Sumiko Taira

Early patterning of the prospective midbrain-hindbrain boundary by the HES-related gene XHR1 in Xenopus embryos.

The molecular mechanisms that govern early patterning of anterior neuroectoderm (ANE) for the prospective brain region in vertebrates are largely unknown. Screening a cDNA library of Xenopus ANE led to the isolation of a Hairy and Enhancer of split- (HES)-related transcriptional repressor gene, Xenopus HES-related 1 (XHR1). XHR1 is specifically expressed in the midbrain-hindbrain boundary (MHB) region at the tailbud stage. The localized expression of XHR1 was detected as early as the early gastrula stage in the presumptive MHB region, an area just anterior to the involuting dorsal mesoderm that is demarcated by the expression of the gene Xbra. Expression of XHR1 was detected much earlier than that of other known MHB genes, XPax-2 and En-2, and also before the formation of the expression boundary between Xotx2 and Xgbx-2, suggesting that the early patterning of the presumptive MHB is independent of Xotx2 and Xgbx-2. Instead, the location of XHR1 expression appears to be determined in relation to the Xbra expression domain, since reduced or ectopic expression of Xbra altered the XHR1 expression domain according to the location of Xbra expression. In functional assays using mRNA injection, overexpression of dominant-negative forms of XHR1 in the MHB region led to marked reduction of XPax-2 and En-2 expression, and this phenotype was rescued by coexpression of wild-type XHR1. Furthermore, ectopically expressed wild-type XHR1 near the MHB region enhanced En-2 expression only in the MHB region but not in the region outside the MHB. These data suggest that XHR1 is required, but not sufficient by itself, to initiate MHB marker gene expression. Based on these data, we propose that XHR1 demarcates the prospective MHB region in the neuroectoderm in Xenopus early gastrulae.

Expression patterns of Xenopus FGF receptor-like 1/nou-darake in early Xenopus development resemble those of planarian nou-darake and Xenopus FGF8.

Fibroblast growth factors (FGFs) mediate many cell‐to‐cell signaling events during early development. Nou‐darake (ndk), a gene encoding an FGF receptor (FGFR)‐like molecule, was found to be highly and specifically expressed in the head region of the planarian Dugesia japonica, and its functional analyses provided strong molecular evidence for the existence of a brain‐inducing circuit based on the FGF signaling pathway. To analyze the role of ndk during vertebrate development, we isolated the Xenopus ortholog of ndk, the vertebrate FGFR‐like 1 gene (XFGFRL1). Expression of XFGFRL1/Xndk was first detected in the anterior region at the late gastrula stage and dramatically increased at the early neurula stage in an overall anterior mesendodermal region, including the prechordal plate, paraxial mesoderm, anterior endoderm, and archenteron roof. This anterior expression pattern resembles that of ndk in planarians, suggesting that the expression of FGFRL1/ndk is conserved in evolution between these two distantly diverged organisms. During the tail bud stages, XFGFRL1/Xndk expression was detected in multiple regions, including the forebrain, eyes, midbrain–hindbrain boundary, otic vesicles, visceral arches, and somites. In many of these regions, XFGFRL1/Xndk was coexpressed with XFGF8, indicating that XFGFRL1/Xndk is a member of the XFGF8 synexpression group, which includes sprouty, sef, and isthmin. Developmental Dynamics 230:700–707, 2004.

Role of crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis

The Xenopus gene crescent encodes a member of the secreted Frizzled-related protein (sFRP) family and is expressed in the head organizer region. However, the target and function of Crescent in early development are not well understood. Here, we describe a role of Crescent in the regulation of convergent extension movements (CEMs) during gastrulation and neurulation. We show that overexpression of Crescent in whole embryos or animal caps inhibits CEMs without affecting tissue specification. Consistent with this, Crescent efficiently forms complexes with Xwnt11 and Xwnt5a, in contrast to another sFRP, Frzb1. As expected, the inhibitory effect of Crescent or Xwnt11 on CEMs is cancelled when both proteins are coexpressed in the neuroectoderm. Interestingly, when coexpressed in the dorsal mesoderm, the activity of Xwnt11 is rather enhanced by Crescent. Supporting this finding, the inhibition of CEMs by Crescent in mesodermalized but not neuralized animal caps is reversed by the dominant-negative form of Cdc42, a putative mediator of Wnt/Ca2+ pathway. Antisense morpholino oligos for Crescent impair neural plate closure and elicit microcephalic embryos with a shortened trunk without affecting early tissue specification. These data suggest a potential role for Crescent in head formation by regulating a non-canonical Wnt pathway positively in the adjacent posterior mesoderm and negatively in the overlying anterior neuroectoderm.

B cells as antigen-presenting cells: Antibody production in vitro against a T-dependent antigen

It was examined whether B cells can serve as antigen-presenting cells (APC) in the antibody response to a T-dependent antigen, trinitrophenyl-ovalbumin (TNP-OVA). B cells purified from mice primed with TNP (TNP-B cells) responded to TNP-OVA in the presence of purified T cells sensitized with OVA (OVA-T cells). OVA-T cells required the addition of APC to proliferate in response to TNP-OVA. APC activity of TNP-B cells in the T-cell proliferation was abolished by 4000 R irradiation. Our experiments also revealed that an antibody response requires more adherent cells than the T-cell proliferation. These results indicate that adherent cells possibly accompanying the T- and B-cell preparations were at a less than functional level. There was genetic restriction between T and B cells for the antibody response. B cells in the pellet fraction of 70% Percoll density sedimentation behaved similarly to the unfractionated TNP-B cells in the antibody response. A T-cell clone specific for human gamma-globulin (HGG) also induced an anti-TNP antibody response in B cells from unprimed mice in the presence of TNP-HGG. These results suggest that B cells are able to elicit an antibody response to a T-dependent antigen in the presence of carrier-primed T cells without the participation of macrophages.

Depletion of mycoplasma from infected cell lines by limiting dilution in 6-methylpurine deoxyriboside

Cells contaminated with mycoplasma were cloned in the presence of 6-methylpurine deoxyriboside. The results showed that a limiting dilution in the culture medium containing the chemical efficiently depletes mycoplasma providing the cells are sensitive to 0.6-1.2 microM 6-methylpurine.

Accessory cell function in a Con A response: Role of Ia and interleukin 1☆

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.

RETRACTED: Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

B cells as antigen-presenting cells: Antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of I-Ab. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-gamma did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.

Secretion and role of interleukin-1-like factor in the antibody response to dinitrophenyl dextran and other type 2 T-independent antigens

Splenic adherent cells (SAC) were found to produce a humoral factor when they were cultured with dinitrophenyl-dextran or some other type 2 T-independent (TI-2) antigens. The factor substituted adherent cells in in vitro antibody responses to TI-2 antigens, and acted in an antigen-nonspecific and H-2-nonrestricted manner. T cells were indicated not to participate in the production of the factor. The factor was eluted from a Sephadex G-75 column with interleukin-1 (IL-1) activity to promote a thymocyte proliferation response to phytohemagglutinin. The molecular weight of the factor was estimated to be 16,000 Da. Both activities of the factor were absorbed by LBRM-33-1A5 cells. These results indicate that SAC secrete IL-1-like factor on direct stimulation by TI-2 antigens and that the secretion of the factor represents a major function of SAC in the antibody response to these antigens.

B cells as accessory cells in a Con A response of a T cell clone

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.