Thyroid hormones were measured by radioimmunoassay in blood plasma and in extracts (butanol/chloroform/ammonia) of pharynx, alimentary canal, and tunic of Phallusia mammillata. Other animals were injected with [125I]T3 and its distribution in the same tissues was determined from 6 to 48 hr after injection. Last, the saturable binding of [125I]T3 to salt-extracted nuclear proteins in the pharynx and alimentary canal was studied in vitro. T4 was found in all tissues examined and in the same order of magnitude (2.7 to 8.4 ng/g) whereas plasma concentration was low (0.2 ng/ml). Tissue T3 concentrations were always much lower than T4 tissue concentrations, being highest in alimentary canal (0.8-1.1 ng/g) and very low in the tunic as well as in plasma, in which T3 was generally below 0.02 ng/ml. The tissue distribution of [125I]T3 was correlated with T3 concentrations. Tissue/plasma ratios were approximately 10 in the alimentary canal, 5 in the pharynx, and 0.18 in the tunic. Saturable binding of T3 to nuclear proteins in the alimentary canal and pharynx was demonstrated. The affinity (Kd) was similar to that found in tissues from other chordates but the maximal binding capacity was much lower. The very low levels of plasma T3 and low T3/T4 ratios may indicate that the endostyle releases primarily T4 into the body fluid. On the other hand, the high levels of T3 and the high T3/T4 ratios in the alimentary canal suggest that this metabolically active target tissue is the main site of the process of deiodination of T4 into T3, a process which has been previously shown in P. mammillata in vivo. The data indicate putative T3 receptors in ascidian tissue, suggesting a physiological role of T3 in protochordates.

Oikopleura dioica has two large subchordal cells which were studied in vivo and with light and electron microscopy. They have fixed positions within the haemocoel of the tail but change their morphology continually by protruding and withdrawing processes in an amoeboid manner. Also the fine structure varies considerably from one animal to the next. The cell surface sometimes indicates a strong pinocytotic activity with many coated pits and vesicles. In other cases there are many small vesicles which are interpreted as exocytotic. They are found both in the cytoplasm close to the plasma membrane and in the haemocoelic fluid. Subchordal cells with no pronounced surface activity have a large amount of rough endoplasmic reticulum, which suggests that they synthesize proteins. The presence of subchordal cells in the tail is correlated with the presence of the ontogenetically related bioluminescent oral gland cells in the pharynx; either both types exist simultaneously or both are lacking. It is speculated that the two cell types are also functionally co-ordinated. Oikopleura albicans has a multitude of tiny subchordal cells which have essentially the same fine structure as the two large Oikopleura dioica cells.

Oikopleura albicans and O. longicauda belong to the two subgenera Vexillaria and Coecaria, respectively. The morphology and ultrastructure of their endostyles were investigated with conventional microscopic procedures as well as with DAB cytochemistry and 125I autoradiography at both light- and electron-microscopic levels. As expected, the general morphology of these endostyles is similar to all hitherto examined endostyles. They possess a ventral portion consisting of alternating glandular and ciliated cell zones, probably serving food capture, and a dorsal region, the corridor. Autoradiographic grains were found mainly in the corridor lumen associated with the apical surface of the two central rows of corridor cells. The same cells also gave strong positive reactions for peroxidase, the iodinating enzyme. Peroxidase activity was found in the apical plasma membrane as well as in the nuclear envelope, rough endoplasmic reticulum, Golgi area and cytoplasmic vesicles. Definitive conclusions concerning an apical uptake and subsequent release into the body fluid of iodinated material could not be made from the present experiments. Our investigations indicate that the two central rows of corridor cells in both subgenera of oikopleurids constitute the protothyroid region, possibly homologous to the vertebrate thyroid gland.

The cellular and subcellular distribution of serotonin (5-hydroxytryptamine, 5-HT) in the endostyle of three species of ascidians, Ciona intestinalis, Corella parallelogramma, Ascidia mentula, was studied by light-(immunoperoxidase) and electron-microscopic (immunogold) immunocytochemistry. At the light-microscopic level 5-HT-like immunoreactivity (5-HT-LI) was exclusively found in cells located in the lateral portion of the endostyle, between zone 7, known to have iodinating capacity, and zone 8, which consists of ciliated cells. At the electron-microscopic level, the 5-HT-immunoreactive cells were found to correspond to cells containing polymorphous, dense granules, 100–300 nm in diameter. The granules were located in the supranuclear cytoplasm facing the endostyle lumen as well as in the infranuclear cytoplasm facing the extracellular space. Quantification showed that the 5-HT-LI was considerably higher (13–67 times) in cytoplasmic areas containing granules as compared to areas devoid of granules. Most, but not all, of the 5-HT-LI was associated with the dense core of the granules. In conclusion, serotonin-containing cells are located in the peripheral portion of the endostyle, between zones 7 and 8. Serotonin is stored in cytoplasmic granules that are present both in the apical and basal cytoplasm. This suggests the possibility that the cells are bipolar and secrete serotonin both in a basal direction to the extracellular space, and in an apical direction to the pharyngeal lumen.

The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electron-microscopic 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 iodine-binding 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.

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.

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.

The endostyles of cephalochordates, ascidians, and larval petromyzontids have the capacity to organify iodine. A similar mechanism in the appendicularian endostyle has hitherto been unknown. Observations in this study of Oikopleura dioica with electron microscopic autoradiography and cytochemistry show that also the appendicularian endostyle has iodinating capacity and that the iodinating cells contain peroxidase, an enzyme responsible for iodination. After incubation in seawater containing 125I−, autoradiography revealed a selective labeling in the dorsal portion of the endostyle. The endostyle of O. dioica is on each side lined by four rows of corridor cells. The autoradiographic grains were mainly located over the endostylar lumen or associated with the luminal surface of the two central rows of corridor cells. These cells, but no other endostylar cells, also showed a positive reaction for peroxidase. The reaction product was distributed along the luminal plasma membrane and was also present in the cytoplasm within rough endoplasmatic reticulum, Golgi apparatus, and vesicles. The selective labeling as well as the cytochemical reaction were abolished by incubation in methimazole, an inhibitor of peroxidase. It is suggested that the two central rows of the corridor cells can be considered as homologs to iodine-binding zones in other endostyles and also as a primitive forerunner to the vertebrate thyroid gland.

The asymmetrical endostyle of Branchiostoma larvae contains two different zones of mucus-producing cells which metamorphose to the paired zones 2 and 4 respectively in the endostyle of the adult. In both the larva and the adult these zones are parts of the food-trapping mechanism. An endostyle zone, which has a position corresponding to that of the paired iodinating zones in the endostyle of the adult, binds iodine selectively. The ultrastructure and labeling pattern indicate that the labeled cells in the larval endostyle belong to functionally different types. In one region of the iodinating zone iodine is mainly bound extracellularly at the apical cell surface. Also in the second region grains are located at the apical cell surface as well as over the cytoplasm and extracellularly at the basal plasma membrane. It is possible that iodination takes place in the lumen close to cells in the first region and that the labeled product is taken up and eventually relased by cells of the second region. Our observations show that this primitive endostyle already has iodinating capacity and may synthesize and release thyroid hormones.

Reissner's fibre (RF) is an extraordinary ancient feature of the chordate central nervous system. In vertebrates the RF material is released by the cells of the subcommissural organ (SCO) situated in the roof of the brain. In cephalochordates the RF forming apparatus is the infundibular organ (IFO), a structure in the floor of the brain vesicle. Both the SCO and the IFO are areas of specialized secretory ependymal cells. Recently the SCO-secretion of many vertebrate species has been studied immunocytochemically by use of different antisera against RF-material from cows. The present investigation with the PAP-technique showed that these antisera give a positive immunocytochemical reaction with the secretion of the IFO of amphioxus. The results indicate molecular similarities of compounds present in both the SCO-secretion and the IFO-secretion. The conception that the SCO and the IFO are descendants from a single large glandular area in the brain vesicle of an ancient chordate ancestor is emphasized by these findings

Light and electron microscopic studies showed that the oral gland cells have two quite different zones. Medially, the basal zone is in contact with body fluids and the endostyle. Its strongly pyroninophile cytoplasm contains the extremely digitated nucleus and numerous small mitochondria. Laterally, the apical zone contacts the epidermis and it may also send a process between epidermal cells and deliver cell fragments into the primordium of the new house. This cell zone contains numerous membranes. It is concluded that the oral gland cells are light producing glands and that the membrane-rich cell fragments which are incorporated into the house wall are the source of the bioluminiscence which has been reported from empty houses. The ontogenetically related subchordal cells have a similar structure and it is possible that also these cells are light producers.