Hideho Uchiyama

Induction of pronephric tubules by activin and retinoic acid in presumptive ectoderm of Xenopus laevis

Correlation between activin and retinoic acid (RA), both of which affect early amphibian development, was studied using Xenopus laevis embryos. In the first set of experiments, two isomers of RA, all‐trans RA and 13‐cis RA, were compared in terms of stability of biological activity against light. Xenopus blastulae were dipped in RA solutions which had either been kept away from light, or had been exposed to light for a few hours. At doses ranging from 10–4to 10–6M, RA elicited head deformity. All‐trans RA, under both dark and light conditions, had similarly potent effects. On the other hand, 13‐cis RA under dark conditions had much weaker effects than it did under light conditions.

Effects of estrogenic hormones on early development of Xenopus laevis

Many chemicals released into the environment have estrogenic activity and can disrupt animal development and the function of endocrine systems. In order to study the effects of estrogens on aquatic animals, we examined the effects of certain estrogens on early development in Xenopus laevis. X. laevis embryos were kept in water containing 10(-10), 10(-9), 10(-7), 10(-6), and 10(-5) M 17 beta-estradiol (E2); 17 alpha-estradiol; diethylstilbestrol (DES); 10(-5) M progesterone (P); or dihydrotestosterone (DHT) beginning at developmental stage 3. Survival rates of the embryos developed in water containing 10(-10)-10(-6) M E2 or DES, all concentrations of 17 alpha-estradiol, and 10(-5) M P or DHT, which were over 70% after stage 48, whereas the rates of the embryos treated with 10(-5) M E2 and DES decreased remarkably after stage 27 and all embryos were dead by stages 42 and 32, respectively. Embryos treated with 10(-5) M E2 showed malformations of the head and abdomen and suppressed organogenesis, including crooked vertebrae at stage 38; the head was smaller and the abdomen was larger than in the controls. Similar effects were observed in embryos developed in 10(-5) M DES but not in 10(-5) M 17 alpha-estradiol, P, or DHT. After 10(-5) M E2 treatment, abnormalities were induced only when the treatment was started before stage 39. However, on day 30 after fertilization, the stage of the embryos treated with 10(-6) M E2 was more progressed than that of the controls. Estrogen receptor (ER 4) mRNA was examined in eggs, embryos, and adult female liver by reverse-transcription polymerase chain reaction. ER4 mRNA was expressed in adult liver, unfertilized and fertilized eggs, and embryos, but ER3 mRNA was not expressed. ER4 mRNA in 10(-6) and 10(-5) M E2-treated embryos showed different expression patterns, which may result from the diverse developmental effects of E2. The present results demonstrate that 10(-5) M E2 and DES induced embryo death and malformations and that ER may be involved in the induction of various developmental defects in X. laevis embryos.

Concentration-dependent inducing activity of activin A

SummaryHuman 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.

Follistatin inhibits the mesoderm-inducing activity of activin A and the vegetalizing factor from chicken embryo

SummaryThe induction of mesoderm is an important process in early amphibian development. In recent studies, activin has become an effective candidate for a natural mesoderm-inducing factor. In the present study, we show that follistatin, an activin-binding protein purified from porcine ovary, inhibits the mesoderm-inducing activity of recombinant human activin A (rh activin A), which is identical to the erythroid differentiation factor (EDF). The quantity of follistatin required for effective suppression of activin was more than three-fold that of activin (w:w). Follistatin also inhibited the mesoderm-inducing activity of the vegetalizing factor purified from chick embryos, suggesting that the vegetalizing factor is closely related to activin.

Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.

GATA factors as key regulatory molecules in the development of Drosophila endoderm

Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent (srp, dGATAb, ABF), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe, and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans, in which a pair of GATA genes, end‐1/3, specifies endodermal fate, and a downstream pair of GATA genes, elt‐2/7, activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes.

Cloning and characterization of the T‐box gene Tbx6 in Xenopus laevis

Tbx6 is a member of the T‐box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6‐expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity.

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.