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Murine embryonic stem (ES) cells are maintained in an undifferentiated state when cultured in medium conditioned by Buffalo rat liver (BRL) cells. BRL conditioned medium (CM) contains a differentiation inhibitory activity (DIA) that is synonymous with leukemia inhibitory factor (LIF). ES cells in monolayer culture can be induced to differentiate by addition of all-trans retinoic acid (RA) to the BRL CM, when they mainly form cells resembling parietal endoderm, or by culture in medium not conditioned by BRL cells. ES cells thus deprived of LIF/DIA differentiate spontaneously to a cell type that expresses Brachyury (T), a marker of early mesoderm. Northern blot analyses have shown previously that transcripts for transforming growth factor beta 1 (TGF-beta 1) are detected in undifferentiated cells while transcripts for TGF-beta 2 and TGF-beta 3 only become detectable after differentiation. We have now determined levels of TGF-beta protein in CM and in the extracellular matrix (ECM) and have used neutralizing antibodies specific for TGF-beta 1 and TGF-beta 2 that do not react with recombinant human TGF-beta 3 to determine the isoform secreted. Using the growth inhibition of mink lung CCL64 cells as a bioassay for TGF-beta activity, we demonstrate that undifferentiated ES cells secrete latent TGF-beta 1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF-beta 2 in both active and latent forms in their CM. Likewise their ECM contains TGF-beta 2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF-beta 1 and TGF-beta 2 isoforms in their CM but TGF-beta-like activity remains after addition of neutralizing antibodies for TGF-beta 1 and TGF-beta 2. This active TGF beta is the major component of the TGF-beta activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF-beta 1 and beta 2 isoforms. The remaining activity in CM correlates with high expression of TGF-beta 3 by Northern blot analysis in these cells. We speculate that TGF-beta 3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF-beta 1 and TGF-beta 2, since it is present in CM only in its active form.

In this article we describe some of the fundamental processes occurring during early murine development, introduce cellular models used to investigate these processes and review some well-known factors that may be involved in their control. These include transforming growth factor beta, retinoic acid and leukaemia inhibitory factor. Refinements to the culture conditions of embryonic stem and embryonal carcinoma cells have enabled us to test the effects of these factors on growth and differentiation and in particular to establish that their interaction may determine the ultimate developmental state of the cell population. Preliminary studies using neutralizing antibodies in embryos are described that suggest that deregulation of normal expression can lead to a failure to implant. Insights into the events underlying normal embryonic development and implantation, yielded by the type of study described here, may contribute to an understanding of the mechanisms causing early embryonic loss and the role of toxicants in this process.

In a search for functions of transforming growth factor-beta during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation proceeds further when the embryoid bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-beta family (TGF-beta 1, TGF-beta 2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10(-8) M and 10(-7) M induced the formation of neurons but in the absence of RA or at concentrations up to 10(-9) M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10(-9) M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10(-9) M RA. TGF-beta s increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-beta isoform added and the muscle cell type formed. TGF-beta 2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-beta s on the formation of mononucleate flat cells was completely abrogated by RA at 10(-9) M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-beta isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-beta 2 were injected into the cavity of mouse blastocysts 3.5 days post coîtum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5-9.5 pc.(ABSTRACT TRUNCATED AT 400 WORDS)

To date, three mammalian TGF-beta isoforms have been identified, each encoded by different genetic loci. Through each is very similar in primary amino acid structure, there are clear differences both in the mature bioactive peptide region and in the latency-associated peptide, which could potentially confer differential biological specificity. As one route to investigate differential biological function in vivo we have used gene specific probes for in situ hybridization studies to examine the distribution of RNA transcripts during mammalian embryogenesis. Mouse embryos from 6 to 14.5 gestational age and human embryos from 32 to 57 days post-fertilization have been probed. A general conclusion from these studies is that each TGF-beta gene has a distinct, through overlapping, pattern of transcript distribution and that this pattern, in most cases, is conserved between mouse and man. We have focused on the biological function the TGF-betas play in certain epithelia and in cardiogenesis, which will be discussed in this presentation.

A monoclonal antibody (3D10) recognizing myosin heavy chain was isolated following immunization with a synthetic peptide sequence of eight amino acids. The antibody reacted with purified rabbit skeletal myosin and light mero-myosin in enzyme-linked immunosorbent assays and Western immunoblotting. A band of approximately 200 kDa was detected in cell extracts of an embryonal carcinoma (EC) cell line (P19EC) and one of its cloned differentiated derivatives, suggesting reactivity against non-muscle myosin. By indirect immunofluorescence, typical myosin banding patterns were observed in cryostat sections of human skeletal and cardiac muscle tissue. In undifferentiated P19EC cells, speckled immunofluorescent staining was observed in the cytoplasm that became organized in cortical rings where the cells made direct contact with each other. These rings consisted of circular bundles of F-actin decorated by myosin. Undifferentiated embryonic stem (ES) cells derived directly from mouse embryos shared the same features, although the pattern was less pronounced. Human testicular primary germ cell tumours showed cortical staining in the embryonal carcinoma component reminiscent of the staining of EC cells in vitro while cytoplasmic staining was observed in tumour cells with a differentiated morphology. In preimplantation embryos, the immunofluorescent staining was observed at cell apices of blastomeres of morula stage embryos. In blastocysts, staining of inner cell mass cells was not detectable. By contrast, various differentiated derivatives of P19EC contained extensive F-actin microfilament bundles throughout the cytoplasm decorated with myosin. Thick stress fibers in filopodious extensions of cells were particularly highly decorated by myosin. Over the nucleus, linear arrays of myosin containing speckled patterns of immunofluorescence were observed that were not associated with F-actin. The same pattern of staining could be observed in trophectoderm cells of the blastocyst. We conclude that embryonic non-muscle myosin is organized in specific patterns depending on the state of differentiation. As the myosin is primarily associated with F-actin we suspect that it forms part of a contractile apparatus that may have significance during embryonic development.

The localization of transforming growth factor type beta 2 (TGF-beta 2) has been followed during preimplantation and early postimplantation murine development using an anti-peptide antibody that specifically recognizes TGF-beta 2. The staining pattern showed that TGF-beta 2 is expressed from the four-cell stage onward and is differentially regulated as cells diverge to various lineages. High levels of staining were found in the trophectoderm of the blastocyst but no staining was observed in the inner cell mass. During postimplantation development the primitive and embryonic ectoderm also lacked detectable staining while visceral endoderm stained well. Parietal endoderm cells also showed positive staining reaction although to a lesser extent than visceral endoderm cells. These findings were confirmed in model systems of the embryo, namely, embryonal carcinoma and embryonic stem cells differentiated to to cells with either visceral or parietal endoderm characteristics. The possible regulatory role of this factor in early embryogenesis is discussed.

An antiserum was prepared against a synthetic peptide corresponding to the first 29 N-terminal amino acid residues of transforming growth factor beta type 2 (TGF beta 2) from porcine platelets. The anti-TGF beta 2 peptide antiserum appeared to be completely specific for TGF beta 2 in several immunological assays, including enzyme-linked immunosorbent assays, immunoblotting and immunofluorescence experiments. Furthermore, this antiserum completely neutralized the growth inhibitory effect of TGF beta 2 on mink lung carcinoma (ML-CC164) cells and the transforming capacity of this factor on quiescent monolayers of NRK cells in the presence of epidermal growth factor. These data indicate that the N-terminal region of TGF beta 2 may be involved in the biological activity of this growth factor. TGF beta 1 was not recognized by the anti-TGF beta 2 peptide antiserum. The specificity of the anti-TGF beta 2 peptide antiserum for TGF beta 2 appeared to be useful in identifying TGF beta 2 produced by different cell systems and will be helpful in determining possible functional differences between TGF beta 1 and TGF beta 2.

Transforming growth factor beta 2 (TGF beta 2) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF beta 2 transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, P10, and ES. Following induction of differentiation, however, TGF beta 2 became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF beta 2 transcripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF beta 2 did not stain P19 EC cells by immunofluorescence but 2-3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF beta 2, while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF beta 2 after plating on a substrate. Neither TGF beta 1 nor TGF beta 2 affected the growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF beta 2 being marginally more effective than TGF beta 1 at the same concentration.

This paper describes the initial immunochemical characterization of specific macrophage-arming factor (SMAF). SMAF is an antigen-specific factor that is released by (sensitized) T lymphocytes after contact with the specific antigen. It renders macrophages specifically cytotoxic. The specificity is dependent on the tumor-mouse combination. In allogeneic systems the specificity is H-2-directed, whereas in the syngeneic systems the specificity is tumor-specific. SMAF has a molecular mass of 65-85 kDa (established by gel filtration). By affinity chromatography SMAF could not be adsorbed with anti-(kappa + lambda light chain) immunoglobulins or anti-IgG from SMAF-containing supernatants. SMAF could be adsorbed with the monoclonal antibody 14-30 (directed against specific T-cell factors), and could be eluted from columns containing the latter. Furthermore, SMAF could also be adsorbed with and eluted from affinity chromatography columns to which specific tumor cell membranes or KCl extracts of these tumor cell membranes were coupled. Other tumor cell membranes could not adsorb SMAF. Together these data show that SMAF is not an antibody but a T-cell factor with an antigen-specific recognition site.