in vascular and airway smooth muscle with airway appearing earlier. Alpha-sm Tm can therefore be used as a good marker of mature differentiated smooth muscle cells. Along with alpha-sm actin and sm-myosin antibodies, alpha-sm Tm is a valuable tool for the study of smooth muscle.     

Smooth muscle actin expression during P19 embryonal carcinoma differentiation in cell culture

P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast-like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast-like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle-specific isoform of myosin heavy-chain mRNA in immortal lines of these fibroblast-like cells. These results suggest that these fibroblast-like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.     

Leiomyosarcomas: three cases with desmin positive tumour cells, lacking ultrastructural features of smooth muscle cells

A combined study of light and electron microscopy and of immunolabelling of three pleomorphic spindle cell sarcomas is presented. The light and electron microscopic features of these sarcomas were most compatible with those described for malignant fibrous histiocytoma (MFH, pleomorphic-storiform subtype). Electronmicroscopically undifferentiated and fibroblast-like cells, fibrohistiocytes and multinucleated histiocytes were observed. Characteristics belonging to smooth muscle cells were absent. By immunostaining, vimentin and desmin could be observed in tumour cells of all three cases, at least on frozen sections. Other markers such as alpha 1-antichymotrypsin, S-100 proteins, laminin, collagen IV and markers specific for skeletal muscle cells (myoglobin, actin and myosin specific for skeletal muscle) could not be demonstrated. These findings indicate that three MFH's are, in fact, poorly differentiated variants of smooth muscle tumours. It is concluded that immunophenotyping is very useful for this type of neoplasm.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Expression of smooth muscle markers in the developing murine lung: potential contractile properties and lineal descent

 Contractile cells in the mammalian lung develop in close association with the outgrowing stem bronchi. Fully differentiated smooth muscle cells are typically found in proximal regions, residing in the substantial muscular walls of the major airways and blood vessels. More distally, cells expressing markers of differentiated smooth muscle cells to a variable degree, and which may therefore possess contractile properties, are to be found scattered around the interstitium. We have investigated the temporal and spatial distribution of smooth muscle lineage markers (smooth muscle myosin mRNA) and of those indicative of contractile function (metavinculin mRNA) in the murine lung. In the smooth muscle layers of the bronchi and major blood vessels, these genes are expressed from the onset of pulmonary budding, concurrently with the appearance of α-smooth muscle actin and calponin proteins. During fetal development, smooth muscle-associated genes and proteins are restricted to this committed smooth muscle population. The first signs of myofibroblast or pericyte differentiation become manifest perinatally, when their expression of α-smooth muscle actin escalates. In the adult lung, such cells may be readily pin-pointed by their positive reaction for metavinculin mRNA, but, at maturity, they do not always coexpress α-smooth muscle actin. Accepted: 3 March 1998