Yoshio Yaoita

Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II

Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells1,2. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin-secreting cells3–6. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin-4, IL-4) from the 2.19 mouse T-cell line7. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF)9–12, which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities10–12: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-primed B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity13. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine.

Induction of Apoptosis and CPP32 Expression by Thyroid Hormone in a Myoblastic Cell Line Derived from Tadpole Tail

During amphibian metamorphosis, the tail and gills that are useful in aquatic life but inappropriate for terrestrial activity are induced to degenerate completely in several days by endogenous thyroid hormone (TH). The dramatic resorption of the tadpole tail has attracted a good deal of attention as an experimental system of cell death, but the mechanism has not been well characterized. To facilitate in vitro analysis, we have established a myoblast cell line (XLT-15) derived from the Xenopus laevis tadpole tail. This cultured cell line died in response to TH and exhibited positive TUNEL reaction and internucleosomal DNA cleavage. Simultaneously, expression of the Xenopus CPP32/apopain/Yama gene was up-regulated by TH in the cell line as it is in regressing tadpole tail, whereas interleukin-1β-converting enzyme (ICE) mRNA is around 1 copy/cell in tail and undetectable in XLT-15 cells. A CPP32/apopain/Yama inhibitor (acetyl-Asp-Glu-Val-Asp-aldehyde) prevented TH-induced apoptosis of XLT-15 cells, but an ICE inhibitor (acetyl-Tyr-Val-Ala-Asp-aldehyde) did not. These results suggested that an increase of CPP32/apopain/Yama gene expression is involved in TH-dependent apoptosis of XLT-15 and tadpole tail resorption during metamorphosis.

Dual mechanisms governing muscle cell death in tadpole tail during amphibian metamorphosis

The tadpole tail, which is twice as long as the body, is induced to resorb completely by thyroid hormone within several days during the anuran metamorphosis. To investigate the underlying mechanism, we undertook two approaches. First, we examined the effect of dominant‐negative thyroid hormone receptor (DNTR) on muscle cell death in vitro. The overexpression of DNTR suppressed the death of a tail‐derived myoblastic cell line induced by thyroid hormone. Second, tadpole tails were injected with a reporter gene and the DNTR expression construct, and the reporter gene expression in muscle cells was followed during the spontaneous metamorphosis. DNTR overexpression inhibited a decrease of the reporter gene expression that began at stage 57 in the control tadpoles but only delayed massive muscle cell death at stage 63 when tails shrink very rapidly. Some remained even a few weeks after the metamorphosis, although most DNTR‐overexpressing cells died by the end of the metamorphosis. These results led us to propose that thyroid hormone induces the suicide of muscle cells (the cell‐autonomous death) in the tail between stage 57 and 62 and that both the murder and suicide mechanisms execute muscle cell death in stage 62–64 to remove muscle promptly and completely. Developmental Dynamics 227:246–255, 2003.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

Generation of albino Xenopus tropicalis using zinc-finger nucleases

To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc‐finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN‐induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.

Expression of matrix metalloproteinase genes in regressing or remodeling organs during amphibian metamorphosis

Several matrix metalloproteinases (MMP) are induced by thyroid hormone (TH) during the climax of amphibian metamorphosis and play a pivotal role in the remodeling of the intestine and the regressing tail and gills by degrading the extracellular matrix (ECM). We compared MMP gene expression levels precisely by quantitative real‐time reverse transcription‐polymerase chain reaction. The expression of MMP genes increases prominently at Nieuwkoop and Faber (NF) stages 60, 60–61 and 62 in the intestine, gills and tail, respectively, when the drastic morphological changes start in each organ. Gene expression analysis in the TH‐treated tadpoles and cell line revealed that MMP mRNAs are upregulated in response to TH quickly within several hours to low levels and then increase in a day to high levels. All TH‐induced MMP genes have TH response elements (TREs). The presence of high affinity TREs in MMP genes correlates with early TH‐induction. Based on these results, we propose that TH stimulates the transcription of MMP genes through TREs within several hours to low levels and then brings about the main increase of mRNAs by TH‐induced transcriptional factors, including TH receptor β, in a cell type‐specific transcriptional environment.

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP‐9TH, similar to the previously reported Xenopus laevis MMP‐9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP‐9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone‐treated tail‐derived myoblastic cultured cells, while MMP‐9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP‐9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP‐9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.

Chromosomal mapping of the mouse IL-4 and human IL-5 genes

We mapped the mouse interleukin (IL)-4 gene on chromosome 11 by restriction fragment length polymorphism using recombinant inbred mouse strains. The human IL-5 gene was mapped on chromosome 5q 23.3-31.1 by in situ hybridization. Because the granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-3 genes were previously mapped on mouse chromosome 11 (within a 230-kb region) and human chromosome 5, the IL-4 and IL-5 genes are likely to cluster on the same chromosomes with the GM-CSF and IL-3 genes in both species.

Comparison of TALEN scaffolds in Xenopus tropicalis

Summary Transcription activator-like effector nucleases (TALENs) are facile and potent tools used to modify a gene of interest for targeted gene knockout. TALENs consist of an N-terminal domain, a DNA-binding domain, and a C-terminal domain, which are derived from a transcription activator-like effector, and the non-specific nuclease domain of FokI. Using Xenopus tropicalis (X. tropicalis), we compared the toxicities and somatic mutation activities of four TALEN architectures in a side-by-side manner: a basic TALEN, a scaffold with the same truncated N- and C-terminal domains as GoldyTALEN, a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain, and a scaffold with the truncated N- and C-terminal domains and an obligate heterodimeric Sharkey nuclease domain. The strongest phenotype and targeted somatic gene mutation were induced by the injection of TALEN mRNAs containing the truncated N- and C-terminal domains and an obligate heterodimeric nuclease domain. The obligate heterodimeric TALENs exhibited reduced toxicity compared to the homodimeric TALENs, and the homodimeric GoldyTALEN-type scaffold showed both a high activity of somatic gene modification and high toxicity. The Sharkey mutation in the heterodimeric nuclease domain reduced the TALEN-mediated somatic mutagenesis.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis.

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue‐specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR‐transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3—the more active form of TH. D2 mRNA was induced in the TR‐overexpressing cells by low levels of TH. D2 promoter contains a TH‐response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cells ability to respond to low levels of T4.