Our goal is to complete a detailed history of Notch 1 activity in the mouse, extending beyond the early embryo into self-renewal and tissue maintenance in the adult. Notch signaling plays an important role in cell fate arbitration, in boundary formation and, less frequently, in fate induction during early development and tissue renewal. Due to early lethality, the role of Notch signaling during and after organogenesis remains to be resolved. Identifying signal-receiving cells, a subset of the Notch protein expressing cells, is key to understanding how Notch regulates cell fate. This goal has broader appeal due to the observation that Notch signals and Amyloid-plaque generating A beta42 peptides are both produced by the same enzymatic activity, presenilin dependent g-secretase. We will obtain an objective characterization of the role Notch core signaling plays in adult vertebrates to gain a better understanding of the possible consequences of the therapeutic use of g-secretase inhibitors. We have generated several reagents to enable a thorough investigation of where Notchl is activated. Cells engaged in Notch signaling can be detected with existing antibodies directed against g-secretase cleaved Notch 1. We developed protocols that permit detection of low levels of this antigen in tissue sections. In conjunction with the effort to map Notch activation using these antibodies, we derived ES cell lines in our laboratory, marked with ROSA-LacZ, that are either Notch 1 deficient or express a proteolysis-deficient Notch 1 allele (N1V->G/N1V->G). Comparing the contribution of these ES lines in different tissues of mouse chimera will map tissues and cell types whose formation in the embryo and renewal adult may not require g-secretase-dependent Notch signals. We propose to correlate Vail 744 antigen detection and ES contributions with two functional assays that will determine the requirements for Notch 1 activity. First, we propose to create a lineage tracer to mark cells experiencing Notch 1 activation via proteolysis. Second, we are generating conditional Not 1 loss- and gain of function alleles. Conditional removal or activation of Notch 1 will permit analysis of its role late in fetal development and in adult tissue renewal.
Aim l: Survey of cells that experience core Notch signaling and express NICD.
Aim2 : Comparison of the developmental potential of N-/- EX cells.
Aim3 : Analyze conditional loss and gain of function Notchl alleles in the adult.
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