A rapid, physiologic protocol for testing transcriptional effects of thyroid-disrupting agents in premetamorphic xenopus tadpoles.

Increasing numbers of substances present in the environment are postulated to have endocrine-disrupting effects on vertebrate populations. However, data on disruption of thyroid signaling are fragmentary, particularly at the molecular level. Thyroid hormone (TH; triiodothyronine, [T.sub.3]) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, [T.sup.3] orchestrates amphibian metamorphosis, thereby providing an exceptional model for identifying thyroid-disrupting chemicals. We combined these two advantages to develop a method for following and quantifying the transcriptional action of [T.sub.3] in Xenopus laevis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to "on-line" imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with TH-responsive elements coupled to either luciferase or green fluorescent protein to follow [T.sub.3]-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pretreatment protocol that induced competence to respond to [T.sub.3] and thus to assess [T.sub.3] effects and [T.sub.3] disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak ([10.sup.12] M) pulse of [T.sub.3] that induced TH receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid-disrupting activity of environmentally relevant concentrations ([10.sup.-8] M) of acetochlor, a persistent herbicide. Key words: acetochlor, endocrine disruption, germinal transgenesis, green fluorescent protein, metamorphosis, somatic gene transfer, thyroid, transcription, Xenopus laevis. Environ Health Perspect 113:1588-1593 (2005). doi:10.1289/ehp.7992 available via http://dx.doi.org/ [Online 11 July 2005]

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The thyroid hormone (TH) triiodothyronine ([T.sub.3]) is critical to vertebrate development and growth, playing vital roles during central nervous system (CNS) development and during organogenesis of heart, muscles, bones, and lungs. In both developing and mature organisms, numerous physiologic functions are regulated by [T.sub.3] availability, including energy metabolism, thermogenesis, pituitary hormone production, and lipogenesis (Yen 2001). The most striking example of [T.sub.3] action in vertebrates is anuran amphibian metamorphosis, one of the best-studied hormone-regulated developmental processes. Amphibian metamorphosis is totally dependent on [T.sub.3] and is associated with dramatic morphologic and physiologic changes, including cell death, division, or differentiation (Dodd and Dodd 1976).

The total dependence of amphibian metamorphosis on TH has logically led to the suggestion that metamorphosis can be used to assess TH disruption. Accordingly, the Xenopus embryonic metamorphosis assay (XEMA) has been proposed by the Organization for Economic Cooperation and Development (OECD) Task Force on Endocrine Disrupters Testing and Assessment as an in vivo assay for identification of substances with potential to disrupt functions of the thyroid system. In this in vivo test, several morphologic and histologic parameters are used to define the potential of a chemical to perturb the thyroid axis (OECD 2004). Because the test covers most of the natural metamorphic process, it takes at least 4 weeks. In contrast, transcriptional responses to [T.sub.3] are much more rapid, with changes being measurable within hours or days. Indeed, [T.sub.3] actions are mainly mediated by their nuclear receptors (TH receptors, TRs), ligand-dependent transcription factors. In vertebrates, two genes encode TRs: TR-[alpha] and TR-[beta] (Mangelsdorf et al. 1995). Generally, TRs form heterodimers with the 9-cis-retinoic acid receptor and interact with comodulator complexes, thereby repressing or activating transcription. Heterodimers bind to TH response elements (or [T.sub.3] responsive element; TRE) in target genes.

We chose to take advantage of the speed of the transcriptional responses to TH and the ease of measuring reporter gene activities, such as luciferase (luc) and green fluorescent protein (GFP), using transgenic approaches. Moreover, both somatic (de Luze et al. 1993; Nakajima and Yaoita 2003; Ulisse et al. 1996) and germinal transgenesis (Coen et al. 2001; Huang and Brown 2000; Marsh-Armstrong et al. 1999; Oofusa et al. 2001) are now widely applied to Xenopus laevis for dissecting

TH-dependent regulations during metamorphosis. The transgenic models we optimized here are based on the fundamental design of composite reporter gene constructs with a hormone-sensitive regulatory region upstream of a fluorescent protein cDNA. We started from the premise that following transcriptional responses in vivo is often marred by high variability. In the case of following TH responses in tadpoles, this variability could be due to variations in endogenous TR levels. Because TR-[beta] is strongly inducible by [T.sub.3] itself, we chose to prime tadpoles to respond to an ulterior [T.sub.3] exposure with a short, weak pulse of [T.sub.3] that was then fully rinsed out. This protocol produced rapid (48 hr), robust, and reproducible responses to TH agonists. Applying this protocol to germinally transgenic tadpoles, we were able to reveal the actions of the preemergent herbicide acetochlor through increased TH responses.

Materials and Methods

Plasmid constructs. The-246 to +130 bp sequence of the TH/bZIP promoter (GenBank accession no. U37375; GenBank 2004) was amplified by polymerase chain reaction (PCR) from X. laevis genomic DNA using the primers 5'-CTGTTATATAGAGGCAGAGGG-3' and 5'-CTATACCTGAATGGGCAGCAG-3', and then cloned into pGEMt-easy vector (Promega, Lyon, France). A SaclI-PstI-digested fragment was excised and cloned into pBluescript (Promega). A SacI-HindIII-digested fragment was excised and cloned into pGL2 basic vector (Promega), producing TH/bZIP-luc.

To obtain the TH/bZIP-eGFP (enhanced GFP) transgene, we proceeded in two steps. We cloned a Sall-ApaI eGFP cDNA and the SV40 polyA signal fragment into the TH/bZIP promoter containing pBluescript. A large Sad-ApaI fragment was excised and cloned into a pBluescript vector with two insulators. To this end, a 1,668-bp fragment of the lysozyme gene (GenBank accession no. X98408) from chicken genomic DNA (Stief et al. 1989) was amplified by PCR using the primer 5'-TGACTCGAGGGATCCATAATATAACTGTACC-3' and 5'-TGAGGTACCAAG CTTAAAAGATTGAAGCAC-3'. One insulator copy was cloned into XhoI and KpnI sites of the pBluescript vector, and the second was cloned into the SmaI site. The complete vector with the two copies of insulators was linearized with EcoRV, and the large Sad-ApaI fragment corresponding to the eGFP cDNA and SV40 polyA signal was inserted.

The [gamma]-crystallin promoter coupled to a RedFP (red fluorescent protein) plasmid was a gift from L. Zimmermann (Medical Research Council, London).

Animals and treatments. We obtained sexually mature X. laevis frogs from d'Elevage de Xenope du Centre National de la Recherche Scientifique (Montpellier, France). Tadpoles were raised in dechlorinated and deiodinated tap water (1:2) and fed with nettle powder (Vallee, Chanzeaux, France). Tadpoles were staged according to Nieuwkoop and Faber (1956; NF staging). The care and treatment of animals used in this study were in accordance with institutional and national guidelines (Sciences et Medecine des Animaux de laboratoire a l'ENVL 2005).

[T.sub.3], 3,5,3'-triiodothyroacetic acid (TRIAC), and acetochlor were purchased from Sigma (St. Quentin Fallavier, France).

Somatic gene transfer and germinal tramgenesis. Somatic gene transfer in Xenopus muscle and brain was performed as described previously (de Luze et al. 1993; Ouatas et al. 1998; Trudeau et al. 2004).

Germinally transgenic tadpoles were produced by restriction enzyme-mediated integration nuclear transplantation according to Kroll and Amaya (1996), with the following modifications: sperm was purified by centrifugation on a two-layer discontinuous Percoll (Sigma) gradient before the permeabilization step, which was performed with digitonin (Sigma) instead of lysolecithin. Two plasmids were used: TH/bZIP-eGFP plasmid and a [gamma]-crystallin promoter coupled to a RedFP plasmid, which is expressed only in the eye. This latter plasmid allows selection of transgenie [F.sub.0] tadpoles during early development before the TH/bZIP driven green fluorescence appears in the tadpole body.

Imaging. Images were captured using an Olympus fluorescent dissecting microscope equipped with an Olympus video camera DP50 (Olympus, Rungis, France). Before photographing, germinally transgenic tadpoles NF stage 52 were anaesthetized in 0.1% tricaine methanesulfonate (MS-222; Sigma) and the skull opened to expose the brain. All pictures were taken with the same parameters (32 x objective and 5-sec exposure time). Quantification was performed using ImageJ software (Rasband 1997). Data are expressed in relative units of fluorescence.

Luciferase activity. Tadpoles were sacrificed by decapitation after anesthesia in 0.1% MS-222. Tissues were dissected, frozen in liquid nitrogen, and stored at -80[degrees]C until assayed according to the manufacturer's instructions (Promega) as previously reported (de Luze et al. 1993). Luciferase activity is expressed as relative light units (RLU). Because in some experiments tadpoles vary in size, luciferase values were normalized against protein content. Protein was measured according to the manufacturer's instructions (BioRad, MarnesLa-Coquette, France).

Statistical analysis of results. In vivo gene transfer results are expressed as mean [+ or -] SE per group. Differences between means were analyzed by Student's t-test or analysis of variance and the Tukey-Kramer test where appropriate. Differences were considered significant at p < 0.05. In many cases, typical experiments are shown, each experiment having been repeated at least twice (with n [greater than or equal to]8 tadpoles/experiment) and providing the same results.

RNA extraction and semiquantitative reverse transcriptase (RT)-PCR analysis. Tadpole tails were harvested into RNA-later (Ambion, Huntingdon, United Kingdom) at 4[degrees]C. Total RNA was extracted using RNAble reagent (Eurobio, Les Ulis, France) following the manufacturer's protocol. Reverse transcription was performed on 2 [micro]g RNA, in 20 [micro]L final volume. Primer hybridization on RNAs was done by mixing total RNAs with specific reverse primers (2 [micro]M each): for TR-[beta] 5'-CTTTTCTATTCTCTCCACGCTAGC-3'; for the internal control Rp18, 5-GACGACCAGTACGACGA-3' (Havis et al. 2003). Mixes were incubated (2 min, 65[degrees]C) and then cooled to room temperature. A 10-[micro]L mix containing 4 [micro]L reverse transcription buffer (Invitrogen, Cergy Pontoise, France), 1 [micro],L dNTP (dATP, dTTP, dCTP, and dGTP, 25 mM each; Pharmacia, Saclay, France), 2 [micro]L 0.1 M dithiothreitol (Invitrogen), and 0.5 [micro]L reverse transcriptase (RT) SuperScript II (5 U/[micro]L; Invitrogen) was added to hybrid primers/RNA before incubation (1 hr, 45[degrees]C). After reverse transcription, we used 2 [micro]L of each cDNA sample and 0.1 [micro]L (0.1 [micro]Ci) of [[alpha]-[sup.32]p] dCTP for PCR in a final volume of 50 [micro]L containing 25 [micro]L PCR Master Mix (Abgene, Courtaboeuf, France), 2 [micro]L of each primer (forward and reverse for the gene of interest and the internal control at 2 [micro]M each; TR-[beta] forward, 5'-ATAGTTAATGCGCCCGAGGGTGGA-3'; and the internal control, RpI8 forward, 5'-AAAGAGAAACTGCTGGC-3'). The PCR reaction consisted of 22 cycles of 60 sec at 94[degrees]C, 1 min at 55[degrees]C, and 1 min at 72[degrees]C (Robocycler; Stratagene, Amsterdam, The Netherlands). Fifteen microliters of PCR products was resolved in 6% acrylamide-tris-borate-EDTA buffer gels and autoradiographed.

Results

Treatment with a rapid, weak pulse of [T.sub.3] induces competence to respond and synchronizes [T.sub.3] transcriptional responses in somatic gene transfer. Our overall aim in these experiments was to exploit the [T.sub.3]-dependent inducibility of the TR-[beta] gene to prime tadpoles so as to have access to a model that could reveal rapid, robust, and reproducible TH transcriptional responses. This priming, or pretreatment procedure, was considered a prerequisite to using premetamorphic (NF stage 54) tadpoles in a reporter gene assay, because intragroup variability can be quite high (de Luze et al. 1993). Using the promoter of the TH/bZIP gene, which is a [T.sub.3] target gene encoding a transcription factor (Furlow and Brown 1999), we compared the effects of a short pulse (24 hr) of [10.sup.-13] M or [10.sup.-12] M [T.sub.3] for the pretreatment against unpretreated, control tadpoles. After a 24-hr rinse period, both sets of tadpoles (pretreated and control) were used for somatic gene transfer in the caudal muscle and exposed to [T.sub.3] ([10.sup.-8 ]M) for 2 days. We found 2-fold increases in mean levels of TH/bZIP expression in caudal muscle of control tadpoles and those given a pretreatment pulse of [T.sub.3] at [10.sup.-13] M (Figure 1B). However, these differences were not significant (10 > 0.05). In contrast, in tadpoles transiently pretreated with [10.sup.-12] M [T.sub.3], 2 days of exposure to [10.sup.-8] M [T.sub.3] induced a nearly 4-fold increase in the transcriptional response of the TH/bZIP-luc construct. This increase was very significant (p < 0.01). Interestingly, the basal levels of transcription from the TH/bZIP promoter decreased as a function of pretreatment pulse concentration, most probably reflecting stronger repression of basal expression by the unliganded TR (Sachs 2004).

[FIGURE 1 OMITTED]

A weak pulse of [T.sub.3] induces TR-[beta] expression in caudal muscle of tadpoles within 6 hr. To verify that the pretreatment protocol was indeed inducing TR-[beta], we followed TR expression in caudal muscle of pretreated tadpoles using semiquantitative RT-PCR. Figure 2 shows that there is a significant, 2.2-fold induction in TR-[beta] expression within 6 hr of exposure of NF stage 54 tadpoles to [10.sup.-12] M [T.sub.3]. Given this finding, and the robust response produced by pretreatment, all the following experiments were performed on animals pretreated with [10.sup.-12] M [T.sub.3] during 24 hr, followed with a rinse of 24 hr before exposure to TH agonists for 48 hr.

[FIGURE 2 OMITTED]

The somatic gene transfer method allows dose-dependent detection of TH agonists in brain and muscle. We used somatic gene transfer with the pretreatment protocol to test transcriptional responses to other TH agonists. Figure 3 shows that pretreated tadpoles exposed to TRIAC (5 x [10.sup.-8] M) for 48 hr displayed a 3-fold increase in transcription from the TH/bZIP promoter (p < 0.05) compared with controls. Similar results were found with 3,5,3',5'-tetraiodothyronine (T4; data not shown).

[FIGURE 3 OMITTED]

In order to test the sensitivity of the somatic gene transfer method and the eventual tissue specificity in transcriptional response to TH agonists, we compared responses in caudal muscle or the brain of pretreated tadpoles. As shown in Figure 4A, in caudal muscle, a 5 x [10.sup.-10] M [T.sub.3] exposure did not significantly increase transcription, but 5 x [10.sup.-9] M and 5 x [10.sup.-8] M [T.sub.3] induced significant responses of 3-fold (p < 0.01) and 5-fold (p < 0.05), respectively. Figure 4B shows results from similar experiments performed using somatic gene transfer in the tadpole brain. All the [T.sub.3] concentrations used ([10.sup.-10] M, [10.sup.-8] M, and [10.sup.-7] M) gave very significant transcriptional responses from the TH/bZIP-luc construct: 3-fold (p < 0.01), 5-fold (p < 0.001), and 5.5-fold (p < 0.001), respectively. Thus, the brain is a more sensitive TH target than is muscle, in terms of transcriptional responses from the TH/bZIP-luc construct.

[FIGURE 4 OMITTED]

The pretreatment protocol can be used on germinal transgenic tadpoles. Because the TH/bZIP construct used in somatic gene transfer experiments had proven suitable for detecting of [T.sub.3] agonists in muscle and brain, we used the same construct in germinal transgenesis (Kroll and Amaya 1996). We prepared a vector with the TH/bZIP-GFP chimeric gene between two copies of the lysozyme chicken gene as insulators (Stief et al. 1989). As a means of verifying transgenesis, we injected a second plasmid containing the [gamma]-crystallin promoter coupled to a RedFP. This plasmid is expressed early in the eye and allows selection of transgenic [F.sub.0] tadpoles before the TH/bZIP-dependent green fluorescence appears in the tadpole body.

We first followed TH/bZIP-driven fluorescence during early development and metamorphosis, focusing on the brain and on limb buds. In limb buds, the transgene is barely expressed at NF stage 51, and expression remains weak until NF stages 61-62. During metamorphosis, the signal intensifies. The transgene is expressed in the CNS at NF stage 51, and the fluorescent signal increases gradually through each metamorphic stage (Figure 5A). However, TH/bZIP expression remains at low enough levels in the brain so as not to interfere with fluorescence induction by TH agonists.

[FIGURE 5 OMITTED]

A number of [F.sub.0] germinally transgenic tadpoles were selected and bred to obtain F1 tadpoles. We next exposed [F.sub.1] transgenic tadpoles to [T.sub.3] ([10.sup.-8] M) using the pretreatment protocol. We confirmed that a short (24 hr) pretreatment with a low concentration of [T.sub.3] ([10.sup.-12] M), followed by a rinse, permits a rapidly detectable fluorescent signal in limb buds (forelimb and hindlimb buds) (Figure 5B), that is, after 48-hr exposure to [T.sub.3] ([10.sup.-8] M). In contrast, if the pretreatment protocol is not used, 4 days of exposure to [T.sub.3] ([10.sup.-8] M) is required to obtain a significant induction (data not shown). Fluorescence was induced in the brain, the olfactory nerves, and the gills (Figure 5B). TRIAC (5 x [10.sup.-8] M) induced similar responses in the brain (data not shown) and limb buds (Figure 5B).

Acetochlor disruption of thyroid signaling can be assessed within 48 hr. Because the pretreatment protocol can be used with germinally transgenic tadpoles to reveal [T.sub.3] effects, we applied it to NF stage 52 tadpoles to assess the potential thyroid-disrupting effects of the herbicide acetochlor. Pretreated tadpoles were exposed to [10.sup.-10] M [T.sub.3] or to [10.sup.- 10] M [T.sub.3] plus [10.sup.-8] M acetochlor for 48 hr. As shown in Figure 6A, the 20% increase in fluorescence in brains of germinally transgenic tadpoles was amplified by addition of [10.sup.-8] M acetochlor (p < 0.001 vs. controls). Figure 6B shows examples of germinally transgenic tadpoles brains that were quantified using ImageJ software.

[FIGURE 6 OMITTED]

Discussion

A central part of this study included establishing a rapid and sensitive method for assaying TH agonist activity within a shorter time frame than the several weeks needed to record TH effects on morphologic changes. This objective required fulfillment of numerous criteria: low background with no interference from endogenous hormone, robust and statistically significant responses, dose dependence, and low threshold. To avoid interference from endogenous hormone, we used euthyroid tadpoles at stages of development where TH levels are naturally low (Leloup and Buscaglia 1977). We had previously found that, when using euthyroid tadpoles in the somatic gene transfer test, a minimum of 4 days was necessary for significant induction of [T.sub.3]-dependent transcription (de Luze et al. 1993). Moreover, variability of responses among individuals was high. We theorized that the delay to response and the range of response levels could be due to lack of competence to respond to TH, possibly due to insufficient TRs in the target tissue, caudal muscle. Indeed, many experimental data have shown that TRs are expressed only at low levels before premetamorphosis, after which TR-[beta] is strongly induced by the [T.sub.3] signal, the TR-[beta] genes in X. laevis having complex promoters containing multiple positive TREs (Urnov and Wolffe 2001). To overcome this TR insufficiency, we chose to treat tadpoles with a brief, weak pulse of [T.sub.3] to induce competence to respond to a later exposure to TH. The logic was that the short pulse of [T.sub.3] should synchronize and harmonize the tadpole responses by up-regulating expression of TR-[beta], and possibly cofactors, thus facilitating TH responses. Further, given that the animals were then rinsed, it was expected that any hormone taken up would be degraded during the 24-hr rinse period, given that the half-life of [T.sub.3] in most vertebrates is around 18-24 hr (Van Middlesworth 1974).

We established that a short priming or pretreatment (24 hr) used with a weak concentration of [T.sub.3] ([10.sup.-12] M) induced an up-regulation of TR-[beta] expression and synchronized responses. These low concentrations and short exposure times do not induce any major morphologic changes in the tadpoles. TR-[beta] transcript levels were increased 2-fold within 6 hr by [10.sup.-12] M [T.sub.3] (Figure 2). These data confirmed previous experiments on whole tail tissue (Havis et al. 2003) or on tail tissues undergoing apoptosis (Helbing et al. 2003). In both cases, the authors observed a significant increase of TR-[beta] expression, however, with much higher [T.sub.3] concentrations ([10.sup.-8] M [T.sub.3] and [10.sup.-7] M [T.sub.3]) than we used.

We next determined whether responses were physiologic. We show that this is the case, in that they are dose dependent in muscle and brain (Figure 4) and are sensitive to known TH agonists, for example, TRIAC (Figure 4). Interestingly, using the TH/bZIP construct we observed a lower threshold to TH agonists in the brain than in muscle. This sensitivity correlates with the main site of TH/bZIP expression, that is, the CNS. Indeed, this gene was first isolated from the diencephalon