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The enteric nervous system is a network of nerves that serve to innervate the gut and control homeostasis, enzyme secretion, and motility. The enteric nervous system develops from the vagal neural crest, migrating from the area of the hindbrain to populate the gut. While we know several major genes that are involved with these processes, we understand little about what genes control the migration of neuronal precursors, what genes drive neuronal differentiation, and what genes drive enteric neuron subtype specification. To determine what these genes are, we aim to isolated enteric neurons and characterize the gene expression profiles of those neurons. To achieve this, it is first necessary to develop a protocol for efficiently and quickly removing viable enteric neurons from the intestinal wall. This thesis investigates initial experiments with enzymatic dissociation protocols to isolate enteric neurons from the adult zebrafish intestine. We describe the methodology used to test the various enzymes and protocols to ascertain which conditions are most favorable for the isolation of enteric neurons from the adult zebrafish gut. Preliminary results suggest a combination of collagenase and papain enzymatic digestion is the most successful strategy for the isolation of enteric neurons from the adult zebrafish intestine. An alternative strategy for identifying genes required for ENS development is to do a reverse genetic screen. The second part of this thesis describes efforts to generate putative mutants using the CRISPR-Cas9 system for ssh1a and coro1ca, two candidate genes that have been shown to influence actin dynamics and cellular motility and may play important roles in neural crest migration and axon guidance during ENS development. We have established three mutant lines for ssh1a and two for coro1ca and have begun characterization of these lines.

Fishing for answers: Isolating enteric neurons and identifying putative ENS mutants