Date of Award

Fall 2024

Language

English

Embargo Period

11-10-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Program

Biomedical Sciences

First Advisor

Prashanth Rangan

Second Advisor

Randall Morse

Committee Members

Joseph Wade, Mark Van Doren, Janice Pata, Randall Morse and Prashanth Rangan

Keywords

Germ cell, maternal, oocyte, transcription, translation, ribosomes, chromatin organization

Subject Categories

Cell and Developmental Biology | Developmental Biology | Genetics | Life Sciences | Molecular Genetics

Abstract

In sexually reproducing organisms, the process of gametogenesis produces mature eggs and sperms required for fertilization to occur. The egg's quality is crucial for the healthy development of the next generation, making oogenesis a tightly regulated process with multiple benchmarks. To investigate the intricacies of oogenesis, we utilized the Drosophila oogenesis system as our primary model. This choice was motivated by several factors. Firstly, the process of egg development in Drosophila is conserved across species, making it a valuable proxy for understanding oogenesis in other organisms, including humans. Secondly, Drosophila melanogaster has long been established as a powerhouse in genetic research, offering a wealth of tools and techniques for precise genetic manipulation. This thesis focuses on one of the main oogenesis benchmarks our lab has found to be highly regulated: the Germ cell to maternal transition (GMT).

During GMT, four key events occur: First, germline stem cells (GSCs) differentiate into mature oocytes, switching off early oogenesis and stem cell associated genes while activating maternal genes necessary for embryonic development. Second, the GSCs transition from a mitotic program to a meiotic program. Third, ribosomes ensure proper translation of transcripts needed for oocyte specification, with any decrease in ribosome numbers or damage leading to arrested oogenesis. Lastly, a selection mechanism determines which mRNAs are maternally deposited into the oocyte, essential for early development when the zygotic genome is in quiescent state.

This thesis explores what regulates the silencing of early oogenesis genes during GSCs differentiation into a mature oocyte and what dictates the timing of this transition. We find that genome organization influences the Nuclear Pore Complex (NPC) formation, which is necessary for proper GSC differentiation and silencing of early oogenesis genes. We also find that appropriate ribosome levels are critical for this transition, as they regulate NPC formation, which is in turn essential for oogenesis. In this thesis, we show how the nucleopore complex is controlled at both transcriptional and translational levels. We examine why such precise control is necessary for GMT to occur and the correct development of the egg, which will give rise to the next generation of offsprings.

Throughout this work, I detail our findings on the interplay between genome organization, NPC formation, and ribosome regulation during GMT. I discuss how these factors work together to ensure successful oogenesis and the production of high-quality eggs capable of launching the next generation.

This thesis builds on work that I contributed to in the lab, examining, how SETDB1, a histone methyltransferase, shapes oocyte identity in Drosophila. We previously showed that as SETDB1 moves to the nucleus, it silences differentiation genes by forming heterochromatin and unexpectedly promotes nuclear pore complex (NPC) formation. These NPCs then anchor SETDB1-created heterochromatin, reinforcing gene silencing. Disrupting this process leads to abnormal gene expression, loss of oocyte identity, and infertility. We proposed a novel feedback loop between heterochromatin and NPCs that drives genetic reprogramming in early oocyte development, offering new insights into cell fate determination.

We then investigated the intricate interplay between genome organization and NPCs formation during the differentiation of germline stem cells to oocytes in Drosophila. We examine how heterochromatin-mediated gene silencing and NPC-facilitated gene anchoring at the nuclear periphery contribute to this cellular transformation. Central to this study is the role of the transcription factor Stonewall (Stwl), which accumulates at the boundaries of active and silenced genomic regions. Our research demonstrates that Stwl is pivotal in both silencing germ cell genes and activating oocyte-specific genes and nucleoporins. This dual action of Stwl reveals a novel feedback mechanism where genome architecture promotes NPC formation, which in turn further modulates genome organization. This thesis argues that this reciprocal relationship between genome structure and NPCs is fundamental to successful cell fate transitions, providing new insights into the molecular mechanisms underlying germline development. Through my work, I focus on the crosstalk between genome organization and NPCs and show that genome organization is required for transcription of NPCs.

I also focused on the role of ribosomes in the formation and maintenance of NPCs. Adequate ribosome levels are required for the translation of NPCs, with any decrease in ribosome numbers or damage leading to arrested oogenesis. We show that disruption of ribosome biogenesis, TOR pathway and elongation machinery cause the downregulation of the translation of NPCs among other factors, leading to the ectopic upregulation of germ cell and early oogenesis genes, heterochromatin changes and mid-oogenesis arrest.

In summary, this work highlights the critical role of GMT in maintaining proper oocyte development and fertility. Our findings expand the current understanding of reproductive biology and provide new perspectives on potential approaches to improve reproductive health and address infertility issues. By elucidating the molecular mechanisms underlying this crucial developmental stage, this research contributes valuable insights to both basic science and potential clinical applications in reproductive medicine.

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This work is licensed under the University at Albany Standard Author Agreement.

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