Ectomesenchymal chondromyxoid tumor of the tongue: insights on histogenesis.

OBJECTIVE: The objective of this study was to investigate the histogenesis of ectomesenchymal chondromyxoid tumors (ECTs) of the tongue. STUDY DESIGN: The biochemical characteristics of a rarely occurring tumor of the tongue were analyzed by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR), and its biological properties were assessed in primary culture in serum-free media. RESULTS: Immunohistochemistry showed that the tumor cells were strongly positive for vimentin, S-100, and glial fibrillary acidic protein (GFAP), but negative for cytokeratin and epithelial membrane antigen. In primary cultures, the cells derived from the ECT were morphologically similar to neuronal cells and expressed Nanog, GFAP, and MAP2. RT-PCR analysis of the surgical specimen was positive for OCT3/4, Sox2, Nanog, MAP2, and CD105 mRNAs. CONCLUSIONS: The results of the present study indicate that ECTs originate from the ectomesenchymal cells of the neural crest and are similar in their molecular and biological characteristics to undifferentiated mesenchymal stem cells.

Modulation of activin A-induced differentiation in vitro by vascular endothelial growth factor in Xenopus presumptive ectodermal cells.

We have previously demonstrated that activin A at low concentrations induced ventral mesoderm including blood-like cells from Xenopus animal caps and that beating heart could be also induced from animal caps treated with 100 ng/ml activin A, suggesting that activin A might be involved in cardiac vasculogenesis. A vascular endothelial growth factor (VEGF) is a powerful mitogen for endothelial cells and is an inducer and regulator of angiogenesis. However, VEGF function in Xenopus development is not clearly identified. In this study, we determined the effect of VEGF on activin A-induced differentiation of animal cap. The VEGF induced duct-like structure composed of Flk-1-positive cells together with the induction of nonvascular tissues, such as neural tissues. This histological result was coincident with our reverse transcriptase-polymerase chain reaction analysis that VEGF together with activin A promoted the expression of Xenopus N-CAM and Xenopus brachyury. This study suggests that VEGF has additional biological activities besides angiogenesis, and arises a different function that VEGF induces stroma cell migration or recruitment that are required for blood vessel formation. This differentiation system will aid in the understanding of angiogenesis during early development.

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen.

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.

Long-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture medium.

Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3-4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro.

Activin A induces craniofacial cartilage from undifferentiated Xenopus ectoderm in vitro.

Activin A has potent mesoderm-inducing activity in amphibian embryos and induces various mesodermal tissues in vitro from the isolated presumptive ectoderm. By using a sandwich culture method established to examine activin A activity, we previously demonstrated that activin-treated ectoderm can function as both a head and trunk-tail organizer, depending on the concentration of activin A. By using activin A and undifferentiated presumptive ectoderm, it is theoretically possible to reproduce embryonic induction. Here, we test this hypothesis by studying the induction of cartilage tissue by using the sandwich-culture method. In the sandwiched explants, the mesenchymal cell condensation expressed type II collagen and cartilage homeoprotein-1 mRNA, and subsequently, cartilage were induced as they are in vivo. goosecoid (gsc) mRNA was prominently expressed in the cartilage in the explants. Xenopus distal-less 4 (X-dll4) mRNA was expressed throughout the explants. In Xenopus embryos, gsc expression is restricted to the cartilage of the lower jaw, and X-dll4 is widely expressed in the ventral head region, including craniofacial cartilage. These finding suggest that the craniofacial cartilage, especially lower jaw cartilage, was induced in the activin-treated sandwiched explants. In addition, a normal developmental pattern was recapitulated at the histological and genetic level. This work also suggests that the craniofacial cartilage-induction pathway is downstream of activin A. This study presents a model system suitable for the in vitro analysis of craniofacial cartilage induction in vertebrates.