Ascidian homologs of mammalian thyroid peroxidase genes are expressed in the thyroid-equivalent region of the endostyle.

The endostyle is a pharyngeal organ for the internal filter feeding of urochordates, cephalochordates, and larval lamprey. This organ is also considered to be homologous to the follicular thyroid gland of higher vertebrates. Thyroglobulin (Tg) and thyroid peroxidase (TPO) are specifically expressed in the thyroid gland of higher vertebrates, and they play an important role in iodine metabolism for the synthesis of thyroid hormones. Previous histochemical observations showed that iodine-concentrating and peroxidase activities were detected in zones 7, 8, and 9 of the ascidian endostyle, suggesting that these zones contains cells that are equivalent to those in the vertebrate follicular thyroid. In order to investigate the molecular developmental mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the thyroid gland, in the present study, we isolated and characterized cDNA clones for TPO genes, CiTPO from Ciona intestinalis and HrTPO from Halocynthia roretzi. Northern blot and in situ hybridization analyses revealed that the expression of the ascidian TPO genes was restricted to zone 7, one of the elements equivalent to the thyroid. These results provide the first evidence at the gene expression level for shared function between a part of the ascidian endostyle and the vertebrate follicular thyroid gland. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:158-169, 1999.     

Distribution of immunoreactive thyroxine in the endostyle and thyroid tissue of the sea lamprey Petromyzon marinus L., 1758

Anti-thyroxine (T₄) immunostaining in the endostyle and thyroid tissue during metamorphosis of sea lamprey Petromyzon marinus suggested that T₄ synthesis occurs on the luminal surface of the apical membrane of selected cells of the endostyle, and that a 'classical' endocrine thyroid function was only evident in post-transformed animals.     

Transformation of the endostyle of the anadromous sea lamprey,Petromyzon marinus L., during metamorphosis. II. Electron microscopy

Electron microscopy was used to follow the transformation of the endostyle to a thyroid gland in the anadromous sea lamprey, Petromyzon marinus L., throughout metamorphosis (stages 1–7). Transformation of the larval (ammocoete) endostyle begins at the first signs of external change (stages 1–2), and the adult form of the gland is reached by stage 5. Only slight modifications of the gland accompany further development to the end of metamorphosis. Development of the thyroid gland involves degeneration, proliferation, and reorganization of the cells in the endostyle, and changes in their fine structure. Ultrastructural changes during early stages are most obvious in the type 1 cells that make up the shrinking glandular tracts, and involves the accumulation of cytoplasmic microfilaments and a variety of cytoplasmic inclusions. The glandular tracts and their cells gradually disappear through autolysis and, apparently, through phagocytosis by neighboring epithelial cells and macrophages. Although the fine structure of the type 2, 3, 4, and 5 cells is not altered in the early stages, by stage 3, many of these cells become either vacuolated, undergo autolysis, or are extruded. Phagocytosis of some of each of these cell types likely occurs. Thyroid follicles are first observed during stage 4. Some of their lumina seem to arise from the accumulation of material in intercellular spaces and from vacuoles among cell clusters. Other lumina may represent a portion of the original lumen of the endostyle. Many follicles appear to be comprised of cells with cytological characteristics similar to those of larval cell types 3 and 2c. Some of the other larval cell types, such as type 5, may also be involved. In young adult lampreys follicles are composed of cuboidal to columnar cells that lack the dilated cisternae of rough endoplasmic reticulum seen in follicular cells of higher vertebrates. Dense collagenous connective tissue surrounding the follicles contains relatively few blood vessels. The transformation process described may have some relevance to our understanding of the development and evolution of the vertebrate thyroid gland.     

Theory on the evolutionary history of lamprey metamorphosis: role of reproductive and thyroid axes

Metamorphosis is a developmental strategy used by only a small number of extant fishes and little is known about its phylogenetic development during the evolution history of this large group of vertebrates. The present report provides a putative evolutionary history of metamorphosis in the lamprey, an extant agnathan with direct descendancy from some of the oldest known vertebrates. The study reviews recent data on the role of the thyroid gland and its hormones in metamorphosis, summarizes some recent views on the evolution of the endostyle/follicular thyroid in lampreys, and provides new data on the content of two gonadotropin-releasing hormones (GnRH-I and -III) in brain during goitrogen-stimulated, precocious metamorphosis. These new data support an earlier viewpoint of a relationship between thyroid and reproductive axes during metamorphosis. It is proposed that the earliest lampreys were paedomorphic larvae and they lived in a marine environment; as such, they resembled in many ways the larvae from which the ancient protochordates, Larvacea, are derived. The iodide-concentrating efficiency of the endostyle was a critical factor in the evolution of metamorphosis and this gland was replaced by a follicular thyroid, for postmetamorphic animals needed to store iodine following their invasion of freshwater. Larval growth and postmetamorphic reproduction in freshwater became fixtures in the lamprey life cycle; a non-parasitic adult life-history type appeared later. The presence among extant lampreys of two different adult life-history types, and examples of the lability of the timing of sexual maturation in some species, imply that there has been a complex interplay between the thyroid and reproductive axes during the evolution of metamorphosis in lampreys. This proposal is consistent with what we know of interplay of these axes in extant adult lampreys and with the long-held viewpoint that thyroid function and sexual maturation are an association with an ancient history.     

Incorporation of Radioiodine into the Developing Endostyle of the Larval Lamprey

Incorporation of radioiodine (¹³¹I⁻) into small larvae of the Japanese river lamprey, 14 days after fertilization, was studied by radioautography. A small amount of radioiodine was incorporated into the larvae and became located in the mid-dorsal region of endostylar cells. The incorporation of radioiodine was greatest in endostylar cells near the junction with the anlage of the endostyle duct. It is possible that these cells form the anlage of the endostyle duct, which enters the endostyle.     

文昌鱼肝盲囊与脊椎动物肝脏起源

早期形态和胚胎学研究结果表明, 文昌鱼哈氏窝、内柱和肝盲囊分别是脊椎动物脑垂体、甲状腺和肝脏的同源器官。文章总结了近年来有关文昌鱼肝盲囊与脊椎动物肝脏之间关系的研究成果, 揭示文昌鱼肝盲囊和脊椎动物肝脏具有同源性, 并证明文昌鱼中存在类似脊椎动物的GH/IGF (Growth hormone/insulin-like growth factor)和TH/THR (Thyroid hormone/thyroid hormone receptor)信号通路, 为脊椎动物肝脏起源于文昌鱼肝盲囊样结构提供了分子水平证据。     

Sequence and developmental expression of amphioxus AmphiNk2–1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain

 We characterized an amphioxus NK-2 homeobox gene (AmphiNk2–1), a homologue of vertebrate Nkx2–1, which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2–1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2–1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions; this pattern is comparable to Nkx2–1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain. Received: 4 September 1998 / Accepted: 24 October 1998     

The ammocoetes endostyle: its oxidative enzymes as an evidence of its homology with the thyroid of higher chordates.

Histochemical methods were used for the demonstration of activity of the following intracellular oxidative enzymes, unstudied hitherto, in the epithelial cells of the endostyle of the river lampre (Lampertr aluviatilis L.) ammocoetes: reduced NAD dehydrogenase (NADD), lactate dehydrogenase (LD), cytochrome oxidase (CO), succinate dehydrogenase (SD), alpha-glycerophosphate dehydrogenase (alphaGPD) and glucose-6-phosphate dehydrogenase (G6PD). The activities of NADD and LD in the iodophil and throidogenic cells of type 3, then of subtype 2c and partly types 4 and 5 of the endostylar epithelium and the hypobranchial duct-lining epithelium were particularly ithe larva proves the possibility of their participation in the formation of the thyroid gland in the period of metamorphosis. In type 1 cells of the ammocoetes, despite their fairly strong enzymatic reactivity, the oxidative activity does not change significantly during the ontogenetic stages examined. The data obtained make it possible to modify the present views on the genesis of the thyroid gland of the adult lamprey, namely, they indicate the participation of the type 6 cells of the hypobranchial duct-lining epithelium in the process of thyroidogenesis.     

Is Lamprey Metamorphosis Regulated by Thyroid Hormones?

Lampreys are one of just a few fishes which have a true (firstor first type) of metamorphosis in their life cycle. In thesea lamprey (Petromyzon marinus), spontaneous metamorphosisis initiated when the size (length and weight), condition factor,and lipid stores reach appropriate levels and coincide withthe postwinter rise in water temperature. The serum levels ofthe thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃),drop dramatically at the onset of metamorphosis and metamorphosiscan be induced with treatment of animals with the goitrogen,KCIO₄, which also results in a decline in serum levels of thyroidhormones. The fact that thyroid hormone treatment can blockspontaneous and induced metamorphosis is support for the viewthat thyroid hormones, particularly T₃, operates like a juvenilehormone in lamprey metamorphosis; this view is counter to therole of thyroid hormones in metamorphosis of other vertebrates.The monodeiodinase pathways, whereby T₄ is converted to T₃ orto the biologically inactive reverse T₃, and even further degradationof T₃, may be a significant mechanism directing metamorphicchange. Lamprey metamorphosis is facultative in that it is initiatedor inhibited depending upon the coordination of a complex integrationof environmental, metabolic and hormonal cues. Thyroid hormonesdo not regulate lamprey metamorphosis in the sense observedin other vertebrate metamorphoses but they are important tothe developmental process. Some of the features of the involvementof thyroid hormones in lamprey metamorphosis may be relatedto the presence of the endostyle in larvae which in turn reflectsthe ancient origins of this vertebrate and perhaps the conservationof an ancient method of induction of metamorphosis. Some cluefor other factors which initiate lamprey metamorphosis may comethrough the examination of inducers of metamorphosis in lowerchordates     

Thyroid-specific enhancer-binding protein/thyroid transcription factor 1 is not required for the initial specification of the thyroid and lung primordia.

Targeted disruption of the homeobox gene T/ebp (Ttf1) in mice results in ablation of the thyroid and pituitary, and severe deformities in development of the lung and hypothalamus. T/ebp is expressed in the thyroid, lung, and ventral forebrain during normal embryogenesis. Examination of thyroid development in T/ebp homozygous null mutant embryos revealed that the thyroid rudiment is initially formed but is eliminated through apoptosis. Absence of T/EBP expression in the lung primordium does not activate apoptosis since a lung tissue, albeit dysmorphic, is nevertheless formed in T/ebp-/- embryos. These results demonstrate that T/EBP is not required for the initial specification of thyroid or lung primordia, but is absolutely essential for the development and morphogenesis of these organs.     

Dorsoventral axis inversion: Enteropneust anatomy links invertebrates to chordates turned upside down

The relationships between chordates with their dorsal nerve cord and other animal groups remain unclear. The hemichordata, specifically the enteropneusta (acorn worms), have been considered a sister group to the chordata. Enteropneusts combine various chordate features (e.g. lateral gill openings, dorsal nerve cord) with features that are usually associated with gastroneuralian invertebrates (e.g. dorsal heart, circumenteric nerve ring, ventral nerve cord). Here we analyse various morphological and functional characteristics that enteropneusts share with either invertebrates or chordates in the light of our recent proposal that the chordata may derive – by bodily dorsoventral inversion – from a gastroneuralian ancestor. We show that many seemingly non-chordate features of enteropneusts will align with similar features in the chordates – provided that we compare the ventral side of an enteropneust to the dorsal side of a chordate. This inversion proposes several interesting and new putative homologies between enteropneusts and acranian chordates, such as between their epibranchial ridge/endostyle (later thyroid gland), their postanal tails, atrial walls, and also between the chordates' dorsal notochord and the enteropneusts' posteroventral pygochord. Significantly, positional homology between notochord and pygochord is also supported by the expression domains of Brachyury orthologs in vertebrates and invertebrates: a Brachyury ortholog is active in the postero ventral mesoderm in Drosophila and in the dorsal mesoderm in chordates. In conclusion, we propose that the anatomy of enteropneusts may serve as a conceptual 'missing link' between gastroneuralian invertebrates and notoneuralian chordates. We discuss whether the enteropneust's dorsoanterior nervous centre plus their ventral trunk cord then corresponds to brain and dorsal nerve cord in the chordata.     

Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey (Lampetra planeri Bl.).

1. The biosynthesis of 18-19S thyroglobulin has been studied in a larval and adult freshwater lamprey (Lampetra planeri Bl.). 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates (125I, [3H]leucine and various [3H]carbohydrates). 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18-19S Tg), containing a small proportion of labeled T3 and T4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. In adults, the biosynthesis of 18-19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18-19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. Some 18-19S Tg is apparently stored in the endostyle.     

Pax2.1 is required for the development of thyroid follicles in zebrafish

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.     

Immunocytochemical localization of thyroglobulin in the endostyle of the anadromous sea lamprey, Petromyzon marinus L

Thyroglobulin (TG) was localized in the endostyle of the anadromous sea lamprey, Petromyzon marinus L. by means of the unlabeled antibody peroxidase-antiperoxidase immunocytochemical method. TG was found localized on the apical surface and within the cytoplasm of type 2c and 3 cells and in some type 5 cells. By identifying the cells of the endostyle immunocytochemically it may be possible to study more readily the events of endostylar transformation during metamorphosis.