Frogs model man: <i>In vivo</i> thyroid hormone signaling during development

Sachs, Laurent M.; Buchholz, Daniel R.

doi:10.1002/dvg.23000

Cited by 37 publications

(35 citation statements)

References 112 publications

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“…Amphibian metamorphosis provides an accessible and dramatic developmental model to circumvent the problems inherent in studying TH signaling during development in mammals (2–4). Metamorphosis requires TH for induction and progression to completion, and it involves tissue remodeling, resorption and growth; therefore, on the cellular level, it entails multiple cell fates.…”

Section: Introductionmentioning

confidence: 99%

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

et al. 2017

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The essential role of thyroid hormone (TH) signaling in mammalian development warrants the examination of man-made chemicals for its disruption. Among vertebrate species, the molecular components of TH signaling are highly conserved, including the thyroid hormone receptors (TRs), their heterodimer binding partners the retinoid-X receptors (RXRs), and their DNA recognition sequences (TREs). This molecular conservation allows examination of potential TH disruption in the tractable, in vivo model system of amphibian metamorphosis. Metamorphosis requires TH signaling for both instigation and progression, and it provides dramatic and well-characterized phenotypes involving different cell fates. Here we describe a quantitative, precocious-metamorphosis assay suite we developed using one-week post-fertilization (PF) Xenopus laevis tadpoles in order to assess disruption of TH signaling. Tadpoles at this developmental stage (Nieuwkoop-Faber (NF)-48) are competent to respond to TH hormone, although not yet producing TH, along many metamorphic pathways, and they are uniform in size. This allowed us to quantify changes in morphology associated with natural metamorphosis (e.g. gill and tail resorption, brain expansion, and craniofacial remodeling) after five days of treatment. Using the same tadpoles from morphological measurements, we quantified a 20-fold increase in TH-induced cellular proliferation in the rostral head region by whole-mount immunocytochemistry. At the molecular level, we used F3-generation tadpoles from a transgenic X. laevis line, which expresses luciferase under the control of a native TRE, to assess the ability of compounds to disrupt TR function. The luciferase reporter showed over 10-fold activation by physiologic concentrations of TH. We used the synthetic TR antagonist NH-3 to demonstrate the feasibility of our assay suite to measure inhibition of TH activity at the level of the receptor. Finally, we assessed the capabilities of suspected TH-disrupting chemicals tetrabrominated diphenyl ether 47 (BDE-47) and tetrabromobisphenol A (TBBPA). We found that BDE-47 displays general toxicity rather than TH disruption, as it did not increase brain width nor affect the TRE-luciferase reporter. However, TBBPA, a suspected TR antagonist, although not effective in antagonizing cell proliferation, significantly inhibited the TRE-luciferase reporter, suggesting that it bears closer scrutiny as a TH disruptor. Overall the assay suite has important advantages over the classical tadpole metamorphosis assays with respect to the uniformity of animal size, small test volume, reproducibility, and short test period. The assays are performed before endogenous TH production and free feeding start, which further reduces complexity and variability.

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Section: Introductionmentioning

confidence: 99%

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

et al. 2017

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“…The Xenopus tadpole is a well-established and valuable vertebrate model for hormone-dependent growth and development, molecular mechanisms of gene expression in vivo, and regeneration in tail, limbs, and spinal cord (Beck et al 2009;Buchholz 2015;Sachs and Buchholz 2017). Most tadpole tissues have been examined to some extent to determine their function during tadpole life and to determine their hormone-dependent changes throughout metamorphosis (Pouyet and Beaumont 1975;Dodd and Dodd 1976;Fox 1983;Gilbert et al 1996;Brown and Cai 2007).…”

Section: Discussionmentioning

confidence: 99%

XenopusTadpole Tissue Harvest

Patmann

Shewade

Schneider

et al. 2017

Cold Spring Harb Protoc

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The procedures described here apply to Xenopus tadpoles from the beginning of feeding through the major changes of metamorphosis and are appropriate for downstream postoperative snap freezing for molecular analysis, fixation for histological analysis, and sterile organ culture. To the uninitiated, the most difficult aspects of tadpole tissue dissections are likely knowing the appearance and location of organs, and the difficulty manipulating and holding tadpoles in place to carry out the oftentimes fine and precise dissections. Therefore, images and stepwise instructions are given for the harvest of external organs (tail, head, eyes, tail skin, back skin, gills, thymus, hind limbs, forelimbs) and peritoneal organs (intestine, pancreas, liver, spleen, lungs, fat bodies, kidney/gonad complex), as well as brain, heart, and blood. Dissections are typically done under a dissection stereomicroscope, and two pairs of fine straight forceps, one pair of fine curved forceps, and one pair of microdissection scissors are sufficient for most tissue harvests. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. Reagents Ethanol (70%) Frog waterRemove chlorine/chloramine from tap water by carbon filtration (Alternatively, reconstitute reverse-osmosis water to 800 μS using Instant Ocean or equivalent). Adjust the pH to 7.5 using sodium bicarbonate.Ice MS-222 solution (0.1% tricaine methanesulfonate [Sigma-Aldrich], in frog water)Add sodium bicarbonate to 0.1% to adjust the pH to 7.0-7.5.Phosphate-buffered saline (0.6×) Dilute 1× PBS 60:40 with reverse-osmosis or distilled water. Dilution is required because the osmolarity of amphibian blood is 62% that of mammalian blood.

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“…(Abdalla et al, 1987) In the present study, we test the hypothesis that equilin acts as a TH agonist in Xenopus laevis tadpoles, which are a widely used animal model for studies involving TH disruption toxicity. Xenopus laevis tadpoles are particularly sensitive to increases in TH signaling because it is the key initiator of metamorphosis (Sachs & Buchholz, 2017). We treated tadpoles 2-4 days of acute exposure to equilin and examined changes in body size, gene expression, and rates of neurogenesis and and cell death in the optic tectum, the functional homolog of the superior colliculus.…”

Section: Introductionmentioning

confidence: 99%

Equilin does not affect thyroid hormone signaling in the developing Xenopus laevis tadpole brain

Bass

Husain

Dahora

et al. 2019

Preprint

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Toxcast/Tox21 is a massive federally run research effort dedicated to better understanding the potential toxicity of thousands of compounds in a high throughput manner. Among this list of compounds is equilin, an estrogen-like compound that was flagged as a potential thyroid hormone agonist. Here we examine if equilin acts like a thyroid hormone agonist on cellular and molecular mechanisms of brain development in Xenopus laevis tadpoles. To examine the effect of equilin, tadpoles were divided into eight groups and received 4 days of exposure. The experimental groups were as follows: 1 µL, 10 µL, and 100 µL of equilin, 1 µL, 10µM, and 100 µM of 17-β estradiol as an estrogen control, 15 µg/mL thyroxine (T 4 ) as a thyroid hormone control, and a no-exposure control. After 4 days of treatment, animals were treated with CldU to label dividing cells for 2hr and then euthanized in MS-222. After fixation, body length was measured and the brains dissected out. IHC was performed on brains for CldU to label proliferating neural progenitor cells. Brains were then whole-mounted and analyzed using confocal microscopy. We found that equilin did not increase the number of dividing progenitor cells in a T 4 -like manner. Instead, equilin decreased proliferation in a dose-dependent manner, as did estradiol. The same paradigm was performed separately staining for caspase-3 and h2ax, finding that equilin increased cell death in contrast to CNTL and T 4 . In another experiment, RNA was extracted from tadpole brains in each group and qPCR was performed to assess change in expression of thyroid hormone-sensitive genes, Equilin did not affect gene expression in a thyroid hormone-like manner. Our data indicate that equilin does not act as a thyroid hormone agonist in the Xenopus laevis nervous system but instead acts similarly to estradiol. Our data strongly suggest that equilin is not a TH disruptor, contrary to the findings of the ToxCast/Tox21 dataset.

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Frogs model man: In vivo thyroid hormone signaling during development

Cited by 37 publications

References 112 publications

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

A multi-tiered, in vivo, quantitative assay suite for environmental disruptors of thyroid hormone signaling

XenopusTadpole Tissue Harvest

Equilin does not affect thyroid hormone signaling in the developing Xenopus laevis tadpole brain

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