Smad expression during kidney development.

Signaling by the transforming growth factor (TGF)-beta superfamily is important during kidney development. Here, we describe the spatial and temporal expression patterns of the Smads, the transcription factors that translate TGF- signals into gene expression. RT-PCR data and in situ hybridization analysis showed that the receptor-regulated (R) Smads (Smad1, -2, -3, -5, and -8), the common partner Smad (Smad4), and the inhibitory (I) Smads (Smad6 and -7) were all expressed during mouse kidney development from embryonic day 12 until the end of nephrogenesis at postnatal day 15. Each Smad had a distinct spatial distribution. All were expressed by mesenchymal cells in the nephrogenic zone and were downregulated once these cells began to epithelialize. The common partner Smad, Smad4, was present in uninduced mesenchymal cells and at ureteric bud tips. The bone morphogenetic-responsive R-Smads, Smad1, -5, and -8, were mainly expressed in the nephrogenic zone, whereas the TGF-- responsive R-Smads were predominantly noted in the medullary interstitium. Expression of the I-Smad Smad7 was also seen in mesenchymal cells in the interstitium. Based on the observed patterns of expression, we speculate that individual or combinations of Smads may play specific roles in cell-fate determination during kidney development.

The human adult cardiomyocyte phenotype.

AIM: Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology. METHODS: Atrial and ventricular cardiomyocytes were obtained from patients undergoing cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. RESULTS: The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions. CONCLUSION: Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.

Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice.

Deletion of the transforming growth factor beta1 (TGF-beta1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF-beta more precisely, we inactivated the TGF-beta type I receptor (TbetaRI) gene by gene targeting. Mice lacking TbetaRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TbetaRI(-/-) yolk sacs, clonogenic assays on yolk sac-derived hematopoietic precursors in vitro revealed that TbetaRI(-/-) mice exhibit normal hematopoietic potential compared with wild-type and heterozygous siblings. Endothelial cells derived from TbetaRI-deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TbetaRI is crucial for the function of TGF-beta during vascular development and can not be compensated for by the activin receptor-like kinase-1 (ALK-1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF-beta signaling via TbetaRI.

Expression of TGF-beta stimulated clone-22 (TSC-22) in mouse development and TGF-beta signalling.

TSC-22 is a highly conserved member of a novel family of transcription factors, that is a direct target of transforming growth factor-beta (TGF-beta) in osteoblastic cells. We have investigated the expression of TSC-22 in detail during mouse development using in situ hybridization. We detected strong expression of TSC-22 in the embryo proper first at embryonic day 8.5 (E8.5), in the primitive heart, intermediate mesoderm and the neural tube. The dynamics of the TSC-22 distribution in the neural tube was particularly striking, with ubiquitous expression rostrally and restriction to neural tissue nearer the floor plate more caudally; between E8.5 and E9.5 the zone of restricted expression extended rostrally. At later stages of development, TSC-22 was detected in the mesenchymal compartment of many tissues and organs, including the lung, trachea, kidney, stomach, intestine, tooth buds, and in precartilage condensations. Furthermore, TSC-22 was highly expressed in the floor plate itself and notochord, and the endothelium lining the blood vessels, in particular the major arteries. Many of these sites have been proposed previously as possible TGF-beta target tissues; the results imply that TSC-22 may also be a direct TGF-beta target gene during mouse embryogenesis. Experiments on TSC-22 expression in embryoid bodies of embryonic stem (ES) cells expressing dominant negative TGF-beta binding receptors initially supported this hypothesis. However, examination of somatic chimeras derived from these same mutant ES cells at nominal E9.5 showed that TSC-22 expression in the heart and neural tube was still detectable despite obvious phenotypic abnormalities. We therefore propose that although TSC-22 may be a direct target of TGF-beta in late development, other factors are likely to be major regulators of expression at earlier stages.

Expression of the inhibitory Smad7 in early mouse development and upregulation during embryonic vasculogenesis.

SMAD proteins are downstream targets of serine/threonine kinase receptors of the transforming growth factor beta (TGF beta) superfamily. Ligands activating these receptors regulate cell growth, differentiation and development in many tissues of various organisms. In mammals eight different Smad genes are known, each with different roles in mediating signalling between plasma membrane and nucleus. Smad6 and Smad7 are inhibitors of TGF beta family signalling. They are both expressed in human adult vascular endothelial cells, particularly after these cells have been subjected to shear stress (Topper et al. [1997] Proc Natl Acad Sci USA 94:9314-9319). Here we show by reverse transcriptase polymerase chain reaction and in situ hybridization that Smad7 mRNA is highly expressed in the developing vascular system of the mouse embryo but is also detectable much earlier in preimplantation embryos and during gastrulation. We also demonstrate by transient transgenesis that overexpression of Smad7 in mouse zygotes inhibits development beyond the 2-cell stage. This confirms earlier conclusions of similar, but complementary, experiments using a dominant negative type II TGF beta receptor demonstrating that TGF beta signalling is required for normal preimplantation development.

Snail is an immediate early target gene of parathyroid hormone related peptide signaling in parietal endoderm formation.

In mouse development, parietal endoderm (PE) is formed from both primitive endoderm (PrE) and visceral endoderm (VE). This process can be mimicked in vitro by using F9 embryonal carcinoma cells (EC) cells, differentiated to PrE or VE cells, and treating these with Parathyroid Hormone related Peptide (PTHrP). By means of differential display RT-PCR, we identified Snail (Sna) as a gene upregulated during the differentiation from F9 PrE to PE. We show that Sna is an immediate early target gene of PTHrP action in the formation of F9 PE cells. Using RT-PCR, we detected Sna transcripts in pre-implantation mouse embryos from the zygote-stage onwards. Sna was strongly upregulated in parallel with type 1 PTH/PTHrP Receptor (PTH(rP)-R1) mRNA in mouse blastocysts plated in culture, concomitant with detection of the PE-marker Follistatin and appearance of PE cells. By radioactive in situ hybridization on sections of mouse embryos, we found Sna expression in the earliest PE cells at E5.5. Sna remained expressed until at least E7.5. At this stage, we also observed clear expression in endoderm cells delaminating from the epithelial sheet of VE cells in the marginal zone. We conclude that PTH(rP)-R1 and Sna are expressed in endodermal cells that change from an epithelial to a mesenchymal phenotype. Since Sna expression has been described at other sites where epithelio-mesenchymal transitions (EMT) occur, such as the primitive streak at gastrulation and in pre-migratory neural crest cells, we hypothesize that Sna is instrumental in the action of PTHrP inducing PE formation, which we propose to be the first EMT in mouse development.

Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice.

We have analysed the function of transforming growth factor beta (TGF-beta) in yolk sac development in mice by generating somatic chimaeras in which the extraembryonic mesoderm, which gives rise to the endothelial and haematopoietic cells of the yolk sac vasculature, is derived from embryonic stem (ES) cells. The ES cells were stably transfected and express either the full-length type II binding receptor or a kinase-deficient mutant of this receptor. Examination of yolk sacs from chimaeras between E8.5 and 9.5, and analysis of marker expression in embryoid bodies from these mutant ES cell lines in prolonged suspension culture demonstrated that (1) a major function of TGF-beta in yolk sac mesoderm is to regulate production and deposition of fibronectin in the extracellular matrix that maintains yolk sac integrity, (2) TGF-beta signalling is not required for differentiation of extraembryonic mesoderm into endothelial cells but is necessary for their subsequent organisation into robust vessels, and (3) TGF-beta signalling must be tightly regulated for the differentiation of primitive haematopoietic cells to take place normally. Together, these results show that defective TGF-beta signalling in the extraembryonic mesoderm alone is sufficient to account for the extraembryonic phenotype reported previously in TGF-beta1(-/-) mice (Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995) Development 121, 1845-1854).

Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1.

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcription-polymerase chain reaction in immature oocytes and in in vitro-produced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

Ectopic expression of the transforming growth factor beta type II receptor disrupts mesoderm organisation during mouse gastrulation.

Transforming growth factor beta (TGFbeta) regulates the cell cycle and extracellular matrix (ECM) deposition of many cells in vitro. We have analysed chimaeric mouse embryos generated from embryonic stem cells with abnormal receptor expression to study the effect of TGFbeta on these processes in vivo and the consequences for normal development. The binding receptor for TGFbeta, TbetaRII, is first detected in the embryo proper around day 8.5 in the heart. Ectopic expression of TbetaRII from the blastocyst stage onward resulted in an embryonic lethal around 9.5 dpc. Analysis of earlier stages revealed that the primitive streak of TbetaRII chimaeras failed to elongate. Furthermore, although cells passed through the streak and initially formed mesoderm, they tended to accumulate within the streak. These defects temporally and spatially paralleled the expression of the TGFbeta type I receptor, which is first expressed in the node and primitive streak. We present evidence that classical TGFbeta-induced growth inhibition was probably the cause of insufficient mesoderm being available for paraxial and axial structures. The results demonstrate that (1) TGFbeta mRNA and protein detected previously in early postimplantation embryos is present as a biologically active ligand; and (2) assuming that ectopic expression of TbetaRII results in no other changes in ES cells, the absence of TbetaRII is the principle reason why the embryo proper is unresponsive to TGFbeta ligand until after gastrulation.

Molecular cloning, genetic mapping, and developmental expression of a bovine transforming growth factor beta (TGF-beta) type I receptor.

A full-length cDNA encoding the bovine transforming growth factor beta (TGF-beta) receptor type I (bT beta R-I) was isolated from a placenta cDNA library. The deduced protein sequence of 499 residues contains a single transmembrane domain, a cysteinerich extracellular domain, and an intracellular kinase domain with predicted serine/threonine specificity. The amino acid sequence is 96% and 95% identical with its human and mouse homologues, respectively. Genetic mapping assigned the TGFBR1 gene to bovine chromosome 8 at a male genetic distance of 2 centimorgan from D8S28. Assuming conservation of gene order, the linkage data define a breakpoint in mammalian chromosome evolution. Both TGF-beta receptor type I and II mRNAs were found to be expressed in bovine oocytes and preimplantation two-cell, four-cell, eight-cell, morula-, and blastocyst-stage embryos, as determined by heminested reverse transcription polymerase chain reaction (RT-PCR). The mRNA expression patterns of TGF-beta receptor types I, II, and III in a variety of bovine organ tissues were examined by Northern blot hybridization, and highest levels were detected in lung and ovary.

Differential expression of BMP receptors in early mouse development.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of polypeptide signaling molecules. They function via binding to two types of transmembrane serine/threonine kinase receptors, type I and type II receptors, that are both necessary for signaling. The expression patterns of the type II BMP receptor (BMPR-II) and three type I BMP receptors (ActR-I, BMPR-IA and BMPR-IB) were examined in preimplantation embryos by means of heminested reverse transcription-polymerase chain reaction (RT-PCR). BMPR-II mRNA was detected in one-cell, two-cell and blastocyst stage embryos. ActR-I exhibited a similar expression pattern. BMPR-IA mRNA however was only detected in blastocysts, whereas BMPR-IB transcripts were detected at all stages from the one-cell zygote to the uncompacted morula, but not in the compacted morula and blastocyst. If translated into proteins, this suggests that different receptor complexes can be formed at different developmental stages. Transcripts for BMPs were not detected in preimplantation embryos, but were detected in the maternal tissues surrounding the embryos. BMPR-II, BMPR-IA and BMPR-IB mRNAs were also detected in undifferentiated and differentiated embryonal carcinoma and embryonic stem cells. In postimplantation embryos BMPR-II transcripts were first detected from 6.0 days post coitum. In situ hybridization analysis revealed that BMPR-II mRNA is ubiquitously expressed in the entire embryo at least until midgestation.

Expression of ALK-1, a type 1 serine/threonine kinase receptor, coincides with sites of vasculogenesis and angiogenesis in early mouse development.

ALK-1 is a type I serine/threonine kinase receptor for members of the TGF-beta superfamily of growth factors; its endogenous ligand is not known. In this study, we have analyzed the temporal and spatial expression pattern of ALK-1 mRNA in mouse embryos from the one-cell zygote until 12.5 dpc using RT-PCR and in situ hybridization. ALK-1 mRNA was first detected in the embryo at 6.5 dpc. From 7.5-8.5 dpc expression was highest at sites of vasculogenesis in both the embryonic and extraembryonic part of the conceptus, in trophoblast giant cells, and in the endothelial lining of the blood vessels in the decidua. From 9.5-12.5 dpc, ALK-1 was found to be expressed in several different tissues and organs, but was highest in blood vessels, mesenchyme of the lung, submucosal layer of the stomach and intestines, and at specific sites of epithelial-mesenchymal interactions. Its expression pattern suggests that ALK-1 is a type I receptor for TGF-beta1 in the developing mouse.

Aberrant platelet-derived growth factor alpha-receptor transcript as a diagnostic marker for early human germ cell tumors of the adult testis.

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.

Human teratocarcinoma cells express functional insulin-like growth factor I receptors.

Using iodinated insulin-like growth factors (IGFs) we have detected receptors for IGF-I at the cell surface of the clonally derived human embryonal carcinoma cell line Tera 2 clone 13. Affinity crosslinking of IGFs to Tera 2 clone 13-derived membrane preparations revealed the presence of proteins with features of both type-I and type-II IGF receptors. Treatment of Tera 2 clone 13 cells with retinoic acid to induce differentiation results in an increased number of cell surface receptors, apparently without altering the ratio of type-I and type-II receptors. In addition, Tera 2 clone 13 IGF-I receptors catalyze (auto)phosphorylation at tyrosine upon IGF-I and insulin binding. These findings suggest that type-I IGF receptors might be involved in mediating the effects of IGFs and insulin upon the proliferation of Tera 2 clone 13 cells.

Transforming growth factor-beta and its receptor are differentially regulated in human embryonal carcinoma cells.

The human embryonal carcinoma cell lines Tera-2 clone 13 and NTera-2 clone D1 can be induced by retinoic acid to differentiate in vitro into neuroectodermal derivatives. The undifferentiated cells are rapidly proliferating and tumorigenic, whereas retinoic-acid-treated cells possess a decreased growth rate, lose their transformed phenotype and show a finite lifespan. Differentiation is accompanied by a marked increase in the levels of mRNA for TGF-beta 1 and TGF-beta 2 and the production of TGF-beta activity. Just like murine embryonal carcinoma cells the growth of Tera-2 clone 13 cells is not affected by the addition of either TGF-beta 1 or TGF-beta 2 to the culture medium. In contrast to published data on murine embryonal carcinoma cells, Tera-2 clone 13 and NTera-2 clone D1 cells bind TGF-beta 1 with high affinity, which is due to the presence of type-III TGF-beta receptors. Furthermore, and again in contrast to murine embryonal carcinoma cells, treatment of the human embryonal carcinoma cells with retinoic acid causes a nearly complete loss of TGF-beta 1 binding sites. These results are discussed in the light of similarities and differences in the regulation of growth and differentiation of human and murine embryonal carcinoma cell lines.

Differentially regulated production of platelet-derived growth factor and of transforming growth factor beta by a human teratocarcinoma cell line.

The human teratocarcinoma stem cell line Tera-2 clone 13 is induced by retinoic acid to differentiate in vitro into endodermal or neuroectodermal cell types. In the absence of externally added growth factors, Tera-2 clone 13 cells proliferated at the same rate as in the presence of serum growth factors. Analysis of serum-free medium conditioned by Tera-2 clone 13 cells showed the presence of a polypeptide immunologically and biochemically related to platelet-derived growth factor (PDGF). In addition transforming growth factor beta (TGF-beta), but no TGF-alpha production could be detected. Tera-2 clone 13 cells specifically expressed high levels of the A-chain mRNA, but not the B-chain mRNA of PDGF. During retinoic acid induced differentiation the level of A-chain mRNA became markedly reduced. In contrast the TGF-beta mRNA levels increased significantly upon differentiation. The implications of these findings are discussed in terms of regulation of growth and differentiation in early embryos as well as in (human) teratocarcinomas.

Cell cycle analysis during retinoic acid induced differentiation of a human embryonal carcinoma-derived cell line.

Several subclones of the human embryonal carcinoma (EC) cell line Tera-2 can be induced to differentiate in monolayer culture by retinoic acid (RA) to a flattened cell type with reduced growth rate. Using a method based on the transition probability model, we have analysed changes in cell cycle kinetics of Tera-2 cells during the differentiation process. Growth inhibition was shown to occur without a lag period and to be partly due to an increase in the duration of the S-phase, but with a relatively greater contribution from an increase in the duration of G1-phase. Since the fraction of the cell population in the G1-phase then doubled, cells accumulated in this part of the cycle. In contrast, the reduced proliferation rate of two murine EC cell lines, PC13 and P19, treated with RA occurs after a lag period of about two cell cycles and is mainly attributable to an increase in the duration of the S-phase. The results illustrate a differential response of human and murine EC cells to growth regulation by RA and again emphasize that although the stem cells of murine teratocarcinomas may provide a useful model, they are not identical to their human counterparts.

Production of transforming growth factors by simian sarcoma virus-transformed cells.

Cellular transformation of normal rat kidney (NRK) cells by simian sarcoma virus (SSV) results in a complete loss of the cellular requirement of externally added polypeptide growth factors for proliferation. Moreover, SSV-transformed NRK cells have a strongly reduced ability to bind both external platelet-derived growth factor and epidermal growth factor, when compared to nontransformed NRK cells. Analysis of serum-free medium conditioned by SSV-transformed NRK cells shows that this cell line secretes both types alpha and beta transforming growth factor (TGF). The level of TGF alpha production (300 ng/liter conditioned medium) by SSV-transformed NRK is among the highest described to date. Since addition of TGF alpha and beta in combination is sufficient to induce phenotypic transformation of NRK cells, it is concluded that although expression of the sis oncogene is essential for transformation, expression of additional genes may be required for the phenotypic alterations accompanying complete cellular transformation.