Investigating the Regulation of Early Development of the Amphipod Crustacean Parhyale hawaiensis

Abstract

The amphipod crustacean Parhyale hawaiensis is an ideal model organism for studying development, evolution, and regeneration. Its ease of rearing in the lab, accessibility of embryos at all developmental stages, large broods produced year-round, and the variety of functional experiments that can be conducted on animals and embryos make it a valuable resource for investigating many biological questions. As an out group to insects, Parhyale offers a platform for studying biological diversity through comparative studies. Recent research has identified a full repertoire of single-copy genes encoding the machinery associated with DNA methylation in the Parhyale genome. This discovery provides an opportunity to explore the role of DNA methylation in early embryogenesis using an invertebrate model system. In this thesis, we present Parhyale hawaiensis as a tractable model to study DNA methylation dynamics during embryonic development. Our aim was to investigate the regulation during early embryo development, describe the maternal-to-zygotic transition (MZT) and zygotic-genome-activation (ZGA) in Parhyale, and explore the potential function of DNA methylation during embryogenesis while examining its regulatory role in gene expression. Parhyale possesses a large genome size of approximately 3.6 Gb, which was initially sequenced by Kao et al. in 2016. An update using Dovetail technology enhanced the assembly by generating large scaffolds butleft significant gaps. Therefore, our objective was to further improve the existing assembly by integrating PacBio data to close the gaps and correct assembly errors. This effort proved successful, as over 70% of the gaps in the assembly were closed, suggesting further coverage would give further improvement. Subsequently, we performed an expression-driven annotation using a wide range of RNA-seq data from various embryonic stages and different adult conditions. The well-annotated genome served as the foundation for analyzing transcriptomic data from early embryonic stages to detect de novo zygotic transcription using intronic RNA signals. We described the transcriptome of early Parhyale embryos and proposed a model for the maternal-to-zygotic-transition (MZT) and zygotic-genome-activation (ZGA) timelines. Our findings demonstrated that zygotic transcription begins as early as 11 hours post-fertilization and occurs in two waves. The minor wave of ZGA in Parhyale commences at the 32-cell stage, while the major wave takes place at the start of the blastodisc formation stage. We discovered that the earliest transcribed genes in Parhyale are typically short, intron-less or intron-poor, and newly evolved. Furthermore, we validated the presence of DNA methylation mediator genes, with a focus on DNMT1, DNMT3, and MBD2/3. We analyzed the expression pattern of DNA methylation machinery genes during the early stages of Parhyale embryogenesis and found that they are provided maternally. To explore the relationship between DNA methylation and gene expression, we correlated our gene expression datasets with methylseq datasets. Our results revealed a positive correlation between gene-body methylation and gene expression levels. Lastly, we conducted functional experiments targeting DNMT1, DNMT3, and MBD2/3 to understand their roles during embryonic development. We utilized CRISPR/Cas9 to generate knockout animals for each of the three genes, revealing that the loss of any of these genes is lethal to embryos. Additionally, we performed RNA interference (RNAi) knockdown on MBD2/3, which also resulted in an early embryonic lethality, confirming its essential role in embryogenesis. Profiling the transcriptome of knockdown embryos revealed that the knockdown of MBD2/3 altered the expression of many genes, including developmental transcription factors with low levels of gene-body methylation.The work presented in this thesis offers a comprehensive understanding of the early embryonic development of Parhyale and emphasizes the crucial role of DNA methylation during embryogenesis. In future, we aim to investigate whether MBD2/3 regulates gene expression through its association with the NuRD complex, and if this occurs in a DNA methylation-dependent, independent, or both manners. Furthermore, the improved assembly and annotation presented in this thesis will greatly facilitate more precise analyses, enabling us to address intriguing questions regarding the promising model organism Parhyale hawaiensis.