Expression profile and thyroid hormone responsiveness of transporters and deiodinases in early embryonic chicken brain development

Molecular and Cellular Endocrinology 349 (2012) 289–297

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Expression profile and thyroid hormone responsiveness of transporters and deiodinases in early embryonic chicken brain development Stijn L.J. Van Herck a, Stijn Geysens a, Joke Delbaere a, Przemko Tylzanowski b, Veerle M. Darras a,⇑ a b

Laboratory of Comparative Endocrinology, Animal Physiology and Neurobiology Section, Department of Biology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium Laboratory of Skeletal Development and Joint Disorders, Division of Rheumatology, Department of Musculoskeletal Sciences, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

a r t i c l e

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Article history: Received 14 July 2011 Received in revised form 21 October 2011 Accepted 12 November 2011 Available online 22 November 2011 Keywords: Thyroid hormone Brain Deiodinase Transporter Development Chicken

a b s t r a c t We used the chick embryo to study the mechanisms regulating intracellular TH availability in developing brain. TH-transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 were expressed in a regionspecific way, well before the onset of endogenous TH secretion. Between day 4 and 10 of development MCT8 and D2 mRNA levels increased, while OATP1C1 and D3 mRNA levels decreased. D2 and D3 mRNAs were translated into active protein, while no D1 activity was detectable. Injection of THs into the yolk 24 h before sampling increased TH levels in the brain and resulted in decreased OATP1C1 and increased MCT8 expression in 4-day-old embryos. A compensatory response in deiodinase activity was only observed at day 8. We conclude that THs are active in the early embryonic brain and TH-transporters and deiodinases can regulate their availability. However, the absence of clear compensatory mechanisms at day 4 makes the brain more vulnerable for changes in maternal TH supply. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Normal brain development in vertebrates is critically dependent on correct thyroid hormone (TH) signalling (for reviews see: Bernal, 2005; Anderson, 2001; Ahmed et al., 2008; Morreale de Escobar et al., 2004b). The early embryo does not have a functional thyroid gland and therefore depends on the THs supplied by the mother. Clinical studies showed that maternal TH deficiency during the first trimester of pregnancy can affect the outcome of human neurodevelopment (Pop et al., 1999; Haddow et al., 1999). Experiments in rats showed that early maternal TH deficiency affects neuronal migration in the cortex (Lavado-Autric et al., 2003), while maternal hyperthyroidism too can disturb foetal brain development (Evans et al., 2002). Experimental data on the mechanisms regulating intracellular TH availability and action prior to the onset of foetal TH secretion, however, remain scarce. TH action can be controlled in individual cells through selective TH uptake and intracellular TH metabolism. The thyroid gland Abbreviations: D1–3, type 1–3 iodothyronine deiodinase; E1, E2, E3, . . ., chicken embryos of 1, 2, 3, . . . days old; LAT, L-type amino acid transporter; MCT, monocarboxylate transporter; OATP, organic anion-transporting polypeptide; T3, 3,5,30 -triiodothyronine; T4, thyroxine; TH, thyroid hormone; TR, TH receptor; QPCR, quantitative polymerase chain reaction. ⇑ Corresponding author. Address: Laboratory of Comparative Endocrinology, Naamsestraat 61, P.O. Box 2464, B-3000 Leuven, Belgium. Tel.: +32 6 23985; fax: +32 6 24262. E-mail address: [email protected] (V.M. Darras). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.11.012

secretes predominantly thyroxine (T4) that is converted to the receptor-active 3,5,30 -triiodothyronine (T3) in different tissues. Binding of T3 to its nuclear receptors (TRs) directly affects transcription of many genes that are important in development (Harvey and Williams, 2002). Transport of T4 and T3 in and out of cells is controlled by several classes of transmembrane transporters, including members of the organic anion transporter family (OATP), L-type amino acid transporters (LATs) and monocarboxylate transporters (MCTs) (Visser et al., 2008). Intracellular activation or inactivation of T4 and T3 in turn is determined by three types of iodothyronine deiodinases, namely D1, D2, and D3 (Gereben et al., 2008). D2 activates T4 to T3 while D3 inactivates T4 and T3 to respectively reverse T3 and T2. The D1 enzyme can stimulate the activating as well as the inactivating pathway but D2 and D3 are considered to be the main deiodinases for TH metabolism in the brain (Horn and Heuer, 2010). Variable expression of these factors allows tight regulation of the intracellular T3 levels and enables the cell to respond to fluctuations in systemic TH levels. During adult life, variations in TH availability typically elicit a strong compensatory response from the deiodinases (Gereben et al., 2008). Studies in rats showed that an increase in TH availability was rapidly followed by an increase in D3 and a decrease in D2 expression in the brain (Tu et al., 1999; Burmeister et al., 1997) while the opposite was true in hypothyroid rats (Tu et al., 1997, 1999). The response of TH transporter expression to changes in TH status is less clear and the effects vary between different transporters, tissue types and developmental stage. In the brain,

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studies in rats showed that OATP1C1 expression was up- or downregulated in the capillaries under hypo- and hyperthyroid conditions, respectively (Sugiyama et al., 2003). The expression of MCT8 in the cerebral cortex however was not affected by hypothyroidism (Babu et al., 2011). In the hypothalamus of prolonged critically ill rabbits with low circulating T3 levels, increased MCT10 and OATP1C1 but not MCT8 mRNA expression was detected (Mebis et al., 2009). In vitro expression analysis of nine different TH transporters in N-Tera-2 cells, having biochemical and developmental characteristics similar to central nervous system precursor cells, did not demonstrate any compensatory response when cells were cultured in T3 depleted media. T3 deficiency even decreased MCT10 and LAT2 mRNA expression (Chan et al., 2011). To increase our understanding of the role of TH during early brain development, detailed information on the presence and activity of TH transporters and deiodinases is essential. In this study we used the chicken model organism. Similar to the human situation the thyroid gland becomes functional around mid embryonic development (Morreale de Escobar et al., 2004a; Thommes, 1987), which in the chicken corresponds to days 9–10 of the 21 day incubation period (Thommes, 1987). We determined the TH concentrations and the ontogenetic expression profiles of TH transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 in brain of 4- to 12-day-old embryos. Subsequently we analysed the expression of these genes under hyperthyroid conditions at E4 and E8 to determine the TH responsiveness of the brain at this time.

extracts as previously described in detail (Reyns et al., 2002, 2003). We separated the entire brain from the eyes and the rest of the surrounding tissues. To meet the limits of our detection system, we used pools of whole brains from 8 individual embryos at E4, from 4 individual embryos at E6, from 2 individual embryos at E8; E10 and E12 brains were not pooled. Homogenization was carried out in methanol and 1500–2000 cpm of [131I]T3 and [125I]T4 were added as internal recovery tracers. Thereafter, chloroform was added to form a solvent mixture of chloroform:methanol (v/v 2:1). After centrifugation, the pellet was re-extracted with the same solvent. Both supernatants were combined and 0.05% CaCl2 was added so that the final chloroform:methanol:water ratio was 8:4:3. The mixture was centrifuged and the lower apolar phase was re-extracted with chloroform:methanol:0.05% CaCl2 (3:49:48) in an amount equal to that removed. The pooled upper polar layers were further purified by anion-exchange chromatography on AG 1-X2 resin columns (Bio-Rad, Nazareth, Belgium). The iodothyronines were eluted with 70% acetic acid, evaporated to dryness and resuspended in RIA buffer. Typical recoveries of extracted THs ranged from 55% to 75% for T3 and from 40% to 60% for T4. The T3 RIA had a detection limit of 2 fmol and an intra-assay variability of 2.2%. The T4 RIA had a detection limit of 5 fmol and an intra-assay variability of 2.8%. For the T3 RIA cross-reactivity with T4 was 0.1–0.5%, whereas for the T4 RIA cross-reactivity with T3 was 3.5%. Hormone levels were expressed as picomol per gram brain tissue. 2.3. Reverse transcription and quantitative PCR (Q-PCR)

2. Methods 2.1. Tissue sampling Chicken embryonic development takes 21 days from fertilisation to hatching. Fertilized eggs from Ross broiler chickens were purchased from a commercial hatchery (Belgabroed, Merksplas, Belgium). The eggs were incubated in a forced draft incubator at 37.5 °C and 50% relative humidity and automatically turned at 45° angles every hour. The start of the incubation was called day 0 (E0). After 4, 6, 8, 10 and 12 days of incubation (E4–E12) eggs were opened and telencephalon, diencephalon, mesencephalon and rhombencephalon were dissected. The meninges and choroid plexus were removed and therefore not included in the Q-PCR analysis, with the exception of the membranes lining the telencephalon at E4 and the mesencephalon at E4 and E6 of which removal was particularly difficult. The rhombencephalon contains both metencephalon and myelencephalon which on day 4 were too small to be analyzed individually. The telencephalon differentiates further into the cerebral hemispheres, the diencephalon represents the later thalamic regions, the mesencephalon differentiates into the optic lobes and the rhombencephalon becomes cerebellum, pons and medulla oblongata (Bellairs and Osmond, 2005). Samples were immediately frozen in liquid nitrogen and stored at –80 °C for later use. Hyperthyroid samples were obtained by injecting a combination of 0.5 lg T3 + 1 lg T4 into the yolk through a small hole in the air chamber, 24 h prior to sampling. The injected dose represents a 5- to 10-fold increase in TH content of the egg yolk and closely resembles the normal T3/T4 ratio (unpublished data and reference Prati et al., 1992). THs were dissolved in 0.1 N NaOH and diluted in 0.9% NaCl. Control eggs were injected with the carrier solution only. The Ethical Committee for animal experiments of the K.U. Leuven approved all experimental protocols. 2.2. Determination of T4 and T3 levels To determine TH availability in the developing brain and the uptake of THs injected in the yolk, we measured T3 and T4 in brain

Total RNA was isolated using TRIzol reagent (Invitrogen, Merelbeke, Belgium) and treated with DNase I (Invitrogen) to remove residual genomic DNA. At E4 the separate brain regions were pooled from 5 individual embryos, at E6 from 3 embryos and for the later stages tissues from individual embryos were used. In the ontogenetic study 6 biological replicates were analyzed for each brain region, in the hyperthyroid experiment (E4 and E8) 5 biological replicates were used. Four microgram of total RNA was reverse transcribed using SuperScript III Reverse Transcriptase and Oligo(dT)12–18 primers (Invitrogen). Four microliter of 1/10 diluted cDNA, corresponding to approximately 80 ng of total RNA, was used for real-time PCR. Primers were chosen using the Primer Express Software v.2.0 (Applied Biosystems, Foster City, CA) and are listed in Table 1. Real-time PCR was performed using an ABI PRISM 7000 Sequence Detection System thermal cycler (Applied Biosystems) in a total volume of 25 ll with 1 Platinum SYBR Green (Invitrogen) and 300 nM of each primer. According to the manufacturer’s instructions, the thermal cycle parameters included 2 min of initial denaturation at 50 °C and 95 °C, followed by 45 cycles of a two step reaction with a combined annealing and elongation step 30 s at 60 °C and a denaturation step 15 s at 95 °C. The amplification program was followed by a dissociation protocol to detect any non-specific amplification. Non-template and water controls were used to detect non-specific amplification. Each experimental and standard sample was analyzed in duplicate. Relative expression values were calculated based on the standard curve with the 7000 System Sequence Detection Software (version 1.2.3; Applied Biosystems) using a 1:5 dilution series of pooled cDNA as the standard. The expression data was normalised according to the method described by Vandesompele et al. (2002). The geNorm software for Microsoft ExcelÒ calculates a normalisation factor based on the geometric means of multiple internal control genes. For this, we used four reference genes: b-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M) and Cyclophilin A. After normalisation, all data were expressed relative to the mean mRNA expression which was assigned the arbitrary value of 1. No common reference sample was included in the ontogeny and hyperthyroid experiments, so direct

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S.L.J. Van Herck et al. / Molecular and Cellular Endocrinology 349 (2012) 289–297 Table 1 Primer sequences for the Q-PCR analyses. Name

Accession no mRNA

Forward sequence (50 ? 30 )

Reverse sequence (50 ? 30 )

Amplicon length

D2 D3 OATP1C1 MCT8 TRa TRb0 TRb2 b-Actin GAPDH B2M Cyclophilin A

NM_204114 NM_001122648 NM_001039097 XM_426274 NM_205313 X17504 X62642 NM_205518 NM_204305 NM_001001750 GQ849480

TGT TTC TGA GCC GCT CCA A CAG GAG GAG AAG GTG ATG TAC CA CAT GGG ACG ATA TCA GTA TGA AAG A CAA CTC CTT CGG GAT CAT CTA CA CTT GAT GGA ATT GCG GTG AAT GCT CCG CGC CCG TG CCC AGC TGC TGG TAG CAA TT ATG GCT CCG GTA TGT GCA A GAA GCT TAC TGG AAT GGC TTT CC GAA CGT CCT CAA CTG CTT CGT GGC TAC AAG GGC TCC TGC TT

ACA CTG GAG TTC GGA GCT TCT C TCT GGA GCC GGG TTT TGT ACT CGA GAG TGG AGT TTG GCT TTT CT AGC CAA CCC ATG CTG TTT TAA CAT TGG CTG CTC TTT CTT TTG C TGG GTA TAT ACC CTG ACA TAC TGC TG CCT GAG CAT CAC AAC TGC TGT A TGT CTT TCT GGC CCA TAC CAA GAT ATC ATC ATA CTT GGC TGG TTT CTC GGC ACG CCA TCC TTC ATC CCG TTG TGG CGC GTA AA

142 100 97 95 111 88 92 120 97 75 77

comparison of the relative values between these experiments is not possible. 2.4. Deiodinase enzyme assays In vitro deiodination activities were determined in homogenates of the different brain regions. Homogenates were prepared from tissue pools of 10 (E4), 6 (E6), 4 (E8) or 2 (E10 and E12) individuals per sample. Each sample was homogenized in 1 ml buffer solution (2 mM EDTA, 100 mM phosphate, and 1 mM DTT, pH 7.2). After centrifugation (1500g, 10 min, 4 °C), the supernatant was used for analysis. One fraction of the supernatant was used for the measurement of protein concentrations by the method of Bradford using dye reagent concentrate (Bio-Rad, Nazareth, Belgium) (Bradford, 1976). Deiodinase assays were performed as described in detail before (Reyns et al., 2005; Darras et al., 1992). For D1 activity the incubation mixture contained 1 mg of protein/ml and 1 lM of rT3 containing 50,000 cpm of radiolabelled [125I]-rT3. D2 activity was assayed in a similar way by incubating 1 mg of protein/ml and 1 nM T4 + 50,000 cpm [125I]-T4 at 37°C for 120 min. A second mixture was prepared with 100 nM T4 to assess possible interference of D1 activity. Only when iodide production was absent or minimal in the presence of 100 nM T4, enzyme activity using 1 nM T4 was considered to be true D2 activity. In the D3 activity test, the incubation mixture contained 0.25 mg of protein/ ml and 1 nM T3 + 150,000 cpm [125I]-T3 as well as 1 lM rT3 and 0.1 mM 6n-propylthiouracil to block possible D1 interference. The mixture was incubated for 120 min at 37°C. T3 to T2 conversion was determined using HPLC separation and an online radioactivity monitor as described before (Darras et al., 1992). Activities were calculated as femtomol of hormone deiodinated per mg protein and per minute. 2.5. Statistical methods

the trend was similar, with a decrease in hormone concentration from E4 to E6 and a gradual increase from E6 onwards. Next, we investigated TH levels in the brain following injection of 0.5 lg T3 + 1 lg T4 in the yolk 24 h before sampling. This resulted in significantly increased T4 levels at all tested time-points. T3 levels were significantly increased at stages E6–E12, but not at E4 (Table 2). To additionally check the TH status of the brain we analysed the expression of TH receptors (TR) TRa, TRb0 and TRb2 in the different brain regions at E4 and E8. This showed a prominent increase in TR expression in most brain regions at E4 and a mild response at E8 (Supplementary Fig. 1).

3.2. Ontogenetic expression profile of TH transporters and response to TH supplementation To determine if the early brain is capable of active regulation of TH uptake, we investigated the mRNA expression dynamics of two TH transporters OATP1C1 and MCT8 using Q-PCR. OATP1C1 mRNA expression in the mesencephalon and rhombencephalon was low during the investigated period. In the telencephalon and diencephalon OATP1C1 expression level was high at E4, and showed a significant decrease towards the later time points (p < 0.0001) (Fig. 2A). MCT8 mRNA expression was significantly higher in the diencephalon compared to the other brain regions at all time points (p < 0.05), and increased significantly towards E10 in all regions (p < 0.001) (Fig. 2B). Detailed analysis following TH supplementation showed that MCT8 and OATP1C1 responded differently to an increase in brain TH levels. At E4 OATP1C1 mRNA expression significantly decreased in the telencephalon (Fig. 3A), while MCT8 mRNA expression significantly increased in the telencephalon, diencephalon and rhombencephalon (Fig. 3C). At E8 no significant changes were observed in the expression of the transporters (Fig. 3B and D).

Statistical analysis was performed using the general linear models procedure in the SAS software (SAS Institute, NC). Data from the ontogeny experiments were analyzed by one-way ANOVA (followed by Scheffé’s post test) to identify statistical significant differences within each brain region over time and for differences between brain regions at a given developmental stage. For single comparisons of data from the hyperthyroid and control brain samples an unpaired T-test was used. p < 0.05 was used throughout as the criterion to accept a statistical significant difference. 3. Results 3.1. Thyroid hormone levels and induction of hyperthyroidism To confirm the availability of THs in the early brain, we first investigated the normal T4 and T3 profile (Fig. 1). For both of them

Fig. 1. Ontogenetic profile of TH levels in the brain during the first half of embryonic development. Concentrations are expressed as pmol T4 or T3 per gram of tissue. Data are presented as mean ± SEM (n = 6).

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Table 2 T3 and T4 levels in brain samples 24 h after injection of 0.5 lg T3 + 1 lg T4 in the yolk. Moment of sampling

Treatment

T4 (pmol/g)

E4

Control TH-injected Control TH-injected Control TH-injected Control TH-injected Control TH-injected

0.95 ± 0.16 2.79 ± 0.20⁄⁄⁄ 0.31 ± 0.06 11.2 ± 3.35⁄ 0.53 ± 0.08 5.56 ± 1.04⁄⁄⁄ 0.79 ± 0.08 6.25 ± 0.77⁄⁄⁄ 0.92 ± 0.12 3.80 ± 0.73⁄⁄

E6 E8 E10 E12

Fold change 2.9 36.5 10.5 7.9 4.1

T3 (pmol/g)

Fold change

0.33 ± 0.04 0.35 ± 0.03 0.14 ± 0.03 1.12 ± 0.27⁄⁄ 0.16 ± 0.01 1.42 ± 0.28⁄⁄ 0.21 ± 0.01 1.87 ± 0.37⁄⁄ 0.41 ± 0.03 1.37 ± 0.11⁄⁄⁄

1.1 8.1 9.0 9.0 3.4

Concentrations are expressed as pmol T3 or T4 per gram of tissue. Data are presented as means ± SEM (n = 6); fold changes were calculated by dividing the means of the TH-injected group by the means of the control group. (Unpaired T-test: ⁄p < 0.05; ⁄⁄p < 0.01; ⁄⁄⁄p < 0.001).

Fig. 2. mRNA expression of OATP1C1 and MCT8 in the different regions of developing chicken brain from E4 to E12. The relative mRNA quantity is expressed as mean ± SEM (n = 6), data are normalized based on the mean expression of four internal control genes (b-actin, GAPDH, B2M and Cyclophilin A).

Fig. 3. Effect of 0.5 lg T3 + 1 lg T4 injection on OATP1C1 and MCT8 mRNA expression in the different brain regions at E4 and E8. The relative mRNA quantity is expressed as mean ± SEM (n = 5), data are normalized based on the mean expression of three internal control genes (b-actin, GAPDH and Cyclophilin A). Brain regions: telen, telencephalon; dienc, diencephalon; mesen, mesencephalon; rhomb, rhombencephalon. (Unpaired T-test comparing control and TH-injected samples in each brain area: ⁄ p < 0.05; ⁄⁄p < 0.01).

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3.3. Ontogenetic expression profile of the deiodinases and response to TH supplementation Following transmembrane TH transport, T3 availability is further regulated by the deiodinases. We therefore analyzed mRNA expression as well as activity of D1, D2, and D3 in the different brain regions. Q-PCR analysis showed the presence of mRNA for the three types of deiodinases at all stages. D2 mRNA expression levels were quite similar in all brain regions and significantly increased over time until E10 (p < 0.0001), after which expression significantly increased further only in the rhombencephalon (Fig. 4B). D2 enzymatic activity followed the same pattern, but kept increasing in all brain areas after E10 (p < 0.0001) (Fig. 4C). D3 mRNA expression decreased as development progressed. This decrease was significant in the telencephalon between E4 and E10–E12 (p < 0.01) (Fig. 4D). D3 activity levels showed significant variation in all brain regions (p < 0.01) and, contrary to the mRNA data, were lowest in the telencephalon and highest in the mesencephalon (Fig. 4E). D1 mRNA was detectable throughout the period studied (Fig. 4A), but did not seem to be translated into active protein as we were not able to detect D1 activity at any stage. Since deiodinases show a compensatory response to hyperthyroidism in late embryonic and posthatch brain, we also analyzed the effect of TH injection on D1, D2, and D3 mRNA expression

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and activity at E4 and E8. Statistical analysis showed significantly increased D2 mRNA levels in the diencephalon and rhombencephalon at E4 (Fig. 5C), while no significant changes were detected at E8 (Fig. 5D). D2 enzymatic activity in contrast, was little affected at E4 (Fig. 6A) but was significantly decreased in the diencephalon and mesencephalon at E8 (Fig. 6B). For D3 we detected a significant increase in mRNA expression in the telencephalon at E4 (Fig. 5E), and a significant decrease in the diencephalon at E8 (Fig. 5F). No changes in D3 enzyme activity occurred following TH supplementation at E4 (Fig. 6C) while D3 activity was significantly increased in the telencephalon at E8 (Fig. 6D).

4. Discussion Thyroid hormones play a crucial role in vertebrate brain development by controlling the expression of TH-regulated genes through binding of T3 to the nuclear TRs. It has been shown that TRa transcripts are present in chicken embryos even before the start of incubation (Flamant and Samarut, 1998), while the distribution of all three known TR isoforms from day 4 of development onwards has been described in detail (Forrest et al., 1990, 1991; Darras et al., 2011). The current view of TH action in the brain is that T4 is activated in glial cells to T3 which is subsequently

Fig. 4. mRNA expression and activity levels of D1, D2, and D3 in the different regions of developing chicken brain from E4 to E12. The relative mRNA quantity is expressed as means ± SEM (n = 6), data are normalized based on the mean expression of four internal control genes (b-actin, GAPDH, B2 M and Cyclophilin A). Deiodinase activity is expressed as femtomoles substrate deiodinated per milligram protein per minute and data are presented as mean ± SEM (E4: n = 3, E6: n = 4, E8: n = 4, E10: n = 5, E12: n = 6).

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Fig. 5. Effect of 0.5 lg T3 + 1 lg T4 injection on D1, D2, and D3 mRNA expression levels in the different brain regions at E4 and E8. The relative mRNA quantity is expressed as mean ± SEM (n = 5), data are normalized based on the mean expression of three internal control genes (b-actin, GAPDH and Cyclophilin A). Brain regions: telen, telencephalon; dienc, diencephalon; mesen, mesencephalon; rhomb, rhombencephalon. (Unpaired T-test comparing control and TH-injected samples in each brain area: ⁄ p < 0.05).

transported to neurons (Alkemade et al., 2011). Deiodinases and TH transporters therefore locally regulate intracellular TH availability and it is important to investigate their presence and distribution in developing brain. In this study we showed that THs are present in the chicken brain at day 4 of embryonic development and that the TH transporters and deiodinases are expressed in a region specific way, well before the onset of embryonic TH function. This result is consistent with mammalian data showing the importance of maternal THs for normal brain development (Morreale de Escobar et al., 2004a). We further showed that the TH transporters and deiodinases respond to increased TH levels and that this response changes according to the developmental stage. Several types of TH transporters have been described in mammals (Visser et al., 2008), but to date little is known about the different avian homologues. The only functionally characterised TH transporter is the T4-specific transporter OATP1C1 in the chicken (Nakao et al., 2006). We investigated the ontogenetic expression pattern of OATP1C1 in chicken brain and also included MCT8, encoded by the chicken slc16a2 gene because in humans MCT8 deficiency is linked to a severe syndrome of psychomotor retardation

(Friesema et al., 2004; Dumitrescu et al., 2004). Our experiments showed that OATP1C1 expression was high in the telencephalon and diencephalon at E4 but strongly decreased thereafter. In contrast, MCT8 mRNA levels showed a gradual increase in all brain regions up to E10, which is around mid incubation. This pattern is different from the one recently described in human foetal cortex during the first half of gestation, where MCT8 expression remained constant while OATP1C1 expression increased towards mid gestation (Chan et al., 2011). OATP1C1 is considered to be very important for the uptake of T4 into the brain and T4 is indeed the major TH available in the yolk. The early differentiation of brain progresses in an anterior–posterior direction, and major changes take place in the forebrain in the early stages of pregnancy/incubation (Morreale de Escobar et al., 2004a; Puelles, 2001). High expression of OATP1C1 in the E4–E6 chicken telencephalon and diencephalon suggests that these regions can take up more T4 specifically at these early stages. However, D3 activity levels are high and neither telencephalon nor diencephalon contains high D2 activity at E4, so it remains unclear whether this can lead to an increase in receptor-active T3.

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Fig. 6. Effect of 0.5 lg T3 + 1 lg T4 injection on D2 and D3 activity levels in the different brain regions at E4 and E8. Deiodinase activity is expressed as femtomoles substrate deiodinated per milligram protein per minute and data are presented as mean ± SEM (E4: n = 3, E8: n = 6). Brain regions: telen, telencephalon; dienc, diencephalon; mesen, mesencephalon; rhomb, rhombencephalon. (Unpaired T-test comparing control and TH-injected samples in each brain area: ⁄p < 0.05; ⁄⁄p < 0.01).

Our experiments showed the presence of the mRNAs for all three types of deiodinases throughout the investigated period. Interestingly, the mRNA expression changes for D1 and D3 were often not reflected in the protein activity levels, suggesting considerable posttranscriptional regulation. During the late stages of chicken embryonic development, D2 is the predominant enzyme producing T3 in the brain as very little or no D1 activity has been detected (Van der Geyten et al., 2002). The present results show that this is also the case for early brain development since no D1 activity could be detected, despite the presence of D1 mRNA. Similar observations regarding D1 expression in the developing brain have also been reported in humans where Chan et al. described D1 mRNA expression, but no D1 activity in the foetal cerebral cortex (Chan et al., 2002). We found a gradual increase in D2 mRNA and enzyme activity in all brain regions from E4 to E12. An increase in D2 mRNA in the brain has previously been described between E7 and E10 (Gereben et al., 2004). During the last week of embryonic development both D2 mRNA and enzyme activity were found to increase in diencephalon, optic lobes, cerebellum and brain stem (Van der Geyten et al., 2002). Taken together these data show that the need for local T3 production increases continuously throughout embryonic brain development. In contrast to D2, D3 mRNA and activity levels showed a more irregular pattern, and enzyme activity did not decrease concomitantly with mRNA expression. Across the different experiments, the D3 enzyme activity levels in the different brain regions seemed to be more consistent than the relative mRNA expression levels, suggesting strong posttranslational regulation. The D3 activity levels were high compared to levels previously found in the same brain regions during the last week of embryonic development, which were between 0.2 and 2 fmol T3 deiodinated/mg protein/min (Reyns et al., 2005; Van der Geyten et al., 2002) compared to 3–30 fmol T3 deiodinated/mg protein/ min in the present study. The high D3 activity levels during early development may serve to protect several areas in the brain from premature T3 signalling as was shown for instance in D3 null mice, where cochlear development is impaired due to premature TR activation by T3 (Ng et al., 2009). Measurement of THs in brain extracts showed that both T3 and T4 concentrations decreased between E4 and E6 and then increased

again. Since the first colloid droplets in the thyroid gland are only observed at E7 (Bellairs and Osmond, 2005) and hormone secretion starts even later, this increase points towards increasing uptake of THs from the yolk. In chickens more than 95% of the maternal THs are stored in the yolk (McNabb and Wilson, 1997), and injection of additional THs in the yolk strongly increased TH levels in the brain. To independently check the hyperthyroid status at the level of gene expression we determined the mRNA expression levels of the different TRs. TRs have been shown to respond to changing TH levels in the posthatch chicken brain (Gereben et al., 1998), and increased TR expression after T3 supplementation has been described in vitro, in N-Tera-2 cells (Chan et al., 2003). Our analysis of TR mRNA expression after TH supplementation showed increased TRa, TRb0 and TRb2 expression in several brain areas at E4 and E8. This confirmed the hyperthyroid status in the brain and showed that a change in TH status affected gene expression already at E4. More detailed analysis of the TH levels after TH supplementation showed that at E4 only T4 increased, and this difference was specific for brain since T3 levels in the rest of the body were significantly increased at E4 (data not shown). The lack of T3 increase in brain could be due to a combination of factors. At this stage, the expression of MCT8 was still relatively low, limiting the capacity for T3 uptake while the expression of OATP1C1, transporting mainly T4, was higher than at later stages. In addition, local T4 to T3 conversion by D2 was still low at E4 and it is generally accepted that this is the main source of T3 in the brain (Calvo et al., 1990). We also noticed that at least until E12, the concentration of T3 in brain was lower than the concentration of T4. This is clearly different from the situation after E16, where there is a clear predominance of T3 over T4 (Reyns et al., 2003). This shift corresponds with the increase in D2 activity and again points towards the importance of D2 in the regulation of T3 levels in developing brain. During adult life in mammals, an increase in TH availability typically elicits a strong compensatory response from the deiodinases (Gereben et al., 2008). Such a link has also been described in posthatch chicken brain. Thyroidectomy increased D2 activity and decreased D3 activity and these changes were counteracted by T4 supplementation (Rudas et al., 1993). This compensatory mechanism was not yet functional at E4, since neither D2 nor D3 activity

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in brain changed following TH injection. At E8, some regions in the brain did react with a decrease in D2 and an increase in D3 activity, suggesting that the potential for homeostatic regulation starts to mature around this period. We realise that the fact that the increase in TH levels was higher at E8 compared to E4 may be a confounding factor in the interpretation of the deiodinase activity results. However, the more prominent changes in mRNA expression of deiodinases as well as TH transporters in E4 compared to E8 (discussed below) indicate that even the E4 brain was definitely hyperthyroid. We found increases in D1, D2, and D3 mRNA expression at E4. These changes clearly do not agree with a compensatory response. Our results further show a decrease in OATP1C1 expression and an increase in MCT8 expression at E4 following TH supplementation. It is difficult to conclude whether or not these changes can be interpreted as a compensatory response, since TH transporters can stimulate cellular TH influx but also efflux (Visser et al., 2008; Sugiyama et al., 2003). A decrease in OATP1C1 could lead to a relative decrease in uptake of T4 into the brain while an increase in MCT8 could help to increase the efflux of excess TH from brain cells. An alternative explanation would be that the observed changes are the result of an acceleration of the normal ontogenetic pattern, as could also be the case for the increase in D2. Since THs are known to stimulate brain development, it is difficult to discriminate between a direct effect of THs on a specific gene and an indirect effect due to a general acceleration of brain development. In conclusion, we have shown that T3, T4 as well as TH transporters and deiodinases are present and active in the embryonic chicken brain at least from day 4 of development, long before the start of embryonic thyroidal hormone secretion. Detailed analysis of OATP1C1, MCT8, D2 and D3 mRNA expression demonstrated that expression levels and ontogenetic profiles may diverge widely among brain regions, possibly reflecting regional differences in the timing of differentiation. The expression of TH transporters and deiodinases changes following TH supplementation but this response is stage- and region specific. While some regulation may occur in E4 embryos at the level of TH transport, compensatory mechanisms at the level of deiodinase activity only occur at E8. Therefore the early embryonic brain may be particularly vulnerable to changes in maternal TH supply.

Disclosure statement The authors have nothing to disclose.

Role of funding source The Research Council of the Katholieke Universiteit Leuven and the Fund for Scientific Research-Flanders are organisations funding scientific research based on grant applications for a specific research topic for a period of 4 years. They do not interfere with how the studies are set up or where and when the results are submitted for publication.

Acknowledgements We gratefully acknowledge the technical assistance of W. Van Ham, L. Noterdaeme and I. Proven in the experiments. This work was supported by the Research Council of the Katholieke Universiteit Leuven (Grant OT/07/036) and by the Fund for Scientific Research-Flanders (Grant G.0455.08).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2011.11.012.

References Ahmed, O.M., El-Gareib, A.W., El-Bakry, A.M., Abd El-Tawab, S.M., Ahmed, R.G., 2008. Thyroid hormones states and brain development interactions. Int. J. Dev. Neurosci. 26, 147–209. Alkemade, A., Friesema, E.C., Kalsbeek, A., Swaab, D.F., Visser, T.J., Fliers, E., 2011. Expression of thyroid hormone transporters in the human hypothalamus. J. Clin. Endocrinol. Metab. 96, E967–E971. Anderson, G.W., 2001. Thyroid hormones and the brain. Front. Neuroendocrinol. 22, 1–17. Babu, S., Sinha, R.A., Mohan, V., Rao, G., Pal, A., Pathak, A., Singh, M., Godbole, M.M., 2011. Effect of hypothyroxinemia on thyroid hormone responsiveness and action during rat postnatal neocortical development. Exp. Neurol. 228, 91–98. Bellairs, R., Osmond, M., 2005. The Atlas of Chick Development. Elsevier, Amsterdam, Boston, pp. xxiv, 470 p. Bernal, J., 2005. Thyroid hormones and brain development. Vitam. Horm. 71, 95– 122. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burmeister, L.A., Pachucki, J., St Germain, D.L., 1997. Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138, 5231–5237. Calvo, R., Obregon, M.J., Ruiz de Ona, C., Escobar del Rey, F., Morreale de Escobar, G., 1990. Congenital hypothyroidism, as studied in rats. Crucial role of maternal thyroxine but not of 3,5,30 -triiodothyronine in the protection of the fetal brain. J. Clin. Invest. 86, 889–899. Chan, S., Kachilele, S., McCabe, C.J., Tannahill, L.A., Boelaert, K., Gittoes, N.J., Visser, T.J., Franklyn, J.A., Kilby, M.D., 2002. Early expression of thyroid hormone deiodinases and receptors in human fetal cerebral cortex. Brain Res. Dev. Brain Res. 138, 109–116. Chan, S., McCabe, C.J., Visser, T.J., Franklyn, J.A., Kilby, M.D., 2003. Thyroid hormone responsiveness in N-Tera-2 cells. J. Endocrinol. 178, 159–167. Chan, S.Y., Martin-Santos, A., Loubiere, L.S., Gonzalez, A.M., Stieger, B., Logan, A., McCabe, C.J., Franklyn, J.A., Kilby, M.D., 2011. The expression of thyroid hormone transporters in the human fetal cerebral cortex during early development and in N-Tera-2 neurodifferentiation. J. Physiol. 589, 2827–2845. Darras, V.M., Visser, T.J., Berghman, L.R., Kuhn, E.R., 1992. Ontogeny of type I and type III deiodinase activities in embryonic and posthatch chicks: relationship with changes in plasma triiodothyronine and growth hormone levels. Comp. Biochem. Physiol. Comp. Physiol. 103, 131–136. Darras, V.M., Van Herck, S.L., Heijlen, M., De Groef, B., 2011. Thyroid hormone receptors in two model species for vertebrate embryonic development: chicken and zebrafish. J. Thyroid Res. 2011, 402320. Dumitrescu, A.M., Liao, X.H., Best, T.B., Brockmann, K., Refetoff, S., 2004. A novel syndrome combining thyroid and neurological abnormalities is associated with mutations in a monocarboxylate transporter gene. Am. J. Hum. Genet. 74, 168– 175. Evans, I.M., Pickard, M.R., Sinha, A.K., Leonard, A.J., Sampson, D.C., Ekins, R.P., 2002. Influence of maternal hyperthyroidism in the rat on the expression of neuronal and astrocytic cytoskeletal proteins in fetal brain. J. Endocrinol. 175, 597–604. Flamant, F., Samarut, J., 1998. Involvement of thyroid hormone and its alpha receptor in avian neurulation. Dev. Biol. 197, 1–11. Forrest, D., Sjoberg, M., Vennstrom, B., 1990. Contrasting developmental and tissuespecific expression of alpha and beta thyroid hormone receptor genes. EMBO J. 9, 1519–1528. Forrest, D., Hallbook, F., Persson, H., Vennstrom, B., 1991. Distinct functions for thyroid hormone receptors alpha and beta in brain development indicated by differential expression of receptor genes. EMBO J. 10, 269–275. Friesema, E.C., Grueters, A., Biebermann, H., Krude, H., von Moers, A., Reeser, M., Barrett, T.G., Mancilla, E.E., Svensson, J., Kester, M.H., Kuiper, G.G., Balkassmi, S., Uitterlinden, A.G., Koehrle, J., Rodien, P., Halestrap, A.P., Visser, T.J., 2004. Association between mutations in a thyroid hormone transporter and severe Xlinked psychomotor retardation. Lancet 364, 1435–1437. Gereben, B., Kollar, A., Bartha, T., Buys, N., Decuypere, E., Rudas, P., 1998. 3,30 ,5Triiodothyronine (T3) uptake and expression of thyroid hormone receptors during the adaptation to hypothyroidism of the brain of chicken. Acta Vet. Hung. 46, 473–485. Gereben, B., Pachucki, J., Kollar, A., Liposits, Z., Fekete, C., 2004. Ontogenic redistribution of type 2 deiodinase messenger ribonucleic acid in the brain of chicken. Endocrinology 145, 3619–3625. Gereben, B., Zeold, A., Dentice, M., Salvatore, D., Bianco, A.C., 2008. Activation and inactivation of thyroid hormone by deiodinases: local action with general consequences. Cell. Mol. Life Sci. 65, 570–590. Haddow, J.E., Palomaki, G.E., Allan, W.C., Williams, J.R., Knight, G.J., Gagnon, J., O’Heir, C.E., Mitchell, M.L., Hermos, R.J., Waisbren, S.E., Faix, J.D., Klein, R.Z., 1999. Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N. Engl. J. Med. 341, 549–555.

S.L.J. Van Herck et al. / Molecular and Cellular Endocrinology 349 (2012) 289–297 Harvey, C.B., Williams, G.R., 2002. Mechanism of thyroid hormone action. Thyroid 12, 441–446. Horn, S., Heuer, H., 2010. Thyroid hormone action during brain development: more questions than answers. Mol. Cell. Endocrinol. 315, 19–26. Lavado-Autric, R., Auso, E., Garcia-Velasco, J.V., Arufe Mdel, C., Escobar del Rey, F., Berbel, P., Morreale de Escobar, G., 2003. Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. J. Clin. Invest. 111, 1073–1082. McNabb, F.M.A., Wilson, C.M., 1997. Thyroid hormone deposition in avian eggs and effects on embryonic development. Am. Zoologist. 37, 553–560. Mebis, L., Debaveye, Y., Ellger, B., Derde, S., Ververs, E.J., Langouche, L., Darras, V.M., Fliers, E., Visser, T.J., Van den Berghe, G., 2009. Changes in the central component of the hypothalamus–pituitary–thyroid axis in a rabbit model of prolonged critical illness. Crit. Care 13, R147. Morreale de Escobar, G., Obregon, M.J., Escobar del Rey, F., 2004a. Maternal thyroid hormones early in pregnancy and fetal brain development. Best Pract. Res. Clin. Endocrinol. Metab. 18, 225–248. Morreale de Escobar, G., Obregon, M.J., Escobar del Rey, F., 2004b. Role of thyroid hormone during early brain development. Eur. J. Endocrinol. 151 (Suppl. 3), U25–U37. Nakao, N., Takagi, T., Iigo, M., Tsukamoto, T., Yasuo, S., Masuda, T., Yanagisawa, T., Ebihara, S., Yoshimura, T., 2006. Possible involvement of organic anion transporting polypeptide 1C1 in the photoperiodic response of gonads in birds. Endocrinology 147, 1067–1073. Ng, L., Hernandez, A., He, W., Ren, T., Srinivas, M., Ma, M., Galton, V.A., St Germain, D.L., Forrest, D., 2009. A protective role for type 3 deiodinase, a thyroid hormone-inactivating enzyme, in cochlear development and auditory function. Endocrinology 150, 1952–1960. Pop, V.J., Kuijpens, J.L., van Baar, A.L., Verkerk, G., van Son, M.M., de Vijlder, J.J., Vulsma, T., Wiersinga, W.M., Drexhage, H.A., Vader, H.L., 1999. Low maternal free thyroxine concentrations during early pregnancy are associated with impaired psychomotor development in infancy. Clin. Endocrinol. (Oxf) 50, 149– 155. Prati, M., Calvo, R., Morreale, G., Morreale de Escobar, G., 1992. L-Thyroxine and 3,5,30 -triiodothyronine concentrations in the chicken egg and in the embryo before and after the onset of thyroid function. Endocrinology 130, 2651–2659. Puelles, L., 2001. Brain segmentation and forebrain development in amniotes. Brain Res. Bull. 55, 695–710.

297

Reyns, G.E., Janssens, K.A., Buyse, J., Kühn, E.R., Darras, V.M., 2002. Changes in thyroid hormone levels in chicken liver during fasting and refeeding. Comp. Biochem. Physiol. 132B, 239–345. Reyns, G.E., Venken, K., Morreale de Escobar, G., Kuhn, E.R., Darras, V.M., 2003. Dynamics and regulation of intracellular thyroid hormone concentrations in embryonic chicken liver, kidney, brain, and blood. Gen. Comp. Endocrinol. 134, 80–87. Reyns, G.E., Verhoelst, C.H., Kuhn, E.R., Darras, V.M., Van der Geyten, S., 2005. Regulation of thyroid hormone availability in liver and brain by glucocorticoids. Gen. Comp. Endocrinol. 140, 101–108. Rudas, P., Bartha, T., Frenyo, L.V., 1993. Thyroid hormone deiodination in the brain of young chickens acutely adapts to changes in thyroid status. Acta Vet. Hung. 41, 381–393. Sugiyama, D., Kusuhara, H., Taniguchi, H., Ishikawa, S., Nozaki, Y., Aburatani, H., Sugiyama, Y., 2003. Functional characterization of rat brain-specific organic anion transporter (Oatp14) at the blood–brain barrier: high affinity transporter for thyroxine. J. Biol. Chem. 278, 43489–43495. Thommes, R.C., 1987. Ontogenesis of thyroid function and regulation in the developing chick embryo. J. Exp. Zool. Suppl. 1, 273–279. Tu, H.M., Kim, S.W., Salvatore, D., Bartha, T., Legradi, G., Larsen, P.R., Lechan, R.M., 1997. Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone. Endocrinology 138, 3359–3368. Tu, H.M., Legradi, G., Bartha, T., Salvatore, D., Lechan, R.M., Larsen, P.R., 1999. Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone. Endocrinology 140, 784–790. Van der Geyten, S., Van den Eynde, I., Segers, I.B., Kuhn, E.R., Darras, V.M., 2002. Differential expression of iodothyronine deiodinases in chicken tissues during the last week of embryonic development. Gen. Comp. Endocrinol. 128, 65–73. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034. Visser, W.E., Friesema, E.C., Jansen, J., Visser, T.J., 2008. Thyroid hormone transport in and out of cells. Trends Endocrinol. Metab. 19, 50–56.