Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Jonathan A. Raper

Abstract

During development, zebrafish olfactory sensory neurons (OSNs) project axons from the olfactory epithelium to neuropil structures in the olfactory bulb (OB) known as protoglomeruli. OSNs expressing either OMP or TRPC2 exclusively project axons to two distinct, complementary sets of protoglomeruli. It is not known how differential protoglomerular targeting of these two populations is maintained. The guidance receptors nrp2a and nrp2b, along with their ligand sema3fa, were identified through RNAseq as potential candidates responsible for differential protoglomerular targeting of OMP- and TRPC2-class OSNs. Nrp2a and nrp2b are expressed in TRPC2-class OSNs, while sema3fa is expressed in OMP-class OSNs and OB. To investigate the involvement of these guidance factors in OSN axon guidance, the protoglomerular targeting fidelity of OSNs expressing OMP:RFP or TRPC2:Venus were assessed in nrp2a, nrp2b, and sema3fa mutants. The roles for these three guidance factors were further investigated through genetic interaction studies and live time-lapse imaging experiments. Targeting fidelity of TRPC2-class OSNs is degraded in nrp2a, nrp2b, and sema3fa mutants with increased rates of misprojecting axons observed in OMP-specific protoglomeruli as well as non-protoglomerular regions within the OB. Genetic interaction experiments suggest that nrp2a and nrp2b are partially redundant and that sema3fa is likely one of multiple guidance cues that acts in the same pathway as nrp2a and nrp2b. Live time-lapse imaging experiments comparing nrp2a and sema3fa mutants to their heterozygous siblings show that the dynamic behavior of TRPC2-class OSN growth cones are altered in mutant animals. The results support a model in which sema3fa functions as a repulsive cue to exclude nrp2a- and/or nrp2b-expressing TRPC2-class OSNs from OMP-specific protoglomeruli as well as form the dorsal-medial OB, adding to a growing list of guidance factors involved in protoglomerular targeting of OSNs. I also describe a protocol for the registration of guidance cue expression data in the 36hpf OB and present an unbiased registration template and model of the 72hpf OB and protoglomeruli. This work highlights the complex interaction of guidance factors involved in protoglomerular targeting of OSNs and lays a foundation for the systematic study of olfactory circuit formation.

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