Document Type

Restricted

Advisor

Deborah Eastman

Publication Date

2016

Comments

Winner of the 2016 Oakes Ames Prize for best honors paper.

Abstract

Ambystoma mexicanum, better known as the Mexican Salamander or Axolotl, is a classic model of vertebrate development and organ regeneration. Our primary research interest is in the activity of the Notch pathway controlling the patterning and differentiation of taste buds in the axolotl. The Notch pathway is a highly regulated juxtacrine signaling pathway. In order to better understand the activity of the pathway in these cells, the levels of expression of various Notch pathway genes during different stages of development is essential. There are also many microRNAs (miRNAs) associated with the post-transcriptional regulation of the Notch pathway. A knowledge base of the expression and regulation of the associated genes must be established to understand the activity and regulation of the Notch pathway in developing taste buds. However, since the axolotl genome has not yet been fully sequenced, it is currently difficult to study gene expression and post-transcriptional regulation in axolotls.

Expressed Sequence Tag (EST) databases, which are comprised of single passes of cDNA sequences reverse transcribed from mRNAs, are commonly generated for species that lack sequenced genomes. However, the single pass on cDNA, in addition to library construction processes, may cause EST data to be unreliable. 5

In this study, we sought to determine and validate a method of microRNA target discovery using an A. mexicanum EST database (kindly provided by R. Voss, University of Kentucky). We established an open reading frame (ORF) for the EST data, then searched the 3’UTR for 7-mer motifs and annotated the found motifs by comparing them to those found in other vertebrates the mirBase.org dataset. We performed qRTPCR on RNA isolated from axolotl embryos to validate the presence of three miRNAs, whose target site motifs we identified in the EST database. We also identified miRNAs and their target sequences in genes of the Notch Pathway.

In addition to our miRNA studies, a transcriptome analysis using RNA-Seq was performed on various stages of development in axolotl oropharyngeal endoderm tissue. This data was used to build de novo transcriptome assemblies for each stage of development. The data was analyzed for RNA-Seq read quality and assembly quality. The results of the transcriptome assembly will be analyzed for differential gene expression.

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The views expressed in this paper are solely those of the author.