For nearly a century, amphibian metamorphosis has served as an important model of how thyroid hormones regulate vertebrate development. Consequently metamorphosis has been studied in a number of ways including: morphologically, developmentally, ecologically, and from an endocrine perspective. Over the last two decades, much has been learned about the molecular basis of anuran (frog) metamorphosis. However, very little is known about the molecular underpinnings of urodele (salamander) metamorphosis. Using the axolotl and axolotl hybrids as models, I present some of the first studies on the gene expression changes that occur during urodele metamorphosis. In Chapter 1, the motivation for the research described in the subsequent chapters is presented and the literature is briefly reviewed. In Chapter 2, the first microarray analysis of urodele metamorphosis is presented. This analysis shows that hundreds of genes are differentially expressed during thyroid hormone-induced metamorphic skin remodeling. Chapter 3 extends the analysis presented in Chapter 2 by showing that the transcriptional patterns associated with metamorphic skin remodeling are robust even when the concentration of thyroid hormone used to induce metamorphosis is varied by an order of magnitude. Chapter 4 makes use of the differentially expressed genes identified in Chapters 2 and 3 to articulate the first model of urodele metamorphosis to integrate changes in morphology, gene expression, and histology. In addition, Chapter 4 outlines a novel application for piecewise linear regression. In turn, Chapter 5 makes use of the model presented in Chapter 4 to demonstrate that full siblings segregating profound variation in metamorphic timing begin to diverge in phenotype early during larval development. In Chapter 6 the conclusions drawn from the research are summarized and future directions are suggested.

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The anuran Xenopus tropicalis tadpole is an attractive model animal in toxicological evaluation of suspected thyroid disrupting xenobiotics. Due to its reliance of a functioning hypothalamic-pituitary-thyroid (HPT) axis for normal metamorphosis, effects on the HPT axis produces apical endpoints, which are easy to measure. More sensitive endpoints of mRNA expression and histological evaluation of the thyroid gland itself provide strong indications of in vivo thyroid endocrine disruption. X. tropicalis is traditionally exposed in groups of 20 animals in four replicates for each treatment group. However, exposing tadpoles individually can provide stronger statistics and a reduction of total animal sacrifice. In this study we have developed and optimized an individual exposure system by a method development study. This method was then applied in an exposure experiment of a suspected thyroid endocrine disruptor, propylparaben (PrP). Prometamorphotic (NF stage 51) X. tropicalis tadpoles were distributed in three treatment groups (0.05, 0.5 and 5.0 mg PrP/L water) and maintained semi-statically for 14 days. Afterwards, apical measurements (body weight (BW), total body length (BL), snout to vent length (SVL) and hind limb length (HLL)) and reached developmental stage were assessed. In addition, mRNA expression of HPT axis relevant genes encoding deiodinase 2 (D2, hepatic tissue), deiodinase 3 (D3, hepatic and tail tissue) and transthyretin (Ttr, hepatic tissue) were measured by quantitative reverse transcription PCR (qRT-PCR). The PrP exposure did not affect general growth and development, but it did cause a downregulation of dio3 and ttr. The downregulation of dio3 could possibly be associated with a reduced serum content of thyroid hormone, while ttr might be connected to a previously described xenoestrogenic effect of PrP in vitro and in fish.

Nanoparticles (NPs) are engineered in the nanoscale (<100nm) to have unique physico-chemical properties from their bulk counterparts. Nanosilver (nAg) is the most prevalent nanoparticle in consumer products due to its strong antimicrobial action and can be released to the environment during product manufacture, usage and disposal. The predicted environmental concentrations are within the North American guidelines for the protection of aquatic life and in drinking water. While nAg toxicity at high concentrations has been well described, the sublethal effects at environmentally-relevant concentrations are relatively unknown. Initial screening in our lab showed nAg was a potential endocrine disrupting chemical (EDC). Amphibian metamorphosis is mediated by thyroid hormone (TH), and nAg perturbed TH-dependent transcriptional responses in the tailfin of bullfrog (Rana catesbeiana) tadpoles. The primary objective of this thesis was to further investigate and characterize the effects of low, environmentally relevant concentrations of nAg on TH-dependent metamorphosis in R. catesbeiana and Xenopus laevis. Two chronic, 28 day in vivo exposures at 0.06 and 6µg/L nAg were conducted with premetamorphic R. catesbeiana tadpoles using TH to induce precocious metamorphosis. Ionic silver (iAg) was also examined to control for the complete dissolution of Ag. Analysis of metamorphic stage progression demonstrated nAg-induced acceleration of hindlimb growth and development. After 6 days of nAg exposure, analysis with quantitative real-time polymerase chain reaction (QPCR) demonstrated nAg-induced disruption of TH-responsive transcripts in a tissue-specific manner. Furthermore, the nAg effects could not be fully explained by iAg, indicating NP-specific disruption. Two chronic, 28 day exposures to 0.018-1.8 µg/L nAg were conducted on X. laevis premetamorphic and prometamorphic tadpoles. nAg was found to significantly bioaccumulate in tadpole tissue after 28 days. Furthermore, nAg increased the hindlimb length during early premetamorphosis and in post-metamorphic juvenile tadpoles. Using an in-house MAGEX microarray and QPCR transcriptional analysis, 7 biomarkers of nAg exposure were validated. Five of these targets showed disruption to their TH-response. Furthermore, the increased mRNA abundance of two peroxidases indicated that nAg generated reactive oxygen species (ROS) even at low, environmental concentrations. This thesis demonstrates that nAg has consistent EDC actions across two distinct amphibian species, and the data suggest that regulatory guidelines for silver may need revision. A X. laevis derived fibroblast-like TH-responsive cell line, XTC-2, was used in conjunction with the 7 biomarkers of nAg exposure to gain mechanistic insight into the role of ROS in TH signaling disruption. Monocultures were created and validated to increase the specificity of TH-response. While the monocultures were successfully created, the biomarkers were not responsive to nAg in this cell line. Additional investigations were made into the relationship between genetic sex and responsiveness to TH. Genetic sexing methods were used to investigate transcriptional differences between males and females during natural and TH-induced metamorphosis. The sexing protocol was optimized and validated successfully. The genetic sex was determined for premetamorphic and prometamorphic X. laevis tadpoles exposed to TH for 48 h. QPCR and microarray analysis were used to identify three markers that demonstrated transcriptional sex-bias during early gonadal differentiation stages. / Graduate / 0307 / 0383 / 0487 / amanda_carew14@yahoo.ca

Thyroid hormones (THs) are essential signaling molecules for the postembryonic development of all vertebrates. THs are capable of initiating a diverse set of developmental programs across multiple tissues. The role of TH in regulating gene expression is well-known, but the initiation of TH signaling is still not fully understood. In amphibians, THs are the sole hormones required for the metamorphosis from tadpole to juvenile froglet. Amphibians are a useful model for studying TH signaling, as they undergo extensive, tissue-specific response programs in response to exogenous TH. The metamorphosis of the American bullfrog, Rana [Lithobates] catesbeiana is temperature sensitive. R. catesbeiana tadpoles do not undergo metamorphosis at cold temperatures (4-5 °C) even in the presence of THs that should otherwise prompt it. However, tadpoles undergo metamorphosis at an accelerated rate when returned to warm temperatures (24-25 °C) forty days after their initial TH exposure. R. catesbeiana tadpoles possess a “molecular memory” of TH exposure which establishes the TH signal at cold temperatures and prompts accelerated metamorphosis after a return to warmer temperatures. The mechanisms of the molecular memory which allow it to uncouple the initiation of TH signaling from the execution of the TH response program are not fully understood. Previous research has established that transcripts encoding transcription factors are a substantial component of the TH-dependent transcriptomic response of cultured tailfin (C-Fin) at cold temperatures. However, not all of these putative transcripts encoding transcription factors required active transcription and translation for their induction, which suggests that the initiation of a TH signal involves mechanisms other than regulating gene expression. Herein, we used quantitative polymerase chain reaction (qPCR) and RNA-Sequencing (RNA-Seq) to investigate the TH-dependent transcriptomic response of the back skin, a tissue that undergoes extensive remodeling during metamorphosis. Cultured back skin (C-Skin) was TH-responsive in warm, cold and temperature shift conditions. Forty-four transcripts underwent significant changes in abundance in response to TH in cold temperatures under which the molecular memory is established. Seven of these transcripts encoded putative transcription factors. Surprisingly, the only TH-responsive transcript significantly changed at 4 °C in both the C-Skin and the previously studied C-Fin was thyroid hormone-induced basic leucine zipper-containing protein (thibz). Thibz has been found to be TH-responsive at cold temperatures in the liver, lung, liver, brain, tailfin and back skin of whole animals, which suggests it may be an important regulator of initiating TH signaling. The lack of overlap in the transcriptomic responses of C-Skin and C-Fin may suggest that even the early initiation of TH signaling has tissue-specificity. Alternately, the molecular memory may include mechanisms that do not require active transcription and translation. Transcripts associated with epigenetic modifications and post-transcriptional changes to mRNA stability were also significantly expressed at 4 °C within the C-Skin. Previous investigation of the putative transcription factors in C-Fin revealed that active transcription and translation was not always required for changes in transcript abundance. Multiple mechanisms may be at play in the TH response at different temperatures. In cold temperatures, TH may modulate mRNA stability to influence transcript abundance as a part of initiating TH signaling without executing metamorphosis. Further research is needed to explore potential alternative mechanisms of establishing the molecular memory and the accelerated metamorphic response. The temperature sensitivity of R. catesbeiana’s TH response is incredibly valuable in investigating mechanisms of early TH signaling during postembryonic vertebrate development. / Graduate