ORCID

0000-0002-3984-541X

Date of Award

5-3-2024

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Developmental, Regenerative, & Stem Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Each cell in the brain has the same genomic sequence, yet they can have vastly different phenotypes and function. This diversity is a result of complex genetic and signaling pathways, and knowing how these are regulated is key to understanding physiological development and how pathogenic dysfunctions arise. Genomic methods such as chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq) have played pivotal roles in dissecting these processes. However, a key limitation is that the cells are destroyed upon observation of their molecular states, which preclude our ability to correlate historical epigenetic information with future readouts of cell function or animal behavior. This dissertation explores technological advancements and their application in studying neurodevelopmental disorders. First, I introduce the design and iterative development of Calling Cards, a method that records transient molecular events, enabling retrospective analysis of gene regulatory elements and gene expression. This is particularly pertinent for analyzing cellular outcomes that are undetermined at the time of a molecular event. To demonstrate the unique insight that this can provide, I apply Calling Cards in the mouse brain to understand how the observed consequences of neurodevelopmental disorders can be associated with historical molecular events. This dissertation is structured as follows: Chapter 1 introduces foundational concepts and context, particularly focusing on epigenetics of the developing brain. Chapter 2 offers a detailed guide to bulk Calling Cards, equipping researchers at all levels to conduct and analyze these experiments independently, featuring enhanced reagents and protocols for improved assay sensitivity and flexibility. This chapter also includes a guided tutorial of custom software for data processing, facilitating broader access and application of this technology. Chapter 3 details the generation of transgenic Calling Cards mouse lines, sharing the insights gained from this approach. Chapter 4 applies bulk Calling Cards to examine epigenetic differences in brain masculinization and their role in sex-dependent gene expression, revealing candidate genomic regions associated with neurodevelopmental disorders may be influenced by perinatal hormonal fluctuations. The dissertation continues into the realm of single-cell genomics, presenting the combinatorial indexing Calling Cards method, which allows for the concurrent analysis of the transcriptome and gene regulatory elements. Chapter 5 is a study that focuses on defining the core phenotype of a syndromic neurodevelopmental disorder. It uses the transcriptomic data to delineate how cortical development goes awry in a bona-fide model of autism and intellectual disability. Chapter 6 delves into the methodological advancements of adapting Calling Cards to the combinatorial indexing platform and shows preliminary analysis of the gene regulatory elements, setting the stage for future research. Finally, Chapter 7 concludes this dissertation by summarizing the key findings and significance of each chapter and proposing potential future directions. In summary, the presented body of work expands our understanding of epigenetic gene regulation in brain development and neurodevelopmental disorders, offering new perspectives on the epigenetic underpinnings of these processes. The advancements in Calling Cards technology presented herein aims to equip the scientific community with innovative tools for exploring biological phenomena across various fields and disciplines.

Language

English (en)

Chair and Committee

Joseph Dougherty

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