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.     

Expression of<Emphasis Type="Italic"> FoxE</Emphasis> and<Emphasis Type="Italic"> FoxQ</Emphasis> genes in the endostyle of<Emphasis Type="Italic"> Ciona intestinalis</Emphasis>

We have analyzed the expression patterns of two Fox genes, FoxE and FoxQ, in the ascidian Ciona intestinalis. Expression of Ci-FoxE was specific to the endostyle of adults, being prominent in the thyroid-equivalent region of zone 7. Ci-FoxQ was expressed in several endodermal organs of adult ascidians, such as the endostyle, branchial sac and esophagus. In the endostyle, the pattern of Ci-FoxQ expression was similar to that of CiTTF-1, being prominent in the thyroid-equivalent regions of zones 7 and 8. Therefore, these Fox genes may perform thyroid-equivalent functions in the ascidian endostyle.Edited by N. Satoh     

Identification of a novel partner of duox: EFP1, a thioredoxin-related protein

H(2)O(2) is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid. ThOX proteins (thyroid oxidase) also called Duox are believed to be responsible for H(2)O(2) generation. Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions. In this study, we demonstrate interactions of Duoxs with TPO and with p22(phox) without any effect on Duox activity. By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1 (EF-hand binding protein 1), one partner of Duoxs that belongs to the thioredoxin-related protein family. EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved. EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes. It interacts also with TPO but not thyroglobulin. Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H(2)O(2) production. EFP1 and Duox mRNA share similar distribution in nine different tissues. These results suggest that EFP1 could be one of the partners in the assembly of the multiprotein complex constituting the thyroid H(2)O(2) generating system but is certainly not sufficient to permit H(2)O(2) generation.     

Expression and functional analysis of Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.

In solitary ascidians the fate of endoderm is determined at a very early stage of development and depends on cytoplasmic factors whose nature has not been determined. We have isolated a member of the NK-2 gene family, Cititf1, from the ascidian Ciona intestinalis, showing high sequence homology to mammalian TITF1. The Cititf1 gene was expressed in all endodermal precursors at the pregastrula and gastrula stages, and is thus the first specific regulatory endodermal marker to be isolated from an ascidian. Cititf1 expression was downregulated at the end of gastrulation to reappear at middle tailbud and larval stages in the most anterior and ventral parts of head endoderm, regions which give rise, after metamorphosis, to the adult endostyle, where Cititf1 mRNA was still present. Microinjection of Cititf1 mRNA into fertilized eggs resulted in tadpole larvae with abnormalities in head-trunk development consequent to the formation of excess endoderm, perhaps due to recruitment of notochord precursors to an endodermal fate. These data suggest that Cititf1 plays an important role in normal endoderm differentiation during ascidian embryogenesis.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

Comparative analysis of thyroxine distribution in ascidian larvae

The ascidian endostyle is a mucus-secreting pharyngeal organ, it has iodine-concentrating activity and the biosynthesis of thyroid hormones has been well documented. According to our recent findings, ascidians possess thyroid hormones, which are localized in mesenchymal cells. We have studied the presence and localization of l-thyroxine (T₄) in Ascidia malaca (Traustedt), Ascidiella aspersa (Müller), Phallusia mamillata (Cuvier) and Ciona intestinalis (Linnaeus) larvae and its involvement in metamorphosis. In vivo treatment of swimming larvae with exogenous T₄ and thiourea (a thyroid hormone synthesis inhibitor), demonstrate the presence of T4 during larval development. These results were confirmed by in vitro experiments utilizing dot blotting, radioimmunoassay and immunoperoxidase staining. The hormone was localized in mesenchymal cells of all four ascidians, spread out in the body cavity, under the adhesive papillae and around the intestine. The presence of TH in mesenchymal cells could be related to blood cells, musculature and heart tissue differentiation. The results suggest that this hormone could be involved in the control of metamorphosis.     

Cell shape, spatial patterns of cilia, and mucus-net construction in the ascidian endostyle

The endostyle of the ascidian Ciona intestinalis secretes a mucus-net composed of regularly spaced longitudinal and transverse mucoprotein filaments. It is a gutter-shaped organ composed of eight different longitudinally aligned epithelial zones numbered 1-8. Each zone, with the exception of zone 7 which is unciliatcd. has a characteristic cell shape and spatial pattern of cilia and microvilli. Zones 1.5 and 8 are composed of multiciliated cells, and zones 2,3,4 and 6 of monociliated cells. Cell apices in zones 2, 3 and 4 are rectangular in cross-section, and bear highly ordered rows of cilia. Spacings between cilia both within and between rows are different in each of these zones, but they are similar to interfilament spacings in the mucus-net. The basic structure of the mucus-net is probably secreted and constructed by secretory cells and cilia in zones 1-4. Further secretion may be added in zones 6 and 7 whilst cilia in zones 5, 6 and 8 propel the net out on to the inner surface of the pharynx where it acts as a food filter. The highly organized and structurally complex pattern of ciliated cpithelia in the aseidian endostyle is surprising when compared with the endostyle of the cephalochordate Branchiostoma lanceolatum, which is composed entirely of monociliated cells that differ very little in structure between epithelial zones.     

Electron-microscopic studies of iodine-binding and peroxidase activity in the endostyle of the larval amphioxus (Branchiostoma lanceolatum)

Summary The asymmetric endostyle in the larval amphioxus (Branchiostoma lanceolatum) was examined by light-and electron-microscopic cytochemistry (peroxidase; incubation in diaminobenzidine) and autoradiography (incubation in ¹²⁵I^-). Compared to the adult the same cellular zones were also found in the larval endostyle, with the exception of zone 1, which was absent. The corresponding adult and larval zones had a similar morphology. All cells in zones 5a, 5b, and 6 were reactive for peroxidase. A reaction product was also present in the lateral 2 to 3 cell rows of zone 3. The dense reaction product was located on the inner surface of membranes of the rough endoplasmatic reticulum, Golgi apparatus and vesicles, and multivesicular bodies as well as on the outer surface of the luminal plasma membrane. An incomplete row of granule-containing, peroxidase-negative cells was located between zones 5b and 6. After incubation of larvae in sea water containing ¹²⁵I^-, autoradiographic grains were selectively concentrated over the lumen at the apical surface of all peroxidase-positive zones. The highest grain density occurred in relation to zone 5a, which in the adult has been recognized as the iodination center. Few grains were located over the cytoplasm. Methimazole, an inhibitor of peroxidase, abolished the cytochemical reaction and the appearance of autoradiographic grains. The observations indicate that iodination in the larval endostyle takes place extracellularly and is catalyzed by peroxidase bound in the plasma membrane.     

Investigation of the contribution from peripheral GnRH-like immunoreactive 'neuroblasts' to the regenerating central nervous system in the protochordate Ciona intestinalis

The neural ganglion of the ascidian Ciona intestinalis regenerates in its entirety within a few weeks of ablation. Here we investigate the role of gonadotropin-releasing hormone-like immunoreactive (GnRH-li) cells in regeneration. Immunocytochemical studies show that in addition to a previously described plexus of GnRH-like neurones located in association with the dorsal strand, the normal adult brain contains GnRH-li neurones. These are predominantly localized to the ventral cortical rind at the posterior of the ganglion. Following ablation, non-process bearing GnRH-li cells appear in the regenerating area within two days. By day 8 post-ablation, process bearing GnRH-li cells are detected close to the regenerating brain. The number of these cells increases at later stages. GnRH-li cells are first detected within the regenerating brain at 14 days post-ablation and their number subsequently increases. These cells are initially concentrated along the ventral surface of the regenerating brain near to the dorsal strand. Double labelling studies with bromodeoxyuridine show that none of the GnRH-li cells are labelled at any stage of regeneration. The data are consistent with a sub-population of the new neurones being derived from GnRH-li neuroblasts born prior to ablation, which migrate from the dorsal strand complex into the regenerating ganglion.     

Fine structure and its functional properties of the endostyle of Ascidians,Ciona intestinalis

Summary For the purpose used in understanding thyroid phylogenesis, the fine structure and the iodine metabolism of the endostyle of Ascidians,Ciona intestinalis, was studied by electron microscopy and electron microscopic autoradiography. There are 8 kinds of zones in the endostyle.Zone 1, 3, and 5 cells, especially zone 1 cells, are characterized by numerous long cilia. These cells which show no indications of protein-secretion but numerous small vesicles and cytoplasmic filaments might play a role in catching and transporting food, absorption of liquid and supporting the endostylar construction.Zone 2, 4, and 6 cells are large and characterized by well developed rough endoplasmic reticulum and numerous electron-dense secretory granules which are considered to be synthesized in the rough endoplasmic reticulum and transported to the Golgi apparatus to mature. They, which are somewhat similar to the pancreatic exocrine cells in fine structure, are believed to secrete the proteinous or mucoproteinous substances which might be related to the digestion of food.Zone 7 and 8 cells which might be homologous to the thyroid cell of the higher vertebrate contains poorly developed rough endoplasmic reticulum, small Golgi apparatus, a few multivesicular bodies, a few lysosomes, and numerous small vesicles. In addition zone 8 cells bear cilia on their apical surface. The cytoplasmic characteristics of these cell types, especially of zone 8 cells, are fairly similar to those of type 2C and type 3 cells of the endostyle of a larval lamprey, though the rough endoplasmic reticulum is not so well developed. By electron microscopic autoradiography numerous silver grains were observed on the apical cell membrane region of zone 7 and 8 cells, especially of zone 8 cells, 1, 4, 6, 16 and 24 hours after immersion in sea water containing¹²⁵I. This fact suggests that the iodination takes place in the apical cell membrane region of these cells. The materials in the endostylar lumen is washed away during the fixation and dehydrating processes of the tissue. Therefore, the possibility of iodination of thyroglobulin-like substances taking place within the endostylar lumen cannot be ruled out. Grains were also found in the multivesicular bodies and lysosomes after 4, 6, 16 and 24 hours, especially 16 and 24 hours. It seems that the organic iodine might be reabsorbed into the cytoplasm of these cells.This investigation was supported by research grant from Dr. Henry C. Buswell Research Fellowship.On leave from Department of Anatomy, Hiroshima University, School of Medicine, as a Visiting Research Professor. The authors wish to express their hearty thanks to Dr. Oliver P. Jones for his valuable criticism.     

Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the Ascidian Herdmania curvata

Ascidians, along with other urochordates, are the most evolutionarydistant group from vertebrates to display definitive chordate-specificcharacters, such as a notochord, dorsal hollow nerve cord, pharynxand endostyle. Most solitary ascidians have a biphasic lifehistory that has partitioned the development of these charactersbetween a planktonic microscopic tadpole larva (notochord anddorsal nerve cord) and a larger sessile adult (pharynx and endostyle).Very little is known of the molecular axial patterning processesoperating during ascidian postlarval development. Two axialpatterning homeobox genes Otx and Cdx are expressed in a spatiallyrestricted manner along the ascidian anteroposterior axis duringembryogenesis and postlarval development (i.e., metamorphosis).Comparisons of these patterns with those of homologous cephalochordateand vertebrate genes suggest that the novel ascidian biphasicbody plan was not accompanied by a deployment of these genesinto new pathways but by a heterochronic shift in tissue-specificexpression. Studies examining the role of all-trans retinoicacid (RA) in axial patterning in chordates also contribute toour understanding of the role of homeobox genes in the developmentof larval and adult ascidian body plans. Our studies demonstratethat RA does not regulate axial patterning in the developingascidian larval neuroaxis in a manner homologous to that foundin vertebrates. Although RA may regulate the expression of someascidian homeobox genes, ectopic application of RA does notappear to alter the morphology of the larval CNS. However, treatmentwith similar or lower concentrations of RA, have a profoundeffect on postlarval development and the juvenile body plan.These changes are correlated to a dramatic reduction of Otxexpression. Through these RA-induced effects we infer that whileRA may regulate the expression of some homeobox genes duringembryogenesis it has a far more dramatic impact on postlarvaldevelopment where regulative processes predominate.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.