Date of Award

12-2011

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Zoology

Advisor

Sharon Ashworth

Second Committee Member

Ian Bricknell

Third Committee Member

Seth Tyler

Abstract

Essential and highly abundant in eukaryotic cells, actin provides the basic framework for cell motility, cell shape, and intracellular organization. The dynamic nature of the actin cytoskeleton is controlled by the distribution of monomeric and filamentous actin in the cell. Actin-binding proteins influence steps involved in cytoskeleton alterations, maintenance, and regulation. Therefore, the actin depolymerizing factor (ADF)/cofilin (AC) protein family plays a vital role in actin regulation. Cofilin 1, an 18 kDa non-muscle AC protein isoform, preferentially binds ADP-actin monomers (Van Troys and others 2008; Lee and Dominguez 2010). Cofilin binds along the actin polymer and weakens lateral contacts within the filament leading to filament severing, actin depolymerization, and as a result increased actin filament turnover (Theriot 1997; McGough and others 1997). Cofilin binding to actin is inhibited by phosphorylation of serine 3 on cofilin (Agnew and others 1995). AC isoform null model organisms have been characterized in Saccharomyces cerevisiae (Moon and others 1993), Dictyostelium discoideum (Aizawa and others 1995), Caenorhabditis elegans (McKim and others 1994), Drosophila melanogaster (Gunsalus and others 1995), and Mus musculus (Ikeda and others 2003; Gurniak and others 2005). These model systems have severe developmental defects, most essential for survival. To investigate the role of cofilin 1 in zebrafish development, we acquired a mixed population of zebrafish embryos from the Zebrafish International Resource Center that were either wildtype or heterozygous for a mutation in the cofilin 1 gene. Insertion of the hi3735aTg viral insert downstream of the first exon interrupts expression of the cfl1 gene (Amsterdam and others 2004). Identified through polymerase chain reaction amplification, heterozygous cfl1+/ hi3736aTg zebrafish were mated to produce a 3:1 phenotypic mixed embryo spawn of wildtype cjfl1+l+ to cfl1h3736aTg/hi3736aTg. PCR of larval DNA extracts from 5dpf zebrafish confirmed a 1:2:1 genotypic ratio of wildtype cfl1+l+ to cfl1+/hi3736aTg to cfl1hi3736aTg/ hi3736aTg offspring (Marquis and others 2009; Preziosi and others 2010). Developmental abnormalities were not apparent in the cofilin 1 mutant until 48 hours post fertilization (hpf) when pericardial edema was first observed. At 72 hpf pericardial edema increased. Craniofacial development was abnormal and atypical eye structure was observed. The cofilin 1 mutant did not develop an inflated swim bladder at 5dpf as did wildtype larvae. The cofilin 1 mutant larvae survived 8 to 12 days post fertilization. These studies demonstrated cofilin 1 was essential to normal zebrafish development. The cofilin 1 zebrafish mutant provides a practical approach to study the significance of cofilin 1 in zebrafish development and to establish Danio rerio as a model for studying cofilin 1 regulation of actin dynamics.

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