Notch is a highly conserved signaling pathway that participates into a lot of developmental events throughout the whole life. In many cases, Notch signaling regulation adopts the lateral inhibition model. Although the general mechanism was revealed, the model of lateral inhibition varies in different developmental contexts in order to ensure different regulating consequence. with such diversity, some details of Notch regulatory mechanism in different contexts remain unclear nowadays. Here we designed a Notch activating system with a photocleavable protein, PhoCl. Firstly, we tested the photocleavage of PhoCl in HeLa cells, the photoconversion and expected protein translocation can be observed after light excitation. Next, we designed two PhoCl-NICD constructs and successfully ex...[ Read more ]

Notch is a highly conserved signaling pathway that participates into a lot of developmental events throughout the whole life. In many cases, Notch signaling regulation adopts the lateral inhibition model. Although the general mechanism was revealed, the model of lateral inhibition varies in different developmental contexts in order to ensure different regulating consequence. with such diversity, some details of Notch regulatory mechanism in different contexts remain unclear nowadays. Here we designed a Notch activating system with a photocleavable protein, PhoCl. Firstly, we tested the photocleavage of PhoCl in HeLa cells, the photoconversion and expected protein translocation can be observed after light excitation. Next, we designed two PhoCl-NICD constructs and successfully expressed them in S2 cells. To further exam the Notch activating ability, we designed a Notch luciferase reporter and co-transfected with our constructs, luciferase assay results indicated that our reporter can indeed response to Notch signaling, and the expression of PhoCl-NICD would not induce ectopic Notch signaling. Finally, we generated embryos that express PhoCl-mCherry-NICD without affecting the normal development of embryos, and light excitation of PhoCl-mCherry-NICD expressed in cellularization embryos showed nucleus-like translocated. Overall, we designed and constructed an in vivo system that activates Notch signaling by optogenetics. Together with the trustable neuroblast delamination model, we will be able to manipulate Notch at a spatiotemporal scale. The optogenetics system we designed will provide a powerful tool for precise spatiotemporal control of Notch signaling activity.