Notch signaling occurs when cell surface Notch receptors are stimulated by Notch ligands on an apposing cell leading to release of the Notch receptor intracellular domain which targets numerous downstream genes. A large variety of cell fate decisions depend on regulated Notch signaling during development and differentiation in mammals. Thus, several human diseases and cancers arise from malfunctioning of Notch signaling pathways. Diseases range from skeletal deformities to heart disease and a major cancer arising from mutations in Notchl is T cell leukemia. The O-fucose glycans attached to the EGF repeats of Notch receptors play critical but ill-defined roles in the regulation of Notch signaling. In their absence mouse embryos die at mid-gestation with defects typical of a loss of signaling through all four Notch receptors. O-fucose on Notch receptors is elongated by the addition of GlcNAc by one of three Fringe GlcNAc-transferases. Dysregulation of Notch signaling by altered Fringe gene expression has been associated with developmental defects and cancer prognosis. Therefore it is very, important to understand molecular mechanisms by which O-fucose glycans and the different Fringe activities regulate Notch signaling. This is a challenge however, because the three Fringe genes are often co-expressed. We propose to simplify the situation by generating embryos, embryonic stem (ES) cells and mouse embryo fibroblasts (MEF) that express a single Fringe gene from its endogenous locus, or from the Lfng locus, or that express no Fringe genes at all. We will use these biological materials in which Notch receptors will carry only O- fucose or O-fucose with GlcNAc transferred by a single Fringe enzyme, to identify roles for each Fringe glycosyltransferase in embryonic development and in T and B cell development.
In Specific aim 1 T and B cell development and immune respones will be investigated in mice or fetal liver cells from embryos that express only Lfng, only Mfng or only Rfng.
Specific aim 2 is to generate mice or embryos expressing only a single Fng activity knocked in to the well-characterized Lfng locus by recombinase mediated cassette exchange. We will determine whether the GlcNAc-transferase activity encoded by each Fringe gene is able to function equivalently when expressed in a regulated fashion at the right time and place in vivo.
Specific aim 3 is to identify mechanisms by which mammalian Notch receptors carrying solely O-fucose signal in embryos, ES and MEF cells, and the effects on ligand binding and signaling of restoring each Fringe activity separately. The combined experiments will identify specific roles for Lfng, Mfng and Rfng and generate valuable mouse strains and cell lines for mechanistic studies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095022-08
Application #
7575716
Study Section
Intercellular Interactions (ICI)
Program Officer
Woodhouse, Elizabeth
Project Start
2002-04-01
Project End
2012-02-28
Budget Start
2009-03-01
Budget End
2010-02-28
Support Year
8
Fiscal Year
2009
Total Cost
$422,605
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Varshney, Shweta; Stanley, Pamela (2017) Notch Ligand Binding Assay Using Flow Cytometry. Bio Protoc 7:
Song, Yinghui; Kumar, Vivek; Wei, Hua-Xing et al. (2016) Lunatic, Manic, and Radical Fringe Each Promote T and B Cell Development. J Immunol 196:232-43
Wang, Weihuan; Yu, Shuiliang; Zimmerman, Grant et al. (2015) Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem Cells 33:2280-93
Tashima, Yuko; Stanley, Pamela (2014) Antibodies that detect O-linked ?-D-N-acetylglucosamine on the extracellular domain of cell surface glycoproteins. J Biol Chem 289:11132-42
Yao, David; Huang, Yuanshuai; Huang, Xiaoran et al. (2011) Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions. Blood 117:5652-62
Visan, Ioana; Yuan, Julie S; Liu, Ying et al. (2010) Lunatic fringe enhances competition for delta-like Notch ligands but does not overcome defective pre-TCR signaling during thymocyte beta-selection in vivo. J Immunol 185:4609-17
Guilmeau, Sandra; Flandez, Marta; Bancroft, Laura et al. (2008) Intestinal deletion of Pofut1 in the mouse inactivates notch signaling and causes enterocolitis. Gastroenterology 135:849-60, 860.e1-6
Stahl, Mark; Uemura, Kazuhide; Ge, Changhui et al. (2008) Roles of Pofut1 and O-fucose in mammalian Notch signaling. J Biol Chem 283:13638-51
Stanley, Pamela (2007) Regulation of Notch signaling by glycosylation. Curr Opin Struct Biol 17:530-5
Shi, Shaolin; Ge, Changhui; Luo, Yi et al. (2007) The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling. J Biol Chem 282:20133-41

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