Gene Expression and Enhancer Discovery in the Neural Crest
Date of Award
Doctor of Philosophy (PhD)
Generation of complex body plans by multicellular organisms requires an intricate program of specialized gene expression in different cells during development. To understand cell identity, it is necessary to know which combinations of genes result in specific cell types, particularly in cell
types that are closely related by developmental lineage. Here, I describe a method to purify and analyze the transcriptomes of several pigment cell types from the zebrafish, Danio rerio. Using this method, I identified several gene combinations that are specifically expressed in each of these cell types. This revealed a dramatic upregulation of several metabolic pathways that generate the guanine-based pigment of iridophores. Another requirement for understanding how unique cell types develop and maintain their identity is the identification of the regulatory features necessary for cell-specific gene expression. Towards this end, I describe a method to
identify the active cis-regulatory elements within enhancers of expressed repeats. I apply this method, termed LTR Enhancer Activity Prediction (LEAP), to predict the necessary transcription viii factor binding sites within a neural crest-specific enhancer derived from a LTR-containing retrotransposon. Using transgenic reporter assays, I demonstrate that LEAP accurately predicts the cis-regulatory positions that confer neural crest-specific enhancer activity of this enhancer. Finally, identifying the dynamics of transcription factor binding in precursor cells that result in specific cell fate decisions will be necessary to understand the complex system of development of multicellular organisms. Thus, I describe the implementation of "Calling Cards", a method developed in the Mitra lab to trace transcription factor binding events during development, in an
intact vertebrate model organism. In this effort I identify the first large-scale genome-wide insertions of DNA transposons via the transient expression of transposase:transcription factor protein fusions in zebrafish embryos, and show that TFIIB-directed Calling Card insertions occur near the transcription start sites of genes. Together, these studies represents steps toward a fuller
understanding of gene expression, gene regulation, and development.
Chair and Committee
Jim Skeath, Ting Wang, Kelly Monk, Zachary Pincus
Higdon, Charles, "Gene Expression and Enhancer Discovery in the Neural Crest" (2015). Arts & Sciences Electronic Theses and Dissertations. 430.