To access this work you must either be on the Smith College campus OR have valid Smith login credentials.

On Campus users: To access this work if you are on campus please Select the Download button.

Off Campus users: To access this work from off campus, please select the Off-Campus button and enter your Smith username and password when prompted.

Non-Smith users: You may request this item through Interlibrary Loan at your own library.

Publication Date

2024-5

First Advisor

Michael J. F. Barresi

Document Type

Honors Project

Degree Name

Bachelor of Arts

Department

Neuroscience

Keywords

zebrafish, neurodevelopment, embryonic development, meteorin, meteorin-like, radial glia, HCR, CRISPR, PCR, morpholino, gene expression patterns, knockout, transgenesis

Abstract

Neurogenesis and gliogenesis are critical processes for neurodevelopment. Radial glial cells (RGCs), an essential progenitor cell population in the central nervous system (CNS), give rise to both neurons and glia throughout embryogenesis in vertebrates. Meteorin (metrn) is a secreted protein, an orphan ligand and a neurotrophic factor. It has been shown to play a regulatory role in RGC differentiation in-vitro (with mice cell cultures). I hypothesize that metrn may act as a unique regulator for RGC development in vivo. To investigate this, I first established the spatiotemporal gene expression patterns of both metrn & its paralog meteorin-like (metrnL) during zebrafish embryogenesis. Using hybridization chain reaction (HCR) procedures, I found that metrn and metrnL are expressed in both complementary and overlapping patterns that include regions of the CNS with known progenitor cell domains. I also conducted a preliminary colocalization study by combining both HCR and immunocytochemistry that revealed that metrn and metrnL do colocalize with RGCs. To be able to track metrn expression in real-time in live zebrafish embryos, I attempted to create a transgenic reporter line. This was done using a plasmid created in the Tol2 Gateway Cloning system to tag the metrn promoter with a GFP reporter. Although these attempts produced some successful transgenesis, the alleles isolated do not show metrn expression patterns in the CNS. Last but not least, I investigated loss of function of metrn by carrying out morpholino knockdown studies and CRISPR promoter KO studies. The morpholinos targeted to disrupt the translation of metrn while the CRISPR targeted to disrupt the transcription of metrn. Our preliminary data on phenotypic characterization also revealed developmental defects in regions with established metrn and metrnL expression. This includes abnormal developmental phenotypes in the brain (hydrocephalus), somite formation (compaction), trunk formation (tail truncation) and pronephros (kidney cyst formation). Moreover, HCR analysis on these metrn morphants revealed that expression of metrnL is altered, suggesting that metrn and metrnL may be working in a feedback mechanism. We posit that understanding how metrn & metrnL regulate RGC development can provide new insight into regulators mediating progenitor cells in the CNS. This would not only have implications in how the brain is formed, but also how neurodevelopmental disorders (including congenital brain cancers) may arise.

Rights

©2024 Shariqa Tasnim Shaila. 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

81 unnumbered pages: color illustrations. Includes bibliographical references (pages 74-81).

Download

Smith Only:
Off Campus Download

Share

COinS