# Gene regulatory network controlling neural crest derived formation of facial cartilage

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2021 · $382,950

## Abstract

In the previous grant period, we explored regulatory differences between cranial and trunk neural crest
cells. Comparative transcriptomics coupled with functional perturbation revealed a premigratory “cranial-specific”
neural crest GRN subcircuit that links anterior identity to ability to differentiate into facial cartilage. Here, we
propose to elucidate the gene regulatory network (GRN) downstream of this cranial-specific subcircuit
that confers ability to differentiate into facial skeleton. The goal is to understand the program underlying
differentiation and pattern formation of craniofacial cartilage. First, we will explore regulatory changes in trunk
neural crest cells after introduction of cranial crest subcircuit genes. Next, we will characterize late-migrating
cranial crest cells as they condense to form facial cartilage at the single cell level to understand GRN changes
as a function of time, at single cell resolution and a functional level. Finally, we propose to identify active
enhancers and their direct inputs in late migrating and condensing cranial crest cells by combining in vivo
electroporation of reporter constructs in the chick embryo with high throughput genomic approaches. To these
ends, we will conduct the following aims:
Specific Aim 1: Effects of “reprogramming” trunk neural crest cell identity. Ectopic expression of cranial
 crest subcircuit genes imbues trunk crest cells with chondrogenic potential after grafting to the head. Here we
 will: characterize transcriptional changes that occur in reprogrammed trunk crest cells over time; test the ability
 of reprogrammed trunk neural crest to form ectopic cartilage in their normal environment; test the ability of the
 cranial subcircuit to confer chondrogenic ability onto ES cells and crestosphere-derived cells.
Specific Aim 2: Transcriptional profiling and functional validation of condensing cranial neural crest
 cells at high cell resolution using single cell RNA-seq and multiplex in situ hybridization. To gain
 insights into how neural crest-derived cells differentiate and what gene regulatory programs control their
 progression to facial skeleton, we propose to perform single cell RNA-seq of condensing cranial crest cells in
 the branchial arches with the goal of identifying candidate GRN components of the cartilage-forming module.
 We will validate expression of candidate transcription factors at high resolution and test their function using
 CRISPR-Cas9 knockout.
Specific Aim 3: Construction of a cranial cartilage gene regulatory network (GRN) by identifying cis-
 regulatory elements and direct downstream targets of the cranial crest-specific subcircuit genes. In
 order to build regulatory linkages in branchial arch neural crest, we will characterize their chromatin landscape
 using low-input Assay for Transposase-Accessible Chromatin (ATAC-seq) optimized for as few as 1500 cells
 per replicate and perform CUT&RUN to confirm direct inputs downstream of subcircuit genes.

## Key facts

- **NIH application ID:** 10187306
- **Project number:** 2R01DE024157-06
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Marianne Bronner
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $382,950
- **Award type:** 2
- **Project period:** 2014-06-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10187306

## Citation

> US National Institutes of Health, RePORTER application 10187306, Gene regulatory network controlling neural crest derived formation of facial cartilage (2R01DE024157-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10187306. Licensed CC0.

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