Date of Completion

5-9-2013

Embargo Period

10-29-2013

Keywords

Genomics, transcriptome, stem cell, neural differentiation, hindbrain, motor neuron

Major Advisor

Brenton R. Graveley

Co-Major Advisor

Blanka Rogina

Associate Advisor

Xue-Jun Li

Associate Advisor

Stormy Chamberlain

Associate Advisor

Ren-He Xu

Associate Advisor

Gordon Carmichael

Field of Study

Genetics and Genomics

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Almost every cell in any organism contains the same genomic content. Different cell types, however, display strikingly different morphologies, behaviors and functions. The central nervous system (CNS) provides an excellent example of cellular diversity. The CNS develops as progenitor cells migrate and mature into thousands of distinct subtypes of neurons and supportive cells.

The cellular transformation from undifferentiated, pluripotent, embryonic stem cell to mature neuron is dictated by changing patterns of gene expression. This process must be tightly regulated to ensure proper development. Much of our understanding of the development of the human CNS has been gleaned from cell culture, specifically using human embryonic stem cells (hESC). hESC are derived from the inner cell mass of blastocyst-stage embryos; they can self-renew indefinitely and differentiate into any mature cell type in the body. The complex architecture of the brain cannot be re-created in a dish, but hESC can be directed to differentiate into specific neuronal subtypes.

I have used RNA-Seq to analyze gene, transcription initiation site, and transcript expression patterns between the H9 and CT2 hESC lines. These experiments identified hundreds of genes and transcripts that are differentially expressed between the ES lines and among different culture systems. I also used RNA-Seq to study transcriptome dynamics as H9 ES cells differentiate into spinal motor neurons. These experiments demonstrated that, during neural differentiation, the expression levels of most genes stay relatively constant, while the number of genes and transcripts demonstrating divergent expression from hES cells increases throughout development. Consistent with previous work, a higher extent of alternative splicing was observed in neurons than in ES or differentiating cells. Semi-quantitative polymerase chain reaction (RT-PCR) validated expression changes for a subset of identified events. More than 90% of tested, identified splicing events were successfully validated by PCR. CNS development involves regulation at the epigenetic, transcriptional, post-transcriptional, translational and post-translational levels. Establishing a comprehensive understanding of the molecular events leading to neural differentiation of ES cells will require an integrated analysis of epigenetics, mRNA and small RNA expression, and a complete proteomic analysis. These datasets and analyses represent a small piece of this incredible puzzle.

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