Muscle satellite cells contribute to muscle regeneration. We have used a Pax3GFP/+ mouse line to directly isolate (Pax3)(green fluorescent protein)-expressing muscle satellite cells, by flow cytometry from adult skeletal muscles, as a... more
Muscle satellite cells contribute to muscle regeneration. We have used a Pax3GFP/+ mouse line to directly isolate (Pax3)(green fluorescent protein)-expressing muscle satellite cells, by flow cytometry from adult skeletal muscles, as a homogeneous population of small, nongranular, Pax7+, CD34+, CD45-, Sca1- cells. The flow cytometry parameters thus established enabled us to isolate satellite cells from wild-type muscles. Such cells, grafted
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The embryonic sites in which progenitors of the hematopoietic lineages first emerge are ideal regions to characterize both the cells and environment needed to initiate blood cell development. For a number of years both the murine yolk sac... more
The embryonic sites in which progenitors of the hematopoietic lineages first emerge are ideal regions to characterize both the cells and environment needed to initiate blood cell development. For a number of years both the murine yolk sac and embryo have been recognized to contain progenitors of B lymphocytes. However, clonal, quantitative in vitro assays, which allow precise observation of
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Research Interests: Transcription regulation of development, Multidisciplinary, Nature, Hematopoiesis, Hematopoietic Stem Cells, and 13 moreLiver, Mice, Animals, Heart, Cell Transplantation, Molecular cloning, Cardiac Output Monitoring, Gene Targeting, B Lymphocytes, B Cell Development, Embryos, Fetal death, and High mobility group proteins
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... Development of B Lymphocytes from Lymphoid Committed and Uncommitted Progenitors. Ana Cumano 3 ,; Barbara L. Kee 1,2 ,; Dale A. Ramsden 1,2 ,; Aaron Marshall 1,2 ,; Christopher J. Paige 1,2 ,; Gillian E. Wu 1,2. Article first... more
... Development of B Lymphocytes from Lymphoid Committed and Uncommitted Progenitors. Ana Cumano 3 ,; Barbara L. Kee 1,2 ,; Dale A. Ramsden 1,2 ,; Aaron Marshall 1,2 ,; Christopher J. Paige 1,2 ,; Gillian E. Wu 1,2. Article first published online: 28 APR 2006. ...
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Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. We... more
Small ubiquitin-like modifiers (SUMOs) are attached to other proteins to regulate their function (sumoylation). We investigated the role of Ubc9, which covalently attaches SUMOs to proteins, in the gastrointestinal tract of adult mice. We investigated the effects of decreased sumoylation in adult mammals by generating mice with an inducible knockout (by injection of 4-hydroxytamoxifen) of the E2 enzyme Ubc9 (Ubc9fl/-/ROSA26-CreERT2 mice). We analyzed the phenotypes using a range of histologic techniques. Loss of Ubc9 from adult mice primarily affected the small intestine. Ubc9fl/-/ROSA26-CreERT2 mice died within 6 days of 4-hydroxytamoxifen injection, losing 20% or less of their body weight and developing severe diarrhea on the second day after injection. Surprisingly, other epithelial tissues appeared to be unaffected at that stage. Decreased sumoylation led to the depletion of the intestinal proliferative compartment and to the rapid disappearance of stem cells. Sumoylation was required to separate the proliferative and differentiated compartments from the crypt and control differentiation and function of the secretory lineage. Sumoylation was required for nucleus positioning and polarized organization of actin in the enterocytes. Loss of sumoylation caused detachment of the enterocytes from the basal lamina, as observed in tissue fragility diseases. We identified the intermediate filament keratin 8 as a SUMO substrate in epithelial cells. Sumoylation maintains intestinal stem cells and the architecture, mechanical stability, and function of the intestinal epithelium of mice.
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Research Interests: Genetics, Flow Cytometry, Transcription Regulation, Signal Transduction, Biological Sciences, and 13 moreNeural Crest, Genetic Testing, Mice, Animals, Genetic Screening, Skeletal Muscle, Muscle development, Reproducibility of Results, Base Sequence, Cell Survival, In Situ Hybridisation, Gene expression profiling, and Histone deacetylases
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Summary In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) re- gion. Here, we address the following question:... more
Summary In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/aorta-gonad-mesonephros (AGM) re- gion. Here, we address the following question: can the P-Sp/AGM environment support hemo- poietic differentiation as well as generate stem cells, and, conversely, are other sites where hemo- poietic differentiation occurs capable of generating stem cells? Although
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T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious... more
T cell maturation in Tcf-1(-/-) mice deteriorates progressively and halts completely around 6 mo of age. During fetal development thymocyte subpopulations seem normal, although total cell numbers are lower. By 4 to 6 wk of age, obvious blockades in the differentiation of CD4- 8- thymocytes are observed at two distinct stages (CD44+ 25+ and CD44- 25-), both of which are normally characterized by extensive proliferation. This lack of thymocyte expansion and/or differentiation was also observed when Tcf-1(-/-) progenitor cells from the aorta-gonad-mesonephros region (embryonic day 11.5), fetal liver (embryonic day 12.5/14.5), and fetal bone marrow (embryonic day 18.5) were allowed to differentiate in normal thymic lobes (fetal thymic organ cultures) or were injected intrathymically into normal recipients. Despite these apparent defects in thymocyte differentiation and expansion, adult Tcf-1(-/-) mice are immunocompetent, as they generate virus neutralizing Abs at normal titers. Further...
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The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and... more
The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-...
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The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that... more
The generation of T cells depends on the migration of hematopoietic progenitor cells to the thymus throughout life. The identity of the thymus-settling progenitor cells has been a matter of considerable debate. Here we found that thymopoiesis was initiated by a first wave of T cell lineage-restricted progenitor cells with limited capacity for population expansion but accelerated differentiation into mature T cells. They gave rise to αβ and γδ T cells that constituted Vγ3(+) dendritic epithelial T cells. Thymopoiesis was subsequently maintained by less-differentiated progenitor cells that retained the potential to develop into B cells and myeloid cells. In that second wave, which started before birth, progenitor cells had high proliferative capacity but delayed differentiation capacity and no longer gave rise to embryonic γδ T cells. Our work reconciles conflicting hypotheses on the nature of thymus-settling progenitor cells.
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LYMPHOCYTES (B and T cells) derive continuously from the same multipotential stem cells that produce myeloid cells, including erythrocytes, granulocytes and macrophages. Tri- and bipotential myeloid intermediates between the... more
LYMPHOCYTES (B and T cells) derive continuously from the same multipotential stem cells that produce myeloid cells, including erythrocytes, granulocytes and macrophages. Tri- and bipotential myeloid intermediates between the multipotential stem cells and later unipotential cells have been identified using clonal methods in culture. Although similar methods have detected committed pre-B cells in mouse fetal liver, earlier progenitors with additional non-B lineage options have not been demonstrated in normal tissues. We report the characterization and purification of fetal liver cells that generate clones containing both macrophages and B cells, identified biochemically and morphologically. The common origin of the two cell types was shown by culture of single precursor cells. Their dual potential and unrearranged immunoglobulin loci place the precursors before exclusive B-lineage commitment in the haematopoietic hierarchy. The availability of such cells in purified form will allow direct study of lineage choice in cells having both lymphoid and non-lymphoid options.
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For many years it has been assumed that the ontogeny of the mammalian hematopoietic system involves sequential transfers of hematopoietic stem cells (HSCs) generated in the yolk sac blood islands, to successive hematopoietic organs as... more
For many years it has been assumed that the ontogeny of the mammalian hematopoietic system involves sequential transfers of hematopoietic stem cells (HSCs) generated in the yolk sac blood islands, to successive hematopoietic organs as these become active in the embryo (fetal liver, thymus, spleen and eventually bone marrow). Very little was known about early events related to hematopoiesis that could take place during the 4.5 day gap separating the appearance of the yolk sac blood islands and the stage of a fully active fetal liver. Experiments performed in birds documented that the yolk sac only produce erythro-myeloid precursors that become extinct after the emergence of a second wave of intra-embryonic HSCs from the region neighbouring the dorsal aorta. The experimental approaches undertaken over the last ten years in the murine model, which are reviewed here, led to the conclusion that the rules governing avian hematopoietic development basically apply to higher vertebrates.
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The repertoire of antibody variable (V) regions has been subject to evolutionary selection, affecting both the diversity of V region genes in the germline and their expression in the B lymphocyte population and its subsets. In ontogeny,... more
The repertoire of antibody variable (V) regions has been subject to evolutionary selection, affecting both the diversity of V region genes in the germline and their expression in the B lymphocyte population and its subsets. In ontogeny, contact with an antigen leads to the expansion of B cells expressing antibodies complementary to it. In a defined phase of B cell differentiation, new sets of V regions are generated from the existing repertoire through somatic hypermutation. Cells carrying advantageous antibody mutants are selected into the memory compartment and produce a stable secondary response upon reexposure to the antigen.
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In most cells, the NF-kappaB transcription factor is sequestered in the cytoplasm by interaction with inhibitory proteins, the IkappaBs. Here, we show that combined IkappaBalpha/IkappaBepsilon deficiency in mice leads to neonatal death,... more
In most cells, the NF-kappaB transcription factor is sequestered in the cytoplasm by interaction with inhibitory proteins, the IkappaBs. Here, we show that combined IkappaBalpha/IkappaBepsilon deficiency in mice leads to neonatal death, elevated kappaB binding activity, overexpression of NF-kappaB target genes, and disruption of lymphocyte production. In IkappaBalpha/IkappaBepsilon-deficient fetuses, B220+IgM+ B cells and single-positive T cells die by apoptosis. In adults, IkappaBalpha-/-IkappaBepsilon-/- reconstituted chimeras exhibit a nearly complete absence of T and B cells that is not rescued by cotransfer with wild-type bone marrow. These findings demonstrate that IkappaBs tightly control NF-kappaB activity in vivo and that increased NF-kappaB activity intrinsically impairs lymphocyte survival. Because reduction or rise of NF-kappaB activity leads to similar dysfunction, they also reveal that only a narrow window of NF-kappaB activity is tolerated by lymphocytes.
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We show by an in vitro approach that multipotent hemopoietic cells can be detected in the body of the mouse embryo between the stages of 10-25 somites (8.5-9.5 days of gestation)--i.e., prior to liver colonization (28-32 pairs of... more
We show by an in vitro approach that multipotent hemopoietic cells can be detected in the body of the mouse embryo between the stages of 10-25 somites (8.5-9.5 days of gestation)--i.e., prior to liver colonization (28-32 pairs of somites). Interestingly, hemopoietic cells appear in parallel in this location, the paraaortic splanchnopleura, and in the yolk sac, where they represent a new generation by reference to the primitive hemopoietic stem cells. Lymphoid cell clones, which could differentiate into mature B cells, were obtained from yolk sac and paraaortic splanchnopleura cell preparations but not from other tissues of the embryonic body. These B-cell precursors were first detected around the stage of 10 somites; thereafter, their initial minute numbers increased in parallel in the yolk sac and the paraaortic splanchnopleura, suggesting that their emergence in the two sites was simultaneous. By single cell manipulation, we show that these precursors can generate B and T lymphocytes and myeloid cells; these precursors can thus be defined as multipotent hemopoietic cells.