Microarray analysis identifies keratin loci as sensitive biomarkers for thyroid hormone disruption in the salamander Ambystoma mexicanum

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Abstract

Ambystomatid salamanders offer several advantages for endocrine disruption research, including genomic and bioinformatics resources, an accessible laboratory model (Ambystoma mexicanum), and natural lineages that are broadly distributed among North American habitats. We used microarray analysis to measure the relative abundance of transcripts isolated from A. mexicanum epidermis (skin) after exogenous application of thyroid hormone (TH). Only one gene had a > 2-fold change in transcript abundance after 2 days of TH treatment. However, hundreds of genes showed significantly different transcript levels at days 12 and 28 in comparison to day 0. A list of 123 TH-responsive genes was identified using statistical, BLAST, and fold level criteria. Cluster analysis identified two groups of genes with similar transcription patterns: up-regulated versus down-regulated. Most notably, several keratins exhibited dramatic (1000 fold) increases or decreases in transcript abundance. Keratin gene expression changes coincided with morphological remodeling of epithelial tissues. This suggests that keratin loci can be developed as sensitive biomarkers to assay temporal disruptions of larval-to-adult gene expression programs. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander, thus setting the stage for future investigations of TH disruption in the Mexican axolotl and other salamanders of the genus Ambystoma.

Introduction

Vertebrates use many of the same hormones to regulate periods of post-embryonic development. For example, thyroid hormone (TH) is essential for initiating and orchestrating amphibian metamorphosis, a developmental period in which a suite of genetic, molecular, cellular, morphological, and physiological changes are induced to transform aquatic larvae into more terrestrial adults. Similarly, TH is essential for initiating normal growth and maturation of organ systems during critical windows of early mammalian development. Many aspects of TH regulation and action are evolutionarily conserved between mammals and amphibians, including thyroid hormone synthesis, secretion, and transport (Barrington, 1962, Larsson et al., 1985, Power et al., 2000), tissue-specific regulation of TH concentration (St. Germain and Galton, 1997, Kester et al., 2004), attenuation of TH affect via synergistic interactions with corticosteroids (Hayes, 1997, Helmreich et al., 2005, Kuhn et al., 2005), TH-mediated regulation of gene expression by steroid nuclear receptor isoforms and accessory factors (Lazar, 1993, Brent, 1994, Shi, 2000, Buchholz et al., 2006), and pituitary regulation of TH release (Denver et al., 2002). The conservation of TH-dependent development among vertebrates indicates that tractable non-mammalian organisms can be exploited as models for TH disruption in human.

Amphibians offer special advantages for studying endocrine disruption of TH and other developmentally important hormones (Tata, 1993). In contrast to working with early mammalian life stages, it is straightforward to administer hormones to free-living amphibian embryos and larvae to study the effects of normal and disrupted endocrine signaling on development (e.g. Kitamura et al., 2005). Hormones and toxicants can simply be added to the water in which amphibians are housed and developmental effects can be measured using either gross morphology (e.g. Degitz et al., 2005) or molecular tools (e.g. Turque et al., 2005). Another advantage is that the amphibian metamorphic program presents some of the same basic questions that confront researchers working on early mammalian development (e.g. Zoeller, 2004): How does a single molecule (TH) orchestrate different cellular-level changes and coordinate developmental processes among multiple tissue types, and What are the consequences of delayed or inadequate delivery of TH during critical windows of development? Indeed, the recent finding that a single quantitative trait locus contributes to both developmental timing variation and clinical hypothyroidism in a non-metamorphosing salamander suggests there is much to be learned from studying amphibians (Voss and Smith, 2005). Over the last few decades, amphibian research has helped conceptualize TH-associated trade-offs in allocations to growth, maturation, and developmental plasticity. This body of work is providing a framework for understanding the consequences of disrupted, fetal and natal development in humans (Amiel-Tison et al., 2004, Crespi and Denver, 2004, Zoeller, 2004).

In addition, amphibians are likely to become important sentinels in determining minimal contaminate levels that threaten human health. Natural populations of amphibians are declining rapidly and there is growing concern that endocrine disrupting chemicals are contributing significantly to their demise (e.g. Reeder et al., 2005). For example, the herbicide Atrazine has been linked to developmental abnormalities, reduced growth, and altered metamorphic timing (Hayes, 2005); such insults can affect larval life history parameters (e.g., age and size at metamorphosis, and future reproductive success) that directly impact long-term population stability (sensu Semlitsch et al., 1988, Wilbur, 1996). To better monitor natural populations for endocrine disruption, it will be important to develop methods that allow comprehensive assessment of the thyroidal axis, from neuroendocrine control to target tissues. Additionally, methods must be able to diagnose TH axis disruptions of development from a multitude of natural and anthropomorphic stressors that can yield convergent phenotypes at the morphological level.

Microarray analysis offers the potential to identify diagnostic gene expression patterns that can differentiate among different types of endocrine disruption. Microarray analysis may also provide the power to differentiate among the several molecular and cellular bases (i.e. sites of action) that are possible for each disrupter type. To date, relatively few microarray resources have been developed and applied for work in amphibians, with all effort directed toward anurans (Crump et al., 2002, Baldessari et al., 2005, Chalmers et al., 2005, Turque et al., 2005). We developed a custom Affymetrix GeneChip to identify and monitor genes that are responsive to TH signaling and endocrine disruption in Ambystoma mexicanum, a salamander with a maturing, model organism infrastructure. Genomic, bioinformatic, and living stock resources are available for ambystomatids and a networking infrastructure is under development (Smith et al., 2005). Ambystomatids are easily reared in the laboratory using standardized culture methods and they offer the advantage of being broadly distributed among North American habitats. We report the results of a study that measured the relative abundance of transcripts isolated from Mexican axolotl (A. mexicanum) epidermis (skin) after exogenous application of TH to whole animals. Our study has identified the first collection of loci that are regulated during TH-induced metamorphosis in a salamander amphibian, and thus set the stage for future investigations of TH disruption in the axolotl and other ambystomatid salamanders.

Section snippets

Salamanders, experimental design, and tissue collection

Salamanders (A. mexicanum) were obtained from a single genetic cross, using adults from an inbred strain that is maintained by Voss's group at the University of Kentucky. Embryos and larvae were reared individually at 20–22 °C in 40% Holtfreter's solution. After hatching, larvae were fed freshly hatched brine shrimp (Artemia sp.) nauplii until they were large enough (3 weeks) to eat California blackworms (Lumbriculus sp.; J.F. Enterprises). At approximately 8 months of age, 24 animals were

Morphological metamorphosis

Although A. mexicanum do not normally undergo metamorphosis in the laboratory, metamorphosis can be induced with T4. Salamanders that were sampled after 2 days of T4 treatment showed no external morphological signs of having initiated metamorphosis (Stage 0, Cano-Martinez et al., 1994); thus, they were morphologically identical to day 0 control salamanders. Salamanders that were sampled after 12 days of T4 treatment showed early morphological signs of metamorphosis (reduction of tail fins and

Discussion

Although there have been several microarray analyses of anuran amphibians, ours is the first using a salamander. The custom GeneChip we developed identified a collection of TH-regulated genes that will enable future studies of post-embryonic development, specifically in the areas of developmental genetics and endocrine disruption. Below, we discuss our results within the context of TH-regulated gene expression as it applies to amphibian epidermal development and metamorphosis using the

Role of skin and microarray analysis in endocrine disruption

Although several biochemical and molecular assays have been proposed as tests for endocrine disruption of the TH axis, morphological endpoint methods are more commonly used in amphibians (e.g. Degitz et al., 2005, Opitz et al., 2005). These methods use pre-prometamorphic and prometamorphic Xenopus laevis tadpoles and developmental staging series to evaluate TH induced changes in morphology and developmental timing variation. Our study shows that similar morphological tests can easily be

Ambystoma as a model for endocrine disruption

Our study sets the stage for future gene expression experiments that promise to yield exacting assays for monitoring endocrine disruption in the laboratory and in nature. This later goal is achievable because genome and bioinformatics resources can be extended from the laboratory model A. mexicanum to ambystomatids that are widely distributed among diverse aquatic habitats in North America (Putta et al., 2004, Smith et al., 2005). For over 35 years, the National Science Foundation has provided

Acknowledgements

We acknowledge the service of Donna Walls in the UK Microarray Facility. This project was made possible by grant numbers 5R24RR016344, 2P20RR016741, and 2P20RR016481-04 from the National Center for Research Resources, a component of the National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Aspects of this project were also made possible by funding from the U.S. National Science Foundation

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