Abstract
The Notch signaling pathway directs cell fate decisions in development of nearly every tissue, plays key
roles in numerous diseases, and represents a major drug target. The pathway uses multiple ligands and
receptors that interact with one another in a promiscuous fashion, as well as Fringe glycosyltransferases
that modulate those interactions. Recent work from our lab has revealed that these interactions comprise
a communication ‘code’ in which different ligands activate different target programs, even through the
same receptors. The code appears to function through a dynamic encoding mechanism, in which different
ligands activate Notch receptors in either a pulsatile or sustained fashion. These different dynamics in turn
selectively activate distinct target programs. Current understanding of the code is limited to just two
ligands and one receptor. By elucidating the full code, covering all ligand-receptor-Fringe combinations,
we will enable quantitative prediction of signaling interactions between cells with arbitrary Notch
component expression profiles across different developmental and disease contexts. To decipher this
code and its functional roles, we will combine cell line engineering, quantitative single-cell time-lapse
imaging, direct control of Notch dynamics using mutant receptors and pharmacological perturbations, and
analysis of Notch dynamics in neural stem cells and chick embryos. In ¿Aim 1¿¿, we will map dynamic
signaling modes (pulsatile or sustained) across a full matrix of Notch receptor, ligand, and Fringe protein
combinations. In ¿Aim 2¿¿, we will analyze dynamic signal decoding by determining whether receptor
intracellular domain composition affects target gene expression, or whether target program specificity
depends only on the strength and dynamics of signaling. Finally, we will analyze the function of the Notch
dynamic code in chick spinal cord development (¿Aim 3¿¿). Specifically, we will use a fluorescent reporter
exclusively activated by Notch, together with a new tissue slice preparation that enables imaging of
individual living cells during spinal cord development, to link Notch dynamics to cell fate determination.
Together, these results will reveal the structure and function of the dynamic code underlying Notch
signaling, and show how it operates in a central vertebrate developmental process.