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 Notch1 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.
Aim1 : 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 Notch1 alleles in the adult.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD044056-01
Application #
6600374
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Javois, Lorette Claire
Project Start
2003-01-13
Project End
2007-12-31
Budget Start
2003-01-13
Budget End
2003-12-31
Support Year
1
Fiscal Year
2003
Total Cost
$344,250
Indirect Cost
Name
Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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Demehri, Shadmehr; Liu, Zhenyi; Lee, Jonghyeob et al. (2008) Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity. PLoS Biol 6:e123
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Lee, Jonghyeob; Basak, Jacob M; Demehri, Shadmehr et al. (2007) Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes. Development 134:2795-806
Yang, Xudong; Tomita, Taisuke; Wines-Samuelson, Mary et al. (2006) Notch1 signaling influences v2 interneuron and motor neuron development in the spinal cord. Dev Neurosci 28:102-17
Ong, Chin-Tong; Cheng, Hui-Teng; Chang, Li-Wei et al. (2006) Target selectivity of vertebrate notch proteins. Collaboration between discrete domains and CSL-binding site architecture determines activation probability. J Biol Chem 281:5106-19

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