Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis,Thalia democratica,Dolioletta gegenbauri and Doliolum nationalis (Tunicata,Thaliacea)

Summary The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electronmicroscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity ¹²⁵I^- over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodinebinding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.     

Zur Feinstruktur und Histochemie des Kiemendarmes und der „Leber“ von Branchiostoma lanceolatum (Pallas)

Zusammenfassung Leberblindsack, Epibranchialrinne und Endostyl von Branchiostoma lanceolatum wurden enzymhistochemisch und elektronenmikroskopisch untersucht. Folgende Fermente wurden nachgewiesen: alkalische und saure Phosphatase, ATP-ase, Aminopeptidase, -Naphthyl-acetat- und Indoxyl-acetat-Esterase, Glucosaminidase, Arylsulfatase, NADH- und NADPH-Diaphorase. Die Epibranchialrinne erwies sich als besonders enzymreich.Die Epithelzellen des Leber-Blindsackes besitzen basal zahlreiche Fetteinschlüsse und oberhalb des Kerns große Granula, die vermutlich Verdauungsenzyme enthalten. Das Epithel der Epibranchialrinne enthält z.T. basal sehr große Vakuolen sowie apikale Sekretionsgranula.Im Endostyl lassen sich verschiedene Zonen nachweisen. In den esterasereichen Zonen 2 und 4/5 finden sich Hinweise auf Proteinsynthese (granuläres E.R. und Sekretionsgranula, bei denen es sich zum Teil um Schleimballen handelt). Zone 4 ist weiterhin besonders durch ihren Gehalt an Lipideinschlüssen gekennzeichnet. In den an saurer Phosphatase reichen Zonen 1, 3 und 6 sind die zilientragenden Zellen in charakteristischen Säulen angeordnet.

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Fine structure and histochemistry of the pharynx and the hepatic caecum of Branchiostoma lanceolatum (Pallas)

Summary Cytochemical and ultrastructural studies have been carried out on the hepatic caecum, hyperpharyngeal groove and endostyle of Branchiostoma lanceolatum. The following enzymes have been demonstrated: acid and alkaline phosphatase, ATP-ase, aminopeptidase, -naphthyl-acetate- and indoxyl-acetate esterase, glucosaminidase, arylsulphatase, NADH-and NADPH-diaphorase. The hyperpharyngeal groove proved to be particularly rich in enzymes.The epithelial cells of the hepatic caecum possess at their bases numerous lipid droplets and in the supranuclear cytoplasm granules which presumably contain digestive enzymes.The epithelium of the hyperpharyngeal groove is characterized by large basal vacuoles and apical secretion granules.The electron microscope confirms the existence of several cell zones in the endostyle. The cells of the esterase rich zones 2 and 4/5 possess a well developed system of granulated E. R. as well as globular inclusions which presumably represent mucus droplets. In zone 4 in addition a great number of lipid inclusions and specific electron dense secretion granules occur. In the acid phosphatase rich zones 1, 3 and 6 the cilia-bearing cells are arranged in typical columns.

Herrn Professor Bargmann danke ich für die Überlassung eines Arbeitsplatzes im Anatomischen Institut Kiel.     

催乳素及其受体样免疫活性在文昌鱼神经系统、哈氏窝和其它组织的分布

用兔抗人催乳素多克隆抗体和鼠抗人催乳索受体单克隆抗体对文昌鱼神经系统、哈氏窝和其它组织进行免疫组织化学研究。结果显示:催乳素免疫活性细胞及催乳素受体定位在文昌鱼脑泡、神经管、哈氏窝、轮器、内柱、消化管和性腺(卵巢和精巢),表明催乳素在文昌鱼有广泛分布,并且从进化观点来看,证明催乳素是一种高度保守的古老激素。双重免疫染色进一步揭示催乳素及其受体免疫反应阳性物质共存于同一卵母细胞胞膜和胞质以及精巢中精原细胞、初级与次级精母细胞和Sertoli细胞。研究结果首次证明了文昌鱼脑泡和哈氏窝以及其它组织能够合成和分泌催乳素,表明像脊椎动物一样,催乳素可能参与调节文昌鱼体内代谢和对环境的适应以及性腺发育,提示文昌鱼可能出现原始的脑泡-哈氏窝(催乳素)-靶细胞调控轴的雏形。本研究为文昌鱼哈氏窝内分泌学以及催乳素的起源与演化提供新的基础资料。     

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     

Ultrastructural localization of the iodination centre in the endostyle of the adult amphioxus (Branchiostoma lanceolatum)

Summary The site of iodination in the endostyle of the adult amphioxus was examined by light-and electron-microscopic autoradiography. In accordance with previous studies, light-microscopic autoradiography showed a distinct accumulation of autoradiographic grains at the apical end of epithelial cells in the lateral part of the endostyle. In the electron microscope two distinct cellular zones were identified in an approximate position of the light-microscopic zone 5. Zone 5a, not previously recognized, was adjacent to zone 4 and consisted of six to nine rows of cells free of characteristic granules. Cells in zone 5b contained large mucous granules and had, in previous ultrastructural studies, been identified as belonging to the typical zone 5. Four or less incomplete rows of granule-containing cells, not observed in previous studies, marked the border between zones 5b and 6. After incubation in ¹²⁵I for 5 min, electron-microscopic autoradiography showed a selective concentration of label to zone 5a, which, thus, corresponds to the iodination centre seen in the light microscope. The grains were associated with cilia and microvilli in the lumen. After longer incubation times (30, 60, 90 min) grains were still concentrated at the surface of zone 5a but were also associated with the surface of zones 5b and 6. Grains were also located over the cytoplasm of all three zones. They were associated with vesicles and lysosome-like structures, suggesting secondary uptake of labelled products by endocytosis. Methimazole, an inhibitor of peroxidase, abolished the autoradiographic reaction. In conclusion, the site of iodination in the endostyle of amphioxus is located in zone 5a, which has not previously been ultrastructurally defined. Iodination in the endostyle is an extracellular process, but secondary uptake by endocytosis appears to occur.     

Presence of cells resembling serotonergic elements in four species of tunicates

Summary Cells resembling serotonergic elements have been demonstrated in the endostyle of four species of tunicates by use of fluorescence histochemistry (glyoxylic acid-induced fluorescence, immunofluorescence), silver-nitrate impregnation and autoradiography. Positive cells were also observed in peripharyngeal bands and intestine.In Ciona intestinalis, a relationship appears to exist during postlarval development between the appearance of fluorescent cells in the endostyle and the development of the ovary.This research was supported in part by a grant from CNRS (ATP JV/DP: N 4696-4697). A portion of these results has been presented as a poster at the Third Colloquium of the European Pineal Study Group (Pécs, Hungary, August 1984)     

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.