Investigating the Role of Fascin-1 in Axonal Development of Drosophila Öffentlichkeit

Abstract

During early neurodevelopment, neurons produce axonal projections that are directed to their specific targets for synaptic connections, a process known as axon guidance. One of the key axon guidance events occurs in the embryonic spinal cord where select sets of sensory and motor neurons send their axons across the midline to the contralateral sides of the spinal cord. Axon guidance depends on the motile tip of axons called the growth cone. The motility of the growth cone depends on the actin cytoskeleton and its regulation by actin-binding proteins. Fascin is a family of actin filament bundling proteins that is found in growth cones and concentrates in actin-based membrane protrusions called filopodia, but how it functions in axon guidance remains unknown. Therefore, studying how fascin molecules contribute to the ability of the growth cone to navigate to its target location is an important question, especially as neurological diseases have been linked to defects in axon guidance. To study the role of fascin in axon development and guidance, we used Drosophila melanogaster embryos as a model system to specifically investigate the formation of the ventral nerve cord. Not only do flies have ventral nerve cords that undergo similar processes to the human spinal cord, but Drosophila also contains a homolog of fascin actin-bundling protein 1 (FSCN1) called singed. Taking advantage of the available genetic tools in Drosophila, we performed the knockdown of singed in Drosophila embryos to determine whether the axonal projections of the ventral nerve cord were altered. Our results showed that singed knockdown appears to have some detrimental effects on overall survival of the embryos, and neuronal specific knockdown of singed resulted in altered axonal projections of the ventral nerve cord. Due to the limited numbers of embryos obtained for singed knockdown, future experiments are still needed to establish the role of singed in axon development in Drosophila.

Table of Contents

Introduction.......................................................................................................................... 1

Background on Axon Development ......................................................................................... 1

The Protein of Interest........................................................................................................... 2

Drosophila melanogaster as a Model Organism........................................................................ 3

Hypothesis............................................................................................................................ 5

Materials and Methods .......................................................................................................... 6

Drosophila melanogaster ...................................................................................................... 6

Embryo Collection................................................................................................................. 6

Preservation of Embryos........................................................................................................ 6

Staining ............................................................................................................................... 7

Imaging ............................................................................................................................... 8

GAL4/UAS System ................................................................................................................ 9

snRNAi and Dicer Enhancement of the Knockdown................................................................. 9

Genetic Lines ..................................................................................................................... 11

Statistical Analysis ............................................................................................................. 12

Results .............................................................................................................................. 13

Discussion ......................................................................................................................... 16

Tables and Figures ............................................................................................................. 21

Figure 1 ............................................................................................................................. 21

Table 1............................................................................................................................... 22

Figure 2 ............................................................................................................................. 23

Figure 3 ............................................................................................................................. 24

Figure 4 ............................................................................................................................. 25

Figure 5 ............................................................................................................................. 27

Figure 6 ............................................................................................................................. 28

Figure 7 ............................................................................................................................. 29

Figure 8 ............................................................................................................................. 30

Figure 9 ............................................................................................................................. 31

References ......................................................................................................................... 32

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Genetics Biology, Neuroscience
Stichwort	Protein Knockdown Fascin Drosophila melanogaster embryos Neuroscience Singed Cell Biology
Committee Chair / Thesis Advisor	Zheng, James, Emory University
Committee Members	Myers, Kenneth, Emory University Roeser, Andrea, Emory University Moberg, Ken, Emory University

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