# Coupling gene regulatory and lineage analysis of the cardiac neural crest

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2020 · $623,632

## Abstract

One of the most unique neural crest populations is the “cardiac neural crest” that contributes to the
outflow tract and outflow septum. Ablation of the cardiac crest in bird embryos causes a heart defect
reminiscent of the human birth defect, persistent truncus arteriosus. In preliminary experiments, we have
performed a transcriptome analysis of early migrating cardiac neural crest cells, isolated by enhancer-based
cell sorting. The results reveal transcription factors (e.g. MafB, Krox20, Lhx1, Id1, Sall3) as well as signaling
molecules and other factors that are selectively enriched in the early migrating cardiac neural crest compared
to other cell populations. Here, we propose to explore the role of factors identified in our screen in the
gene regulatory network that imbues the cardiac neural crest with its unique identify. Loss- and gain-of-
function experiments will be used to functionally test the role of these factors and their position in a cardiac
crest-specific gene regulatory module. In addition, we will perform cell lineage analysis using retrovirally
encoded fluorophores to follow cell fate and gene expression of clonally related cardiac neural crest cells. The
following specific aims will be performed:
Aim 1: Testing regulatory connections of genes expressed in early migrating cardiac neural crest
 cells. With our preliminary genome-wide analysis of the active transcriptome of cardiac neural crest cells in
 hand, we will perform loss-of-function experiments to perturb gene function and establish the order of gene
 activity in the cardiac neural crest. Starting with MafB, we will perturb function of the transcription factors
 and analyze effects on expression of known neural crest genes as well as new genes uncovered in our
 screen. In this way, we can assemble a functional gene battery in the early migratory cardiac neural crest.
Aim 2: Transcriptional profiling of individual cardiac neural crest cells using single cell RNA-seq and
 multiplex single molecule fluorescent in situ hybridization (smFISH). To gain a comprehensive view of
 the gene expression profile of individual cardiac crest cells, we will perform single cell RNA-seq on several
 hundred cells per time point sorted from the cardiac crest. To perform a similar analysis with the advantage
 of providing spatial information, we have devised an adaptation of smFISH called Spatial Genomic Analysis
 (SGA) that will be performed on tissue sections of carefully staged embryos, enabling simultaneous analysis
 of the expression of 35 probes selected from cardiac crest genes identified in our transcriptome dataset.
Aim 3: Retrovirally mediated clonal analysis coupled with Spatial Genomic Analysis (SGA) to examine
 the cell lineage and fate of individual chick cardiac neural crest. To determine the developmental
 potential of individual cardiac neural crest cells to contribute to the cardiovascular system, we will perform
 multi-color clonal analysis of the cardiac neural crest regio...

## Key facts

- **NIH application ID:** 9939681
- **Project number:** 5R01HL140587-03
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Marianne Bronner
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $623,632
- **Award type:** 5
- **Project period:** 2018-08-15 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9939681

## Citation

> US National Institutes of Health, RePORTER application 9939681, Coupling gene regulatory and lineage analysis of the cardiac neural crest (5R01HL140587-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9939681. Licensed CC0.

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