Nutrient Dependent Regulation Of The Transcriptomic Dynamics In Hematopoietic Progenitors Of Drosophila And Humans

Abstract

Abstract Introduction In the intricate landscape of hematopoietic progenitor development, a complex framework of regulatory mechanisms orchestrates the dynamic changes in gene expression profiles. My thesis embarks on an investigation towards the identification novel mechanistic underpinnings imposed onto the intrinsic regulatory elements like micro RNAs and extrinsic microenvironmental cues that fine tune the expression of transcriptomic landscapes of developmentally relevant cell type “The Hematopoitic Progenitors”. Central to this exploration is the unveiling of the autophagy of microRNAs (miRNAs) that contribute to the fine tuning of a cell’s transcriptomic landscape. MiRNAs are small RNA molecules that play a crucial role in regulating gene expression 1 . They are involved in various biological processes, including development, differentiation, and disease progression 2 . Understanding the turnover of miRNAs is essential for unraveling their regulatory mechanisms and their impact on cellular processes and disease biology. While the biogenesis and mechanisms of action of miRNAs have been extensively studied, the turnover of miRNAs and its relationship with mRNA autophagy is an aspect that is yet to be fully deciphered. Research on miRNA turnover has raised questions about the stability of miRNAs. Some studies suggest that miRNAs are typically stable, while others propose that they are subject to active regulation 3,4 . The turnover of miRNAs is a dynamic process that involves their degradation and clearance from the cell which in turn can impact their abundance and, consequently, their regulatory activity 5 . The thesis illuminates the intricate interplay of miRNAs undergoing autophagic degradation, thus unveiling a previously uncharted aspect of regulation in hematopoietic progenitor gene expression. This discovery underscores the intricate nature of cellular control mechanisms during the process hematopoietic amplification in the progenitor state. With an account toward evolution, the thesis conducts a comparative analysis of Drosophila and human hematopoietic systems. By dissecting shared and distinct aspects of intrinsic and extrinsic regulation, the study provides a comprehensive view towards some of the novel mechanistic underpinnings that guide hematopoietic transcriptomes across species. As the research navigates through the complexities of gene expression control, it sets the stage for xvfuture inquiries to not only deepen our comprehension of hematopoiesis but also provides a framework for targeting general developmental and gene regulatory networks. The spatial positioning of cells within a tissue or microenvironment has emerged as a critical determinant of cellular behavior and functionality, shaping the intricate landscape of transcriptomic heterogeneity 6 . The advent of advanced single-cell RNA sequencing technologies has revolutionized our ability to capture and analyse the spatially driven transcriptomic heterogeneity at unprecedented resolution 7 . However, performing single-cell sequencing, while a powerful tool for unraveling cellular heterogeneity at the molecular level, comes with the limitation of destroying the spatial information that is inherent within intact tissue samples. This loss of spatial context is a trade-off inherent to the process. Beyond the cellular boundaries, the spatial positioning of hematopoietic progenitors is also an important factor to consider as a crucial extrinsic regulator. Investigating the unique spatial contexts within which these cells exist, this thesis underscores the profound impact of spatial dependencies on the availability of extrinsic factors. Implementing techniques that can decipher the aspect of spatial heterogeneity can provide a powerful handle to identify variance emerging in transcriptomic landscapes due to cellular positioning, thereby steering factors like lineage commitment and differentiation trajectories in these cell populations. Consequently, in response to the inherent challenge of retaining spatial information for the process of high-throughput sequencing, our endeavors encompass the refinement of methodologies aimed at pinpointing and quantifying spatially anisotropic tissue configurations, particularly within intricate structures such as the lymph gland. Therefore, we have developed and standardized a pioneering methodology that seamlessly integrates Laser Capture Microdissection (LCM) with advanced sequencing techniques, meticulously designed to uphold the spatial fidelity of samples under scrutiny.