Development of an optimized synthetic Notch receptor as an in vivo cell–cell contact sensor

Abstract

Detection and manipulation of direct cell-cell contact in complex tissues is a fundamental and challenging problem in many biological studies. Here, we report an optimized Notch-based synthetic receptor (synNQ) useful to study direct cell-cell interactions in Drosophila. With the synNQ system, cells expressing a synthetic receptor, which contains Notch activation machinery and a downstream transcriptional activator, QF, are activated by a synthetic GFP ligand expressed by contacting neighbor cells. To avoid cis-inhibition, mutually exclusive expression of the synthetic ligand and receptor is achieved using the "flippase-out" system. Expression of the synthetic GFP ligand is controlled by the Gal4/UAS system for easy and broad applications. Using synNQ, we successfully visualized cell-cell interactions within and between most fly tissues, revealing previously undocumented cell-cell contacts. Importantly, in addition to detection of cells in contact with one another, synNQ allows for genetic manipulation in all cells in contact with a targeted cell population, which we demonstrate in the context of cell competition in developing wing disks. Altogether, the synNQ genetic system will enable a broad range of studies of cell contact in developmental biology.

Communication codes in developmental signaling pathways

“…For example, the extracellular ligand-binding domains and the intracellular effector domains of Notch can both be replaced with engineered alternative domains to create synthetic receptors that sense arbitrary inputs and activate arbitrary target genes . These and related synthetic communication systems have been used for controlling immune cell responses , engineering synthetic multicellular patterns (Toda et al, 2018) and mapping direct cell-cell contact and neural connections (He et al, 2017;Huang et al, 2016Huang et al, , 2017. By demonstrating the possibility of orthogonal multiplexing, they provoke the question, which we address in the section 'Sensing ligand combinations': why do so many natural pathways appear to use many-to-many ligandreceptor specificities?…”

Protein binders and their applications in developmental biology

“…SynNotch receptors have also been used in the field of developmental biology to ask more basic questions. Two studies (He et al, 2017;Huang et al, 2016) reported that optimized SynNotch receptors can indeed be activated by direct cell-cell contact in vivo in Drosophila. Both studies used the SynNotch system to reveal such direct cell interactions using binary expression systems to express the ligand (GFP) in one subset of cells, while expressing the SynNotch receptor (harbouring a GFP nanobody and an intracellular transcription activation domain) in another subset of cells (He et al, 2017;Huang et al, 2016).…”

“…Two studies (He et al, 2017;Huang et al, 2016) reported that optimized SynNotch receptors can indeed be activated by direct cell-cell contact in vivo in Drosophila. Both studies used the SynNotch system to reveal such direct cell interactions using binary expression systems to express the ligand (GFP) in one subset of cells, while expressing the SynNotch receptor (harbouring a GFP nanobody and an intracellular transcription activation domain) in another subset of cells (He et al, 2017;Huang et al, 2016). Depending on the downstream response of the SynNotch receptor, this setup can reveal cell-cell contacts by inducing a detectable response in the interacting cells (such as the expression of a fluorescent protein; He et al, 2017;Huang et al, 2016) or can act as a synthetic signalling system that induces the expression of a POI (Huang et al, 2016).…”

“…Both studies used the SynNotch system to reveal such direct cell interactions using binary expression systems to express the ligand (GFP) in one subset of cells, while expressing the SynNotch receptor (harbouring a GFP nanobody and an intracellular transcription activation domain) in another subset of cells (He et al, 2017;Huang et al, 2016). Depending on the downstream response of the SynNotch receptor, this setup can reveal cell-cell contacts by inducing a detectable response in the interacting cells (such as the expression of a fluorescent protein; He et al, 2017;Huang et al, 2016) or can act as a synthetic signalling system that induces the expression of a POI (Huang et al, 2016). Without doubt, the power of this system will be further explored, as it is possible to encode novel transcriptional circuits in the responding cell, as reported in the experiments described above, and reveal direct cellcell interactions in vivo that would be difficult to detect otherwise.…”

Cell-cell contact-induced gene editing/activation in mammalian cells using a synNotch-CRISPR/Cas9 system

“…The synthetic Notch (synNotch) receptor is a recently developed cell-cell contact sensing platform, which contains a customized extracellular sensor module, a transmembrane core domain of native Notch, and a customized intracellular responder module (Morsut et al, 2016). Because of its extraordinary flexibility in terms of customizable sensing/response behaviors, the synNotch receptor serves as a powerful tool for cell engineering (Roybal et al, 2016a;Roybal et al, 2016b;He et al, 2017). In the current study, we combined the synNotch receptor with the CRISPR/Cas9 system to develop a cell-cell interaction inducible gene regulated tool.…”