Degree Name

Department

Physiology and Developmental Biology

College

Life Sciences

Defense Date

2021-12-08

Publication Date

2021-12-17

First Faculty Advisor

Arminda Suli

First Faculty Reader

Michael Stark

Honors Coordinator

Roy Silcox

Keywords

Deep Brain Photoreceptors, Non-Visual Opsins, Opsin, Photoreceptors, Deep Brain, Zebrafish

Abstract

Deep brain photoreceptors (DBPs) are non-visual photoreceptors found in the brains of non-mammalian vertebrates like zebrafish. While using optogenetic techniques in our lab to photo-activate the ear (by expressing the light-gated channel: channelrhodopsin) in efforts of identifying optic tectum (OT) neurons that respond to and integrate auditory and visual stimuli, we found that violet light was able to elicit OT neuronal activity. OT activity was also detected in cases when visual and auditory stimulation was not possible due to removal of the eyes and absence of the exogenously expressed channelrhodopsin, respectively. Analyzing OT single-cell sequencing data of 7day post fertilization (dpf) larvae generated in our lab, we found that genes in the opsin family including Tmtops2b, Opn4xb, and rhodopsin are present in this data set, possibly indicating the presence of DBPs in the OT. In this project, we aimed to use the CRISPR-Cas9 technology to knockout Tmtops2b, Opn4xb, and rhodopsin to determine if these genes are partly responsible for the violet light elicited activity in the OT, suggesting that DBPs have a role in OT function. To determine the targeting efficacy of the short-guide RNAs (sgRNAs) for each gene in vitro, I PCR-amplified the target genomic regions and by incubating them with Cas9 and their respective sgRNAs, I was able to successfully show that these sgRNAs are efficacious. Using the same approach, I showed that sgRNAs against the Atoh7 gene, which affects the development of retinal ganglion cells, therefore, giving us the ability to remove visual input and create essentially blind larvae, are equally efficacious. Additionally, I helped establish a transgenic zebrafish line, which expresses a nuclear calcium indicator in the OT, allowing us to better follow OT neuronal activity. Using this line, I show that violet light elicits neuronal activity in all layers of the OT, especially 30-60um from the dorsal side of the OT. In the future, we will use these sgRNAs and Cas9 to knock out Tmtops2b, Opn4xb, and rhodopsin in vivo in zebrafish larvae and determine if these genes contribute to the OT neuronal activity upon violet light activation. This will be the first step in establishing a potential function of DBPs in zebrafish OT.