The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Junctional complexes of the branchia and gut of the tunicate,Pyrosoma atlanticum (Pyrosomatida,Thaliacea)

Summary The branchia of pyrosomes has been found to be like that of ascidians in that it shares with the latter, besides the presence of peribranchial chambers, stigmata bordered by clusters of seven rows of flattened cells, each bearing a single row of long cilia. The intercellular junctions between ciliated cells of the branchial basket and between the cells lining the gut, in pyrosomes, have been studied in thin sections and by freeze-fracture. In both tissues tight and gap junctions are present, but the former are much more extensive in the gut. The gap junctions in the branchial basket exhibit some atypical features. Moreover, although there are extensive zonulae adhaerentes between the ciliated cells of the branchial basket, there are none between the epithelial cells of the intestinal tract. This feature of the branchiae, together with the alignment of their cells into highly ordered rows of seven cells, are similar to those found in some groups of ascidians. The evolutionary relationships between pyrosomes and the aplousobranch ascidians are considered in the light of these results.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

An electron microscopic study of early gonadogenesis in the hermaphroditic appendicularian Oikopleura gracilis (Tunicata,Oikopleuridae)

The postembryonic development of the gonad in the hermaphroditic appendicularian O. gracilis was studied using transmission electron microscopy. The primordial germ cells were detected first in 10-h-old larvae and represent migrating primordial germ syncytium (mPGS) localized in the hemocoel of the tail/trunk junction and several haemocoel areas of the digestive compartment. The mPGS consisted of primordial germ nuclei (PGN) 2 μm in diameter, and elongate somatic-line nuclei 1.8 μm in diameter. In 12.5-h-old juveniles the gonad primordium 40 × 90 μm in size, was separated by a narrow space of haemocoel between the gut and the epidermis of the reproductive compartment. The gonad primordium consisted of the central syncytial part of primordial germ nuclei (PGN), enclosing a single layer of somatic epithelium. In 3-day-old juveniles, the gonad was differentiated into testis and ovary. The testis, 400 × 550 μm in size, is a syncytium of spermatogonial nuclei, covered by a single layer of somatic epithelium. The ovaries, 350 × 850 μm in size, consist of a syncytium with nurse nuclei and meiotic nuclei. The hermaphroditic gonad originates from extragonadal mPGS. Early gonadogenesis in appendicularians has ultrastructural features in common with early gonadogenesis in ascidians.     

Gut ultrastructure of the appendicularian Oikopleura dioica (Tunicata)

Abstract. The appendicularians, planktonic tunicates, possess a specialized, external filtering system that captures food particles <1 μm in size. In this work the alimentary canal of Oikopleura dioica has been studied by serial sections of whole animals and ultrastructure. The gut includes a dorsal esophagus, a bilobed saccular stomach, and a curved intestine, divided into vertical, mid-, and distal intestine (or rectum). No multicellular glands or cellular proliferative centers were found. Three main cell types were recognized, ciliated microvillar cells, globular cells and gastric band cells, with specializations reflecting different physiological roles in the various regions. Ciliated microvillar cells, the most diffuse, are considered to be involved in food propulsion, fecal pellet formation, absorption, and nutrient storage. Pinocytotic features and vacuoles suggest that absorption of macromolecules and intracellular digestion occur in the globular cells of the stomach and rectum. The large gastric band cells of the left lobe have typical features of intense protein synthesis and probably produce enzymes for extracellular digestion. Diffuse interdigitations of many cells enormously increase the plasmalemma surface and may be involved in liquid/ion exchange. Despite the apparent structural simplicity of the gut epithelium, O. dioica efficiently processes food to fulfill the energy requirements of its exceptionally rapid life-cycle.     

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.     

Morphological aspects of fertilization in Phallusia (Ascidia) nigra (Ascidiacea,Tunicata)

The spermatozoa of Phallusia (Ascidia) nigra have an elongated head (approximately 5 m in length) in which a nucleus and a single mitochondrion are located side by side. There is no midpiece. The apex of the head is wedge-shaped. Acrosomal vesicles (approximately 55–65 nm in diameter) and moderately electron-dense material (MEDM) are present between the plasmalemma and the nuclear membranes in the anterior tip of the head. The MEDM occupies a central position and three or four acrosomal vesicles are seen in a line alongside it. The acrosomal vesicles disappear as the sperm makes contact with the surface of the chorion. Gamete fusion most likely occurs between a small process extending from the peripheral margin of the sperm apex and the egg surface, resulting in incorporation of the sperm into the egg from the anterior region of its head.     

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.     

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata,Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.     

Effect of monosodium glutamate on the neural complex of Ciona intestinalis (Tunicata)

Summary Short-term treatment of the ascidian (tunicate) Ciona intestinalis with monosodium glutamate produces a transient decrease in methionine-enkephalin-like immunoreactivity of neurones in the nervous ganglion. Moreover, it causes vacuolisation of the cells in the neural complex, particularly in the neural gland. Similar damages occur after ovariectomy. These results suggest that the ovary exerts an indirect influence on the neural gland via the nervous ganglion, and that the methionine-enkephalin-like substance could be the responsible neuromediator.A portion of these results has been presented as a poster at the 10th International Symposium on Comparative Endocrinology, Copper Mountain, Colorado, USA (July 1985).     

Septate junctions in the cephalic epidermis of turbellarians (Bipalium)

Summary In the epidermis of turbellarians septate junctions of the pleated sheet type have been demonstrated in conventional thin sections and freeze fractured preparations. The structure of these junctions entirely agrees with that found in molluscs and arthropods.Financially supported by DFG (Sto 75/3,4; We 380/5)     

Distribution of peroxidase and iodination activity in the endostyles of Oikopleura albicans and Oikopleura longicauda (Appendicularia,Chordata)

Summary 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 identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

Ultrastructure of the body cavities in juveniles and adults of the appendicularian Oikopleura gracilis (Tunicata,Chordata) suggests how the heart may have evolved in tunicates

The organization of the body cavities is an important morphological trait that can be used for establishing the phylogenetic relationships between different groups of animals. In the present study, the hemocoel and coelomic systems of 10‐hr‐old juveniles and adults of the hermaphroditic oikopleurid Oikopleura gracilis were examined using light and transmission electron microscopy. The trunk hemocoel in 10‐hr‐old juveniles was represented by small clefts containing layers of extracellular matrix of adjacent tissues or interstices with migrating primordial germ syncytium. The wide hemocoel in the tail contained extracellular strands, subdividing the hemocoel into hemal sinuses. In adults, a large hemocoel appeared in the trunk and tail, and also contained extracellular strands. The hermaphroditic gonad was surrounded by its own lining, separating it from the hemocoel. The gamete‐filled cavity in the ovary and testis appeared only at late‐stage gonadogenesis, when the pre‐spawning reduction of syncytium occurred in the gonads. The true coelom in 10‐hr‐old juveniles and adults was represented by the pericardium. The lining of the pericardium consisted of myoepithelial and peritoneal cells. In the myoepithelial cells of 10‐hr‐old juveniles, myofibrils had been formed. The myoepithelial cells of adults had several parallel rows of completely differentiated myofibrils. The substantial reduction of the coelomic and circulatory systems in O. gracilis evidently results from the extreme shortening of ontogeny in appendicularians. Development in O. gracilis from early juvenile to adult involves the following steps, which also suggest how the tunicate heart may have evolved: a single‐layered coelomic sac gives rise to a grooved pericardium with an open hemal sinus (simple heart). In ascidians, this simple heart in turn gives rise to a closed tubular, double‐layered heart–pericardial complex, with a separate pericardial cavity and a closed heart, whose wall is formed by specialized myocardium.     

Fine structure of the abdominal epidermis of the adult mudpuppy,Necturus maculosus (Rafinesque)

Summary The ventral epidermis of adult Necturus maculosus has been studied using electron and light microscopy. Many larval characteristics of amphibian epidermal structure are retained in adult Necturus. The epidermis is a stratified epithelium consisting of four cell layers and five cell types. Major differences compared with other adult amphibians are: (1) the absence of a well defined moulting cycle together with an apparently diminished synthetic and mitotic activity in the stratum germinativum; (2) an outermost cell layer (stratum mucosum) that is unkeratinized and appears to synthesize a mucous layer; and (3) numerous large club-shaped Leydig cells which span the epidermis between the cells of the stratum germinativum and stratum mucosum. The apical region of the stratum granulosum and stratum mucosum cells shows evidence of extensive synthesis. The stratum mucosum appears to be involved in the secretion of vesicular contents onto the outermost surface of the epithelium. The external surfaces of the stratum mucosum cells possess numerous microridges which are supported by an intricate network of cytofilaments in the apical region of these cells. The significance of these features is discussed in relation to the physiology and ecology of this species.     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

The formation of intercellular junctions in insect stem cell progeny (cockroach intestinal epithelium)

Summary The stages in the development of intercellular junctions have been followed in the mesenteric caecal cells of the cockroach midgut, where two types of mature cell, the columnar and the secretory, exist. Nests of undifferentiated replacement cells occur at intervals along the basal lamina, consisting of central, dividing cells and peripheral semi-lunar cells; the former act as proliferative stem cells to give rise to either pre-columnar or pre-secretory cells. The semi-lunar cells are pre-columnar and produce an attenuated process which gradually projects up to the luminal surface, producing microvilli and a dense extracellular substance en route. Intercellular gap junctions appear between these maturing columnar cell borders first, while septate junctions differentiate later; these are assembled from two different sets of intramembranous particle which become organized into either plaques or rows in parallel alignment, possibly mediated by actin filaments and microtubules. The pre-secretory cells, which are much fewer in number, remain associated only with the basal lamina and never reach the lumen; they develop into one of three distinct mature secretory cell types which release their secretory product in different ways. Offprint requests to: N.J. Lane     

Cleavage pattern,gastrulation, and neurulation in the appendicularian, <Emphasis Type="Italic">Oikopleura dioica</Emphasis>

The appendicularian, Oikopleura dioica is a chordate. Its life cycle is extremely short—approximately 5 days—and its tadpole shape with a beating tail is retained throughout entire life. The tadpole hatches after 3 h of development at 20°C. Here, we describe the cleavage pattern and morphogenetic cell movements during gastrulation and neurulation. Cleavage showed an invariant pattern. It is basically bilateral but also shows various minor left–right asymmetries starting from the four-cell stage. We observed two rounds of unequal cleavage of the posterior-vegetal B-line cells at the posterior pole. The nature of the unequal cleavages is reminiscent of those in ascidian embryos and suggests the presence of a centrosome-attracting body, a special subcellular structure at the posterior pole. The representation of the cell division pattern in this report will aid the identification of each cell, a prerequisite for clarifying the gene expression patterns in early embryos. Gastrulation started as early as the 32-cell stage and progressed in three phases. By the end of the second phase at the 64-cell stage, every vegetal cell had ingressed into the embryo, and animal cells had covered the entire embryo by epiboly. There was no archenteron formation. In the anterior region, eight A-line cells were aligned as a 2 × 4 array along the anterior–posterior axis and become internalized during the 64-cell stage. This process was considered to correspond to neurulation. The simple and accelerated development of Oikopleura, nevertheless giving rise to a conserved chordate body plan, is advantageous for studying developmental mechanisms using molecular and genetic approaches and makes this animal the simplest model organism in the phylum Chordata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987