Systematic Screening and Expression Analysis of the Head Organizer Genes in Xenopus Embryos

AbstractWe describe here a systematic screen of an anterior endomesoderm (AEM) cDNA library to isolate novel genes which are expressed in the head organizer region. After removing clones which hybridized to labeled cDNA probes synthesized with total RNA from a trunk region of tailbud embryos, the 5′ ends of 1039 randomly picked cDNA clones were sequenced to make expressed sequence tags (ESTs), which formed 754 tentative unique clusters. Those clusters were compared against public databases and classified according to similarities found to other genes and gene products. Of them, 151 clusters were identified as known Xenopus genes, including eight organizer-specific ones (5.3%). Gene expression pattern screening was performed for 198 unique clones, which were selected because they either have no known function or are predicted to be developmental regulators in other species. The screen revealed nine possible organizer-specific clones (4.5%), four of which appeared to be expressed in the head organizer region. Detailed expression analysis from gastrula to neurula stages showed that these four genes named crescent, P7E4 (homologous to human hypothetical genes), P8F7 (an unclassified gene), and P17F11 (homologous to human and Arabidopsis hypothetical genes) demarcate spatiotemporally distinct subregions of the AEM corresponding to the head organizer region. These results indicate that our screening strategy is effective in isolating novel region-specific genes

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer

Polycomb Limits the Neurogenic Competence of Neural Precursor Cells to Promote Astrogenic Fate Transition

14 páginas, 8 figuras, 1 tabla suplementaria, 1 documento suplementario -- PAGS nros. 600-613During neocortical development, neural precursor cells (NPCs, or neural stem cells) produce neurons first and astrocytes later. Although the timing of the fate switch from neurogenic to astrogenic is critical for determining the number of neurons, the mechanisms are not fully understood. Here, we show that the polycomb group complex (PcG) restricts neurogenic competence of NPCs and promotes the transition of NPC fate from neurogenic to astrogenic. Inactivation of PcG by knockout of the Ring1B or Ezh2 gene or Eed knockdown prolonged the neurogenic phase of NPCs and delayed the onset of the astrogenic phase. Moreover, PcG was found to repress the promoter of the proneural gene neurogenin1 in a developmental-stage-dependent manner. These results demonstrate a role of PcG: the temporal regulation of NPC fateThis work was supported by Grant-in-Aid for Scientific Research (A) and on Priority Areas-Molecular Brain Science from the MEXT of Japan (20022010Peer reviewe

NKT-TCR-transfected γδT cells show cytotoxicity against glycolipid-expressing target cells and K562 cells.

<p>(A, B) To test the lysis of ligand-loaded target cells, the cytotoxic activity of γδT (A) or γδT-NKT-TCR(+) cells (B) against CD1d-HEK293, CD1d-HEK293/Gal, CD1d-HEK293/Zol was assessed by LDH assay at an E:T ratio of 10. (C, D) γδT (C) or γδT-NKT-TCR(+) cells (E) were stimulated by CD1d-HEK293/Gal, CD1d-HEK293/Zol or both and then assessed for cytotoxicity against K562. Data are mean ±SEM from triplicates and representative of 4 healthy donors with similar results. (**p<0.01, ***p<0.005)</p

NKT-TCR mRNA transfection confers functionality.

<p>As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131477#pone.0131477.g002" target="_blank">Fig 2</a>, T cells were electroporated with NKT-TCR mRNA. (A) Six hours after NKT-TCR mRNA electroporation, Vα24⁺Vβ11⁺ cells (left panel) and Vα24^-Vβ11^- cells (right panel) were sorted. (B) Each population was exposed to plate-bound CD1d-dimer loaded with α-GalCer for the indicated periods of times. As positive control, PBMCs were activated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) for 5 min. Cells were then lysed and pERK1/2 and ERK1/2 were detected by Western blotting (upper panel). The bar diagram shows a densitometric analysis of the phosphorylated ERK signal from the upper band normalized by the corresponding total ERK signal (lower panel). (C) Sorted 1x10⁵ Vα24⁺Vβ11⁺ cells (top), Vα24^-Vβ11^- cells (middle) or NKT line (bottom) were cocultured with 1x10⁴ CD1d-HEK293 loaded with or without α-GalCer for 24 h. IFN-γ and IL-4 production in the culture supernatants was analyzed by ELISA. Data are mean ±SEM from triplicates and representative of 5 healthy donors with similar results. (***p<0.005 for CD1d-HEK293/Gal vs. CD1d-HEK293/non and T only)</p

Transfection efficacy of NKT-TCR mRNA into primary T cells.

<p>PBMCs were cultured for 3 days in the presence of anti-CD3 and anti-CD28 Ab. Then, they were electroporated with NKT-TCR mRNA. Subsequently, the expression of NKT-TCR was analyzed at indicated times after mRNA electoroporation. The expression of NKT-TCR was analyzed using anti-CD3 and anti-6B11 antibodies. Representative flow cytometry data are shown (A) as well as mean percentages ±SEM from 5 healthy donors (B).</p