Thyroid hormones are key players in regulating brain development. Thus, transfer of appropriate quantities of thyroid hormones from the blood into the brain at specific stages of development is critical. The choroid plexus forms the blood-cerebrospinal fluid barrier. In reptiles, birds and mammals, the main protein synthesized and secreted by the choroid plexus is a thyroid hormone distributor protein: transthyretin. This transthyretin is secreted into the cerebrospinal fluid and moves thyroid hormones from the blood into the cerebrospinal fluid. Maximal transthyretin synthesis in the choroid plexus occurs just prior to the period of rapid brain growth, suggesting that choroid plexus-derived transthyretin moves thyroid hormones from blood into cerebrospinal fluid just prior to when thyroid hormones are required for rapid brain growth. The structure of transthyretin has been highly conserved, implying strong selection pressure and an important function. In mammals, transthyretin binds T4 (precursor form of thyroid hormone) with higher affinity than T3 (active form of thyroid hormone). In all other vertebrates, transthyretin binds T3 with higher affinity than T4. As mammals are the exception, we should not base our thinking about the role of transthyretin in the choroid plexus solely on mammalian data. Thyroid hormone transmembrane transporters are involved in moving thyroid hormones into and out of cells and have been identified in many tissues, including the choroid plexus. Thyroid hormones enter the choroid plexus via thyroid hormone transmembrane transporters and leave the choroid plexus to enter the cerebrospinal fluid via either thyroid hormone transmembrane transporters or via choroid plexus-derived transthyretin secreted into the cerebrospinal fluid. The quantitative contribution of each route during development remains to be elucidated. This is part of a review series on ontogeny and phylogeny of brain barrier mechanisms.

Iodothyronine deiodinases are important mediators of thyroid hormone (TH) action. They are present in tissues throughout the body where they catalyse 3,5,3 0 -triiodothyronine (T 3 ) production and degradation via, respectively, outer and inner ring deiodination. Three different types of iodothyronine deiodinases (D1, D2 and D3) have been identified in vertebrates from fish to mammals. They share several common characteristics, including a selenocysteine residue in their catalytic centre, but show also some type-specific differences. These specific characteristics seem very well conserved for D2 and D3, while D1 shows more evolutionary diversity related to its Km, 6-n-propyl-2-thiouracil sensitivity and dependence on dithiothreitol as a cofactor in vitro. The three deiodinase types have an impact on systemic T 3 levels and they all contribute directly or indirectly to intracellular T 3 availability in different tissues. The relative contribution of each of them, however, varies amongst species, developmental stages and tissues. This is especially true for amphibians, where the impact of D1 may be minimal. D2 and D3 expression and activity respond to thyroid status in an opposite and conserved way, while the response of D1 is variable, especially in fish. Recently, a number of deiodinases have been cloned from lower chordates. Both urochordates and cephalochordates possess selenodeiodinases, although they cannot be classified in one of the three vertebrate types. In addition, the cephalochordate amphioxus also expresses a non-selenodeiodinase. Finally, deiodinase-like sequences have been identified in the genome of non-deuterostome organisms, suggesting that deiodination of externally derived THs may even be functionally relevant in a wide variety of invertebrates.

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Exposure to appropriate levels of thyroid hormones (THs) at the right time is of key importance for normal development in all vertebrates. Type 3 iodothyronine deiodinase (D3) is the prime TH-inactivating enzyme, and its expression is highest in the early stages of vertebrate development, implying that it may be necessary to shield developing tissues from overexposure to THs. We used antisense morpholino knockdown to examine the role of D3 during early development in zebrafish. Zebrafish possess 2 D3 genes, dio3a and dio3b. Here, we show that both genes are expressed during development and both contribute to in vivo D3 activity. However, dio3b mRNA levels in embryos are higher, and the effects of dio3b knockdown on D3 activity and on the resulting phenotype are more severe. D3 knockdown induced an overall delay in development, as determined by measurements of otic vesicle length, eye and ear size, and body length. The time of hatching was also severely delayed in D3-knockdown embryos. Importantly, we also observed a severe disturbance of several aspects of development. Swim bladder development and inflation was aberrant as was the development of liver and intestine. Furthermore, D3-knockdown larvae spent significantly less time moving, and both embryos and larvae exhibited perturbed escape responses, suggesting that D3 knockdown affects muscle development and/or functioning. These data indicate that D3 is essential for normal zebrafish embryonic and early larval development and show the value of morpholino knockdown in this model to further elucidate the specific role of D3 in some aspects of vertebrate development.

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Methimazole (MMI) is an anti-thyroid drug used in the treatment of chronic hyperthyroidism. There is, however, some debate about its use during pregnancy as MMI is known to cross the mammalian placenta and reach the developing foetus. A similar problem occurs in birds, where MMI is deposited in the egg and taken up by the developing embryo. To investigate whether maternally derived MMI can have detrimental effects on embryonic development, we treated laying hens with MMI (0.03% in drinking water) and measured total and reduced MMI contents in the tissues of hens and embryos at different stages of development. In hens, MMI was selectively increased in the thyroid gland, while its levels in the liver and especially brain remained relatively low. Long-term MMI treatment induced a pronounced goitre with a decrease in thyroxine (T 4 ) content but an increase in thyroidal 3,5,3 0 -triiodothyronine (T 3 ) content. This resulted in normal T 3 levels in tissues except in the brain. In chicken embryos, MMI levels were similar in the liver and brain. They gradually decreased during development but always remained above those in the corresponding maternal tissues. Contrary to the situation in hens, T 4 availability was only moderately affected in embryos. Peripheral T 3 levels were reduced in 14-day-old embryos but normal in 18-day-old embryos, while brain T 3 content was decreased at all embryonic stages tested. We conclude that all embryonic tissues are exposed to relatively high doses of MMI and its oxidised metabolites. The effect of maternal MMI treatment on embryonic thyroid hormone availability is most pronounced for brain T 3 content, which is reduced throughout the embryonic development period.

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Chicken and zebrafish are two model species regularly used to study the role of thyroid hormones in vertebrate development. Similar to mammals, chickens have one thyroid hormone receptor α (TRα) and one TRβ gene, giving rise to three TR isoforms: TRα, TRβ2, and TRβ0, the latter with a very short amino-terminal domain. Zebrafish also have one TRβ gene, providing two TRβ1 variants. The zebrafish TRα gene has been duplicated, and at least three TRα isoforms are expressed: TRαA1-2 and TRαB are very similar, while TRαA1 has a longer carboxy-terminal ligand-binding domain. All these TR isoforms appear to be functional, ligand-binding receptors. As in other vertebrates, the different chicken and zebrafish TR isoforms have a divergent spatiotemporal expression pattern, suggesting that they also have distinct functions. Several isoforms are expressed from the very first stages of embryonic development and early chicken and zebrafish embryos respond to thyroid hormone treatment with changes in gene expression. Future studies in knockdown and mutant animals should allow us to link the different TR isoforms to specific processes in embryonic development.