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

> **NIH NIH DP2** · UNIVERSITY OF PENNSYLVANIA · 2022 · $1,462,500

## Abstract

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.

## Key facts

- **NIH application ID:** 10473135
- **Project number:** 1DP2NS131787-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Bushra Raj
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,462,500
- **Award type:** 1
- **Project period:** 2022-09-22 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10473135

## Citation

> US National Institutes of Health, RePORTER application 10473135, Genomic tools for massively parallel recording of signaling activity at cellular resolution in a brain-wide manner (1DP2NS131787-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10473135. Licensed CC0.

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