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. ? ? ?
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