Robert B' Page, James R' Monaghan, Amy K' Samuels, Jeramiah J' Smith, Christopher K' Beachy and S' R

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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, Christopher K. Beachy
and S. Randal Voss
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To begin characterization of gene expression
during salamander metamorphosis Hypothesis no
variation in gene expression during metamorphosis
For comparison to endocrine disrupted
metamorphosis
To correlate patterns of gene expression with
morphological, developmental, and life history
malformations during endocrine disruption
To identify loci that will be useful in field
diagnostics of normal and endocrine disrupted
metamorphosis
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A phylogeny of model craniates that have a genome
project
WHY SALAMANDER?
--phylogenetic place
--natural history
--experimental possibilities and husbandry
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28 days after TH immersion
epithelial membrane protein 1
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CONTROL
T4 IMMERSION
no TH
50 nM TH
0 days
The promise and pain of genomic data 4844 loci
for analysis
2 days
12 days
28 days
One-factor ANOVA on expression intensity --over
time in control (no effects) --over time in TH
treatment (YES effects) --comparison of control
and TH treatment at each timepoint (YES
effects)
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Volcano Plot
4844 down to 325
P 0.001
P 0.01
P 0.05
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Volcano Plot
4844 to 325
to 123 loci for consideration
2-fold change in gene expression
(And supplement with significant BLASTX to human
genome)
P 0.001
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Dark circles represent the mean of three samples
? SE. (a) keratin 12, (b) keratin 14, (c)
keratin 15, (d) keratin 17, (e) keratin 24, (f)
keratin 4, (g) keratin 6A.
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Keratins Candidates for PCR Verification

• Why Keratins?
• - Several (7) from family meet all of our 3
  criteria
• - An additional 3 family members meet 2 of our 3
  criteria
• - Effect size is large (gt 1000 fold)
• - Likely to be useful in characterizing the
  process of normal metamorphosis

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Five replicates per treatment Total 150
salamanders for analysis, with GeneChips, PCR, or
rt PCR
Growth- PIT Fat - Metamorphosis
-THY Reproduction - GONAD
Morphological assays
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Comparisons with standard metamorphic GEP with
ATR-influenced GEP in tiger salamander
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