Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor)

Summary Recently the mesoderm-inducing effects of the transforming growth factor (TGF-) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF- family has mesoderm-inducing ability.     

A mesoderm-inducing factor produced by WEHI-3 murine myelomonocytic leukemia cells is activin A

The first inductive interaction in amphibian development is mesoderm induction, during which a signal from the vegetal hemisphere of the blastula-staged embryo induces mesoderm from overlying equatorial cells. Recently, a number of 'mesoderm-inducing factors' (MIFs), which may be responsible for this interaction, have been discovered. Examples of these MIFs include members of the fibroblast growth factor family as well as members of the TGF-beta superfamily such as TGF-beta 2. In addition to these purified factors, several new sources of mesoderm-inducing activity have been described. One of the most potent of these is the murine myelomonocytic leukemia cell line WEHI-3. Even at high dilutions, conditioned medium from WEHI-3 cells induces isolated Xenopus animal pole regions to form a variety of mesodermal cell types. In this paper we show by several criteria, including N-terminal amino acid sequencing, Northern blotting and various functional assays, that the WEHI-MIF is activin A. Activins are known to modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and to cause the differentiation of two erythroleukemia cell lines. Our results, along with recent data from other laboratories, indicate that these molecules may also act in early development in the formation of the mesoderm.     

Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.     

Concentration-dependent inducing activity of activin A

Summary Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3–50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development. Offprint requests to: M. Asashima     

Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos

Mesoderm of early vertebrate embryos gradually acquires dorsal–ventral polarity during embryogenesis. This specification of mesoderm is thought to be regulated by several polypeptide growth factors. Bone morphogenetic protein (BMP), a member of the TGF-β family, is one of the regulators suggested to be involved in the formation of ventral mesoderm. In this paper, the nature of the endogenous BMP signal in dorsal–ventral specification was assessed in early Xenopus embryos using a dominant negative mutant of the Xenopus BMP receptor. In ectodermal explant assays, disruption of endogenous BMP signaling by the mutant receptor changed the competence of the explant cells to mesoderm-inducing factors, activin and basic fibroblast growth factor (bFGF), and led to formation of neural tissue without mesoderm induction. This result suggests that endogenous BMP acts as a ventral mesoderm modifier rather than a ventral mesoderm inducer, and that interactions between endogenous BMP and mesoderm-inducing factors may be important in dorsal–ventral patterning of embryonic mesoderm. In addition, the induction of neural tissue by inhibition of the BMP signaling pathway also suggests involvement of BMP in neural induction.     

A mesoderm-inducing factor is produced by Xenopus cell line

Inductive interactions play a major role in the diversification of cell types during vertebrate development. These interactions have been extensively studied in amphibian embryos (usually Xenopus laevis) where the earliest is mesoderm induction, in which an equatorial mesodermal rudiment is induced from the animal hemisphere under the influence of signal from the vegetal hemisphere. The molecular basis of mesoderm induction is unknown, although Tiedemann has isolated a protein form 9- to 13-day chick embryos that has the properties one would expect of a mesoderm-inducing factor. However, the relevance of this molecule to the events of early amphibian development is unclear, and it is a matter of some importance to discover a Xenopus mesoderm-inducing factor. In this paper I show that the Xenopus XTC cell line secretes mesoderm-inducing activity into the culture medium. Isolated animal pole regions cultured in XTC-conditioned medium differentiate into muscle and notochord, while controls form 'atypical epidermis'. Three different cell lines -XL, XL177 and KR- secrete no such activity indicates that the active principle is heat stable, trypsin sensitive, nondialysable, and has an apparent relative molecular mass of about 16,000. Work is in progress to characterize the activity further and to discover whether the mesoderm-inducing factor is also present in normal embryos.     

Inhibition of mesoderm formation by follistatin

 Mesoderm induction requires interaction between cells of the animal and vegetal hemispheres of the embryo. Several molecules have been proposed as candidates for mesoderm-inducing signals, with activin a particularly strong candidate. However, it has not been possible to inhibit mesoderm formation in vivo by specifically blocking activin action. Follistatin is able to inhibit the action of activin but not that of the mature region of Vg1, a member of the transforming growth factor β family. Follistatin therefore provides a useful tool for distinguishing between signalling by these two factors. We have overexpressed Xenopus follistatin mRNA and analysed the expression of several mesodermal markers. Our results show an inhibition of mesodermal formation by follistatin in a concentration-dependent manner, showing the requirement of activin for mesodermal induction. Received: 22 August 1997 / Accepted: 16 January 1998     

Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm.

The potent mesoderm-inducing factors activin and FGF are present as maternally synthesized proteins in embryos of X. laevis. We show that activin can act on explanted blastomeres to induce at least five different cell states ranging from posterolateral mesoderm to dorsoanterior organizer mesoderm. Each state is induced in a narrow dose range bounded by sharp thresholds. By contrast, FGF induces only posterolateral markers and does so over relatively broad dose ranges. FGF can modulate the actions of activin, potentiating them and broadening the threshold-bounded dose windows. Our results indicate that orthogonal gradients of activin and FGF would be sufficient to specify the main elements of the body plan.     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

Mesoderm induction in amphibians and chick

Induction is a process in which the developmental pathway of one cell is controlled by signals emitted from another. Mesoderm induction is the first inductive interaction in theXenopus enbryo and probably occurs in all vertebrates. It is a very important event as it is implicated in the regulation of morphogenesis. Nieuwkoop first demonstrated the importance of vegetal endoderm in inducing the mesoderm. Slack and co-workers incorporated the information obtained from experimental embryology in a “three signal” model for mesoderm induction in amphibians (signals arising from ventral vegetal hemisphere, dorsal vegetal hemisphere and the organizer). More recent research has resulted in the detection of mesoderm inducing factors which are members of FGF and TGF--β families. Activin, a member of the TGF-β family, has been shown to induce differential gene expression and cell differentiation in a concentration-dependent manner giving credence to the theory of morphogen gradients. Study of mesoderm induction in the chick embryo is much more difficult due to several reasons. Novel experimental approaches, however, have been used which point to the role of activin and FGF in chick mesoderm induction. The demonstration of mesoderm inducing activity of activin and FGF in other groups of vertebrates, particularly the chick embryo brings out the possibility of a universal mechanism of mesoderm induction being operative in all the vertebrates.     

Activin redux: specification of mesodermal pattern in Xenopus by graded concentrations of endogenous activin B

Mesoderm formation in the amphibian embryo occurs through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial cells. The first candidate mesoderm-inducing factor to be identified was activin, a member of the transforming growth factor type beta family, and it is now clear that members of this family are indeed involved in mesoderm and endoderm formation. In particular, Derrière and five nodal-related genes are all considered to be strong candidates for endogenous mesoderm-inducing agents. Here, we show that activin, the function of which in mesoderm induction has hitherto been unclear, also plays a role in mesoderm formation. Inhibition of activin function using antisense morpholino oligonucleotides interferes with mesoderm formation in a concentration-dependent manner and also changes the expression levels of other inducing agents such as Xnr2 and Derrière. This work reinstates activin as a key player in mesodermal patterning. It also emphasises the importance of checking for polymorphisms in the 5' untranslated region of the gene of interest when carrying out antisense morpholino experiments in Xenopus laevis.     

Induction of differentiation of the human promyelocytic cell line HL-60 by activin/EDF.

A human promyelocytic cell line, HL-60, treated with activin/EDF was found to differentiate into monocyte/macrophage-like cells. This was shown not only by morphology but by the loss of myeloperoxidase granules and the appearance of nonspecific esterase. Dose-dependent inhibition of the differentiation by follistatin, an activin-binding protein, confirmed that it was indeed caused by activin. Thus, activin/EDF exerts its effect on hematopoietic cells not only on erythroid differentiation but also on at least a part of myeloid cell differentiation.     

Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction.

The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cells and in the presence of a protein synthesis inhibitor, indicating that this is an "immediate-early" response to mesoderm induction.     

Two essential processes in the formation of a dorsal axis during gastrulation ofCynops embryo

The isolated upper marginal zone from the initial stage ofCynops gastrulation is not yet determined to form the dorsal axis mesoderm: notochord and muscle. In this experiment, we will indicate where the dorsal mesoderm-inducing activity is localized in the very early gastrula, and what is an important event for specification of the dorsal axis mesoderm during gastrulation. Recombination experiments showed that dorsal mesoderm-inducing activity was localized definitively in the endodermal epithelium (EE) of the lower marginal zone, with a dorso-ventral gradient; and the EE itself differentiated into endodermal tissues, mainly pharyngeal endoderm. Nevertheless, when dorsal EE alone was transplanted into the ventral region, a secondary axis with dorsal mesoderm was barely formed. However, when dorsal EE was transplanted with the bottle cells which by themselves were incapable of mesoderm induction, a second axis with well-developed dorsal mesoderm was observed. When the animal half with the lower marginal zone was rotated 180° and recombined with the vegetal half, most of the rotated embryos formed only one dorsal axis at the primary blastopore side. The present results suggest that there are at least two essential processes in dorsal axis formation: mesoderm induction of the upper marginal zone by endodermal epithelium of the lower marginal zone, and dorsalization of the upper dorsal marginal zone evoked during involution.     

Regulated expression of the retinoblastoma gene product by fibroblast growth factor but not by activin during mesoderm induction in Xenopus

 The retinoblastoma (RB) gene is a tumor suppressor gene that plays an important role in cell cycle arrest and in the terminal differentiation of skeletal myoblasts. Differentiation into muscle occurs in Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF) or activin A. We examined expression of the RB gene product (pRB) during mesoderm induction in vivo and in vitro. We show that hypo- and hyper-phosphorylated forms of pRB are present during early development and that expression of both forms increases significantly during the blastula stage, concomitant with mesoderm induction. Further investigation revealed that pRB is enriched in the presumptive mesoderm of the blastula stage embryo. In animal cap explants induced by Xenopus bFGF (XbFGF), pRB expression levels increased approximately tenfold while no increase was observed in explants induced by activin. However, when explants were induced by XbFGF in the presence of sodium orthovanadate, a compound previously shown to synergize with FGF to produce more dorsal ”activin-like” inductions than FGF alone, only a slight increase in pRB expression was observed. Furthermore, upregulation of pRB during mesoderm induction in vitro displayed an inverse correlation with expression of XFKH1, a marker for notochord. These results suggest that pRB may be important for patterning along the dorsoventral axis. Received: 22 February 1996 / Accepted: 20 September 1996     

Elucidation of the role of activin in organogenesis using a multiple organ induction system with amphibian and mouse undifferentiated cells in vitro

Studies performed over the last century have clarified the mechanisms of organ and tissue formation. Mesoderm formation is one of the most important events in early body pattern determination during embryogenesis. In 1988, we found that activin A has mesoderm-inducing activity. As activin A could induce dorsal mesoderm formation, unlike fibroblast growth factor and bone morphogenetic protein, this factor was thought to be the molecular entity of the Spemann-Mangold organizer. Subsequently, the mechanisms of early embryogenesis have been clarified using molecular biological techniques, resulting in the identification of many genes that are involved in organ and tissue development. This finding that activin A could induce dorsal mesoderm formation spurred research into the application of agents that induce organs and tissues in vitro . In this regard, we have shown that many organ types can be induced by activin A in vitro . Moreover, we have found that other types of organs can be induced by changing the conditions of treatment. To date, more than 20 different types of tissues and organs have been successfully induced from Xenopus undifferentiated cells in vitro . In recent years, we have applied these protocols to mouse embryonic stem cells, and we have successfully induced several tissues, such as the pancreas and cardiomyocytes. We are also investigating how the pluripotency of undifferentiated stem cells is regulated. In this review, we summarize the current knowledge regarding activin as a mesoderm-inducing factor and its application for the induction of tissues and organs from undifferentiated cells. Moreover, we provide some examples of in vitro tissue differentiation from mouse embryonic stem cells, which may prove useful in regenerative medicine.     

Identification of the two types of specific receptor for activin/EDF expressed on Friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friends leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500-20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15-0.4 nM and 1.5-3.0 nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50-200 fold lower than that of activinA. Transforming growth factor-beta had no binding ability to the activin receptors.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.