Date of Award

Summer 8-15-2017

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

The seemingly limitless capacities of mammals to sense, respond, and manipulate their environments stems from their structurally and functionally diverse nervous systems. Establishing these complex behaviors requires the integration of many biological phenomena including, morphogenetic gradients, cell-cell signaling, transcriptional networks, cell migration and epigenetic gene regulation. As mammalian development progresses, these pathways coordinate the production of highly specialized neuronal and glial cells, that connect and communicate with another in an even more complex manner. While evolution has shaped a multitude of pathways to produce numerous favorable traits, it has also created an intricate system vulnerable to disease. The loss of different types of neurons, each responsible for specialized biochemical communications within the brain and spinal cord, results in a wide variety of neurological and neurodegenerative diseases. Unfortunately, many of these diseases are uniquely human and cannot be wholly studied in model organisms such as Mus musculus or Drosophila melanogaster. Further, their location and absolute necessity precludes isolation directly from patients. Fortunately, advances in our understanding of genetic pathways that specify neuronal cell fates during development have enabled the directed differentiation of embryonic and induced pluripotent stem cells (iPSCs) into specific neuronal subtypes. This knowledge has been further leveraged to directly convert (reprogram) non-neuronal somatic cells into neurons, bypassing the induction of pluripotency. Specifically, ectopically expressing small non-coding microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), with transcription factors in human adult fibroblasts is sufficient to generate functionally mature neuronal subtypes. These direct conversion modalities may prove invaluable in the study of late-onset neurodegenerative diseases, as the original age of human fibroblasts is maintained in converted neurons in contrast to the cellular rejuvenation observed in iPSCs. However, little is known about the epigenetic and molecular events that accompany direct cell-fate conversion limiting the utility of these features. Further, the capacity of miRNAs alone to overcome cell fate barriers has largely been unexplored. Within this thesis I provide mechanistic insights into the cell-fate pioneering activity of miR-9/9*-124. These results demonstrate that miRNAs induce remodeling of chromatin accessibilities, DNA methylation and the transcriptome leading to the generation of functionally excitable neurons. Surprisingly, during neuronal reprogramming, miR-9/9*-124 opens neuronal gene loci embedded in heterochromatic regions while simultaneously repressing fibroblast loci, revealing how miRNAs may overcome the cell-fate barrier that exists in human fibroblasts. These findings led to the discovery of a miRNA-induced permissive neurogenic ground state capable of generating multiple, clinically relevant neuronal subtypes. As such, I show that the addition of motor neuron factors, ISL1 and LHX3, can function as terminal selectors to specify neuronal conversion to a highly enriched population of human spinal cord motor neurons. Altogether, the work contained within this thesis identifies miRNA-mediated epigenetic remodeling events underlying direct neuronal conversion of human fibroblasts and a modular platform capable of generating multiple, clinically relevant neuronal subtypes directly from patients.

Language

English (en)

Chair and Committee

Andrew S. Yoo

Committee Members

Robert Mecham, Shelly Sakiyama-Elbert, David Gutmann, Samantha Morris,

Comments

Permanent URL: https://doi.org/10.7936/K7HM57V3

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