The enteric nervous system (ENS), the largest portion of the peripheral nervous system, is derived from
neural crest populations referred to as “vagal” and “sacral”, arising from the neck and tail regions, respectively.
However, much less has been published about sacral than vagal neural crest. To rectify this knowledge gap,
we propose to: 1. characterize the temporal sequence of migration and differentiation of sacral neural
crest cells into neuronal subtypes in the hindgut; 2. transcriptionally profile the sacral neural crest as at
multiple time points and compare with that of vagal neural crest; 3. test function of transcription factors
that may drive sacral neural crest cell fate choice. The results will enable us to test whether vagal and sacral
neural crest cells give rise to similar or distinct types of enteric neurons and elucidate the influence of the
intestinal environment on their differentiation. We will characterize the chick sacral neural crest using a novel
lineage labeling technique of Replication Incompetent Avian (RIA) retroviruses that enables us to specifically
target and isolate by FACS the sacral neural crest-derived population to perform the following aims:
Specific Aim 1: Retrovirus-mediated lineage analysis of the chick sacral compared with vagal neural
crest: We will label the neural tube caudal to somite 27 with RIA retroviruses that permanently label sacral neural
crest cells in order to follow their long term fate. Preliminary results suggest that sacral neural crest-derived cells
populate the post-umbilical gut and differentiate into cholinergic motor neurons as well as tyrosine hydroxylase
positive cells. Sacral neural crest will be compared with vagal neural crest and interactions between the two
populations examined. Finally, clonal relationships between sacral crest cells will be characterized.
Specific Aim 2: Single cell RNA-seq of sacral neural crest-derived cells in the post-umbilical gut. To
elucidate gene regulatory programs controlling progressive differentiation of sacral neural crest cells into neurons
and glia in the hindgut, we propose to characterize the transcriptional profile of sacral crest-derived cells FACS
sorted from the post-umbilical gut and processed by single cell (sc) RNA-seq at embryonic days (E) 2.5, E6, E8,
10, 15, 21 (prehatching); these will be compared with vagal crest-derived cells at comparable stages. Preliminary
scRNA-seq data on E10 suggest that there are differences in neuronal subtypes produced by sacral vs vagal
crest. scRNA-seq will enable us to sample different neuronal subtypes and infer developmental trajectories.
Specific Aim 3: Role of transcriptional regulators into differentiation of sacral neural crest into neuronal
and glial subtypes. Focusing on transcription factors that are present in the enteric precursor cluster (e.g.
Nfatc1, Foxn2, Sox4, Sox21, Elf2, Znf536), we will test whether sacral neural crest-enriched transcription factors
are critical for mediating proliferation, migration within the gut, and/or cell fate decisions. To this end, we will
perform targeted loss of function in the sacral neural crest using a single-plasmid CRISPR-Cas9 strategy and
examine subsequent effects on distribution and/or differentiation of sacral neural crest-derived cells.
