Notch3 in generation and maintenance of the T lineage.

Summary

Principal Investigator: Howard T Petrie
Abstract: PROJECT SUMMARY The thymus produces new T lymphocytes throughout life to maintain peripheral homeostasis and immune function. Unlike other tissues that undergo steady-state differentiation, the thymus contains no self-renewing stem or progenitor cells, and instead depends on constant recruitment of marrow-derived progenitors that circulate in the blood. The earliest intrathymic progenitors are multi-potent, but notably, lack B lineage potential, and thus do not correspond to any known progenitor in the marrow. Microenvironmental conditions inside the thymus induce these multipotent progenitors to adopt the T lineage fate, and to asymmetrically differentiate into multiple different T lineages. While Notch1 (N1) has been shown to play a key role in T lineage specification, it is inadequate to explain the complex process of generating multiple T lineages in the thymus. In conjunction with PHS award R21AI53739, we sought to identify other putative regulators of T lineage differentiation. Among the genes that we identified was Notch3 (N3). N3 knockout mice had already been generated and were found by other to be overtly normal, a finding that was confirmed in our laboratory. Surprisingly, however, we found that N3 deficiency results in a progressive, age-related degeneration of T progenitor activity in bone marrow. This phenotype is consistent with a human disease (CADASIL) associated with N3 deficiency (a heritable mutation), which is characterized by age-related (adult-onset) degeneration of vascular smooth muscle cells and recurrent strokes. Our current findings reveal N3 mutation to be the first known genetic defect leading to age-related degeneration of T lineage precursors in bone marrow. Further, the specificity of this requirement for N3 in marrow suggests that N3 may represent a marker for the long sought-after precursor to T lineage cells in marrow. Concisely stated, the goals of this project are 1) to expand and finalize for publication our finding that N3 is required to maintain T progenitor activity in marrow;2) to identify N3-expressing cells in marrow, and compare their lineage potentials to those of early intrathymic progenitors (notably, for the presence of B lineage potential);3) to ascertain whether N3 has a role in the thymus as well as the marrow, and to what extent this function overlaps with that of N1;and 4) to identify the targets of N3 signaling in the T lineage, and thus begin to define its molecular function. The approaches involve in vivo and in vitro assays for T lineage potential in bone marrow in mice at various ages;lineage tracing the progeny of marrow cells that signal through N3, using a N3:Cre fusion protein knock-in to conditionally activate a fluorescent reporter;in vivo and in vitro assessment of lineage potential in the reporter-positive cells;intrathymic deletion of N3, and intrathymic deletion of N1 at intermediate stage in N3-deficient mice;assessment of N3 signaling activity in thymus and marrow progenitors, using a N3:Gal3 fusion protein knock-in; and assessment of gene expression in cells that signal through N3, as well as their counterparts in young N3 knockout mice.
Funding Period: 2009-03-15 - 2015-02-28
more information: NIH RePORT

Top Publications

  1. pmc Spatial mapping of thymic stromal microenvironments reveals unique features influencing T lymphoid differentiation
    Ann V Griffith
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
    Immunity 31:999-1009. 2009
  2. ncbi Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes
    Mahmood Mohtashami
    Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada
    J Immunol 185:867-76. 2010
  3. pmc Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis
    Jianjun Shi
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL, USA
    Blood 118:2511-9. 2011
  4. ncbi Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth
    Ann V Griffith
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
    Aging Cell 11:169-77. 2012
  5. pmc Activation kinetics and off-target effects of thymus-initiated cre transgenes
    Jianjun Shi
    The Scripps Research Institute, Jupiter, Florida, United States of America
    PLoS ONE 7:e46590. 2012

Research Grants

Detail Information

Publications5

  1. pmc Spatial mapping of thymic stromal microenvironments reveals unique features influencing T lymphoid differentiation
    Ann V Griffith
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
    Immunity 31:999-1009. 2009
    ..Our findings compel reexamination of how cell migration, lineage specification, and proliferation are controlled by thymic architecture and provide an in-depth resource for global characterization of this control...
  2. ncbi Direct comparison of Dll1- and Dll4-mediated Notch activation levels shows differential lymphomyeloid lineage commitment outcomes
    Mahmood Mohtashami
    Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada
    J Immunol 185:867-76. 2010
    ....
  3. pmc Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis
    Jianjun Shi
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL, USA
    Blood 118:2511-9. 2011
    ....
  4. ncbi Persistent degenerative changes in thymic organ function revealed by an inducible model of organ regrowth
    Ann V Griffith
    Department of Cancer Biology, The Scripps Research Institute, Jupiter, FL 33458, USA
    Aging Cell 11:169-77. 2012
    ..Our findings indicate that while quantitative regrowth of the thymus is achievable, the changes associated with aging persist, including potential negative implications for autoimmunity...
  5. pmc Activation kinetics and off-target effects of thymus-initiated cre transgenes
    Jianjun Shi
    The Scripps Research Institute, Jupiter, Florida, United States of America
    PLoS ONE 7:e46590. 2012
    ....

Research Grants30

  1. GATA3 regulation in transcriptional networks during hematopoietic cell developmen
    James Douglas Engel; Fiscal Year: 2013
    ....
  2. Bcl11b in early T cell development
    Ellen V Rothenberg; Fiscal Year: 2013
    ....