Identification of differentially expressed thyroid hormone responsive genes from the brain of the Mexican Axolotl (Ambystoma mexicanum)

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Abstract

The Mexican axolotl (Ambystoma mexicanum) presents an excellent model to investigate mechanisms of brain development that are conserved among vertebrates. In particular, metamorphic changes of the brain can be induced in free-living aquatic juveniles and adults by simply adding thyroid hormone (T4) to rearing water. Whole brains were sampled from juvenile A. mexicanum that were exposed to 0, 8, and 18 days of 50 nM T4, and these were used to isolate RNA and make normalized cDNA libraries for 454 DNA sequencing. A total of 1,875,732 high quality cDNA reads were assembled with existing ESTs to obtain 5884 new contigs for human RefSeq protein models, and to develop a custom Affymetrix gene expression array (Amby_002) with approximately 20,000 probe sets. The Amby_002 array was used to identify 303 transcripts that differed statistically (p < 0.05, fold change > 1.5) as a function of days of T4 treatment. Further statistical analyses showed that Amby_002 performed concordantly in comparison to an existing, small format expression array. This study introduces a new A. mexicanum microarray resource for the community and the first lists of T4-responsive genes from the brain of a salamander amphibian.

Introduction

Thyroid hormone (TH) is essential for normal development of the mammalian brain. Maternal, fetal, and neonatal TH levels are closely regulated in temporal and spatial contexts to affect proper cell migration, proliferation, and differentiation, and to orchestrate synaptogenesis and myelination of neurons (Anderson et al., 2003, Koibuchi, 2008, Patel et al., 2011). Later in life, thyroid hormone affects the activity of neuroendocrine axes that regulate reproduction, appetite, behavior, stress response, and longevity (Ooka and Shinkai, 1986, Shi et al., 1994, Gussekloo et al., 2004, Kong et al., 2004, Brambilla et al., 2006, Leggio et al., 2008, Duval et al., 2010, Krassas et al., 2010). That so many fundamental biological processes are associated with the action of a single molecule reflects in part the molecular mechanisms through which TH operates. It has been known for some time that TH interacts with nuclear receptors (TRα or TRβ) and cofactors to regulate transcription directly (Oppenheimer et al., 1974, Samuels et al., 1974, Sap et al., 1986, Weinberger et al., 1986), and recent studies are beginning to use unbiased approaches to identify TH-regulated genes in the brain (e.g. Royland et al., 2008, Das et al., 2009). Much less is known about the way TH signals via “non-genomic” mechanisms, including membrane receptors, transporter molecules, and cytoplasmic receptors that elicit changes in cells through signaling pathways (Caria et al., 2009; reviewed by Davis et al., 2008, Furuya et al., 2009, Cheng et al., 2010). Identification of additional TH-regulated genes and molecular mechanisms will require continued studies of traditional models and development of new models.

Many mechanisms that are associated with TH synthesis, activation, and transcriptional regulation are evolutionarily conserved among vertebrates, and these presumably function during homologous stages of development (Tata, 1993, Tata, 2006). Thus, it is possible to investigate mechanisms of mammalian brain development in more experimentally tractable organisms with free-living embryonic and juvenile phases. Anuran amphibians in particular have been extensively studied because it is straightforward to induce metamorphosis in tadpoles with TH and investigate how TH affects developmental changes in vivo through interactions with TRs, accessory co-factors, and changes in chromatin state (e.g. Shi, 2000, Das et al., 2010, Bilesimo et al., 2011). TH levels increase precipitously during anuran metamorphosis to regulate the development of adult tissue and organ systems from pre-existing larval structures and progenitor cell populations. In humans, TH levels similarly increase as the brain matures during the perinatal stage of early development (Brown et al., 2005). Thus, the study of anuran metamorphosis may identify TH-dependent mechanisms that are critical for normal human brain development. However, there are limitations in using anuran models to study the actions of TH. Although it is straightforward to administer TH, dosing regimes are generally administered at developmental stages when TH levels are relatively low in tadpoles. Because genes maybe differentially responsive to TH as a function of age, precocious administration of TH may activate or repress genes that are not typical of normal development and metamorphosis. Also, when tadpoles of spontaneously metamorphosing anurans are exposed to TH, they are already developing toward a metamorphic endpoint; thus anurans do not provide a true negative control for evaluating TH signaling.

The Mexican axolotl (Ambystoma mexicanum) provides an alternative amphibian model to investigate TH signaling (Page et al., 2007, Page et al., 2008, Page et al., 2009). While many amphibians undergo an obligate metamorphosis, A. mexicanum juveniles fail to produce enough TH to induce metamorphosis (Kuhn and Jacobs, 1989, Galton, 1991). As a result, A. mexicanum retain juvenile traits into the adult stage of life, an adaptation that has been termed pedomorphosis. Importantly, metamorphosis can be induced in A. mexicanum by simply adding the thyroxine form of TH (T4) to the water (Page and Voss, 2009). Thus, developmental events can be induced within the context of a natural, hypothyroid condition at juvenile or adult stages of life. In A. mexicanum, paedomorphosis is associated with an unidentified genetic factor that affects developmental timing, response to T4, and hypothalamic-pituitary-thyroid function (Voss and Smith, 2005, Galton, 1991; Rosenkilde and Ussing, 1996; Kuhn et al., 2005).

In previous studies, we used microarray analysis to show that metamorphosis is precisely and reliably induced in A. mexicanum using 5 or 50 nM T4 (Page et al., 2007, 2008). We also reported an integrative model of epidermal gene expression and whole-animal anatomical metamorphosis (Page et al., 2009). Here, we report on a study that used highly parallel 454 DNA sequencing to discover genes from the A. mexicanum brain. The resulting sequence reads were assembled with pre-existing ESTs from Sal-Site (www.ambystoma.org) to design a 2nd generation custom Affymetrix expression array (Amby_002). This new microarray platform was then used to identify genes that are differentially expressed in the A. mexicanum brain after treatment with T4. Our study enhances the A. mexicanum model by providing a new microarray resource and new EST contigs that increase the total number of Ambystoma-human non-redundant orthologous sequences to > 15,000. Also, our study provides the first lists of T4-responsive genes from a salamander amphibian, including genes that are predicted to function in neural developmental and physiological processes within the brain.

Section snippets

Animals and tissue sampling

Nine A. mexicanum juvenile siblings were obtained from the Ambystoma Genetic Stock Center at the University of Kentucky and reared under the same laboratory conditions to approximately 130 days post hatching. At this age, individuals are immature with respect to gonad maturation and have surpassed the time that metamorphosis typically occurs in related species. Three individuals were anesthetized in 0.02% benzocaine and the brains and pituitaries of each were removed, flash frozen in ethanol and

Identification of genes from A. mexicanum brain using 454 DNA sequencing

To generate new ESTs from A. mexicanum brain, cDNAs were synthesized for three brain samples: a brain isolated from a 133 day old individual (D0_454), and brains isolated from 141 and 151 day old individuals that had been reared for 8 and 18 days in 50 nM T4, respectively (D8_454; D18_454). Overall 2.06 × 106 reads were generated and this yielded approximately 584,000–699,000 high-quality sequence reads for each sample, with an average of 374 base pairs in length (Table 1). Typically, 30–50% of

Discussion

Amphibians provide excellent models to investigate the effects of thyroid hormone on development. An important advantage in using A. mexicanum over anuran models is the ability to reliably stimulate the onset of metamorphosis in juveniles or adults. This study is the first to use A. mexicanum to identify genes that are expressed differentially in the brain after inducing metamorphosis with T4. Below, we discuss the approach taken to identify new brain transcripts and measure their abundance

Conclusion

Amphibian metamorphosis provides an excellent model to study TH-responsive periods of development that are homologous among vertebrates. This study introduces a new A. mexicanum microarray resource for the community and the first lists of T4-responsive genes from the brain of a salamander amphibian. The Mexican axolotl (A. mexicanum) is an unusual, hypothyroidic salamander that is available from genetically homogenous laboratory stocks maintained at University of Kentucky. It will be important

Acknowledgments

We thank Robert Page and Meredith Boley for inducing metamorphosis in the A. mexicanum juveniles that were used in this experiment and for collecting brain tissues. The research was supported by grants R24-RR016344 and P20-RR16481 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). The project also used resources developed under Multidisciplinary University Research Initiative grant (W911NF-09-1-0305) from the Army Research Office, and

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