Nervous system evolution : a molecular genetic characterization of neuronal cell types in saccoglossus kowalevskii

My dissertation aims to understand fundamental principles in neural organization in the hemichordate S. kowalevskii because it offers an opportunity to investigate whether all nervous systems are built on fundamentally different organizational principles or whether there are shared commonalities despite gross anatomical differences in body plan and nervous system structure. S. kowalevskii is a hemichordate species belonging to the class Enteropneusta. Hemichordates diverged from chordates during the precambrian more than 620 million years ago and is a key system to study nervous system evolution because it shares a conserved anterior-posterior gene regulatory network (GRN) that patterns the central nervous system (CNS) in chordates despite clearly divergent basic organization principles of nervous system organization. Conservation even extends to the role of developmental enhancers of key ligands with important roles in brain regionalization like Sonic Hedgehog. The first published papers describing nervous system organization in S. kowalevskii appeared in the late 1850s, but the field remains divided between scientists arguing for a CNS or a more nerve net-like nervous system. My dissertation aims to provide modern molecular genetic approaches to aid in the discussion. In order to investigate the role that the anterior-posterior GRN plays on neural cell type organization in S. kowalevskii, I aimed to first characterize the expression profile of multiple neural markers. Using the sequenced genome, chip-seq and ATAC-seq data, and access to hundreds of already cloned transcripts, I have visualized the expression of neural markers by in situ hybridization and using commercially available antibodies for neurotransmitter. Despite the presence of a neural plexus throughout the ectoderm, I found that neuropeptide and neurotransmitter markers generally localize to cells in the proboscis, with large sensory neurons visible throughout the proboscis ectoderm, but with some concentration at the dorsal proboscis base. Expression profiles for neuropeptides and genes involved in their synthesis show distinct expression patterns. Some neuropeptides are concentrated in large cells at the proboscis, others show more specificity towards the two nerve cords on the dorsal and ventral sides. Immunohistochemistry against two major neurotransmitters show a similar regionalization pattern. Serotonin-positive cells (α-5HT) are concentrated along the proboscis and collar and dorsal cells at the trunk project ventrally towards the cord. GABA-positive cells (α-GABA) show a similar concentration at the proboscis base and anterior collar but with a more diffuse axon network along the trunk. This data so far suggests that like in vertebrates, neural cell types are well regionalized along the A-P axis in hemichordates. Next, I successfully adapted the I-SceI meganuclease-mediated transposon system to generate transgenic animals that have mosaic expression of GFP in a variable subset of neurons. When known regulatory information for a specific cell type is cloned next to GFP, and injected into fertilized eggs, it becomes incorporated into the genome. This technique improves axonal tracing in transgenic animals by providing detailed resolution to the cell morphology, dendritic and axonal projections. The mosaic incorporation works to reduce the number of labeled neurons because the transgene becomes incorporated only in a subset of blastomeres early in development. My work has successfully used a regulatory region directly upstream of the Synapsin1, GAT, Nurr1, and the TH loci in the genome to label specific neural populations with fluorescent proteins. My work has already successfully characterized numerous features of the nervous system in S. kowalevskii including evidence for the regionalization of neural cell types, identification of cell type location and their morphologies, visualization of direct neuron-to-neuron connections, and the tracing of projections across both local and long distances towards a potential integration

Keywords: Biology.
Advisor: Lowe Christopher (Associate professor of biology)
Gilly William
Luo Liqun