Investigating the role of egg-3 by characterizing the egg-3(as40) phenotype in C. elegans
Date
2024
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Publisher
University of Delaware
Abstract
Egg activation is a process whereby a mature oocyte transitions from meiosis to mitosis and changes to a new cellular state that supports embryogenesis. In C. elegans, egg activation is regulated by a complex of proteins which include EGG-3, EGG-4, EGG-5, CHS-1 and MBK-2. EGG-3 is a member of the protein tyrosine phosphatase like (PTPL) family that has been proposed to act as a scaffolding molecule to regulate the activity and localization of the above-mentioned proteins. ☐ My research goal is to characterize the temperature-sensitive mutant egg-3(as40) to further elucidate the role of EGG-3 in the oocyte-to embryo transition. The progeny of egg-3(as40) hermaphrodites showed signs of a developmental delay, and I investigated the possibility that this phenotype was caused by egg-3 expression occurring later in development. There was no discernible variation in the developmental rates of the heterozygous and homozygous progeny when compared suggesting that the developmental delay is due to maternally provided EGG-3 and not expression of egg-3 in later stages of embryonic or larval development. RT-PCR results also validated the germline-restricted expression of egg-3. Moreover, an eggshell permeability assay was performed which showed that the eggshell of egg-3(as40) was permeable to the lipophilic dye, FM4-64, at the restrictive temperature suggesting that there is a defect in the formation of the eggshell layer of the mutant. Calcofluor White staining also showed that the chitin layer fails to form in the mutants at 25°C. I have conducted two rounds of suppressor screens and isolated a total of six potential suppressors of egg-3(as40). While egg-3(as40) mutants produce an average of less than 1 progeny at 25°C, the suppressor lines produce an average of 5-24 progeny. I have also performed genetic analysis to determine the dominant or recessive nature of these suppressors and found that all six suppressors are dominant. In the future, we plan to use whole genome sequencing to map these suppressors to their genetic loci.
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Keywords
Egg activation, Protein tyrosine phosphatase, Embryogenesis, Mutants