Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation

A clone HT29-18 has been isolated from the parent cell line HT-29, which derived from a human colon adenocarcinoma (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141). This clone is able to differentiate as the parent cell line does. Differentiation occurs when glucose is replaced by galactose in the culture medium (Pinto, M., M.D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196). We demonstrate here that the differentiated cloned population HT29-18/gal is heterogenous: although 90% of the cells show morphological characteristics of "absorptive cells", only 20-30% of them display sucrase-isomaltase in their apical microvillar membranes. About 10% of the entire cell population consists of cells containing mucous granules similar to intestinal goblet cells. We have isolated two subclones, HT29-18-C1 and HT29-18-N2, from the differentiated HT29-18/gal cells. HT29-18-C1 cells show morphological characteristics of polarized absorptive cells, when growing either in glucose- or in galactose-containing media, but the sucrase-isomaltase is not expressed in the cells grown in glucose-containing medium. The clone HT29-18-N2 is also polarized in both culture conditions and is similar to globlet cells in vivo. It grows as a monolayer, exhibits tight junctions, and contains numerous mucous granules whose exocytosis can be triggered by carbachol, a parasympathomimetic drug. We conclude that the clone HT29-18 first isolated was a multipotent cell population from which we isolated several subclones that differentiate either as absorptive (HT29-18-C1) or as mucous (HT29-18-N2) cells. In contrast to the parent HT-29 cell line, the subclones retain most of their differentiated properties in glucose-containing medium.     

Mesenchymal maintenance of distal epithelial cell phenotype during late fetal lung development

Classical tissue recombination experiments have reported that at early gestation both tracheal and distal lung epithelium have the plasticity to respond to mesenchymal signals. Herein we examined the role of epithelial-mesenchymal interactions in maintaining epithelial differentiation at late (E19-E21, term = 22 days) fetal gestation in the rat. Isolated distal lung epithelial cells were recombined with mesenchymal cells from lung, skin, and intestine, and the homotypic or heterotypic recombinant cell aggregates were cultured for up to 5 days. Recombining lung epithelial cells with mesenchyme from various sources induced a morphological pattern that was specific to the type of inducing mesenchyme. In situ analysis of surfactant protein (SP)-C, SP-B, and Clara cell secretory protein (CCSP) expression, as well as SP-C and CCSP promoter transactivation experiments, revealed that distal lung epithelium requires lung mesenchyme to maintain the alveolar, but not bronchiolar, phenotype. Incubation of lung recombinants with an anti-FGF7 antibody resulted in a partial inhibition of mesenchyme-induced SP-C promoter transactivation. Immunoreactivity for Delta and Lunatic fringe, components of the Notch pathway that regulates cell differentiation, was downregulated in the heterotypic recombinants. In contrast, Hes1 mRNA expression was increased in these recombinants. Cumulatively, these results suggest that at late fetal gestation, distal lung epithelial cells are not fully committed to a specific phenotype and still have the plasticity to respond to various signals. Their alveolar phenotype is likely maintained by Notch/Notch ligand interactions and mesenchymal factors, including FGF7.     

Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos

Pigment cell precursors in the vegetal plate of late mesenchyme blastulae of the sea urchin Strongylocentrotus purpuratus begin to express a cell surface epitope recognized by the monoclonal antibody SP-1/20.3.1. When one-quarter gastrulae are dissociated into ectodermal and mesenchymal fractions, most SP-1/20.3.1 immunoreactive cells separate into the mesenchymal fraction, whereas at the full gastrula and all later stages almost all epitope-bearing cells are in the ectodermal fraction. Exposure of embryos to sulfate-free seawater p-nitrophenyl beta-D-xyloside, and tunicamycin, all of which prevent primary mesenchyme migration, does not inhibit SP-1/20.3.1 immunoreactive cells from distributing similarly to those in controls, although pigment synthesis is completely inhibited in sulfate-free conditions. Time-lapse video sequences reveal that pigment cells, and a small set of rapidly migrating, SP-1/20.3.1 immunoreactive amoeboid cells that appear in the pluteus, remain closely associated with the ectodermal epithelium during most of larval development. Transmission electron microscopy observations of plutei show pigment cells tightly apposed to the ectodermal epithelium at discontinuities in the basal lamina and sandwiched between the basal lamina and the epithelial cells. It is concluded that SP-1/20.3.1 immunoreactive mesenchymal cells invade the ectodermal epithelium and may use migratory substrates other than those used by primary mesenchymal cells.     

Pancreatic mesenchyme regulates epithelial organogenesis throughout development

The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation, and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor (DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly, while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally impaired. To further elucidate signals required for mesenchymal cell function, we eliminated β-catenin signaling and determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis. The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and survival by identifying β-catenin signaling as an essential mediator of this process. These results have implications for developing strategies to expand pancreas progenitors and β-cells for clinical transplantation.     

Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.     

AN ULTRASTRUCTURAL STUDY OF EARLY MORPHOGENETIC EVENTS DURING THE ESTABLISHMENT OF FETAL HEPATIC ERYTHROPOIESIS

Morphogenetic events are described which characterize early stages of the interaction between mesenchyme and expanding epithelial cell cords derived from the hepatic endodermal diverticulum in the C57BL/6J mouse. This interaction culminates in the differentiation of hepatic epithelial and hematopoietic tissues. No basement membrane separates the presumptive hepatic epithelial cells from the adjacent mesenchyme, while intercellular attachments, both adherent junctions and desmosomes, are established transiently between heterologous cell types across this epithelio-mesenchymal interface. Yolk sac-derived erythroblasts found in the primitive liver are distinguished morphologically from endogenous hepatic erythroid cells; they are confined to the vascular compartment and are not, apparently, precursors for hepatic erythropoiesis. The earliest recognizable endogenous hepatic hematopoietic cells appear, extravascularly, among those mesenchymal cells in intimate contact with the endodermal epithelium between the 10¼ and 10½ gestational day. Definitive erythropoiesis commences between the 10½ and 11th fetal days. The ultrastructure of these primitive hepatic erythroid cells (proerythroblasts) and their transition to more mature forms (basophilic and polychromatophilic erythroblasts) are described.     

Variations in cytotoxicity and isoenzyme patterns of uncloned and cloned cultures of axenic Entamoeba histolytica

Five clones of axenic Entamoeba histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. The clone HMI-C121 was more cytotoxic to the cultured Baby Hamster Kidney (BHK) cells while all other clones were significantly (P less than 0.001) less cytotoxic as compared to the cloned HMI-C121 and uncloned E. histolytica (HMI). The uncloned Indian axenic E. histolytica (KCG:0986:11) as well as E. histolytica (NIH:200) cultures were significantly (P less than 0.001) less cytotoxic to cultured BHK cells. No difference in the electromobility of maleate NADP oxidoreductase (ME) or glucophosphate isomerase (GPI) isoenzyme in the lysates of all the cloned and uncloned cultures of E. histolytica was observed. The clones HMI-C121, HMI-C131, HMI-G143 and HMI-C144 had three bands of hexokinase (HK) while all uncloned cultures and one of clones, HMI-C145 had only two bands. Though cloned and uncloned cultures had a single PGM band, the relative electromobility (rf) of phosphoglucomutase (PGM) for clone HMI-C131, HMI-C143 HMI-C144 was relatively less (rf 0.075) and these were also significantly (P less than 0.001) less cytotoxic to BHK cells as compared to clone HMI-C121. It is felt that axenic E. histolytica culture consists of several populations (clones) and expression of isoenzymes pattern or cytotoxic potentials would depend upon the population which predominantly multiples and outgrows other populations in the culture system.     

BEN/DM-GRASP/SC1 expression during mouse facial development: differential expression and regulation in molars and incisors

The cell adhesion molecule BEN/DM-GRASP/SC1 is expressed in a variety of tissues during embryogenesis. Here, we studied the expression pattern of BEN/DM-GRASP/SC1 in different organs involved in facial mouse development, especially in the developing teeth. BEN/DM-GRASP/SC1 was expressed in nose, whisker, gland, and tongue epithelia, as well as in myogenic mesenchyme. In molars, BEN/DM-GRASP/SC1 was firstly expressed in the condensed mesenchyme and thereafter expression was confined to mesenchymal cells of the dental follicle. In contrast, in incisors, transient BEN/DM-GRASP/SC1 expression was restricted to epithelium. In tissue recombination experiments, BEN/DM-GRASP/SC1 expression in mesenchyme was activated by molar, but not incisor epithelium.     

Establishment and characterization of hybrid rat mast cells

Rat peritoneal mast cells (RPMC) and rat basophilic leukemia (RBL) cells are representative of connective tissue-type (CTMC) and mucosal-type (MMC) mast cells, respectively. Using polyethylene glycol, we have fused RPMC with 6-thioguanine resistant, HAT (hypoxanthine, aminopterin, thymidine) sensitive RBL-CA10.7 or RBL-CK2 cells, yielding several hybrid rat mast cell lines (HRMC). The hybridomas exhibited different size and cytoplasmic granularity when compared with parental cell lines. Analysis of both high (Fc epsilon RI) and low affinity (Fc epsilon RL) receptors for IgE revealed that the hybrid lines had more variable receptor patterns than the parent lines. Three hybridoma lines were chosen for further study. Differential histochemical staining with alcian blue and safranin O dyes indicated the hybrids to be predominantly of the MMC type: however, a few cells of one of these uncloned hybridomas were found to be of the CTMC type. Attempts to isolate the CTMC hybridomas yielded one culture which was predominantly of the CTMC phenotype and in a number of other cultures, cells were found expressing simultaneously both the CTMC and the MMC phenotype. After 3 weeks in culture, however, all hybridomas, including those which were cloned further, expressed only the MMC histochemical phenotype. This was found to correlate with the presence of rat mast cell protease II (RMCPII) and the absence of RMCPI in all hybridomas, as detected by Western blot analysis. In addition, the histamine content of all cells was significantly lower than that of the parent RPMC. Most hybrid mast cells expressed both Fc epsilon RI and Fc epsilon RL which in some cases exhibited significant variations in the Mr. These results indicate that somatic cell hybrids expressing the MMC and CTMC phenotype can be produced by the fusion of RBL and RPMC. The CTMC phenotype, however, is unstable, and possible reasons for this are discussed.     

Epithelial Invagination: Adhesive Properties of Cells Can Govern Position and Directionality of Epithelial Folding

The aim of the present study was to investigate the mesenchymal influence on cultured epithelioid cells originating from an already differentiated intestine. Epithelioid cell cultures of 6-day-old suckling rat intestine were established by sequential trypsinizations of the mucosa. Embryonic intestinal monolayers of quail cells (13 days) were used as control because of their natural cell marker. Six to thirty days after plating, both types of epithelioid cells were associated in heterospecific combination with 5½-day-old chick embryonic small intestinal mesenchyme, after removal of the endoderm by collagenase treatment. In order to test the differentiation capabilities of the associations, they were grafted for 10–12 days into 3-day-old chick embryos. The results show that in such an in vivo culture system, the chimeric associations gave rise to well differentiated intestinal structures indicating that the epithelioid cell cultures derived from late embryonic or neonatal intestine will go through organotypic differentiation when recombined with an appropriate mesenchyme.     

Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model

Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem- or progenitor- cells for islet transplantation.     

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.     

DNA of avian myeloblastosis-associated virus type 2 integrates at multiple sites in the chicken genome.

The cellular sites of integration of the avian myeloblastosis-associated virus type 2 (MAV-2) DNA have been examined by Southern blot analysis of cellular DNA from infected cloned and uncloned chicken embryonic fibroblasts. Provirus-cell juncture fragments were not detected in restriction enzyme digests of DNA from MAV-2-infected uncloned cells. However, each MAV-2-infected cell clone examined produced a unique set of junctive bands. Thse findings indicate that multiple sites of integration exists for MAV-2 proviruses in cellular DNA.     

Canonical Wnt signaling regulates smooth muscle precursor development in the mouse ureter

Smooth muscle cells (SMCs) are a key component of many visceral organs, including the ureter, yet the molecular pathways that regulate their development from mesenchymal precursors are insufficiently understood. Here, we identified epithelial Wnt7b and Wnt9b as possible ligands of Fzd1-mediated β-catenin (Ctnnb1)-dependent (canonical) Wnt signaling in the adjacent undifferentiated ureteric mesenchyme. Mice with a conditional deletion of Ctnnb1 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional obstruction of the ureter. Histological analysis revealed that the layer of undifferentiated mesenchymal cells directly adjacent to the ureteric epithelium did not undergo characteristic cell shape changes, exhibited reduced proliferation and failed to differentiate into SMCs. Molecular markers for prospective SMCs were lost, whereas markers of the outer layer of the ureteric mesenchyme fated to become adventitial fibroblasts were expanded to the inner layer. Conditional misexpression of a stabilized form of Ctnnb1 in the prospective ureteric mesenchyme resulted in the formation of a large domain of cells that exhibited histological and molecular features of prospective SMCs and differentiated along this lineage. Our analysis suggests that Wnt signals from the ureteric epithelium pattern the ureteric mesenchyme in a radial fashion by suppressing adventitial fibroblast differentiation and initiating smooth muscle precursor development in the innermost layer of mesenchymal cells.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.     

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney

During kidney development and in response to inductive signals, the metanephric mesenchyme aggregates, becomes polarized, and generates much of the epithelia of the nephron. As such, the metanephric mesenchyme is a renal progenitor cell population that must be replenished as epithelial derivatives are continuously generated. The molecular mechanisms that maintain the undifferentiated state of the metanephric mesenchymal precursor cells have not yet been identified. In this paper, we report that functional inactivation of the homeobox gene Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia and depletion of the progenitor cell population within the metanephric mesenchyme. Failure to renew the mesenchymal cells results in severe renal hypoplasia. Gain of Six2 function in cortical metanephric mesenchymal cells was sufficient to prevent their epithelial differentiation in an organ culture assay. We propose that in the developing kidney, Six2 activity is required for maintaining the mesenchymal progenitor population in an undifferentiated state by opposing the inductive signals emanating from the ureteric bud.     

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations

To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.     

Endodermal Hedgehog signals modulate Notch pathway activity in the developing digestive tract mesenchyme

The digestive tract epithelium and its adjoining mesenchyme undergo coordinated patterning and growth during development. The signals they exchange in the process are not fully characterized but include ligands of the Hedgehog (Hh) family, which originate in the epithelium and are necessary for mesenchymal cells to expand in number and drive elongation of the developing gut tube. The Notch signaling pathway has known requirements in fetal and adult intestinal epithelial progenitors. We detected Notch pathway activity in the embryonic gut mesenchyme and used conditional knockout mice to study its function. Selective disruption of the Notch effector gene RBP-Jκ (Rbpj) in the mesenchyme caused progressive loss of subepithelial fibroblasts and abbreviated gut length, revealing an unexpected requirement in this compartment. Surprisingly, constitutive Notch activity also induced rapid mesenchymal cell loss and impaired organogenesis, probably resulting from increased cell death and suggesting the need for a delicate balance in Notch signaling. Because digestive tract anomalies in mouse embryos with excess Notch activity phenocopy the absence of Hh signaling, we postulated that endodermal Hh restrains mesenchymal Notch pathway activity. Indeed, Hh-deficient embryos showed Notch overactivity in their defective gut mesenchyme and exposure to recombinant sonic hedgehog could override Notch-induced death of cultured fetal gut mesenchymal cells. These results reveal unexpected interactions between prominent signals in gastrointestinal development and provide a coherent explanation for Hh requirements in mesenchymal cell survival and organ growth.     

Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis to carcinogenesis

The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme.