Genomic tools for massively parallel recording of signaling activity at cellular resolution in a brain-wide manner

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PROJECT SUMMARY/ABSTRACT Signaling pathways have been implicated in a myriad of functions including mediating cell fate decisions, proliferation, migration, and spatial patterning, among other roles. Traditionally, these have been studied in specific cell types, at a limited number of timepoints, and in small numbers of cells. This presents three limitations: 1) Our understanding of these pathways has relied on “bulk analysis” of cell populations, which dilutes the signaling effects of individual cells. 2) Live imaging techniques preserve cellular resolution but lack the scalability to extend to whole tissues or organs. 3) Measurements at one or two timepoints do not capture the changing roles of these pathways at various developmental stages. Thus, we need to shift from small-scale “local” snapshots of signaling activities to large-scale “global” snapshots. Here I describe a novel technology that uses CRISPR/Cas tools to record signaling inputs in each cell’s genome and reads these signals at high throughput with single-cell RNA sequencing (scRNA-seq). As a proof-of-concept, I will apply this tool to investigate signaling events in the zebrafish brain. Several critical signaling pathways have been identified that influence neural progenitor fates and regulate spatial patterning of brain regions. In this study, I focus on the Notch and Fgf signaling pathways to test and validate the technology. The Notch pathway is an important mechanism that maintains the balance between neural progenitor cell proliferation and differentiation into neuronal cell types. Fgf signaling has multiple functions in the brain including forebrain development, spatial patterning and modulating left-right asymmetry. The first part of the proposal describes tools to record signaling activity in the zebrafish. It has 5 core components: 1) It uses 3 Cas orthologous proteins to independently record signals. 2) It has temporal inducibility of Cas activation. 3) It enables continuous signal recording at multiple timepoints. 4) It facilitates recording of multiple signaling inputs. 5) It can be scaled to whole tissues and organs and preserves the resolution of single cells. In the second part, I describe how the technology will provide new biological insights into Notch and Fgf signaling. I will investigate 1) whether Notch signaling in progenitors correlates with which types of neurons the progenitors differentiate into; 2) whether Notch-mediated asymmetric divisions reduced the cell heterogeneity within a progenitor niche; 3) how and when the activities of Notch and Fgf intersect to regulate neurogenesis. The combination of CRISPR/Cas-mediated signal barcoding and scRNA-seq provides a powerful platform for rapid, scalable and high-resolution investigation of signaling activity during development. I envision that this technology will provide a framework for innovation in tissue engineering, modeling disease and cancer, and studying adult regeneration.