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C Clare Blackburn

Publications and source records attributed to C Clare Blackburn.

12 recordsLinked to original sources
PubMed2006

Bmp4 and Noggin expression during early thymus and parathyroid organogenesis.

The thymus and parathyroids originate from the third pharyngeal pouches, which form as endodermal outpocketings in the pharyngeal region beginning on embryonic day 9 (E9.0) of mouse development. Using organ-specific markers, we have previously shown that thymus and parathyroid-specific organ domains are established within the primordium prior to formation of the organs proper: Gcm2 expression defines the prospective parathyroid cells in the dorsal pouch from E9.5, while Foxn1 is expressed in the thymus domain from E11.25. Bmp (bone morphogenetic protein) signaling has been implicated in thymic epithelial cell differentiation and thymus organogenesis. In the present study, we report expression patterns of Bmp4 and Noggin, a Bmp4 antagonist, in the third pharyngeal pouch using two lacZ transgenic mouse strains. Results from this gene expression study revealed localization of Bmp4 expression to the ventral region of the third pharyngeal pouch endoderm at E10.5 and E11.5, in those cells that will express Foxn1 and form the thymus. Conversely, the expression of Noggin was confined to the dorsal region of the pouch and primordium at these stages, and thus appeared to be co-expressed with Gcm2 in the parathyroid domain. This represents the first detailed study of Bmp4 and Noggin expression during the early stages of thymus and parathyroid organogenesis.

Animals↗
PubMed2006

Identification of a tandem duplicated array in the Rhox alpha locus on mouse chromosome X.

The Rhox family of homeobox-containing genes maps to three gene dense clusters, alpha, beta, and gamma, on the mouse X chromosome. Rhox genes are expressed primarily in reproductive and extra-embryonic tissues and may be regulated by colinear mechanisms. Uniquely among family members, Rhox4 is also expressed in the developing and adult thymus, where its striking restriction to the third pharyngeal pouch has suggested a role in lineage specification. In this article we describe a previously unreported duplication within the Rhox alpha locus that contains seven copies of Rhox4 and eight copies of Rhox2 and Rhox3 in a tandem array. We further show that all seven Rhox4 copies are expressed, although preferential expression occurs and differs between tissues. In contrast to reproductive tissues, we found no evidence of colinear expression of the Rhox alpha cluster during thymus development. All subspecies of mice examined contained multiple copies of Rhox4. However, the only predicted ortholog of Rhox4, rat Rhox4, is present in a single copy, suggesting that the duplications arose at the time of rat and mouse lineage divergence. Finally, no changes in Rhox4 expression were detected in mice with defects in thymus organogenesis, placing it upstream or outside of established transcriptional pathways.

Amino Acid Sequence↗
PubMed2005

A critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to Leishmania mexicana.

Recognition of pathogen-associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage-specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome-wide transcriptional profiling of DC infected with wild-type or mutant promastigotes or wild-type amastigotes revealed that wild-type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1-/- parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild-type but not lpg1-/- promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild-type-infected animals. These in vivo and in vitro results suggest an LPG-dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down-regulate PAMP expression in mammalian hosts.

Animals↗
PubMed2005

In vivo and in vitro assays of thymic organogenesis.

We describe two complementary methods for the study of early thymus organogenesis in the mouse. The first is an in vitro technique for lineage analysis, where a chosen population of cells within the mouse embryo is labeled with a fluorescent cell tracker dye. The embryos are then transferred to whole embryo culture for a defined period, after which time the location of the labeled cells is determined with respect to the developing thymus. In the second method, an in vivo assay is used to determine the ability of a specific tissue type to form a structurally and functionally normal thymus. This method uses an ectopic grafting technique where the embryonic pharyngeal endoderm containing the prospective thymus tissue is carefully isolated and transplanted under the kidney capsule of an adult mouse. Together, these techniques have allowed the cell types that make a physical contribution to the formation of the thymic epithelium to be identified.

Animals↗
PubMed2004

Functional evidence for a single endodermal origin for the thymic epithelium.

T cell development depends critically on several distinct thymic epithelial cell types that are organized into two main compartments: cortex and medulla. The prevailing hypothesis suggests that these derive from ectoderm and endoderm, respectively. Here we show that lineage analysis provides no evidence for an ectodermal contribution to the thymic rudiment. We further demonstrate, via ectopic transplantation, that isolated pharyngeal endoderm can generate a functional thymus containing organized cortical and medullary regions and that this capacity is not potentiated by the presence of pharyngeal ectoderm. These data establish that the cortical and medullary thymic epithelial compartments derive from a single germ layer, the endoderm, thus refuting the 'dual-origin' model of thymic epithelial ontogeny.

Animals↗
PubMed2003

Uncompromised generation of a specific H-2DM-dependent peptide-MHC class II complex from exogenous antigen in Leishmania mexicana-infected dendritic cells.

Leishmania infection inhibits the capacity of macrophages (MPhi) to present antigens to CD4(+) T cells. Relocation of MHC class II and H-2DM to the parasitophorous vacuole (PV) and their subsequent degradation by the parasite may contribute to this defect. Dendritic cells (DC) are critical for initiation of primary T cell responses. DC can process Leishmania antigen and elicit Leishmania-specific T cells, but it is unknown whether exposure to Leishmania impairs this capacity. In particular, it is not clear whether DC containing live parasites efficiently process and present antigens. We investigated the ability of mouse bone marrow-derived DC infected with L. mexicana to generate pigeon cytochrome c (PCC) peptide-MHC class II complexes, using the mAb D4, which recognizes PCC(89-104) H-2E(k), and the PCC-specific T cell hybridoma 2B4. We show that H-2DM-dependent complex generation is not compromised by infection and that complexes are fully recognized by specific T cells. We further show that in contrast to infected MPhi, in infected DC cytoplasmic H-2DM is not down-regulated and not relocated to the parasite-containing vacuole. This observation may explain the continued ability of infected DC to present PCC, and also indicates differences in the habitat of these intracellular parasites in DC compared to MPhi.

Animals↗
PubMed2003

New serum-free in vitro culture technique for midgestation mouse embryos.

Current in vitro culture methods for mouse embryos are critically dependent on specially prepared rodent serum. Rodent serum requires careful preparation and stringent assessment of serum quality, while commercially available whole embryo culture serum is expensive and shows considerable lot variability. Thus, preparation and testing of suitable serum represents a considerable investment of time and resources, particularly for laboratories with only short-term embryo culture requirements. In addition, serum supplementation of culture medium may introduce unknown serum components that could interfere with interpretation of experimental results, especially where the study is geared towards analysis of a specific growth factor. Here we describe the composition of a standardized serum free culture medium comprised of commercially available stem cell media supplements. With this method, we have successfully cultured midgestation stage mouse embryos and demonstrated, using both morphological and gene expression criteria, that these embryos exhibited proper developmental progression. We believe this method to be a significant advance in whole embryo culture technology that will be of particular use to laboratories needing to utilize whole embryo culture to study midgestation organogenesis.

Animals↗
PubMed2003

A developmental look at thymus organogenesis: where do the non-hematopoietic cells in the thymus come from?

The origins of the non-hematopoietic cell types that comprise the thymic stroma remain a topic of considerable controversy. Three recent studies, using lineage analysis and other methods to determine the developmental potential of specific cell types within the thymus, have provided strong evidence of a single endodermal origin for all thymic epithelial cells. Together with other investigations that merge immunological and developmental biology approaches, these studies have suggested a new model of thymus organogenesis, and have begun to uncover the molecular pathways that control this process.

Animals↗
PubMed2002

Rapid constitutive generation of a specific peptide-MHC class II complex from intact exogenous protein in immature murine dendritic cells.

MHC class II molecules present peptides, derived largely from exogenous antigens, to CD4+ T cells. Complex-generation occurs mainly in the specialized late endosomal MHC class II-rich compartment (MIIC) vesicles of antigen-presenting cells (APC). Dendritic cells (DC) have been reported to store intact antigen in MIIC until the receipt of an activation signal, when they process it into peptide-MHC class II complexes. However, constitutive migration of DC from the periphery to secondary lymphoid organs has been observed, and antigen presentation by nonactivated DC is proposed to play a role in the induction of tolerance to peripheral antigens. Thus, constitutive peptide-MHC class II complex generation must also occur in DC in immunologically quiescent situations. We have used a monoclonal antibody that detects a specific peptide-MHC class II complex to directly demonstrate constitutive complex generation in immature murine DC. Protein-derived peptide-MHC class II complexes were detected by flow cytometry at the DC surface within 1 h of antigen exposure in the absence of an exogenous activation signal, and could be detected by confocal microscopy in the MIIC within 5 min of antigen exposure. This processing activity was endotoxin independent. These data provide evidence for constitutive peptide-MHC class II complex generation in immature DC, and thus support a role for this activity in the induction of peripheral tolerance.

Animals↗
PubMed2002

Identification and characterization of thymic epithelial progenitor cells.

T cell differentiation and repertoire selection depend critically on several distinct thymic epithelial cell types, whose lineage relationships are unclear. We have investigated these relationships via functional analysis of the epithelial populations within the thymic primordium. Here, we show that mAbs MTS20 and MTS24 identify a population of cells that, when purified and grafted ectopically, can differentiate into all known thymic epithelial cell types, attract lymphoid progenitors, and support CD4(+) and CD8(+) T cell development in nude mice. In contrast, other epithelial populations in the thymic primordium can fulfill none of these functions. These data establish that the MTS20(+)24(+) population is sufficient to generate a functional thymus in vivo and thus argue strongly that all thymic epithelial cell types derive from a common progenitor cell.

Animals↗
PubMed2002

One for all and all for one: thymic epithelial stem cells and regeneration.

It has long been believed that the thymic epithelial microenvironment originates from both the endodermal and ectodermal germ cell layers. However, a growing body of evidence indicates that such a dual origin is not the case, and that the diverse thymic epithelial populations all develop from a common epithelial stem cell. This article explores these data, investigates the identity of such cells and the signals that might control their expansion and differentiation, and considers the possibility of stem cell transplantation for thymic regeneration.

Animals↗