Transcript PPT - Department of Biology

Effect of thyroid hormone concentration on the transcriptional response
underlying induced metamorphosis in the Mexican axolotl (Ambystoma)
Robert B.
1
Page ,
S. Randal
1Department
1
Voss ,
Amy K.
1
Samuels ,
Jeramiah J.
1
Smith ,
Srikrishna
1
Putta ,
and Christopher K.
2*
Beachy
of Biology and Spinal Cord and Brain Injury Research Center, University of Kentucky; and
2Department of Biology and Amphibian Growth Project, Minot State University
RESULTS
INTRODUCTION
Genomic approaches offer great potential to biomonitoring studies. First, functional genomics
(e.g., microarray analysis) can be used to gain mechanistic perspective about complex
transcriptional responses to environmental toxicants (e.g., cadmium) in non-model organisms
(i.e., the tiger salamander in North Dakota, Ambystoma tigrinum). This discovery-driven
approach is an excellent tool especially when complemented with complex laboratory
functional genomics experiments (e.g. qPCR analysis) on the salamander model axolotl
(Ambystoma mexicanum) for which a growing genomics infrastructure exists (Smith et al.,
2005; Page et al., 2008). Second, while most genomics projects have provided primarily
descriptive data, projects can use genomics approaches to directly test hypotheses.
PCA representation of GeneChip results.
Comparison of differentially expressed genes.
Environments can show continuous and discrete variation in cadmium levels. Very few
organisms experience more than one level of exposure to this geographic variation. However,
salamanders have a metamorphic life history with two stages that results in exposure to
aquatic-borne cadmium (in larvae) and soil-based cadmium (in transformed salamanders).
This makes salamander an excellent system for use in genomic biomonitoring studies. We
used microarray analysis to determine the standard transcription profile across THinduced metamorphosis in the axolotl. This standard will serve in comparison to disrupteddevelopment transcription profiles.
These recovered gene expression profiles differ because some genes are expressed in larval
amphibians and turned off at metamorphosis, some are turned on at metamorphosis, and
some genes are only up- or down-regulated during metamorphic remodeling. For the purposes
of presentation, LD, QCLD, and QLVD are lumped as “down” and LU, QLVU, and QLCU are
lumped as “up.”
METHODS AND PROTOCOL
These are scatterplots of the 30 GeneChips based on the rotations of
their first two principal components (PC1 and PC2). PCA was
performed on all 3688 probe-sets that were available from
significance testing. PC1 and PC2 account for 89.2% and 6.2% of
the variance respectively. Cano-Martinez development stages are
listed in parentheses in the legend.
USING SALAMANDER IN BIOMONITORING AND
CHARACTERIZING DISRUPTED DEVELOPMENT
Influence of TH on metamorphosis in axolotl.
Salamanders are immersed in T4 (thyroxine)
solution, and metamorphosis can be assessed
morphologically in several ways: reduction of tail fin,
loss of gill fimbrae, loss of gill bars, change in
pigmentation. Additional morphological indicators
can be later assessed, e.g., cranial morphogenesis
can be inspected by clearing and staining
salamanders. The morphogenetic changes can
then be correlated with transcription profiles.
Generalized regression patterns. These are
generalized regression patterns recovered by the
methodology describe in Liu et al. (2005).
Abbreviations are as follows: LD = linear down, LU =
linear up, QLCD = quadratic linear concave down,
QLCU = quadratic linear concave up, QLVD = quadratic
linear convex down, QLVU = quadratic linear convex up,
QC = quadratic concave, and QV = quadratic convex.
Our results allow for the first bioinformatic comparison of
transcription profiles during TH-induction of metamorphosis
between salamander and frog (Xenopus). We chose to
compare our results with salamander skin and frog intestine.
We chose this comparison because both systems feature
substantial remodeling of ephithelium. We found that only
13% of the genes that were differentially expressed in axolotl
were expressed in Xenopus. Of these, fewer than half were
expressed in the same direction as in Xenopus, e.g., a
majority of the genes that were down-regulated in axolotl
were up-regulated in Xenopus and vice versa.
REFERENCES
Buchholz, D.R., R.A. Heimeier, B. Das, T. Washington, Y.-B. Shi. 2007. Pairing morphology with gene expression and identification of a core set of TH-induced
genes across tadpole tissues. Developmental Biology 303:576-590.
Acknowledgments. Aspects of this projects were supported by NIH Grant
Numbers P20RR-016741 and P20RR-16481 from the INBRE program of
NCRR, NIH Grant Number 5-R24-RR016344-06 from NCRR, and NSF Grant
Number IBN-0242833 of the CAREER Award Program. Ryan Winburn
provided the cadmium content analyses. Donna Walls and the University of
Kentucky Microarray Core Facility processed the GeneChips associated with
this study.
Cano-Martinez, A., A. Vargas-Gonzalez, and M. Asai. 1994. Metamorphic stages in Ambystoma mexicanum. Axolotl Newsletter 23:64-71.
Page, R.B., S.R. Voss, A.K. Samuels, J.J. Smith, S. Putta, and C.K. Beachy. 2008. Effect of thyroid hormone concentration on the transcriptional response
underlying induced metamorphosis in the Mexican axolotl (Ambystoma). BMC Genomics 9:78.
Smith, J.J., S. Putta, J.A. Walker, D.K. Kump, A.K. Samuels, J.R. Monaghan, D.W. Weisrock, C. Staben, and S.R. Voss. 2005. Sal-site: integrating new and existing
ambystomatid salamander research and informational resources. BMC Genomics 6:181.
Somji, S., J.H. Todd, M.A. Sens, S.H. Garrett, and D. A. Sens. 2000. Expression of Heat Shock Protein 60 in Human Proximal Tubule Cells Exposed to Heat,
Sodium Arsenite and CdCl2. Toxicol Lett. 115:127-136.
Metamorphosis as stress signalling event.
Metamorphosis represents a developmental
threshold in salamanders that is thyroidregulated and is a stressful life-history
transition, i.e., metamorphosing salamanders
lose mass, dehydrate, and are susceptible to
predation. This transition may be subject to
more intensive cadmium accumulation. This
is interesting because microarray analysis of
axolotls undergoing TH-induced
metamorphosis down-regulate MT during the
transformation. Several additional
biomarkers may be especially useful then in
characterizing disrupted development in
salamanders, e.g, keratin 6A is downregulated and hsp70 is up-regulated across
the metamorphic transformation. Keratin 6A
is associated with cadmium-induction of
several cancers in human (Somji et al., 2004)
and transcription of hsp70 is a known general
stress response.
Salamanders as bioaccululators. Cadmium content
analysis of soil, water and salamanders in each habitat
indicate that salamanders are accumulators of cadmium.
In both aquatic and terrestrial habitats, salamanders exhibit
higher liver cadmium content than the habitats that they are
found in. This supports the idea that salamanders are
accumulators of environmental cadmium and represent an
excellent and novel biomonitoring species. Tiger
salamanders are at the top of the food web in both
systems. No other vertebrate species exhibits these
characteristics.