Biomedical subjects

D Cado

Publications and source records attributed to D Cado.

30 records · Page 2Linked to original sources

PubMed1997

Notch activity influences the alphabeta versus gammadelta T cell lineage decision.

The choice between the alphabeta or gammadelta T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as gammadelta T cells than as alphabeta T cells, implying that reduced Notch activity favors the gammadelta T cell fate over the alphabeta T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional gammadeltaTCR gene rearrangements to adopt the alphabeta T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the alphabeta versus gammadelta T cell lineage decision.

Animals↗

PubMed1997

A targeted mutation at the T-cell receptor alpha/delta locus impairs T-cell development and reveals the presence of the nearby antiapoptosis gene Dad1.

Locus control regions are cis gene regulatory elements comprised of DNase I-hypersensitive sites. These regions usually do not stimulate transcription outside of a chromosomal context, and therefore their ability to regulate the expression of genes is thought to occur through the modification of chromatin accessibility. A locus control region is located downstream of the T-cell receptor (TCR) alpha/delta locus on mouse chromosome 14. This locus control region is known to drive T-cell-specific TCR alpha transcription in transgenic mice. In this report, we describe a targeted deletion of this locus control region and show that this mutation acts at a critical checkpoint in alphabeta T-cell development, between the TCR-intermediate and TCR-high stages. Our analysis further reveals that the antiapoptosis gene Dad1 is at the 3' end of the TCR alpha/delta locus and that Dad1 is required for embryogenesis. We show that mouse Dad1 has a broader expression pattern than the TCR genes, in terms of both tissue and temporal specificity. Finally, we report that the chromatin between TCR alpha and Dad1 is DNase I hypersensitive in a variety of cell types, thus correlating with Dad1 expression and raising the possibility that Dad1 regulatory sequences reside in this region.

Alleles↗

PubMed1996

An activated form of Notch influences the choice between CD4 and CD8 T cell lineages.

Notch is a transmembrane receptor that controls cell fate decisions in Drosophila and whose role in mammalian cell fate decisions is beginning to be explored. We are investigating the role of Notch in a well-studied mammalian cell fate decision: the choice between the CD8 and CD4 T cell lineages. Here we report that expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision.

Animals↗

PubMed1996

Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice.

Animals↗

PubMed1995

A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection.

The transcription factor Nur77, an orphan member of the nuclear hormone receptor superfamily, is highly expressed during T cell receptor-signaled apoptosis, suggesting a possible role for Nur77 in negative selection. We examined this by generating two sets of transgenic mice. In one set of mice, a dominant-negative Nur77 mutant is constitutively expressed and the other in which wild-type Nur77 protein is constitutively expressed in developing thymocytes. We report that inhibition of endogenous Nur77 by the dominant-negative mutant perturbed T cell development and inhibited antigen-induced negative selection in F5T cell receptor transgenic mice. Constitutive expression of wild-type Nur77 protein induced apoptosis in developing thymocytes, resulting in a decreased number of thymocytes and mature T cells. Together, these data support a role for Nur77 in the downstream signaling events in antigen-induced negative selection.

Animals↗

PubMed1995

The role of short homology repeats and TdT in generation of the invariant gamma delta antigen receptor repertoire in the fetal thymus.

Fetal thymic and adult epithelial V gamma 3+ and V gamma 4+ T cells express gamma delta antigen receptors (TCR) with invariant junctions lacking N nucleotides. Using transgenic recombination substrates, we show that di- or trinucleotide repeats, either in the coding region or in P elements, have strong effects on the site of recombination. In other mice bearing a terminal deoxynucleotidyl transferase (TdT) transgene under the control of the CD2 promoter, we found that the frequency of canonical junctions was markedly reduced with a concomitant increase in in-frame noncanonical junctions with N nucleotides. Together, our results show that short homology repeats direct the site of rearrangement and thus play a critical role in the generation of gamma delta T cell receptor canonical junctions. Increased TdT activity in V gamma 3+ T cells has a inhibitory effect on junctional homogeneity in these cells.

Animals↗

PubMed1995

A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.

We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.

Animals↗

PubMed1994

A role for the cytoplasmic tail of the beta chain of CD8 in thymic selection.

The CD8 coreceptor plays a critical role in the recognition of foreign antigens by mature T cells and in the development of class I-restricted T cells. CD8 can be expressed on the surface of T cells as either a heterodimer composed of an alpha and beta chain, or as a homodimer composed of two alpha chains. In this report, we show that a CD8 beta transgene that lacks a cytoplasmic domain can suppress expression of wild-type endogenous CD8 beta and act as a dominant negative mutation. We show that this dominant negative CD8 beta transgene interferes with the development of mature CD8 T cells to different extents depending on the individual class I-restricted TCR. These data suggest CD8 beta plays a role in thymic development, and that different class I-restricted TCRs differ in their dependence on the cytoplasmic tail of CD8 beta.

Animals↗

PubMed1994

A locus control region in the T cell receptor alpha/delta locus.

A locus control region (LCR) is a set of cis-acting elements that regulate chromatin accessibility of a gene locus. In the T cell receptor (TCR) alpha/delta locus, an LCR might regulate the differential tissue and developmental expression and the rearrangement of TCR alpha and delta genes. We have localized a region 3' of the TCR alpha/delta locus containing eight T cell-specific nuclease hypersensitive domains (HS-1 to HS-8), which fit the characteristics of an LCR. In transgenic mice, a TCR alpha gene linked to this region is expressed at a high level, independent of the site of integration and correlates with gene copy number. The transgene is expressed in the alpha beta but not the gamma delta T cell subset and is activated at the right time during development. Proper LCR function requires the region containing HS-2 to HS-6. We propose a model of LCR competition to explain the differential regulation of TCR alpha versus delta genes during development.

Animals↗

PubMed1993

Selection is not required to produce invariant T-cell receptor gamma-gene junctional sequences.

Recombination of V-, D- and J-gene segments can generate an enormous diversity of T-cell antigen receptor (TCR) gene sequences. Although many gamma delta T cells fully exploit this diversification process, those in the epidermal and vaginal epithelium do not, predominantly expressing invariant gamma delta receptors in which the V-(D)-J junctional sequences in almost all the productive rearrangements are identical. The almost exclusive use of identical TCRs by cells in these sites is thought to reflect recognition of a stress-induced autologous antigen. To explain the prevalence of the invariant junctional sequences, it has been proposed that thymic selection operates on a population of originally diverse progenitor cells, resulting in a homogeneous repertoire. Alternatively the invariant sequences may result from biases in the recombination machinery in the fetal thymic progenitors of these cells. We report here the use of mice into which mutated TCR gamma-gene rearrangement substrates have been introduced as transgenes to demonstrate directly that the canonical TCR V gamma 3-J gamma 1 and V gamma 4-J gamma 1 sequences occur at high frequency in the absence of the possibility of selection for the protein products.

Animals↗

PubMed1993

Two enhancer regions in the mouse En-2 locus direct expression to the mid/hindbrain region and mandibular myoblasts.

An En-2/lacZ gene fusion containing 9.5 kb of En-2 genomic DNA was capable of directing lacZ expression in an En-2-specific manner both temporally and spatially during embryogenesis and in the adult. lacZ expression was confined in the embryo to cells within the mid/hindbrain and mandibular arch regions and in the adult to cells of the molecular and granular layers of the cerebellum, and within the pons and colliculi regions. Interestingly, in the adult, transgene expression patterns within the cerebellum in two lines appeared to mark distinct anterior-posterior compartments. Analysis of the expression pattern of this transgene, in fetal and adult mice lacking a functional En-2 protein, provided evidence that the En-2 gene in mouse is not autoregulated. Deletion analysis of the En-2 genomic region and the use of a heterologous promoter identified two enhancer-containing regions of 1.5 and 1.0 kb in length, 5' of the transcribed sequences, which independently directed expression in the embryo to either the mid/hindbrain region or mandibular myoblasts, respectively. The 1.5 kb fragment contains the most anterior neural enhancer and the 1.0 kb fragment, the earliest myogenic enhancer thus far characterized. These En-2-specific regulatory regions can now be used in a biochemical analysis to identify proteins important in anterior-posterior patterning of the vertebrate CNS and in the specification of muscle identity as well as in a mutational analysis to direct expression of other developmentally important genes to these regions.

Animals↗

PubMed1991

Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis.

Treatment with retinoic acid (RA) is known to produce complex teratogenic effects in vertebrates, and its presence in the developing embryo as an endogenous substance has led to the suggestion that RA might be a natural morphogenetic agent. Although our understanding of the molecular mechanism of RA action has improved considerably with the identification of nuclear receptors for RA (RARs) and RA-responsive genes, the exact relationship between the proposed morphogenetic activity of RA and its teratogenic effects remains to be characterized. Here, we show that a RA response element (RARE) present in the RAR beta gene can direct specific spatial and temporal expression of an hsplacZ transgene during mouse embryogenesis. In the early embryo, the transgene is expressed in a specific anterior-posterior domain that is completely obliterated by treatment of pregnant mice with teratogenic doses of RA. The expression of the transgene becomes more restricted as organogenesis progresses and mimics closely the reported expression of the RAR beta gene. These results suggest that, in vivo, some of the morphogenetic effects of RA could be mediated through localized transcriptional activity controlled by the various RARs. The specific pattern of expression of the RAREhsplacZ transgene does not correlate with the proposed sites of action of RA as defined by its teratogenic effects but does support a role for RA in early anterior-posterior patterning along the body axis.

Animals↗