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Publication Date

2019

First Advisor

Michael Barresi

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Biological Sciences

Keywords

Zebrafish, Mutagenesis, CRISPR, Development, Axon guidance

Abstract

During early embryonic development, neuronal projection of axons across the midline of the forebrain connect the two halves of the nervous system by forming structures called commissures. How do pioneering axons know where to cross the midline to form such uniform structures like commissures? Zebrafish postoptic commissure formation has been shown to require a combination of signaling gradients and potential interactions with a “glial bridge” (Barresi et al. 2005). Midline crossing of commissural axons and glial bridge positioning relies on the appropriate expression of a set of repellent guidance cues, Slits, and their associated receptors, Robos (Barresi et al. 2005; Rasband, Hardy, and Chien, 2003). Expression of slit2/3 is thought to condense the POC by providing surround repulsion (Barresi et al. 2005; Long et al. 2004; Rasband, Hardy, and Chien, 2003). We have found that contrary to the published effects of slit2/3, knockdown of slit1a suggests that slit1a may act as a positive guidance cue for commissural axons, particularly due to its expression overlapping with the region of axonal midline crossing and the glial bridge. Utilizing a new computational methodology called ∆SCOPE (Schwartz, Schnabl et al., 2019), we were able to quantify the complex commissural phenotypes in slit1a overexpressed wildtype forebrains and in the achiasmatic yot mutant. To investigate the effects of the loss of slit1a, we have generated an early stop slit1a knockout line, which does not exhibit a commissural phenotype potentially due to compensation via the upregulation of other genes triggered by the production of mutant mRNA (Stanier et al., 2019). To avoid transcriptional adaptation in newly designed mutants, we have generated ‘promoterless’ slit mutants by excising all of the upstream transcriptional elements of the slit genes using CRISPR-­‐Cas9 mediated mutagenesis. ∆SCOPE analysis of the promoterless slit1a mutant and slit1a overexpression phenotypes supports a novel, non-­‐canonical positive role for Slit1a in promoting commissural midline crossing in the zebrafish diencephalon.

Rights

©2019 Cassie Lynne Kemmler Access limited to the Smith College community and other researchers while on campus. Smith College community members also may access from off-campus using a Smith College log-in. Other off-campus researchers may request a copy through Interlibrary Loan for personal use.

Language

English

Comments

78 pages : illustrations (some color) Includes bibliographical references (pages 71-78)

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