Angiotensin (AT) II, the bioactive octapeptide in the renin-angiotensin system that plays a key role in cardiovascular homeostasis, exerts its multiple effects through the different types of AT receptors, AT1a, AT1b, and AT2. Previously, we showed chronic hypotension in angiotensinogen (the precursor of AT)-deficient mice and a dramatic increase in renin mRNA levels in its kidney, but it remains unclear which types of AT receptors regulate the blood pressure and renin gene expression. In order to elucidate the physiological roles of AT1a receptor, we generated mutant mice with a targeted replacement of the AT1a receptor loci by the lacZ gene. In the heterozygous mutant mice, the strong lacZ staining was found in the glomerulus and juxtaglomerular apparatus of the renal cortex, which coincided with that of the signals detected by in situ hybridization. Chronic hypotension was observed in the heterozygous and homozygous mutant mice, with 10 and 22 mm Hg lower systolic blood pressure, respectively, than that of wild-type littermates. Both levels of renin mRNA in the kidney and plasma renin activity were markedly increased only in the homozygous mutant mice. These results demonstrated that an AT1a-mediated signal transduction pathway is, at least in part, involved in the regulation of blood pressure and renin gene expression.

Endothelial-mesenchymal transformation (EMT) is a critical event in the generation of the endocardial cushion, the primordia of the valves and septa of the adult heart. This embryonic phenomenon occurs in the outflow tract (OT) and atrioventricular (AV) canal of the embryonic heart in a spatiotemporally restricted manner, and is initiated by putative myocardially derived inductive signals (adherons) which are transferred to the endocardium across the cardiac jelly. Abnormal development of endocardial cushion tissue is linked to many congenital heart diseases. At the onset of EMT in chick cardiogenesis, transforming growth factor (TGFbeta)-3 is expressed in transforming endothelial and invading mesenchymal cells, while bone morphogenetic protein (BMP)-2 is expressed in the subjacent myocardium. Three-dimensional collagen gel culture experiments of the AV endocardium show that 1) myocardially derived inductive signals upregulate the expression of AV endothelial TGFbeta3 at the onset of EMT, 2) TGFbeta3 needs to be expressed by these endothelial cells to trigger the initial phenotypic changes of EMT, and 3) myocardial BMP2 acts synergistically with TGFbeta3 in the initiation of EMT.

Corresponding author A.Okuda and A.Fukushima contributed equally to this workWe have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.

Transforming growth factor-β (TGFβ) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGFβ as a large latent TGFβ complex containing mature TGFβ, latency associated peptide, and latent TGFβ-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGFβ protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGFβ1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40–100 nm extracellular fibrillar structure and 5–10-nm microfibrils. The anti–LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGFβ proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGFβ as a large latent TGFβ complex.

The POU transcription factor Oct-3/4 has been shown to be critical for maintaining embryonic stem (ES) cell character. However, the molecular mechanisms underlying its function remain elusive. We have previously shown that among the POU transcription factor family of proteins, Oct-3/4 alone is able to bind to the regulatory region of the UTF1 gene bearing a variant octamer sequence together with Sox-2. Here, we demonstrate using Oct-3/4-Oct-6 chimeras that there is a precise correlation between the ability of proteins to form a complex on the UTF1 enhancer with Sox-2 and the ability to maintain the stem cell state in ES cells. Different chimeric proteins show differential abilities to form a Sox-2-containing complex on the UTF1 regulatory region, with a decrease in efficiency of the complex formation accompanied by a decrease in the level of UTF1 expression and the rate of cell proliferation. Overexpression of UTF1 in these slow-growing cells was able to restore their proliferation rate to wild-type levels. Moreover, UTF1 was also observed to have an effect on teratoma formation. These results suggest a molecular pathway by which Oct-3/4 induces rapid proliferation and tumorigenic properties of ES cells through activation of the UTF1 gene.

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Transformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1510-1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFbeta3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFbeta3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFbeta3 function in atrioventricular canal mesenchyme formation. In situ hybridization and immunohistochemistry showed little or no TGFbeta3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFbeta3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFbeta3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFbeta3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFbeta3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFbeta2 protein, either alone or in combination with TGFbeta3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFbeta3 in response to a myocardially derived signal other than TGFbeta3, (ii) atrioventricular canal endocardial TGFbeta3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFbeta3 alone or in combination with TGFbeta2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture.

During early cardiac morphogenesis, outflow tract (OT) and atrio‐ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle‐like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor β3 (TGFβ3) is an essential element in the progression of endothelial‐transformation into mesenchyme. TGFβs are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle‐like cells. Using a monoclonal antibody against smooth muscle‐specific alpha‐actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGFβ3 initiates the expression of SMA, the pre‐migratory AV endothelial monolayer was cultured with or without chicken recombinant TGFβ3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre‐migratory endothelial cells cultured in complete medium (CM199) that contained TGFβ3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial‐mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGFβ3. The induction of the expression of SMA by TGFβ3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial‐mesenchymal transformation. Dev. Dyn. 209:296–309, 1997. © 1997 Wiley‐Liss, Inc.