The distribution of neuropeptide Y-like immunoreactivity in the ascidian Styela plicata.

The occurrence of neuropeptide Y-like substances has been verified in the nervous system and alimentary tract of the ascidian Styela plicata. Neuropeptide Y-like immunoreactivity is present in a few small neurons and in a network of beaded nerve fibres of the cerebral ganglion. Neuropeptide Y-like immunoreactive material can be also localized in the endostyle and in a few cell bodies of the branchial walls. Moreover, immunofluorescent endocrine-like cells of the "open" type occur in the gastric folds. Finally, some possible functions of the ascidian neuropeptide Y are discussed.     

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.     

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.     

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.     

Iodine-concentrating cells in the endostyle of Ammocoetes

Summary The iodine-concentrating cells in the endostyle were examined in four larvae of petromyzon planeri. The visceral cells (types 2c, 3, and 4) were provided with cilia and contained granules with a characteristic content of concentric lamellae. Type 3 was particularly rich in ergastoplasm. The parietal cells (type 5) were short with a polymorphous content indicating a resorptive rather than a synthetic function. Based on the ultrastructure of the cells it has not been possible to decide which group forms the follicular epithelium in the petromyzone after metamorphosis, but it is most likely type 3 with its protein-synthetizing apparatus.     

Endostyle cell recruitment as a frame of reference for development and growth in the Urochordate Oikopleura dioica

In models of growth and life history, and in molecular and cell biology, there is a need for more accurate frames of reference to characterize developmental progression. In Caenorhabditis elegans, complete fate maps of cell lineage provide such a standard of reference. To be more widely applicable, reference frames should be easier to measure while still providing strong predictive capacity. Towards this aim, we have analyzed growth of the endostyle in the appendicularian Oikopleura dioica at the cellular level, and measured its response to temperature and food availability. Specifically, we test the hypothesis that age of a specific developmental stage in O. dioica can be predicted from the number of endostyle cells and temperature. We show that the endostyle grows by recruiting cells from the posterior tip into the lateral arms of the organ in an anterior-posterior orientation and that the rate of increase in lateral arm endostyle cells is temperature-dependent but unresponsive to nutritional intake. Endostyle cells therefore serve as an accurate and easily measured marker to describe developmental progression. Conceptually, such a method of characterizing developmental progression should help bridge life-history events and molecular mechanisms throughout organismal aging, facilitating cross-disciplinary understanding by providing a common experimental framework.     

Identification of the endostyle as a stem cell niche in a colonial chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.     

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.     

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.     

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.     

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.     

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.     

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.     

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