Naoto Ueno

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

SummaryRecently the mesoderm-inducing effects of the transforming growth factor β (TGF-β) 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-β family has mesoderm-inducing ability.

Genes for bone morphogenetic proteins are differentially transcribed in early amphibian embryos

We have previously demonstrated that activin, a member of the TGF-beta family, has a potent mesoderm-inducing activity in Xenopus embryos. In the course of screening for activin-related genes from Xenopus, we have cloned cDNAs for Xenopus homologue of BMP-2, -4 and -7. Northern blot analysis revealed that these BMP genes are maternally encoded and differentially regulated after fertilization. Alkaline phosphatase-inducing assay using the recombinant BMP proteins has shown that at least BMP-2 and -4 have similar activity to mammalian counterparts.

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.

Activin as a cell differentiation factor

Activin, originally discovered as a polypeptide hormone that is capable of stimulating follicle-stimulating hormone secretion from pituitary cells in vitro, has recently been found to have a much wider range of biological activities. There are a number of reports of activin action as a cell differentiation factor on various types of cells rather than as a modulator of hormone secretion, as predicted initially, based on its structural similarity to transforming growth factor-beta. Studies of the distribution of activin and its receptor in a variety of tissues and its wide-ranging actions clearly illustrates its multifunctional properties. In particular, activin has been shown to be a potential regulator of early development of Xenopus laevis. Observation of activin effect in embryogenesis is of general importance to our understanding of the role of the family of growth factors in developmental processes.

Structure and sequence analysis of the human activin beta A subunit gene.

The cloned genomic DNA containing the human activin beta A subunit gene were analyzed by restriction endonuclease mapping, Southern blotting and DNA sequencing. The activin gene is composed of two exons interrupted by the 9-kb intron. The TATA, CCAAT and CT-stretch sequences were found in the 5-flanking region of the gene. An intronic sequence contained SV40 enhancer core element in the vicinity of the exon 1. In the 3-flanking region, we identified eight consensus polyadenylation sequences, five ATTTA motifs, CA element consisting of (CA)14, AP-1 binding site and two SV40 enhancer core elements. A dot matrix analysis revealed the high degree of conservation between the human and rat sequences within the 3-flanking region, suggesting a possible functional significance.

Multiple genes for Xenopus activin receptor expressed during early embryogenesis

Four distinct cDNAs for activin receptor designated as XSTK2, 3, 8 and 9 have been cloned from a Xenopus laevis cDNA library. The protein structures deduced from the cDNAs have shown that they all have a putative extracellular ligand‐binding domain, a single transmembrane domain and cytoplasmic Ser/Thr kinase domain, except that XSTK2 is extremely similar to the XSTK3 gene but lacks a carboxyl‐terminal part of the kinase motif. Northern blot analysis showed that all transcripts are maternally inherited. The levels or transcript for XSTK2, 3 and 8 appeared to fluctuate during early development while those for XSTK9 maintain constant.

A carboxyl-terminal truncated version of the activin receptor mediates activin signals in early Xenopus embryos

The function of a carboxyl‐terminal truncated version of the Xenopus activin receptor, encoded by a previously isolated gene XSTK2, was investigated in early embryos. The transcript corresponding to the truncated receptor gene was detected throughout embryonic development although the temporal expression pattern was different from that of an intact receptor. Injection of X5TR2 mRNA into early embryos resulted in the formation of a duplicated body axis. Mesoderm induction as evaluated by the activation of the α‐actin gene in presumptive ectoderm (animal cap) treated with exogenous activin was significantly enhanced by the injection of XSTK2 mRNA. These results suggest that the truncated receptor is capable of transmitting the activin signal to the same extent as the native receptor.

Localization of thymosin β4 to the neural tissues during the development of Xenopus laevis, as studied by in situ hybridization and immunohistochemistry

Abstract Thymosin β4, a polypeptide of 5 kDa, is known to have capacity to regulate actin polymerization by binding to an actin monomer. Distribution ofXenopus laevis thymosin β4 (XTβ4) in the developingXenopus larva was examined by means of in situ hybridization and immunohistochemistry. Analysis with in situ hybridization revealed that XTβ4 mRNA becomes gradually localized to the neural tissues, notochord and inner epidermis during neurula stages. Intense accumulation of XTβ4 mRNA was observed in the ganglions of cranial nerves and in the dorsal region of the spinal cord from stage 26 and onwards. XTβ4 immunoreactivity (XTI) was observed in larvas at all developmental stages later than stage 26, tail bud embryo. Immunoreactivity was initially distributed to the ganglion of cranial nerve V and Rohon-Beard cells. As the development progressed, the XTI appeared in other neuronal groups. By late tadpole stages (stages 42–47) the XTI was found in the pineal body, oculomotor and trochlcar motoneurons of the midbrain, various neurons in the rhombencephalon, ganglions of cranial nerves V, VII/VIII and IX/X. In the spinal cord the XTI was observed in Rohon-Beard cells, dorsal root ganglion cells, motoneurons and other spinal cord neurons. Immunoreactivity was seen in both cell bodies and axons of the neurons. These findings suggest that thymosin β4 plays a role in the development of neurons, especially of sensory neurons.

Identification of Bone Morphogenetic Protein-2 in Early Xenopus laevis Embryos

Polyclonal antibodies capable of reacting with amphibian bone morphogenetic protein (BMP-2 and -4) were raised in rabbits by immunization with a synthetic 21 amino acid peptide which corresponds to a sequence residing in the mature protein of Xenopus BMP-2 (xBMP-2). The antibodies recognized an embryonic BMP as well as mammalian and bacteria expressed recombinant xBMPs. The antibodies detected, under reducing conditions, a 30 kDa protein in the extract of oocytes and embryos during early development. Interestingly, acidification of the extract from each developmental stage yielded a protein band of smaller molecular weight of 18 kDa, which is similar in size to reduced form of mature BMPs purified from mammalian species. Two-dimensional electrophoresis employed to examine the molecular weight of unreduced forms using the antibody, revealed that both molecular forms are monomeric in the embryos. The result suggests that at least BMP-2 mRNA previously detected in early embryos, is translated into peptide but the dimerization may be incomplete or strictly limited in these embryos.

Regulation of activin βA mRNA level by cAMP

We demonstrated the presence of five species of the activin beta A mRNA in human placenta and one major RNA associated with two minor RNAs of the activin in the fetal membrane. We investigated the effect of 8-bromo-cAMP (8-Br-cAMP) on accumulation of activin beta A subunit mRNA in human fibrosarcoma HT1080 cells. Although low levels of the activin mRNA were detectable in the untreated cells, the one main RNA species was predominantly accumulated by 8-Br-cAMP. We propose that generation of multiple activin mRNAs in the fetal membrane and cAMP-treated HT1080 cells is presumably due to a cell-specific alternative polyadenylation.