Generation of ziwi:GFP-UtrCH Transgenic Zebrafish line for Monitoring Actin Dynamics in Fertilizing Eggs
Date Issued
2015
Date
2015
Author(s)
Lin, Ssu-Ting
Abstract
Actin is one of the most abundant proteins in eukaryote and is an essential component of cytoskeleton. Filamentous actin (F-actin), assembled by polymerization of globular actin (G-actin) monomers, is involved in many crucial physiological functions, such as the formation and maintenance of cell shape, cell motility, cell division and intracellular trafficking. Consequently, the development of tools for monitoring F-actin dynamic is important to understand the functional roles of actin during development and other physiological processes. Fertilization starts with sperm contacting eggs, and followed by dramatic changes inside egg to form a complex muticellular organism. In teleost, there is a specific sperm entry site called micropyle, a dimple-like structure consisting of circular tuft of microvilli and containing a meshwork of actin filaments. Furthermore, the F-actin network of unfertilized egg rearranges immediately to the egg cortex during/after fertilization in species such as sea urchin and mouse. Some studies also indicate that the fine regulation of actin filament may be related to calcium wave trigger by fertilization. I was interested in studying actin dynamic during fertilization and early embryogenesis. Due to the lack of a suitable F-actin reporting line in zebrafish, I used a ziwi promoter to drive the maternal expression of green fluorescent protein-utrophin (GFP-UtrCH) fusion proteins. The ziwi promoter is a germ cell-specific promoter and GFP-UtrCH is an F-actin probe, which can bind F-actin by its calponin homology domain. In various development stages of Tg(ziwi:GFP-UtrCH) embryos, the fluorescent protein signal faithfully represent the distribution of F-actin that resembles the rhodamine phalloidin staining patterns. I also performed in vitro fertilization using the unfertilized egg from Tg(ziwi:GFP-UtrCH) and observed dramatic reorganization of cortical actin. This transgenic line is thus an effective tool to study the regulation of F-actin dynamics during fertilization and subsequent development.
Subjects
Zebrafish
Actin dynamics
Fertilization
Utrophin
Transgenic fish
Type
thesis
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