C 2014 Wiley Periodicals, Inc.
Birth Defects Research (Part B) 101:347–354 (2014)
Research Article
Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish Masha Reider∗ and Victoria P. Connaughton Department of Biology, American University, Washington, District of Columbia
Thyroid hormones are required for vertebrate development, and disruption of the thyroid system in developing embryos can result in a large range of morphologic and physiologic changes, including in the eye and retina. In this study, our anatomic analyses following low-dose, chronic thyroid inhibition reveal that both methimazole (MMI) exposure and rearing temperature affect eye development in a time- and temperature-dependent fashion. Maximal sensitivity to MMI for external eye development occurred at 65 hr postfertilization (hpf) for zebrafish reared at 28°C, and at 69 hpf for those reared at 31°C. Changes in eye diameter corresponded to changes in thickness of two inner retinal layers: the ganglion cell layer and the inner plexiform layer, with irreversible MMI-induced decreases in layer thickness observed in larvae treated with MMI until 66 hpf at 28°C. We infer that maximal sensitivity to MMI between 65 and 66 hpf at 28°C indicates a critical period of thyroid-dependent eye and retinal development. Furthermore, our results support previous work that shows spontaneous escape from MMI-induced effects potentially due to embryonic compensatory actions, as our data show that C 2014 embryos treated beyond the critical period generally resemble controls. Birth Defects Res (Part B) 101:347–354, 2014. Wiley Periodicals, Inc.
Key words: retina; thyroid; zebrafish; development; methimazole
INTRODUCTION Thyroid hormones are involved in the development of all major organ systems in vertebrates, including the central nervous system (Denver, 1998; Chan and Kilby, 2000; Power et al., 2001). Maintenance of euthyroid conditions is crucial during pregnancy as the developing fetus requires maternally supplied thyroid hormones throughout development. Consequently, maternal thyroid hormone deficiency or disruption can result in neurologic cretinism and other severe defects (Zoeller and Crofton, 2000; Obregon et al., 2007). Clinical interventions are critical for mitigating the wide range of adverse effects of abnormal levels of thyroid hormones. Common drugs used to treat hyperthyroid conditions are propylthiouracil (PTU), methimazole (MMI), and carbimazole, the latter being rapidly metabolized to MMI after consumption (Jansson et al., 1985; Diav-Citrin and Ornoy, 2002). Both PTU and MMI are goitrogens that block thyroid hormone synthesis by suppressing the activity of thyroid peroxidase, the enzyme that facilitates the binding of iodine to tyrosine and the subsequent binding of iodinated tyrosine to thyroglobulin (Ohtaki et al., 1996). When either compound is taken during pregnancy, it crosses the placenta and impacts fetal development. Importantly, exposure to
either compound during pregnancy has been associated with some birth defects, particularly when maternal thyroid hormone levels fail to remain stable (Diav-Citrin and Ornoy, 2002). Thyroid hormones (T3 and T4) are synthesized in the thyroid gland that is located near the trachea in mammals and the ventral aorta in fishes. Thyroid hormones, thyroid hormone receptors, and deiodinase enzymes are important for proper eye and retinal development. Deiodinases either inactivate T3 or convert T4 to T3, thus playing a crucial role in the regulation and stability of thyroid hormone levels in target tissues. For example, thyroid hormone deficiencies in rats are associated with decreased eye volume and optic nerve size, and the thinning of some retinal layers (Gamborino et al., 2001; Sevilla Romero et al., 2002; Pinazo-Duran et al., 2011). Misregulation of thyroid hormone levels, as shown in deiodinase 3 knockout mice, can ∗ Correspondence to: Masha Reider, Department of Biology, Hurst Hall 101, American University, 4400 Massachusetts Avenue, NW, Washington, DC 20016. E-mail: [email protected] Grant sponsor: American University Received 01 July 2014; Accepted 23 July 2014
Published online in Wiley Online Library (wileyonlinelibrary.com/journal/ bdrb) DOI: 10.1002/bdrb.21118
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lead to 80% reduction of cone photoreceptors (Ng et al., 2001; 2010), and knockout of tshr (thyroid stimulating hormone receptor) delays onset of M cone expression in mice (Lu et al., 2009). In chicks, expressions of thyroid receptors and deiodinases are directly associated with the progression of retinal development (Trimarchi et al., 2008). Further, genetic studies show that external administration of T3 changes expression of retina-related genes (Peleyo et al., 2012), and evidence suggests that the thyroid receptor 2 (tr2) gene is important for correct cone opsin expression (Suzuki et al., 2013). A recent study in zebrafish found that phenylthiourea, a pigment suppressor that affects thyroxine levels, reduces lens and eye size (Li et al., 2012). As in humans, exposure to MMI or PTU in zebrafish directly affects the thyroid system, decreasing intrafollicular T4 concentrations (Thienpont et al., 2011) and inhibiting iodine uptake (Brown, 1997). However, these compounds do not begin to inhibit thyroid hormone synthesis in zebrafish embryos until after the embryonic thyroid system is mature. Before this time, the embryo receives maternally derived thyroid hormones through the yolk. Thus, the onset time of sensitivity to MMI and other thyroid synthesis inhibitors can provide information about the timing of thyroid system maturity, which is reported to occur between 60 and 72 hr (Alt et al., 2006), as well as the timing of thyroid-dependent development in specific tissues. Zebrafish eye development begins at approximately 14 hr postfertilization (hpf) and is complete at approximately 72 hpf (Schmitt and Dowling, 1994, 1999). At 30 hpf, the first ganglion cell axons form the optic nerve, and by 48 hpf, these processes have reached the tectum (Burrill and Easter, 1994). New layers of bipolar cells can be seen at 60 hpf in the retina, while photoreceptors (rods and cones) continue to increase in size, and both plexiform layers become distinct (Schmitt and Dowling, 1999). Synaptic ribbons are present at 70 hpf (Schmitt and Dowling, 1999), which corresponds to the approximate time that the embryo develops a functional thyroid system (Brown, 1997; Elsalini and Rohr, 2003; Alt et al., 2006). By 4 to 5 days postfertilization, the retina is thought to be fully functional. Retinal development as well as overall embryogenesis is a temperature-dependent process in zebrafish (Kimmel et al., 1995). While the standard rearing temperature for zebrafish embryos is 28°C to 29°C, they maximally tolerate up to 41.7°C (Lawrence, 2007). Comparison of embryos reared at 25°C and 33°C revealed that higher temperatures can accelerate rate of development (Kimmel et al., 1995). The developmental interactions between temperature and other systems, such as the thyroid axis, have yet to be assessed. One goal of the current work was to examine the effects of chronic low-dose MMI exposure on visual system development in zebrafish as measured by anatomic changes in eye diameter and retinal layer thickness. In particular, our hypothesis was that low-dose MMI treatment would produce zebrafish embryos with smaller eyes and thinner retinal layers compared to unexposed control animals. We aimed to correlate changes in external eye diameter with changes in inner retinal layers. A second goal was to assess whether duration of treatment impacted these
MMI-induced eye and retinal defects. We predicted that zebrafish exposed to MMI until later ages would produce more severe phenotypes than those in treatment for less time. Our third goal was to determine the effect of rearing temperature on sensitivity to thyroid hormone disruption. We reasoned that MMI-treated animals reared at a slightly higher temperature (31°C) may show decreased sensitivity to MMI when compared to those reared at the standard rearing temperature of 28°C to 29°C. Thus, results from the current study set out to elucidate (1) whether embryonic thyroid hormones are required for eye and retinal layer development immediately before 72 hpf in zebrafish, (2) if changes in retinal layer thickness are correlated with changes in overall eye diameter in MMI-treated fish, (3) if MMI-induced effects depend on duration of treatment, and (4) whether rearing temperature may impact retinal development and/or interact with the thyroid system in this process. Given the known need for balanced thyroid hormone levels and concerns raised about thyroid-targeting endocrine disruptors found in global water supplies (Brechner et al., 2000; Weyer and Riley, 2001; Jugan et al., 2009; Shi et al., 2009; Yan et al., 2012), the results of these experiments will be relevant to species affected by environmental thyroid disruption as well as clinical studies examining the effects of developmental MMI exposure.
MATERIALS AND METHODS Experimental Conditions Zebrafish (Danio rerio) adults were maintained and spawned in the Fish Facility at American University, following established protocols (Westerfield, 2000) and in compliance with American University’s Institutional Animal Care and Use Committee policies. Treatment and control solutions were changed daily. To identify specific developmental ages that are most sensitive to MMI-induced deformities of the eye and retina, embryos were chronically exposed to MMI and removed from treatment between 60 and 72 hpf. This age range was selected because retinal layers are present and clearly defined, synapses are functional (Schmitt and Dowling, 1999), and endogenous synthesis of thyroid hormones is reported to begin (Brown, 1997; Elsalini and Rohr, 2003). Thus, this design allowed us to (1) observe the effect of MMI exposure at defined larval ages to identify possible critical time points that are most sensitive to MMI exposure and (2) determine if removal from MMI treatment allowed recovery of any drug-induced effects. To determine if increased rearing temperature could compensate for any effects of MMI exposure, embryos were exposed to MMI or control conditions at either 28°C or 31°C, resulting in 4 treatments: (1) system water (control) at 28°C, (2) system water (control) at 31°C, (3) 0.3 mM MMI at 28°C, and (4) 0.3 mM MMI at 31°C. The concentration of MMI (#46429) used, 0.3 mM, was selected for comparing our findings with other reports that examine the effects of low-dose MMI on zebrafish development (Brown, 1997; Liu and Chan, 2002; Lam et al., 2005). This concentration is also reported to be the “highest nontoxic dose” (Brown, 1997), though more recent studies have used significantly higher doses (100 mM, Komoike et al., 2013). Birth Defects Research (Part B) 101:347–354, 2014
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Experimental Design Embryos (N = 300) were placed into one of the four treatment/temperature conditions within 1 hr of fertilization. Starting at 60 hpf, a subset of embryos was removed from all four treatment groups, washed in distilled water, and placed into separate Petri dishes with system water. These embryos were then allowed to develop undisturbed until 72 hpf. This procedure was repeated hourly until all animals were euthanized (MS-222) and fixed using 4% paraformaldehyde at 72 hpf. This experiment was replicated using embryos from three separate spawns.
Measurements and Analyses To determine if MMI exposure and/or temperature affect visual system development, external eye diameter and retinal layer thickness were measured. In particular, we examined the inner retinal layers—ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL)—because synapses between bipolar and ganglion cells are forming at these experimental ages (Schmitt and Dowling, 1999), and inner retinal layer thicknesses are reported to be altered following MMI exposure in rats (Gamborino et al., 2001; Sevilla-Romero et al., 2002). To measure external eye diameter, larvae (N = 300) were placed on their side and measured along the anterior-to-posterior axis of the eye. By 72 hpf, all treated and controls were hatched and thus dechorionation was not required. For retinal layer analysis, a subset of fixed larvae (n = 10–20 per experimental condition per time point) was equilibrated in 30% sucrose overnight and 20 m thick cryosections were then obtained along the anterior–posterior axis. Sections were collected on silane-coated microscope slides and kept in the −80°C freezer until needed. To identify retinal layers, slides were warmed to room temperature and then stained with 4’,6diamidino-2-phenylindole. Retinal layers were measured through the midline of the eyecup in the vitread–sclerad direction in central retina behind the lens (Fig. 1). Images were captured using an Olympus BX61WI microscope (Olympus; Waltham, MA) Metamorph (version 7.7); measurements were calculated using Image J (version 1.46). Each measurement was made twice and averaged to decrease error. Effects of age removed from MMI treatment, and temperature on external eye diameter and retinal layer thicknesses were statistically analyzed using a univariate general linear model (SPSS 21.0.0). To account for known confounding effects of body size, mean body length per time point and treatment was measured as total length from the anterior-most point of the snout to the end of the tail, with segmented lines used to account for spinal or tail curvature (Image J). This was included in our statistical model as a covariate, as previously shown in general linear models with a covariate confound (Robinson et al., 1998). Post hoc Bonferroni t-tests were performed as needed.
RESULTS General Observations Survival rate was high (approximately 95–98%) in all groups, and hatching times for treated and controls were Birth Defects Research (Part B) 101:347–354, 2014
Fig. 1. Representative micrograph of the retina of a 72 hpf zebrafish. Retinal layer thickness was measured in the vitread– scleral direction in central retina behind the lens. Labels indicate how measurements were obtained. PL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 25 m.
similar, with all fish hatching between 48 and 72 hpf. MMI-treated and control fish did not display any obvious differences in characteristics, such as pigmentation, movement, and overall behavior.
External Eye Diameter Larvae reared at 28°C were significantly affected by MMI treatment (p < 0.05) as a function of age (p < 0.01). Furthermore, a significant treatment by temperature interaction was observed (p < 0.01) and indicates that the embryos from each temperature group responded differently to MMI treatment. Overall, our analysis at 72 hpf showed maximal sensitivity to MMI in zebrafish larvae treated until 65 and 72 hpf in the 28°C group, and until 69 hpf in the 31°C group (Fig. 2). MMI treatment until those time points produced nonrecoverable effects of MMI. At 28°C, eye diameter (N = 100) measurements showed unexpected patterns as a function of when the larvae were removed from MMI (Fig. 2A). We hypothesized that the longer the embryos remained in treatment, the more severe the MMI-induced effects. Similarly, the earlier the embryos were removed from MMI treatment (and thus the longer they have to recover in system water), the greater the recovery was expected. We found that larvae removed from treatment at the earliest experimental time points (60–64 hpf) had eye diameter measurements statistically similar to control larvae. However, embryos removed from treatment at 65 hpf had significantly smaller eye diameters than controls (p < 0.05; Fig. 2A), indicating that these embryos were unable to recover from the effects of MMI exposure.
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Fig. 2. The effects of MMI exposure and rearing temperature on external eye diameter. Larvae were chronically exposed to MMI and reared at either (A) 28°C or (B) 31°C. At 60 hpf, and hourly until 72 hpf, a subset of larvae was removed from MMI and placed into control water until 72 hpf to allow recovery. All larvae were collected at 72 hpf, euthanized in tricaine (MS-222), and fixed in 4% paraformaldehyde for later analysis. Eye diameters were measured in the anterior–posterior direction from the lateral side of the embryo/larva. Data are presented as means ± SE (n = 10–20). Asterisks indicate significant difference from control measurements. hpf, hours postfertilization.
Similarly, embryos exposed to MMI until the final 72 hpf time point had significantly smaller eye diameters (approximately 195 m) than controls (approximately 204 m), p < 0.01. In contrast, embryos reared in MMI until 66 to 71 hpf had eye diameters comparable to controls, an unexpected finding. Zebrafish embryos reared in 31°C (N = 200) showed that MMI treatment until 69 hpf produced significantly smaller eye diameter measurements (approximately 194 m) than untreated larvae (approximately 207 m; Fig. 2B). Interestingly, embryos exposed to MMI until 70 hpf had increased eye diameter measurements compared to embryos removed from treatment at either 71 or 72 hpf (p < 0.01). This supports the data observed in the 28°C rearing group that showed that the initial maximal sensitivity to MMI was followed by measurements that were comparable to controls. Taken together, the above data suggest that eye diameter is sensitive to MMI exposure and that this sensitivity is maximal when embryos were treated until 65 and 69 hpf when reared at 28°C and 31°C, respectively. In general, treatment for longer and shorter than these ages resulted in measurements similar to controls.
Retinal Layer Thickness
Fig. 3. Changes in retinal layer thickness in MMI treated and control larvae at 72 hpf. (A) Thickness of the GCL was decreased by MMI exposure, though not significant (p = 0.06). However, a significant treatment by temperature interaction effect was observed (p < 0.05), indicating that while control larvae have a thicker GCL when reared in the higher temperature, MMI treatment can reverse this effect. (B) Thickness of the inner plexiform layer (IPL) showed a clear temperature effect in control fish (p < 0.05), with thicker IPLs observed in retinas of larvae reared at 31°C compared to larvae reared at 28°C. No significant difference in IPL thickness was observed in MMI-treated retinas. (C) Inner nuclear layer (INL) thickness measurements revealed a significant treatment effect in the 31°C group (p < 0.01), with decreased layer thickness in MMI treated retinas, and a significant temperature effect in the treated group (p < 0.001). The patterns of change in the INL are similar to those observed in GCL measurements. Error bars represent ± SE. Horizontal lines above the bars indicate significant differences.*p < 0.05; **p < 0.01; ***p < 0.0001.
To determine if there were corresponding changes in retinal lamination that may account for the observed MMI-induced changes in eye diameter, we measured Birth Defects Research (Part B) 101:347–354, 2014
THYROID AND TEMPERATURE EFFECTS IN ZEBRAFISH EYE DEVELOPMENT the thickness of individual retinal layers in 72 hpf MMI-exposed and control larvae reared at both temperatures (Fig. 3) and analyzed embryos after they were exposed to MMI until 66, 70, or 72 hpf (Fig. 4). For the latter analysis, we used only larvae from the 28°C rearing group. These ages were selected to correspond to our external eye diameter data, both statistically significant and nonsignificant, so that we could determine if similar patterns exist for retinal layer measurements. Additionally, these time points correspond to critical times of retinal layer development (Schmitt and Dowling, 1999). Trends in the retinal layer data showed that both temperature and treatment play a role (either individually or as an interaction) in the development of some retinal layers (Fig. 3). Furthermore, we found that layer thickness varied across the ages examined (Fig. 4), suggesting that these retinal layers are differentially sensitive to MMI and/or that there are different critical periods of sensitivity in this tissue. GCL. GCL thickness measurements recorded at 72 hpf from control larvae reared at 28°C averaged 17 m. This value was larger (18–19 m) in control larvae reared at 31°C, a nonsignificant difference indicating no temperature effect (p = 0.1). GCL thickness measurements in MMI-treated larvae at this age were smaller than controls, though not significant (p = 0.06), averaging approximately 15 m in the 28°C rearing group and approximately 12 m in the 31°C rearing group (Fig. 3A). However, a significant temperature by treatment interaction was observed (p < 0.05), suggesting that treated and control larvae respond differently to rearing temperature. When examining measurements from the 28°C group only, the thinnest GCL measurement was observed in larvae exposed to MMI until 66 hpf (11.3 m), a statistically significant difference when compared to controls (Fig. 4A, p < 0.01). GCL thickness in embryos exposed to MMI until 70 hpf was significantly larger than both controls at this age (p < 0.05) and embryos exposed to MMI for the full 72 hr (p < 0.05). Thus, after 70 hr of continual MMI treatment, MMI-induced decreases in GCL thickness were no longer evident. IPL. IPL thickness at 72 hpf was significantly greater in larvae reared at 31°C than in larvae reared at 28°C (p < 0.05). Specifically, control embryos from the 28°C group had an IPL thickness of approximately 6 m, while controls reared at 31°C had an IPL thickness averaging 8 m (Fig. 3B). This indicates that for untreated embryos, a slightly higher rearing temperature can significantly increase the thickness of this retinal layer. IPL thicknesses were smaller in MMI-treated fish, regardless of rearing temperature; however, these differences were not significant (p = 0.18). In addition, the combined effect of rearing temperature and MMI exposure was also insignificant, suggesting a lack of a temperature/MMI interaction for this synapse layer. As observed for GCL measurements, IPL thickness in animals reared at 28°C was smallest in larvae exposed to MMI until 66 hpf (approximately 4.5 m; Fig. 4B). While the age of removal from MMI treatment was not statistically significant for these measurements, examination of the regression data suggests a positive linear relationship between time exposed to MMI and thickness of the IPL (R2 = 0.97). These trends are the opposite of what was hyBirth Defects Research (Part B) 101:347–354, 2014
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Fig. 4. Measurements of retinal layers in MMI-treated larvae exposed until 66, 70, and 72 hpf. (A) GCL thickness measurements were significantly smallest in larvae exposed to MMI until 66 hpf compared to controls (p < 0.01). A significant increase in GCL thickness was observed at 70 hpf (p < 0.05), followed by a significant decrease in thickness at 72 hpf (p < 0.05). (B) IPL thickness measurements also showed the smallest value in retinas exposed to MMI until 66 hpf. However, measurements of this retinal layer are not significantly different. The data suggest that more time in system water inversely affects eye size—a finding that is opposite of what we predicted. (C) INL thickness measurements also did not produce statistically significant results, suggesting that MMI exposure does not affect this retinal layer during our measured time points. Values are means ± SE. Horizontal bars indicate significant differences with *p < 0.05; **p < 0.01; ***p 95%) throughout the duration of the study. Second, general characteristics of MMI exposed larvae—pigmentation, overall behavior—were the same as controls. Third, we do not believe that the reduction in eye diameter in MMI-exposed larvae was due to a developmental delay, as larvae within this treatment typically hatched at the same time, or earlier, than controls, and body size was controlled for within our general linear model. Taken together, our results suggest that the effects of MMI observed in this study are due to its specific action on the newly developed thyroid gland and not the result of general teratogenicity of this compound. In fact, our results indicate a low MMI concentration of 0.3 mM is sufficient to significantly reduce eye diameter and thin the inner retina, which is unlikely to be the sole target of MMI toxicity. This is in agreement with initial MMI studies in zebrafish, which report this low concentration to be effective for overall growth patterns (Brown, 1997; Liu and Chan, 2002; Lam et al., 2005), reduction of zebrafish intrafollicular T4 levels by approximately 50% (Thienpont et al., 2011), and inhibition of iodine uptake by approximately 95% (Brown, 1997). Based on our above data and the documented specificity of MMI action on the zebrafish thyroid gland, we are confident that MMI-induced decreases in T3/T4 synthesis underlie our results. Clinical relevance of the current findings extends from retinal development in hypothyroid pregnancies to aquatic organisms in polluted water containing endocrine disruptors, as this study used very low, environmentally relevant, doses of MMI to elicit our effects. Results from the present study further support the importance of thyroid hormones in neuronal development. Studies have shown that visual acuity of hyperthyroid rats is superior to control animals (Brunjez and Alberts, 1981), suggesting that thyroid disruption can impact neuronal function and behavior. Our studies suggest that this can occur at low levels of MMI, and future studies in our laboratory will examine behavioral consequences of hypothyroid development in zebrafish.
ACKNOWLEDGMENTS The authors thank American University for the funding of this project, and Dr. Dan Fong for providBirth Defects Research (Part B) 101:347–354, 2014
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ing comments on the manuscript. American University graduate student Mellon and Helmlinge awards were used in the completion of this project.
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Birth Defects Research (Part B) 101:347–354, 2014
Birth Defects Research (Part B) 101:347–354 (2014)
Research Article
Effects of Low-Dose Embryonic Thyroid Disruption and Rearing Temperature on the Development of the Eye and Retina in Zebrafish Masha Reider∗ and Victoria P. Connaughton Department of Biology, American University, Washington, District of Columbia
Thyroid hormones are required for vertebrate development, and disruption of the thyroid system in developing embryos can result in a large range of morphologic and physiologic changes, including in the eye and retina. In this study, our anatomic analyses following low-dose, chronic thyroid inhibition reveal that both methimazole (MMI) exposure and rearing temperature affect eye development in a time- and temperature-dependent fashion. Maximal sensitivity to MMI for external eye development occurred at 65 hr postfertilization (hpf) for zebrafish reared at 28°C, and at 69 hpf for those reared at 31°C. Changes in eye diameter corresponded to changes in thickness of two inner retinal layers: the ganglion cell layer and the inner plexiform layer, with irreversible MMI-induced decreases in layer thickness observed in larvae treated with MMI until 66 hpf at 28°C. We infer that maximal sensitivity to MMI between 65 and 66 hpf at 28°C indicates a critical period of thyroid-dependent eye and retinal development. Furthermore, our results support previous work that shows spontaneous escape from MMI-induced effects potentially due to embryonic compensatory actions, as our data show that C 2014 embryos treated beyond the critical period generally resemble controls. Birth Defects Res (Part B) 101:347–354, 2014. Wiley Periodicals, Inc.
Key words: retina; thyroid; zebrafish; development; methimazole
INTRODUCTION Thyroid hormones are involved in the development of all major organ systems in vertebrates, including the central nervous system (Denver, 1998; Chan and Kilby, 2000; Power et al., 2001). Maintenance of euthyroid conditions is crucial during pregnancy as the developing fetus requires maternally supplied thyroid hormones throughout development. Consequently, maternal thyroid hormone deficiency or disruption can result in neurologic cretinism and other severe defects (Zoeller and Crofton, 2000; Obregon et al., 2007). Clinical interventions are critical for mitigating the wide range of adverse effects of abnormal levels of thyroid hormones. Common drugs used to treat hyperthyroid conditions are propylthiouracil (PTU), methimazole (MMI), and carbimazole, the latter being rapidly metabolized to MMI after consumption (Jansson et al., 1985; Diav-Citrin and Ornoy, 2002). Both PTU and MMI are goitrogens that block thyroid hormone synthesis by suppressing the activity of thyroid peroxidase, the enzyme that facilitates the binding of iodine to tyrosine and the subsequent binding of iodinated tyrosine to thyroglobulin (Ohtaki et al., 1996). When either compound is taken during pregnancy, it crosses the placenta and impacts fetal development. Importantly, exposure to
either compound during pregnancy has been associated with some birth defects, particularly when maternal thyroid hormone levels fail to remain stable (Diav-Citrin and Ornoy, 2002). Thyroid hormones (T3 and T4) are synthesized in the thyroid gland that is located near the trachea in mammals and the ventral aorta in fishes. Thyroid hormones, thyroid hormone receptors, and deiodinase enzymes are important for proper eye and retinal development. Deiodinases either inactivate T3 or convert T4 to T3, thus playing a crucial role in the regulation and stability of thyroid hormone levels in target tissues. For example, thyroid hormone deficiencies in rats are associated with decreased eye volume and optic nerve size, and the thinning of some retinal layers (Gamborino et al., 2001; Sevilla Romero et al., 2002; Pinazo-Duran et al., 2011). Misregulation of thyroid hormone levels, as shown in deiodinase 3 knockout mice, can ∗ Correspondence to: Masha Reider, Department of Biology, Hurst Hall 101, American University, 4400 Massachusetts Avenue, NW, Washington, DC 20016. E-mail: [email protected] Grant sponsor: American University Received 01 July 2014; Accepted 23 July 2014
Published online in Wiley Online Library (wileyonlinelibrary.com/journal/ bdrb) DOI: 10.1002/bdrb.21118
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lead to 80% reduction of cone photoreceptors (Ng et al., 2001; 2010), and knockout of tshr (thyroid stimulating hormone receptor) delays onset of M cone expression in mice (Lu et al., 2009). In chicks, expressions of thyroid receptors and deiodinases are directly associated with the progression of retinal development (Trimarchi et al., 2008). Further, genetic studies show that external administration of T3 changes expression of retina-related genes (Peleyo et al., 2012), and evidence suggests that the thyroid receptor 2 (tr2) gene is important for correct cone opsin expression (Suzuki et al., 2013). A recent study in zebrafish found that phenylthiourea, a pigment suppressor that affects thyroxine levels, reduces lens and eye size (Li et al., 2012). As in humans, exposure to MMI or PTU in zebrafish directly affects the thyroid system, decreasing intrafollicular T4 concentrations (Thienpont et al., 2011) and inhibiting iodine uptake (Brown, 1997). However, these compounds do not begin to inhibit thyroid hormone synthesis in zebrafish embryos until after the embryonic thyroid system is mature. Before this time, the embryo receives maternally derived thyroid hormones through the yolk. Thus, the onset time of sensitivity to MMI and other thyroid synthesis inhibitors can provide information about the timing of thyroid system maturity, which is reported to occur between 60 and 72 hr (Alt et al., 2006), as well as the timing of thyroid-dependent development in specific tissues. Zebrafish eye development begins at approximately 14 hr postfertilization (hpf) and is complete at approximately 72 hpf (Schmitt and Dowling, 1994, 1999). At 30 hpf, the first ganglion cell axons form the optic nerve, and by 48 hpf, these processes have reached the tectum (Burrill and Easter, 1994). New layers of bipolar cells can be seen at 60 hpf in the retina, while photoreceptors (rods and cones) continue to increase in size, and both plexiform layers become distinct (Schmitt and Dowling, 1999). Synaptic ribbons are present at 70 hpf (Schmitt and Dowling, 1999), which corresponds to the approximate time that the embryo develops a functional thyroid system (Brown, 1997; Elsalini and Rohr, 2003; Alt et al., 2006). By 4 to 5 days postfertilization, the retina is thought to be fully functional. Retinal development as well as overall embryogenesis is a temperature-dependent process in zebrafish (Kimmel et al., 1995). While the standard rearing temperature for zebrafish embryos is 28°C to 29°C, they maximally tolerate up to 41.7°C (Lawrence, 2007). Comparison of embryos reared at 25°C and 33°C revealed that higher temperatures can accelerate rate of development (Kimmel et al., 1995). The developmental interactions between temperature and other systems, such as the thyroid axis, have yet to be assessed. One goal of the current work was to examine the effects of chronic low-dose MMI exposure on visual system development in zebrafish as measured by anatomic changes in eye diameter and retinal layer thickness. In particular, our hypothesis was that low-dose MMI treatment would produce zebrafish embryos with smaller eyes and thinner retinal layers compared to unexposed control animals. We aimed to correlate changes in external eye diameter with changes in inner retinal layers. A second goal was to assess whether duration of treatment impacted these
MMI-induced eye and retinal defects. We predicted that zebrafish exposed to MMI until later ages would produce more severe phenotypes than those in treatment for less time. Our third goal was to determine the effect of rearing temperature on sensitivity to thyroid hormone disruption. We reasoned that MMI-treated animals reared at a slightly higher temperature (31°C) may show decreased sensitivity to MMI when compared to those reared at the standard rearing temperature of 28°C to 29°C. Thus, results from the current study set out to elucidate (1) whether embryonic thyroid hormones are required for eye and retinal layer development immediately before 72 hpf in zebrafish, (2) if changes in retinal layer thickness are correlated with changes in overall eye diameter in MMI-treated fish, (3) if MMI-induced effects depend on duration of treatment, and (4) whether rearing temperature may impact retinal development and/or interact with the thyroid system in this process. Given the known need for balanced thyroid hormone levels and concerns raised about thyroid-targeting endocrine disruptors found in global water supplies (Brechner et al., 2000; Weyer and Riley, 2001; Jugan et al., 2009; Shi et al., 2009; Yan et al., 2012), the results of these experiments will be relevant to species affected by environmental thyroid disruption as well as clinical studies examining the effects of developmental MMI exposure.
MATERIALS AND METHODS Experimental Conditions Zebrafish (Danio rerio) adults were maintained and spawned in the Fish Facility at American University, following established protocols (Westerfield, 2000) and in compliance with American University’s Institutional Animal Care and Use Committee policies. Treatment and control solutions were changed daily. To identify specific developmental ages that are most sensitive to MMI-induced deformities of the eye and retina, embryos were chronically exposed to MMI and removed from treatment between 60 and 72 hpf. This age range was selected because retinal layers are present and clearly defined, synapses are functional (Schmitt and Dowling, 1999), and endogenous synthesis of thyroid hormones is reported to begin (Brown, 1997; Elsalini and Rohr, 2003). Thus, this design allowed us to (1) observe the effect of MMI exposure at defined larval ages to identify possible critical time points that are most sensitive to MMI exposure and (2) determine if removal from MMI treatment allowed recovery of any drug-induced effects. To determine if increased rearing temperature could compensate for any effects of MMI exposure, embryos were exposed to MMI or control conditions at either 28°C or 31°C, resulting in 4 treatments: (1) system water (control) at 28°C, (2) system water (control) at 31°C, (3) 0.3 mM MMI at 28°C, and (4) 0.3 mM MMI at 31°C. The concentration of MMI (#46429) used, 0.3 mM, was selected for comparing our findings with other reports that examine the effects of low-dose MMI on zebrafish development (Brown, 1997; Liu and Chan, 2002; Lam et al., 2005). This concentration is also reported to be the “highest nontoxic dose” (Brown, 1997), though more recent studies have used significantly higher doses (100 mM, Komoike et al., 2013). Birth Defects Research (Part B) 101:347–354, 2014
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Experimental Design Embryos (N = 300) were placed into one of the four treatment/temperature conditions within 1 hr of fertilization. Starting at 60 hpf, a subset of embryos was removed from all four treatment groups, washed in distilled water, and placed into separate Petri dishes with system water. These embryos were then allowed to develop undisturbed until 72 hpf. This procedure was repeated hourly until all animals were euthanized (MS-222) and fixed using 4% paraformaldehyde at 72 hpf. This experiment was replicated using embryos from three separate spawns.
Measurements and Analyses To determine if MMI exposure and/or temperature affect visual system development, external eye diameter and retinal layer thickness were measured. In particular, we examined the inner retinal layers—ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL)—because synapses between bipolar and ganglion cells are forming at these experimental ages (Schmitt and Dowling, 1999), and inner retinal layer thicknesses are reported to be altered following MMI exposure in rats (Gamborino et al., 2001; Sevilla-Romero et al., 2002). To measure external eye diameter, larvae (N = 300) were placed on their side and measured along the anterior-to-posterior axis of the eye. By 72 hpf, all treated and controls were hatched and thus dechorionation was not required. For retinal layer analysis, a subset of fixed larvae (n = 10–20 per experimental condition per time point) was equilibrated in 30% sucrose overnight and 20 m thick cryosections were then obtained along the anterior–posterior axis. Sections were collected on silane-coated microscope slides and kept in the −80°C freezer until needed. To identify retinal layers, slides were warmed to room temperature and then stained with 4’,6diamidino-2-phenylindole. Retinal layers were measured through the midline of the eyecup in the vitread–sclerad direction in central retina behind the lens (Fig. 1). Images were captured using an Olympus BX61WI microscope (Olympus; Waltham, MA) Metamorph (version 7.7); measurements were calculated using Image J (version 1.46). Each measurement was made twice and averaged to decrease error. Effects of age removed from MMI treatment, and temperature on external eye diameter and retinal layer thicknesses were statistically analyzed using a univariate general linear model (SPSS 21.0.0). To account for known confounding effects of body size, mean body length per time point and treatment was measured as total length from the anterior-most point of the snout to the end of the tail, with segmented lines used to account for spinal or tail curvature (Image J). This was included in our statistical model as a covariate, as previously shown in general linear models with a covariate confound (Robinson et al., 1998). Post hoc Bonferroni t-tests were performed as needed.
RESULTS General Observations Survival rate was high (approximately 95–98%) in all groups, and hatching times for treated and controls were Birth Defects Research (Part B) 101:347–354, 2014
Fig. 1. Representative micrograph of the retina of a 72 hpf zebrafish. Retinal layer thickness was measured in the vitread– scleral direction in central retina behind the lens. Labels indicate how measurements were obtained. PL, photoreceptor layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 25 m.
similar, with all fish hatching between 48 and 72 hpf. MMI-treated and control fish did not display any obvious differences in characteristics, such as pigmentation, movement, and overall behavior.
External Eye Diameter Larvae reared at 28°C were significantly affected by MMI treatment (p < 0.05) as a function of age (p < 0.01). Furthermore, a significant treatment by temperature interaction was observed (p < 0.01) and indicates that the embryos from each temperature group responded differently to MMI treatment. Overall, our analysis at 72 hpf showed maximal sensitivity to MMI in zebrafish larvae treated until 65 and 72 hpf in the 28°C group, and until 69 hpf in the 31°C group (Fig. 2). MMI treatment until those time points produced nonrecoverable effects of MMI. At 28°C, eye diameter (N = 100) measurements showed unexpected patterns as a function of when the larvae were removed from MMI (Fig. 2A). We hypothesized that the longer the embryos remained in treatment, the more severe the MMI-induced effects. Similarly, the earlier the embryos were removed from MMI treatment (and thus the longer they have to recover in system water), the greater the recovery was expected. We found that larvae removed from treatment at the earliest experimental time points (60–64 hpf) had eye diameter measurements statistically similar to control larvae. However, embryos removed from treatment at 65 hpf had significantly smaller eye diameters than controls (p < 0.05; Fig. 2A), indicating that these embryos were unable to recover from the effects of MMI exposure.
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Fig. 2. The effects of MMI exposure and rearing temperature on external eye diameter. Larvae were chronically exposed to MMI and reared at either (A) 28°C or (B) 31°C. At 60 hpf, and hourly until 72 hpf, a subset of larvae was removed from MMI and placed into control water until 72 hpf to allow recovery. All larvae were collected at 72 hpf, euthanized in tricaine (MS-222), and fixed in 4% paraformaldehyde for later analysis. Eye diameters were measured in the anterior–posterior direction from the lateral side of the embryo/larva. Data are presented as means ± SE (n = 10–20). Asterisks indicate significant difference from control measurements. hpf, hours postfertilization.
Similarly, embryos exposed to MMI until the final 72 hpf time point had significantly smaller eye diameters (approximately 195 m) than controls (approximately 204 m), p < 0.01. In contrast, embryos reared in MMI until 66 to 71 hpf had eye diameters comparable to controls, an unexpected finding. Zebrafish embryos reared in 31°C (N = 200) showed that MMI treatment until 69 hpf produced significantly smaller eye diameter measurements (approximately 194 m) than untreated larvae (approximately 207 m; Fig. 2B). Interestingly, embryos exposed to MMI until 70 hpf had increased eye diameter measurements compared to embryos removed from treatment at either 71 or 72 hpf (p < 0.01). This supports the data observed in the 28°C rearing group that showed that the initial maximal sensitivity to MMI was followed by measurements that were comparable to controls. Taken together, the above data suggest that eye diameter is sensitive to MMI exposure and that this sensitivity is maximal when embryos were treated until 65 and 69 hpf when reared at 28°C and 31°C, respectively. In general, treatment for longer and shorter than these ages resulted in measurements similar to controls.
Retinal Layer Thickness
Fig. 3. Changes in retinal layer thickness in MMI treated and control larvae at 72 hpf. (A) Thickness of the GCL was decreased by MMI exposure, though not significant (p = 0.06). However, a significant treatment by temperature interaction effect was observed (p < 0.05), indicating that while control larvae have a thicker GCL when reared in the higher temperature, MMI treatment can reverse this effect. (B) Thickness of the inner plexiform layer (IPL) showed a clear temperature effect in control fish (p < 0.05), with thicker IPLs observed in retinas of larvae reared at 31°C compared to larvae reared at 28°C. No significant difference in IPL thickness was observed in MMI-treated retinas. (C) Inner nuclear layer (INL) thickness measurements revealed a significant treatment effect in the 31°C group (p < 0.01), with decreased layer thickness in MMI treated retinas, and a significant temperature effect in the treated group (p < 0.001). The patterns of change in the INL are similar to those observed in GCL measurements. Error bars represent ± SE. Horizontal lines above the bars indicate significant differences.*p < 0.05; **p < 0.01; ***p < 0.0001.
To determine if there were corresponding changes in retinal lamination that may account for the observed MMI-induced changes in eye diameter, we measured Birth Defects Research (Part B) 101:347–354, 2014
THYROID AND TEMPERATURE EFFECTS IN ZEBRAFISH EYE DEVELOPMENT the thickness of individual retinal layers in 72 hpf MMI-exposed and control larvae reared at both temperatures (Fig. 3) and analyzed embryos after they were exposed to MMI until 66, 70, or 72 hpf (Fig. 4). For the latter analysis, we used only larvae from the 28°C rearing group. These ages were selected to correspond to our external eye diameter data, both statistically significant and nonsignificant, so that we could determine if similar patterns exist for retinal layer measurements. Additionally, these time points correspond to critical times of retinal layer development (Schmitt and Dowling, 1999). Trends in the retinal layer data showed that both temperature and treatment play a role (either individually or as an interaction) in the development of some retinal layers (Fig. 3). Furthermore, we found that layer thickness varied across the ages examined (Fig. 4), suggesting that these retinal layers are differentially sensitive to MMI and/or that there are different critical periods of sensitivity in this tissue. GCL. GCL thickness measurements recorded at 72 hpf from control larvae reared at 28°C averaged 17 m. This value was larger (18–19 m) in control larvae reared at 31°C, a nonsignificant difference indicating no temperature effect (p = 0.1). GCL thickness measurements in MMI-treated larvae at this age were smaller than controls, though not significant (p = 0.06), averaging approximately 15 m in the 28°C rearing group and approximately 12 m in the 31°C rearing group (Fig. 3A). However, a significant temperature by treatment interaction was observed (p < 0.05), suggesting that treated and control larvae respond differently to rearing temperature. When examining measurements from the 28°C group only, the thinnest GCL measurement was observed in larvae exposed to MMI until 66 hpf (11.3 m), a statistically significant difference when compared to controls (Fig. 4A, p < 0.01). GCL thickness in embryos exposed to MMI until 70 hpf was significantly larger than both controls at this age (p < 0.05) and embryos exposed to MMI for the full 72 hr (p < 0.05). Thus, after 70 hr of continual MMI treatment, MMI-induced decreases in GCL thickness were no longer evident. IPL. IPL thickness at 72 hpf was significantly greater in larvae reared at 31°C than in larvae reared at 28°C (p < 0.05). Specifically, control embryos from the 28°C group had an IPL thickness of approximately 6 m, while controls reared at 31°C had an IPL thickness averaging 8 m (Fig. 3B). This indicates that for untreated embryos, a slightly higher rearing temperature can significantly increase the thickness of this retinal layer. IPL thicknesses were smaller in MMI-treated fish, regardless of rearing temperature; however, these differences were not significant (p = 0.18). In addition, the combined effect of rearing temperature and MMI exposure was also insignificant, suggesting a lack of a temperature/MMI interaction for this synapse layer. As observed for GCL measurements, IPL thickness in animals reared at 28°C was smallest in larvae exposed to MMI until 66 hpf (approximately 4.5 m; Fig. 4B). While the age of removal from MMI treatment was not statistically significant for these measurements, examination of the regression data suggests a positive linear relationship between time exposed to MMI and thickness of the IPL (R2 = 0.97). These trends are the opposite of what was hyBirth Defects Research (Part B) 101:347–354, 2014
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Fig. 4. Measurements of retinal layers in MMI-treated larvae exposed until 66, 70, and 72 hpf. (A) GCL thickness measurements were significantly smallest in larvae exposed to MMI until 66 hpf compared to controls (p < 0.01). A significant increase in GCL thickness was observed at 70 hpf (p < 0.05), followed by a significant decrease in thickness at 72 hpf (p < 0.05). (B) IPL thickness measurements also showed the smallest value in retinas exposed to MMI until 66 hpf. However, measurements of this retinal layer are not significantly different. The data suggest that more time in system water inversely affects eye size—a finding that is opposite of what we predicted. (C) INL thickness measurements also did not produce statistically significant results, suggesting that MMI exposure does not affect this retinal layer during our measured time points. Values are means ± SE. Horizontal bars indicate significant differences with *p < 0.05; **p < 0.01; ***p 95%) throughout the duration of the study. Second, general characteristics of MMI exposed larvae—pigmentation, overall behavior—were the same as controls. Third, we do not believe that the reduction in eye diameter in MMI-exposed larvae was due to a developmental delay, as larvae within this treatment typically hatched at the same time, or earlier, than controls, and body size was controlled for within our general linear model. Taken together, our results suggest that the effects of MMI observed in this study are due to its specific action on the newly developed thyroid gland and not the result of general teratogenicity of this compound. In fact, our results indicate a low MMI concentration of 0.3 mM is sufficient to significantly reduce eye diameter and thin the inner retina, which is unlikely to be the sole target of MMI toxicity. This is in agreement with initial MMI studies in zebrafish, which report this low concentration to be effective for overall growth patterns (Brown, 1997; Liu and Chan, 2002; Lam et al., 2005), reduction of zebrafish intrafollicular T4 levels by approximately 50% (Thienpont et al., 2011), and inhibition of iodine uptake by approximately 95% (Brown, 1997). Based on our above data and the documented specificity of MMI action on the zebrafish thyroid gland, we are confident that MMI-induced decreases in T3/T4 synthesis underlie our results. Clinical relevance of the current findings extends from retinal development in hypothyroid pregnancies to aquatic organisms in polluted water containing endocrine disruptors, as this study used very low, environmentally relevant, doses of MMI to elicit our effects. Results from the present study further support the importance of thyroid hormones in neuronal development. Studies have shown that visual acuity of hyperthyroid rats is superior to control animals (Brunjez and Alberts, 1981), suggesting that thyroid disruption can impact neuronal function and behavior. Our studies suggest that this can occur at low levels of MMI, and future studies in our laboratory will examine behavioral consequences of hypothyroid development in zebrafish.
ACKNOWLEDGMENTS The authors thank American University for the funding of this project, and Dr. Dan Fong for providBirth Defects Research (Part B) 101:347–354, 2014
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ing comments on the manuscript. American University graduate student Mellon and Helmlinge awards were used in the completion of this project.
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