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The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann's organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2.

Abstract —Although smooth muscle cells (SMCs) are critical components of the circulatory system, the regulatory mechanisms of SMC differentiation remain largely unknown. In the present study, we examined the mechanism of SMC differentiation by using Xenopus laevis SM22α (XSM22α) as an SMC-specific marker. XSM22α cDNA contained a 600-bp open reading frame, and the predicted amino acid sequences were highly conserved in evolution. XSM22α transcripts were first detected in heart anlage, head mesenchyme, and the dorsal side of the lateral plate mesoderm at the tail-bud stage, possibly representing the precursors of muscle lineage. At the tadpole stage, XSM22α transcripts were restricted to the vascular and visceral SMCs. XSM22α was strongly induced by basic fibroblast growth factor (FGF) in animal caps. Although expressions of Xenopus cardiac actin were not affected by the expression of a dominant-negative FGF receptor, its injection dramatically suppressed the XSM22α expression. These results suggest that XSM22α is a useful molecular marker for the SMC lineage in Xenopus and that FGF signaling plays an important role in the induction of XSM22α and in the differentiation of SMCs.

We characterized Xenopus SIP1 (XSIP1), Smad interacting protein, from activin-treated animal caps by differential screening. The XSIP1 is very similar to mouse SIP1 in the protein coding region including the zinc finger domain and homeodomain. The expression pattern was analyzed by RT-PCR and whole mount in situ hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateral edge of neural plate and, in the tailbud stage, in the forebrain, neural tube, and eye. Overexpression of XSIP1 at the animal caps resulted in activation of anterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induced enlargement of neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos were short-tailed. Our findings suggest that XSIP1 is a transcriptional repressor, which may be involved in the activin-dependent signal pathway.

The kidney is a favorite subject of developmental biologists. In the past decade, we have gained a great deal of knowledge about the regulation of kidney development. This review describes the organization of the kidney, the molecules involved in the regulation of kidney development, and a unique protocol for kidney culture. The pronephros, mesonephros, and metanephros represent three distinct renal organs which function, in succession, as the vertebrate excretory system.1 All three such kidneys are derived from the intermediate mesoderm and develop in a well defined temporal and spatial sequence. The pronephros, which consists of a tubule and excretory organ, is established earliest and functions as the simplest of the excretory organs in vertebrates. The amphibian pronephros, a simple organ which provides renal function in the early embryo, is conceptualized in the present study as a model system for devising experimental strategies for the in-vitro cultivation of donor organs and tissues for subsequent transplantation into vertebrate hosts. Combined activin and retinoic acid treatment of blastula-stage ectoderm cultured for 3 days yielded a pronephros-like structure. Its functional capacity was confirmed by transplanting it into late neurula-stage host embryos from which the natural pronephros rudiments had been removed. The donor tissue differentiated into a pronephros-like structure, and the host embryos were able to suppress the edema ordinarily caused by removal of the natural pronephric rudiments. Thus, this provides a model system for analyzing the experimental conditions and relevant principles for the cultivation of functional vertebrate organs for transplantation.

We report here the cloning and expression of the Xenopus orthologue of the T-box transcription factor gene Tbx2 (optomotor-blind in Drosophila). Tbx2 is first detected in the ventral mesodermal cells just above the yolk plug at late gastrula. At the neurula stage it is strongly expressed in the cement gland, dorsal root ganglia, and otic vesicle region. At the tailbud stage strong Tbx2 expression is observed in the dorsal part of the optic cup and trigeminal ganglia, and it is also expressed in the branchial arches, heart anlage, nasal pit, proctodeum, and the region around the pronephros.

The sal (spalt) gene family is characterized by unique double zinc finger motifs and is conserved among various species from Drosophila to humans. Here we report a new Xenopus member of this family, Xsal-3. It is 38% homologous at the amino acid level to the previously reported Xenopus homologue of the spalt gene, Xsal-1. Alternatively spliced Xsal-3 transcripts give rise to RNAs coding either two or three double zinc fingers, and the longer form is expressed maternally. Xsal-3 is expressed in the neural tube, the mandibular, hyoid, and branchial arch, and the pronephric duct, which is different from the expression pattern of Xsal-1. These findings suggest that Xsal-3 may have distinct roles in early Xenopus development.

The discovery that peptide growth factors are capable of embryonic induction has led to rapid growth in our understanding of this process. One of these factors, activin, can control mesoderm formation inXenopus animal caps in a dose-dependent manner. Activin may also participate in kidney and heart organogenesis in combination with other factors. Further, animal caps treated with a high concentration of activin clearly show organizer actions, which are closely related to the fundamental body plan along the anteroposterior axis. These in vitro experiments will serve as an excellent model system for analysis of cell differentiation, organogenesis, and morphogenesis at the molecular level.

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.

Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing "blood-like cells". Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination.

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin-treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin-treated exp ants. Co-treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin-treated explants.

The inductive interactions between activin-induced and non-induced cells were investigated in newt animal cap explants. A wide range of concentrations of activin A (0.1–100 ng/ml) induced mesodermal tissues in the animal caps, but at generally low frequencies. Animal caps treated with 100 ng/ml of activin A, on the other hand, differentiated solely into nonspecific endoderm. At this concentration, various mesodermal tissues were induced in addition to endoderm as the animal caps increased in size. They were more frequently induced in explants in which the activin-treated animal caps were combined with untreated animal caps. Central nervous systems were frequently induced in sandwich explants with larger amounts of untreated animal caps. Differentiation of endodermal organs such as the liver, the pancreas and the intestine in the long-term cultured sandwich explants was confirmed by electron microscopy. Lineage tracing of the combination and sandwich explants revealed that the activin-treated anim...

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment.

Recently, several proteins which have a follistatin module have been isolated. One of them, the follistatin-related protein (FRP), is encoded by TSC-36 (TGF-β-stimulated clone 36) in mouse, originally isolated as a cDNA clone up-regulated by TGF-β1 in mouse osteogenic MC3T3E1 cells. To determine the physiological role of FRP in early Xenopus embryonic development, we cloned the Xenopus FRP (xFRP) cDNA. The resulting cDNA clone was a secreted glycoprotein consisting of 299 amino acid residues with about 70% similarity to the mammalian and avian FRPs. Northern blotting analysis revealed that xFRP gene expression started at stage 10, the onset of gastrulation, gradually increased during the blastula and neurula stages and was sustained through the tail-bud stage. Whole-mount in situ hybridization analysis showed the localization of xFRP mRNAs in the Spemann organizer, notochord, neural floor plate, hypochord and somite. The similarities with the pattern of expression of Xenopus follistatin mRNA suggests that xFRP may play a role in neuralization.

The differentiation patterns of animal cap explants from the Japanese salamanders Hynobius lichenatus and Hynobius nigrescens were examined after exposure to various concentrations of activin A. A wide range of concentrations of activin A (0.5-100 ng/ml) induced various mesodermal tissues such as ventral mesoderm, somitic muscle, and notochord. At concentrations higher than 50 ng/ml, yolk-rich endodermal tissue was induced in many of the explants. Activin A is also known to have mesoderm-and/or endoderm-inducing activity on the animal caps of Xenopus laevis and Cynops pyrrhogaster, but their response patterns are slightly different. The mode of differentiation of activin-treated Hynobius animal caps was compared with that of Xenopus and Cynops in relation to the structure of the animal caps.

We have demonstrated that the tissue differentiation patterns along the dorsoventral and anteroposterior axes can be controlled by a combination of activin A, concanavalin A (Con A), and retinoic acid. Xenopus blastula animal caps, normally fated to form epidermal tissues, differentiated into ventral mesoderm tissues such as coelomic epithelium and blood-like cells following treatment with activin A (0.5 ng/ml). Dorsal mesoderm tissues like muscle and notochord, were induced by graded addition of Con A. Conversely, Con A (1 mg/ml) induced anterior neural tissues, forebrain accompanied by eyes and cement glands, in the animal caps. Posterior neural tissues, hindbrain with ear vesicles and spinal cord, were induced by graded addition of activin A. Retinoic acid was also capable of shifting the Con A-induced anterior neural tissues to more posterior tissue phenotypes, however, its caudalizing activity was slightly different from that of activin A. These results suggest that the concentration gradien...

We report the isolation and characterization of a Xenopus sequence, XNLRR-1, that is closely related to a gene for mouse neuronal leucine-rich repeat protein (NLRR-1). The cDNA clone is 4179 bp long and encodes a putative transmembrane glycoprotein of 718 amino acids, containing 12 leucine-rich repeats followed by one C2-type immunoglobulin-like domain and one fibronectin type-III repeat. XNLRR-1 is transcribed mainly in the developing eye area and the ventricular zone from diencephalon to hindbrain and slightly in spinal cord in Xenopus tadpoles. The similarity of the XNLRR-1 gene to other known cell adhesion molecules, together with the expression pattern, suggests that XNLRR-1 is involved in interactions at the neuronal cell surface.

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.

Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3beta (GSK-3beta). This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway. We designated this protein Axil for Axin like. Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication. Axil was phosphorylated by GSK-3beta. Axil bound not only to GSK-3beta but also to beta-catenin, and the GSK-3beta-binding site of Axil was distinct from the beta-catenin-binding site. Furthermore, Axil enhanced GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation.

In beginning of muscle development, determination is induced in the mesoderm, and then differentiation occurs with accumulation of muscle structural proteins. Mesoderm cells differentiate to many type cells, but the direct signaling activator for muscle determination is still unknown. In this paper we report some of the conditions required for determination of muscle. Muscle determination during Xenopus development was found to be marked by Xmyf-5 and XmyoD expression, but not by expression of Xmyogenin or Xmrf4. Xmyf-5 and XmyoD expression was first detected in the early gastrula stage. Xmyf-5 expression was first detected on the dorsal side, whereas XmyoD was initially expressed on the ventral side. Subsequently, expression of both genes was strongly induced on the dorsal side. The expression of Xmyf-5 and XmyoD did not continue to increase on the ventral side when it was separated from the dorsal side, although muscle originates from the both sides. These findings suggest that a continuous inc...

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae-early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops.

A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis.

Abstract The parasitic nematode, Rhabdias tokyoensis, Wilkie, 1930, which is found primarily in the saccular lungs of the Japanese newt, Cynops pyrrhogaster, Boie, 1826, is described from the clinical and ultrastructural perspectives. Newts are commonly infected with 1-3 nematodes per lung and often appear healthy, but may present in a wasted state and rarely, if the parasites have penetrated the lungs, will appear grossly bloated with air. This distention alters the newt diving behavior due to impaired buoyancy control. The nematodes induce ulcerative lesions on the inner, pulmonary surface and, if they escape from the lungs, may attach to abdominal organs such as the liver. Rhabdias tokyoensis has minor annulations in the head region, with an oral cavity devoid of crown or lips, and with narrow, sub-cuticular, bacillary band-like longitudinal ridges, as observed by scanning electron microscopy (SEM). Cuticular wrinkling due to dehydration is evident by SEM. Transmission electron microscopy revealed that the cuticle was distinctive, with a particularly thick inner cortex, two thin laminations comprising the basal, fibrous layer, and a convoluted, outer cortex which reflected the surface wrinkling observed by scanning electron microscopy. Cells lining the intestinal tract had apical surfaces lined by microvilli and both large (2.5 μm) and small (0.3 μm) electron dense secretory granules. Somatic muscles encircling the periphery of the nematode body were typical for this genus. The life cycle, prevalence in the natural environment, and possible intermediate hosts for R. tokyoensis remain unknown. Infection of Japanese newts by R. tokyoensis, which otherwise are in good health, may not significantly impair this primarily aquatic amphibian because it relies much on integumentary respiration and the nematode-damaged, primitive saccular lungs do not undergo significant hemorrhage.

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells. Stem Cells 2014;32:3099–3111

The importance of epithelial–mesenchymal interaction on the development of gastro-intestinal (GI) organs has been repeatedly reported, but its molecular mechanism has not been fully understood though several factors including hepatocyte growth factor and endothelin-3 have been shown to mediate it. Activins have been demonstrated to play important roles in the regulation of organogenesis in vertebrates, but their roles in the regulation of growth and differentiation of GI organs remain to be solved. In the present study, we examined expression of activins in developing rat GI tract, and found that inhibin bA encoding activin A was specifically expressed by GI mesenchymes, while inhibin bB encoding activin B was expressed by both epithelial and mesenchymal components. We then examined the effect of activin A on the growth of fetal rat GI epithelial cells in primary culture. We found that activin A inhibited the growth of forestomach and glandular stomach epithelial cells while it stimulated the growth of colonic epithelial cells. These results suggest that activin A secreted from GI mesenchymes region-specifically regulates the growth of attaching epithelial cells. We thus conclude that activin A mediates epithelial-mesenchymal interaction in the developing GI tract.

Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

Renal lineages including kidney are derived from intermediate mesoderm, which are differentiated from a subset of caudal undifferentiated mesoderm. The inductive mechanisms of mammalian intermediate mesoderm and renal lineages are still poorly understood. Mouse embryonic stem cells (mESCs) can be a good in vitro model to reconstitute the developmental pathway of renal lineages and to analyze the mechanisms of the sequential differentiation. We examined the effects of Activin A and retinoic acid (RA) on the induction of intermediate mesoderm from mESCs under defined, serum-free, adherent, monolayer culture conditions. We measured the expression level of intermediate mesodermal marker genes and examined the developmental potential of the differentiated cells into kidney using an ex vivo transplantation assay. Adding Activin A followed by RA to mESC cultures induced the expression of marker genes and proteins for intermediate mesoderm, odd-skipped related 1 (Osr1) and Wilm’s Tumor 1 (Wt1). These differentiated cells integrated into laminin-positive tubular cells and Pax2-positive renal cells in cultured embryonic kidney explants. We demonstrated that intermediate mesodermal marker expression was also induced by RA receptor (RAR) agonist, but not by retinoid X receptor (RXR) agonists. Furthermore, the expression of these markers was decreased by RAR antagonists. We directed the differentiation of mESCs into intermediate mesoderm using Activin A and RA and revealed the role of RAR signaling in this differentiation. These methods and findings will improve our understanding of renal lineage development and could contribute to the regenerative medicine of kidney.

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices.

Chemokine molecules play important roles in the immune system. However, several chemokine molecules are expressed during early development before the immune system is established. Using reverse transcription�polymerase chain reaction (RT-PCR) and overexpression of chemokine molecules, we identified and characterized Xenopus laevis CXC-type chemokine ligands (XCXCL13L1, XCXCL13L2, XCXCLa, XCXCLb, XCXCLd, and XCXCLe) and receptors (XCXCR1/2, XCXCR3, XCXCR5, XCXCR6, and XCXCRa) during early development. The CXC-type ligands have low identity with genes for human CXC ligands (CXCL). With the exception of XCXCRa, the CXC receptors (CXCR) identified in the present study had high (~40%�65%) identity with human CXCR genes. Although the expression patterns for the CXCL and CXCR genes differed, transcript levels for all genes were very low during early embryogenesis. Overexpression of XCXCL13L1, XCXCL13L2, XCXCLa, XCXCR3, XCXCR6, and XCXCRa interfered with gastrulation and neural fold closure. The results of the present study suggest that several chemokine molecules are related to cell movements during early morphogenesis.

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells.

Most kidney nephron components, including glomeruli and renal tubules, derive from the metanephric mesenchyme. The overall differentiation into each component finishes at birth, but the molecular events linking the perinatal and adult kidneys remain elusive. Dullard was cloned from Xenopus kidneys, and encodes a phosphatase that negatively regulates BMP signalling. Here we report that Dullard deletion in the murine metanephric mesenchyme leads to failure of nephron maintenance after birth, resulting in lethality before adulthood. The nephron components are lost by massive apoptosis within 3 weeks after birth, leading to formation of a large hollow with a thin-layered cortex and medulla. Phosphorylated Smad1/5/8 is upregulated in the mutant nephrons, probably through cell-autonomous inhibitory effects of Dullard on BMP signalling. Importantly, administration of the BMP receptor kinase inhibitor LDN-193189 partially rescued the defects caused by Dullard deletion. Thus, Dullard keeps BMP signalling at an appropriate level, which is required for nephron maintenance in the postnatal period.

Cell surface glycans show dynamic changes during cell differentiation. Several glycans are useful biomarkers of tumors, stem cells, and embryogenesis. Glycomic studies have been performed using liquid chromatography and mass spectrometry, which are powerful tools for glycan structural analysis but are difficult to use for small sample sizes. Recently, a lectin microarray system was developed for profiling cell surface glycome changes to terminal carbohydrate chains and branch types, using sample sizes of a few micrograms. In this study, we used the lectin microarray system for the first time to investigate stage-specific glycomes in Xenopus laevis embryos. Unsupervised cluster analysis of lectin microarray data indicated that glycan profiles changed sequentially during development. Nine lectin probes showed significantly different signals between early and the late-stage embryos: 4 showed higher signals in the early stages, and 5 exhibited higher signals in the late stages. The gene expression profiles of relevant glycosyltransferase genes support the lectin microarray data. Therefore, we have shown that lectin microarray is an effective tool for high-throughput glycan analysis in Xenopus embryogenesis, allowing glycan profiling of early embryos and small biopsy specimens.

Dullard/Ctdnep1 is a member of the serine/threonine phosphatase family of the C-terminal domain of eukaryotic RNA polymerase II. Embryos lacking Dullard activity fail to form primordial germ cells (PGCs). In the mouse, the formation of PGCs is influenced by BMP4 and WNT3 activity. Although Dullard is reputed to negatively regulate BMP receptor function, in this study we found mutations in Dullard had no detectable effect on BMP4 and p-Smad activity. Furthermore Dullard mutations did not influence the dosage-dependent inductive effect of Bmp4 in PGC formation. However, Dullard may function as a positive regulator of WNT signalling. Combined loss of one copy each of Dullard and Wnt3 had a synergistic effect on the reduction of PGC numbers in the compound heterozygous embryo. In addition, loss of Dullard function was accompanied by down-regulation of WNT/β-catenin signalling activity and a reduction in the level of Dishevelled 2 (Dvl2). Therefore, Dullard may play a role in the fine-tuning of WNT signalling activity by modulating the expression of ligands/antagonists and the availability of Dvl2 protein during specification of the germ cell lineage.

The kidney has been used as a model organ to analyze organogenesis. In in vitro experiments using Xenopus blastula ectoderm, the development of pronephric tubules (the prototype of the kidney) may be induced by treatment with activin A and retinoic acid (RA). The present study examined whether pronephric tubules induced in ectodermal explants exhibited similar characteristics to those of normal embryos at the molecular level. The experimental conditions required for high frequency induction (100%) of pronephric tubule formation from presumptive ectoderm without the development of muscle and notochord were determined. The developmental expression of the pronephros marker genes Xlim-1 and Xlcaax-1 was examined in induced pronephric tubules. After treatment with 10 ng/mL activin A and 10−4 mol/L RA, only pronephric tubules were induced at a high frequency. Induced pronephric tubules showed the same timing and patterns of expression for the marker genes Xlim-1 and Xlcaax-1 as normal embryos. These results suggest that the in vitro development of pronephric tubules induced in the presumptive ectoderm by activin A and RA parallels normal development at the molecular level.

AXenopus laevisoocyte cDNA library was screened with a PCR-generatedX. laevisvitellogenin (VTG) receptor probe and a 3.6 kb cDNA clone containing the entire open reading frame, and 5′ and 3′ noncoding regions were isolated. The deduced amino acid sequence was 72% homologous to the chicken VLDL/VTG receptor, and the characteristic domains were highly conserved. Ligand binding analysis confirmed that the cloned receptor wasXenopusVTG-specific. Although Northern blotting analysis revealed that this gene was expressed as a major transcript of 3.6 kb inXenopusovary, weak but significant expression was observed in other tissues by RT-PCR analysis. The fact that major expression of the gene occurs in the ovary suggests that it has an important function in this organ.

Determination (inducing) factors, the extracellular matrix, signaling pathways, transcription factors and genes interact in pattern formation and neural induction. Genes can either be activated or repressed. The animalvegetal and dorso–ventral polarities are determined in very early developmental stages. Factors of the TGF-β superfamily in a graded distribution are involved in the determination of endoderm, mesoderm and ectoderm. The differentiation of mesoderm also depends on the animal ectoderm. Neural inducing factors have been partially purified.

An antibody against the Xenopus activin/inhibin beta(B) subunit (94-107) was raised in a rabbit. Using this antibody, the distribution of activin/inhibin beta(B) immunoreactivity in the pituitary of adult X. laevis was studied. Beta(B) immunoreactivity was detected in gonadotrophs, thyrotrophs, and somatotrophs under light microscopy. Electron microscopy revealed that a beta(B)-immunoreactive substance exists in LH, TSH, and GH granules, in contrast to findings in the rat and goldfish. These results indicate that the expression of activin/inhibin beta(B) in pituitary cells is not consistent among vertebrate species.

AstroNewt experiment explores the effects of earth gravity on the early development of Japanese red-bellied newt, Cynops pyrrhogaster. Since female newts keep spermatophore in cloaca, fertilized eggs could be obtained without mating. Fertilization of newt's egg occurs just prior to spawning, so that gonadotrophic cues applied to females in orbit leads to laying eggs fertilized just in space. A property of newt being kept in hibernation at low temperature may be of great help for the space experiment carried out with much limited resources. A general outline of the AstroNewt project is shown here in addition to some technical advances for the development of the project. Experimental schemes of two space experiments (IML-2 in summer 1994 and unmanned SFU at the beginning of 1995) are also shown.

Somatic stem/progenitor cells are known to be present in most adult tissues. However, those in the lung have limited abilities for tissue regeneration after serious damage as a result of chronic disease. Therefore, regenerative medicine using exogenous stem cells has been suggested for the treatment of progressive lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis. Embryonic stem (ES) cells and induced pluripotent stem cells, with their potent differentiation abilities, are promising sources for the generation of various tissue cells. In this study, we investigated the effects of various differentiation-inducing growth factors on the differentiation of lung cells from ES cells in vitro. Several factors, including activin, nodal, and noggin, significantly promoted the induction of Nkx2.1-positive lung progenitor cells when cells were cultured as embryoid bodies. Bone morphogenetic protein (BMP) 4 signaling controls the lineage commitment of lung cells along the proximal-distal axis. BMP4 promotes the induction of distal cell lineages of alveolar bud, such as Clara cells and mucus-producing goblet cells. These results suggest that several developmentally essential factors, including nodal/activin and BMP signaling, are important in the control of the differentiation of lung epithelial cells from mouse ES cells in vitro.

Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here, we demonstrate that a recombinant lectin probe, rBC2LCN, a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells, is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in culture medium containing fluorescent dye-labeled rBC2LCN. Fluorescent dye-labeled rBC2LCN was also successfully used to separate live pluripotent stem cells from a mixed cell population by flow cytometry.

Embryonic induction plays an important role in establishing the fundamental body plan during early amphibian development. The factors mediating this embryonic induction have, however, only recently been discovered. In the mid-1980's, certain peptide growth factors belonging to the FGF and TGF-beta families were found to have a mesoderm-inducing effect on isolated Xenopus blastula ectoderm. The study of embryonic induction subsequently expanded rapidly and knowledge at the molecular level has gradually accumulated. One of these peptide growth factors, activin, a member of the TGF-beta superfamily, is present maternally in the Xenopus early embryo and induces various mesodermal and endodermal tissues in isolated presumptive ectoderm. After exposure of presumptive ectoderm to activin, many genes are expressed in the same manner as in normal embryogenesis. Ectoderm treated with activin can induce a complete secondary embryo, the same as the organizer does in transplantation experiments. These findings suggest that activin is one of the first induction signals responsible for establishing the embryonic body plan in early amphibian development. In this article we shall review to what extent we can control the embryonic body plan in vitro, referring to some significant findings in this field.

Pre-mated adult female newts and embryos have been flown on the International Microgravity Laboratory-2 (IML-2) Space Shuttle flight in 1994 (Wiederhold et al., 1992b). With the specimens available from this flight, the calcification of otoliths, ulna, radius and backbone of the flown larvae and adult newts were analyzed. The experiments presented here studied the development of the otoliths on the ground. Otoliths of living newts, from embryo to adult, were observed in situ with the application of a new X-ray and bio-imaging analyzer system. For the establishment of this method, newts at different developmental stages were used. An imaging plate temporarily stores the X-ray energy pattern at the bio-imaging analyzer. A latent image on the imaging plate was transformed into a digital time series signal with an image reader. Acquired digital information was computed with the image processor. The processed information was recorded on film with an image recorder, in order to visualize it on an enlargement computed radiograph. To analyze development of the otoliths, photo-stimulated luminescence level was detected by an image analyzer, using transmitted X-ray photons. A single clump of otoconia could first be seen at stage 33. Stage-36 embryos first have distinguishable otoliths, with the utricle in front and saccule behind. Our results show that this X-ray method detects the otoliths equally as well as sectioning. In the newt, the mandibular/maxillary bone formed before the spine. It is suspected that for the newt embryo, living in water, feeding becomes necessary prior to support of the body.

Midkine (MK) is a heparin-binding growth factor and forms a novel protein family together with another member, pleiotrophin (PTN)/heparin-binding growth-associated molecule (HB-GAM). A cDNA clone isolated from Xenopus laevis specifies for the Xenopus counterpart of MK (XMK), and the mode of XMK expression was studied by in situ hybridization and Northern blot analysis. XMK was first expressed at stage 11 (middle gastrula) and was located in the neural anlage at stage 12 (late gastrula). Through stage 13 to 15 (early neurula), XMK expression was restricted to the neural folds. At stage 23 (tailbud stage), XMK was predominantly localized in the brain and neural tube. At the larva stage, XMK expression was again restrictedly observed in the brain, the optic vesicles, the otic vesicle, and the spinal cord, all of which are derivatives of the neural tube, as well as in the branchial arches, derivatives of the cranial neural crest. Comparing the mode of MK expression between Xenopus and the mouse, we propose that MK plays evolutionally conserved roles in neurogenesis and development of the craniofacial architecture of ectomesenchymal origin. We also found that XMK was expressed in various adult organs; strong expression was observed in the brain, the eye and the spinal cord, all of which showed intense MK expression at the larva stage.

Single animal hemisphere blastomeres isolated from the eight-cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32-cell stage) of normal development. The competence gradually increased during the morula stages.

The sequence in which the otoliths and semicircular canals and their associated sensory epithelia appear and develop in the newt are described. Three-dimensional reconstruction of serial sections through the otic vesicle of newt embryos from stages 31 through 58 demonstrate the first appearance, relative position and growth of the otoliths. A single otolith is first seen in stage 33 embryos (approximately 9 days old); this splits into separate utricular and saccular otoliths at stage 40 (13 days). The lateral semicircular canal is the first to appear, at stage 41 (14 days). The anterior and posterior canals appear approximately one week later and the vestibular apparatus is essentially fully formed at stage 58 (approximately 5 weeks). The data reported here will serve as ground-based controls for fertilized newt eggs flown on the International Microgravity Laboratory-2 Space Shuttle flight, to investigate the influence of microgravity on the development of the gravity-sensing organs.

Activin possesses mesoderm-inducing activity, erythroid-differentiating activity, and follicle-stimulating hormone-releasing activity. The chemical structures of the activin molecule are formed by a combination of two beta-subunit peptides of inhibin. Inhibin is a dimer consisting of an alpha and beta subunit. To examine the mesoderm-inducing activity of these substances, we tested several configurations including: (1) two types of alpha-subunit peptide; (2) two types of inhibin A and B dimer; (3) beta A-subunit peptide monomer; (4) three types of activins A, AB and B, and (5) follistatin (activin-binding protein) by the animal cap assay using Xenopus laevis ectoderm, and by the erythroid-differentiating factor (EDF) test. Activins, which are composed of dimeric inhibin beta A- or beta B-subunit peptides, had the highest mesoderm-inducing and EDF activities. The monomeric beta A-subunit peptide exhibited mesoderm-inducing and EDF activities that were much lower than activin A. The inhibitory effect of follistatin on mesodermal induction by the beta A-subunit peptide was also lower than that of activin. Both inhibins A and B had very weak mesoderm-inducing activity and no EDF activity. The two types of inhibin alpha-subunit monomer had little mesoderm-inducing activity and no EDF activity. The mesoderm induction caused by activin A was not suppressed by the addition of the alpha-subunit monomer and inhibin. The mesoderm-inducing activity in relation to the chemical structures of the monomeric and/or dimeric inhibin alpha and beta A subunits is discussed.

Skeletal muscle represents a plentiful and accessible source of adult stem cells. Skeletal-muscle-derived stem cells, termed satellite cells, play essential roles in postnatal growth, maintenance, repair, and regeneration of skeletal muscle. Although it is well known that the number of satellite cells increases following physical exercise, functional alterations in satellite cells such as proliferative capacity and differentiation efficiency following exercise and their molecular mechanisms remain unclear. Here, we found that functional overload, which is widely used to model resistance exercise, causes skeletal muscle hypertrophy and converts satellite cells from quiescent state to activated state. Our analysis showed that functional overload induces the expression of MyoD in satellite cells and enhances the proliferative capacity and differentiation potential of these cells. The changes in satellite cell properties coincided with the inactivation of Notch signaling and the activation of Wnt signaling and likely involve modulation by transcription factors of the Sox family. These results indicate the effects of resistance exercise on the regulation of satellite cells and provide insight into the molecular mechanism of satellite cell activation following physical exercise.

Adult stem cells play an essential role in mammalian organ maintenance and repair throughout adulthood since they ensure that organs retain their ability to regenerate. The choice of cell fate by adult stem cells for cellular proliferation, self-renewal, and differentiation into multiple lineages is critically important for the homeostasis and biological function of individual organs. Responses of stem cells to stress, injury, or environmental change are precisely regulated by intercellular and intracellular signaling networks, and these molecular events cooperatively define the ability of stem cell throughout life. Skeletal muscle tissue represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle contains myogenic satellite cells and muscle-derived stem cells that retain multipotent differentiation abilities. These stem cell populations have the capacity for long-term proliferation and high self-renewal. The molecular mechanisms associated with deficits in skeletal muscle and stem cell function have been extensively studied. Muscle-derived stem cells are an obvious, readily available cell resource that offers promise for cell-based therapy and various applications in the field of tissue engineering. This review describes the strategies commonly used to identify and functionally characterize adult stem cells, focusing especially on satellite cells, and discusses their potential applications.

Naturally fertilized Microhyla ornata and Xenopus laevis embryos at dorsal lip of blastopore stage were exposed to 0.3, and 0.6% sodium chloride for high salinity treatment and dilute hydrochloric acid and sodium hydroxide for treatment with low and high pH, respectively. After treatment for different durations, embryos were studied morphologically and using in situ hybridization with selected genes important for normal development and for coping with environmental stress. Altered salinity and pH caused defects in axis formation and neural tube closure, delay in hatching, abnormal swimming of tadpoles and even developmental arrest. This was accompanied by significant decrease in the expression of selected development-regulating genes like goosecoid (required for gastrulation movements), brachyury (mesodermal marker gene), noggin (involved in neural induction), NCAM (required for neural cell adhesion) and MyoD (essential for muscle development), and considerable increase in the transcription of stress response genes hsp30 and hsp70. Altering the expression of embryonic genes could be one of the mechanisms through which environmental factors influence development of amphibian embryos.

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N- and O-linked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from α-2–3 to α-2–6, and the expression of α-1–2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a “glycome shift” upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs.