The cardiac neural crest is a unique population of multipotent precursors that arise in the hindbrain and then migrates away to populate parts of the heart and outflow tract. Defects in the cardiac neural crest have been linked to DiGeorge and CHARGE syndromes. These disorders are characterized by conotruncal, valvular, and septal defects, and affect almost 35000 babies born in the United States every year. The goal of this proposal is to expand our understanding of the cardiac NC gene regulatory network (GRN) and its role in cardiovascular development by implementing single cell RNA-seq and ATAC-seq in the cardiac NC. Our previous population level transcriptome analysis has identified the transcription factors MafB and Krox20 as being specifically expressed in the migratory cardiac NC, but no other NC populations. Interestingly, their expression was lost after knock-down of Ets1, a transcription factor expressed in the cardiac NC of developing mouse, chicken, and frog embryos, suggesting a possible role for Ets1 in maintaining cardiac NC identity through direct or indirect interactions with MafB and Krox20. I hypothesize that Ets1, MafB, and Krox20 are a part of a cardiac crest-specific sub-circuit that imbues the cardiac NC with its unique identity. I propose to test this hypothesis via two specific aims:Aim 1: Single-cell RNA-seq to identify novel factors expressed in the cardiac NC: I will implement single-cell RNA-seq (scRNA-seq) technology in the chick cardiac NC. For genes that are enriched in this population, I will perform targeted CRISPR/Cas9-mediated knockout to investigate their role in NC specification, migration, survival, and proliferation. I will then compare the transcriptomes of NC cells with and without Ets1, MafB, and Krox20 to identify a putative gene regulatory network, which I will perturb to explore long-term effects on cardiovascular development.Aim 2: Characterization of cis-regulatory elements modulating expression of cardiac NC genes: Using an unbiased high-throughput approach (ATAC-seq), I will identify active enhancers governing the expression of Ets1, MafB, and Krox20 specifically in the cardiac NC. I will then dissect these enhancer elements to identify minimal enhancers. Lastly, using CRISPR/Cas9, I will mutate potential transcription factor binding sites to identify transcriptional inputs into the expression of Ets1, MafB, and Krox20.
|Program type||Predoctoral Fellowship|
|Effective start/end date||07/01/2018 → 06/30/2020|