Gene regulatory programs underlying differences in neural crest developmental potential in the head and neck

Proposal Summary The neural crest is an embryonic cell population which gives rise to varied derivates along the developing vertebrate body axis. Neural crest from the cranial region forms the craniofacial skeleton, while subunits of the adjacent vagal neural crest form parts of cardiac mesenchyme and the enteric nervous system. The physical boundaries between these regions have been established via quail-chick grafting experiments which demonstrate that, prior to neural crest emigration from the neural tube, the fates of each region are non- interchangeable. Few previous studies have assessed gene regulation differences by subregion to explain this phenomenon, and even fewer have looked at single-cell resolution or at premigratory stages. We aim to address this gap in knowledge by using high-resolution modern techniques to interrogate the subtle differences in gene expression and regulation between the cranial, cardiac, and enteric subregions of the premigratory neural crest in the head and neck. We hypothesize that slight modifications in gene expression programs between these neighboring populations may be amplified in order to bias each subpopulation to a unique, non-interchangeable fate. Ultimately, this work will aid in our understanding of the mechanisms which establish fate boundaries between neural crest subpopulations of varying axial levels, leading to the loss of ectomesenchymal potential in more posterior regions of the body. Aim 1: Transcriptional profiling of premigratory head and neck neural crest. Previous work to address the transcriptional state of neural crest in the head and neck has typically taken a bulk-sequencing approach, worked at migratory stages, or pooled cells from multiple axial levels. Here, we propose using a plate-based single-cell RNA sequencing method on premigratory neural crest cells at cranial versus cardiac versus enteric axial levels. We will identify transcription factors which are differentially expressed in the neural crest between these levels and assess their role in regulating cell fates from our regions of interest. Aim 2: Determine transcriptional regulation controlling boundary setting in premigratory head and neck neural crest. Previous studies on regulatory circuits within the premigratory neural crest have primarily focused on cranial neural crest. We aim to understand the differences in transcriptional regulation before emigration which establish different fate potentials. We will perform ATAC-sequencing on premigratory neural crest cells from the cardiac and enteric regions to compare with published premigratory cranial crest data. We will identify putative enhancers for transcription factors of interest and computationally identify their regulatory inputs to assess differences in gene regulatory circuits across axial levels.