Department of Biology and Salamander Genome Project Department of Biology and Amphibian Growth Project Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in salamanders (Ambystoma) Robert B. Page, James R. Monaghan, Amy K. Samuels, Jeramiah J. Smith, and Christopher K. Beachy and S. Randal Voss To begin characterization of gene expression during salamander metamorphosis Hypothesis: no variation in gene expression during metamorphosis For comparison to endocrine disrupted metamorphosis To identify loci that will be useful in field diagnostics of normal and endocrine disrupted metamorphosis To correlate patterns of gene expression with morphological, developmental, and life history malformations during endocrine disruption THE GOAL Skin Liver brain Collect mRNA cDNAMICROARRAY Grow axolotls (Ambystoma mexicanum) Basically….. THE PLAN For our microarrays, we use Affymetrix GeneChips to quantify gene expression for approximately 4800 genes, many of which are homologous to human genes. This allows for a comparison to the human genome, and establishes salamander as an excellent model system to understand the role of xenobiotics in human health issues. HOW A MICROARRAY WORKS theoretically… …and an example of ours (based on the animal on the above cover!) AND OUR EXPERIMENT 0 days 2 days 12 days 28 days CONTROL IMMERSION IN T 4 21 animals (3 per treatment), thus 21 GeneChips used for analysis. epithelial membrane protein 1 is upregulated in this metamorphosing salamander 28 days after TH immersion There are approximately 4800 genes for examination on this GeneChip (or “microarray”). This work is funded by This work succeeds because of the continued work of the members of the Amphibian Growth Project: Drew Henry, Janel Richter, Ken Cabarle, Claude Ouedraego, Jeremiah Johnson, Heather Modrow, Judd Entzel and (especially) Karen Pocha-Melby. PCR verification of microarray SOME OF OUR MICROARRAY RESULTS Genes listed here are those that were significant at P < 0.001, exhibited at least a two-fold change in expression when compared to controls, and also have a match to the human genome. There are many other significant patterns of gene expression that do not meet these three criteria and are subject to further analysis. This is a “housekeeping” gene, i.e, it is expressed at the same levels all the time and is used as a control and baseline to evaluate up- and down-regulation of the other PCR products. We used PCR methods as a secondary verification of our microarray results. We chose to examine keratin gene expression; these genes were an important part of the identified genes presented above and should be ideal for field diagnostics of endocrine-disrupted metamorphic development (see below). Red arrows indicate significant up-regulated metamorphic genes (“turned on genes”) and green arrows indicated down- regulated metamorphic genes (“turned off genes”). GREEN IS “OFF GENES”; RED IS “ON” GENES. no TH 50 nM TH 0 days 2 days 12 days 28 days 5 nM TH 0  g/L (ppb) 1  g/L (ppb) 5  g/L (ppb) Atrazine dosage COMPARING THE STANDARD Gene Expression Profile (GEP) WITH AN ENDOCRINE DISRUPTED GEP The establishment of the gene expression profile during normal induction of metamorphosis now allows for a comparison to endocrine-disrupted metamorphosis. This project is ongoing; please visit this experiment at the AGP in Moore 232 and 214.