Early mammalian development before the oocyte-to-embryo transition is under 'maternal control' from factors deposited in the cytoplasm during oocyte growth, synthesized independent of de novo transcription. Maternal mRNAs encode proteins necessary for early embryo development. Two elements in the mRNA 3’untranslated region (UTR), the cytoplasmic polyadenylation element (CPE) and the hexanucleotide (AAUAAA) are involved in the control of translation of specific mRNAs during meiotic maturation. Despite advances in understanding the translational regulation during meiotic maturation, regulation at the 1-cell stage has not been explained. More studies are required to explain this complex mechanism of temporal mRNA recruitment after fertilization. Maternal mRNAs translated at different stages were examined to understand how specific maternal mRNAs are synthesized and stored, what are these maternal mRNAs, which maternal mRNAs are translated, and how these maternal mRNAs are temporally regulated. Polysomal mRNAs from eggs and 1-cell embryos were analyzed by microarray analysis and this indicated that temporally significant biological activities were encoded by mRNAs recruited at different stages of development. The mRNAs recruited in eggs were involved in homeostasis and transport mechanisms and those recruited in zygotes were involved in biosynthesis and metabolic activities. These data indicated that there is a temporal regulation of maternal mRNAs to meet the different biological requirements of the embryos. After the identification of temporally translated mRNAs, experiments were performed to understand the mechanism underlying temporal translation. The prevalence of the CPE differed between the two mRNA populations translated i.e., egg and 1-cell stage polysomal mRNAs. CPEs were present in ~53% of transcripts at the 1-cell stage compared to ~86% at the MII stage. This indicated that novel motifs other than CPEs regulate translation of mRNAs at the 1-cell stage. Truncation and deletion experiments were conducted using chimeric mRNAs based on one mRNA that was enriched in the 1- cell polysomes (Bag4). These experiments led to the identification of two regulatory regions that control translation at the 1-cell stage, an 80 nt region and a 43 nt region with different regulatory motifs. The 80 nt region is involved in activation of translation and the 43 nt region has an inhibitory effect on translation at the MII and early 1-cell stage. These results provide a detailed picture of how specific maternal mRNAs are prevented from undergoing translation at the MII stage and how the effect of inhibition is eliminated by the late 1-cell stage. / Biochemistry

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.