Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma) Robert B. Page1,

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Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma) Robert B. Page1, S. Randal Voss1, Amy K. Samuels1, Jeramiah J. Smith1, Srikrishna Putta1, and Christopher K. Beachy2* 1Department of Biology and Spinal Cord and Brain Injury Research Center, University of Kentucky; and 2Department of Biology and Amphibian Growth Project, Minot State University

Comparisons of standard metamorphic gene expression and morphology profile with endocrine-disrupted profile in salamander Ambystoma mavortium (tigrinum) Ambystoma mexicanum metamorphosis Paedomorphosis (= metamorphic failure) + 5 nM TH metamorphosis

Basically….. skin liver brain tail Collect mRNA cDNA microarray Grow salamanders in experimental design skin liver brain tail Collect mRNA cDNA microarray

cDNA T

28 days after immersion in 5 nM TH epithelial membrane protein 1

DESIGN CONTROL T4 IMMERSION T4 IMMERSION no TH 5 nM TH 50 nM TH 0 days 2 days 12 days 28 days

15 DAYS POST- EXPOSURE control 50 nM TH 5 nM TH

ANALYSIS PART I: UNIVARIATE ANALYSIS A series (4844) of one-factor ANOVAs comparing expression level among treatment groups (I.e., 0, 5, and 50 nM TH) Linear and quadratic regression analyses Used to identify genes for multivariate analysis

Volcano Plot representing univariate analyses 4844 to 802 loci for consideration (= DEGs) P = 0.05 2X up or down expression relative to controls

MULTIVARIATE ANALYSIS ANALYSIS PART II: MULTIVARIATE ANALYSIS Principal Components Analysis Bioinformatic comparison to other microarray databases: metamorphosis in Xenopus

day 28 day 12 day 2 day 0

day 12 day 28 day 2 day 0

day 28 day 12 day 28 day 12 day 2 day 2 day 0

A phylogeny of model craniates that have a genome project mouse human chick Xenopus axolotl zebrafish Mammalia Amniota Lissamphibia Tetrapoda Osteichthyes A phylogeny of model craniates that have a genome project WHY SALAMANDER? --phylogenetic place --natural history --experimental possibilities and husbandry

Bioinformatic Comparison with Xenopus TH-skin (this study, 2008, BMC Genomics 9:78) TH-intestine (Buccholz et al., 2007, Dev. Biol. 303:576-590) 802 DEGs 1997 DEGs n = 111 EXPRESSION PATTERN % of genes Common DEGs

To view microarray database, visit Finishing up -TH dose influence timing of onset -Sets of transcription responses -Diversity in expression To view microarray database, visit -www.ambystoma.org