The influence of tunichrome and other reducing compounds on tunic and fin formation in embryonic Ascidia callosa Stimpson

Tunic in 46-hr-old Ascidia callosa larvae reared from dechorionated neurulae is either markedly reduced in thickness or absent altogether. The epidermis is fragile and cuticular fins fail to develop. Dechorionated neurulae treated with tunichrome and other reducing compounds (e.g., glutathione, ascorbate) show an enhancement in tunic formation and rudimentary fin development. UV absorbance spectra of extracts from unfertilized eggs, late tail-bud embryos, and tadpole larvae indicate that tunichrome may be present in all developmental stages. Experiments with neurulae in which the chorion was punctured with tungsten needles but not removed signify that the test cells are the most likely source of tunichrome. Results are consistent with the hypothesis that tunichrome is involved in the natural processes of tunic morphogenesis in ascidian embryos.     

Larval Tunic and the Function of the Test Cells in Ascidians

Abstract In normal ascidian development, cuticular fins begin to form at the late tailbud stage and are fully formed at hatching. When one or several neurulae were manually demembranated (follicle cells, vitelline coat and test cells removed) and cultured in seawater they failed to form caudal fins. Fins were normal when the follicle cells alone were removed. The shape of the fins was normal when demembranation was delayed to the late tailbud stage. Does demembranation cause the loss of an essential factor produced by the embryos themselves or do the test cells provide a factor for fin morphogenesis? Demembranated neurulae of Ascidia callosa were cultured in groups ranging in size from 2 to 80 in 1 ml volumes of seawater. The mean lengths of the caudal fins increased with group size. In larger groups, some embryos developed fins that were normal in shape and as long as undemembranated controls. Results were similar with Corella inflata. These experiments suggest that a diffusible substance from the embryos facilitates fin morphogenesis and that test cells are not required. Test cells deposit ‘ornaments’ on the tunic in some species. In other species no ornaments are produced. Ten families are compared. It is proposed that the test cells make the tunic hydrophilic.     

Implication of Catecholamines During Spawning in Marine Bivalve Molluscs

Summary

During the tail-bud stage of Ascidiella aspersa embryogenesis, the test cells or innermost cells of the egg envelope manifest locomotive activities. Light microscopy further reveals that the adhesive behaviour of test cells changes in the course of embryogenesis. Mechanical dechorionation experiments performed on 846 embryos demonstrate that up to the tail-bud stage all test cells are attached to the inner surface of the chorion. The embryo is completely devoid of test cells. At the onset of larval tunic secretion, increasing numbers of test cells settle on the embryo until all test cells adhere to it. This switch in adhesive properties is completed within 65 min. The hatched larva carries the entire complement of test cells until the onset of metamorphosis. SEM and observations show that test cells do not establish direct cell-to-cell contacts to ectodermal cells but attach to the larval tunic.     

Initial stages of tunic morphogenesis in the ascidian Halocynthia: a fine structural study

Tunic morphogenesis in embryos of the ascidian Halocynthia roretzi was examined by scanning and transmission electron microscopy. For this purpose it was necessary to modify the classical embedding procedure. Soon after reaching the initial tail-bud stage, tunic deposition is initiated on the dorsal side of the embryo. As soon as the embryo is completely covered by the tunic, larval fins are formed. The test cells settle onto the embryo. At this stage only the outer cuticle and the outer tunic compartment have appeared. Tunic morphogenesis is accompanied by ultrastructural modifications of the epidermis characteristic of secreting cells. Cytochemical investigations reveal polysaccharide glycogen-like material in the lumen of epidermal lacunae and in the outer compartment of the tunic. Our observations strongly suggest that this material is stored in the lacunae and discharged into the outer compartment. The significance of fluffy osmiophilic material that appears at the early tail-bud stage and enlaces the whole embryo is discussed.     

Ascidian Larvae: The Role of Test Cells in Preventing Hydrophobicity

Abstract Ascidian test cells co-differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species, their vacuoles contain submicroscopic granules or filaments called ‘ornaments’ and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species, the test cells are lost at hatching. In other species, the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species, the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during midembryogenesis. After completing their development, the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.     

Behavior and cellular morphology of the test cells during embryogenesis of the ascidian Halocynthia roretzi

During the early stages of embryogenesis of the ascidian Halocynthia roretzi the test cells creep exclusively on the inner surface of the chorion. Concomitant with elongation of the embryonic tail, however, the test cells begin to gather around the embryo and finally cover the whole embryo. The time at which the test cells surround the embryo almost coincides with that of initiation of larval tunic formation. Scanning electron microscope observations revealed that the test cells extend numerous cytoplasmic processes or pseudopodia. During larval tunic formation, the test cells compose a net by intertwining their filopodia, and the cell net covers the whole embryo.     

Self and non-self recognition between gametes of the ascidian,Halocynthia roretzi

Summary The self-sterility ofHalocynthia roretzi from Mutsu Bay, Japan, was examined. This sterility is strict and not a single egg can be fertilized in self-sterile animals. Less than 2% of the animals were self-fertile (with 100% cross-fertility). All heterologous sperm can fertilize all eggs, although there are pairs of individuals in which the coelomocytes recognize each other as self. Eggs deprived of follicle cells cannot be fertilized by either autologous or heterologous spermatozoa. Detached autologous or heterologous follicle cells can reattach to the chorion in calcium-enriched sea water and the reconstituted eggs recover their ability to be fertilized. A mosaic egg can therefore be obtained, which consists of oocyte, test cells and chorion originating from one individual and follicle cells from another. The mosaic egg was used to determine the site of recognition of self and non-self. The results indicate that the recognition resides in the chorion and/or test cells, probably the chorion. The relationship between somatic alloreactivity, previously found in coelomocytes ofH. roretzi, and gamete reactivity is discussed.     

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea). 1: demonstration of polysaccharides

The distribution of carbohydrates was demonstrated in the embryonic, larval, and juvenile tunics of Halocynthia papillosa. An enzyme-gold marker (cellobiohydrolase-Au) was used to identify cellulose on ultrathin sections. This is the first time this biopolymer has been detected in the embryonic or larval tunic of an ascidian. Cellulose is present from the initial tail-bud stage onwards, as soon as the outer compartment of the tunic appears. Both compartments of the larval tunic also contain non-cellulosic polysaccharides, as demonstrated by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Our observations point to two types of cellulose synthesis. One occurs during the embryonic and larval stages, when glycogen-like material is stored in epidermal intracellular lacunae and discharged into the tunic where it is presumably used to synthesize cellulose throughout the depth of the tunic. The second occurs from the onset of metamorphosis onwards, just above the apical plasmalemma of epidermal cells, like cellulose biogenesis in plants.     

Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, Ascidiacea)

The larvae of the ascidian Ciona intestinalis from which the chorion with the test cells and follicle cells were removed developed normally without the test cells until the early tailbud stage. A number of round-shaped cells morphologically similar to the test cells but with different lectin affinities and autofluorescence, then appeared on the neck region of the demembranated embryos. The new cells had three different types: round, particulate, and granular, and these cells increased in number after the late tailbud stage. The morphology of the adhesive papillae, tunic layers and epidermis of the demembranated larvae was similar to that of control larvae; however, the affinity to lectins was different in the swimming period. Control larvae attached to the substratum after the swimming period, resorbed the tail completely and underwent rotation of the visceral organs. Conversely, rotation occurred before completion of tail resorption in the demembranated larvae. Furthermore, the metamorphic events progressed more slowly in the demembranated larvae. These results suggest that the test cells play important roles in normal development and morphogenesis of ascidian larvae. Received: 4 December 1998 / Accepted: 9 April 1999     

THE EFFECTS OF CONTRASTING MODES OF FERTILIZATION ON LEVELS OF INBREEDING IN THE MARINE INVERTEBRATE GENUS CORELLA

A simple difference in the body design of two species of marine urochordates in the ascidian genus Corella suggested that these species may differ in their mating systems. The two coexisting species share common life-history traits and morphology with the exception of a difference in body design that affects site of fertilization and embryonic development. Corella inflata has internal fertilization and embryonic development, while C. willmeriana has external fertilization and embryonic development. The natural mating system of these two species of solitary ascidians was inferred by comparing the relative survival of selfed and outcrossed fertilizations in the laboratory. Corella inflata, the internal fertilizer, showed no difference in survival between selfed and outcrossed fertilizations at any developmental stage through metamorphosis and early juvenile development. In contrast, self-fertilized crosses of C. willmeriana had significantly lower survival than outcrossed fertilizations even at the earliest scorable developmental stages. These results suggest that C. inflata may inbreed frequently in nature, while viable C. willmeriana offspring are primarily a result of outcrossing. The internally-fertilizing species, C. inflata, showed approximately 10% male sterility in laboratory crosses despite apparent morphological hermaphroditism. The externally-fertilizing, commonly outcrossing species, C. willmeriana, showed no difference in fertility between genders.     

DNA sequence of a 3.8 kilobase pair region controlling Drosophila chorion gene amplification

During Drosophila oogenesis, two clusters of chorion genes and their flanking DNA sequences undergo amplification in the ovarian follicle cells. Amplification results from repeated rounds of initiation and bidirectional replication within the chorion gene regions, possibly from a single origin, producing nested replication forks. Previously we have shown that following reintroduction into the Drosophila genome, a specific 3.8 kilobase pair DNA segment from the amplified third chromosome domain could induce developmentally regulated amplification at its site of insertion. Here we present the complete nucleotide sequence of this amplification control element and of genes encoding the chorion structural proteins s18-1 and s15-1, which are contained within it. Sequences that may be involved in the regulation of chorion gene amplification and expression are identified.     

Test cell migration and tunic formation during posthatching development of the larva of the ascidian, Ciona intestinalis

Morphological changes in the tunic layers and migration of the test cells during swimming period in the larva of the ascidian, Ciona intestinalis , were observed by light and electron microscopy. The swimming period was divided into three stages. In stage 1, further formation of juvenile tunic layer started only in the larval trunk and neck region. In stage 2, the layer became swollen in the ventral and dorsal sides of the neck region and in stage 3, the swelling expanded backward. Concomitantly with these changes, the outermost larval tunic layer (outer cuticular layer), which had been formed before hatching, also swelled in the neck region in stage 2 and formed two humps in stage 3, although the layer did not change in the tail region during the swimming period. Test cells that were present over the entire larval tunic layer in stage 1 began to move from the surface of the fin toward that of the side of the body in stage 2, and finally gathered to form six bands running radially from the anterior end to the posterior end of the trunk region and aligned along the lateral sides of body in the tail region in stage 3. In electron microscopic observations, pseudopodia protruding from the test cells invaded the larval tunic, following which they extended proximate to the juvenile tunic in the trunk region. In the tail region, which had no juvenile tunic layer as that described, the pseudopodia invaded and remained adjacent to the surface of the epidermis or the sensory cilia protruded from the epidermis. Metamorphosis of the larvae, further tunic formation, degradation of adhesive papilla, attachment of larva to the substratum and tail resorption commenced after these morphological changes occurred. The possible role of the test cells in metamorphosis is discussed.     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

Ultrastructural studies on oogenesis in symphyla

Summary In the oocytes of Hanseniella nivea, cortical granules are formed in the peripheral ooplasm during late stages of oogenesis. Single Golgi elements are involved in the process. Concurrent with the formation of cortical granules is the appearance of a chorion on the oocyte surface. Precursors of this envelope are most likely synthesized by follicle cells.     

The role of the follicular epithelium in growing eggs of a dipteran insect during late oogenesis and cleavage

The gall midge Heteropeza pygmaea can reproduce by means of paedogenesis (i.e., larval parthenogenesis). In that process, follicles are produced that develop while floating in the hemocoele of the mother larva. A chorion is not formed at the end of oogenesis, and the growing embryos remain enveloped by the follicular epithelium. To investigate possible adaptations of the follicular epithelium to this unusual egg development, its ultrastructure has been studied during late oogenesis and cleavage. Earlier investigations had shown that the follicle cells are provided with a specifically arranged microtubular frame, which may be responsible for the anisometric growth of the egg. The present work shows that the follicle cells are always joined by desmosomes and septate junctions. During development, the septate junctions increase their surface and change their orientation to become parallel to the longitudinal egg axis, thus increasing the resistance of the follicle cells to being torn apart by growth tensions. The total surface of the follicular epithelium increases during development. Well-developed nucleoli in the nuclei and numerous ribosomes in the cytoplasm of follicle cells indicate a high level of synthetic activity. This activity may be required to support the increase in the membrane surface and the establishment of the microtubular frame. Lipid droplets, glycogen, and different inclusions in the follicle cells may represent nutrient and energy reserves. Structures indicating a quantitative significant transfer of nutrients from the follicle cells to the egg were not found.     

A new cellulosic structure, the tunic cord in the ascidianPolyandrocarpa misakiensis

Summary A specialized structure of tunic cord inPolyandrocarpa misakiensis is investigated by electron microscopy. The tunic cord is a cord-like coiled structure of 5–30 m in diameter and 0.1–9.0 mm in length. The tunic cords originate and elongate from the dorsal tunic, and their termini have a swollen and ornamented structure. Scanning and transmission electron micrographs and the electron diffractogram show that the tunic cords are composed of bundled microfibrils of cellulose I with high crystallinity. The tunic cord is completely surrounded by single-layered epidermal cells, which have been found as the site of cellulose biosynthesis. A number of tunic cords are connected to the internal tunic of the siphon by forming eyelet structures at their termini. These observations suggest that the tunic cords act as a connector between dorsal and internal tunic of the siphon.     

Nervous system of ascidian larvae: Caudal primary sensory neurons

Summary In larvae of Diplosoma macdonaldi one sensory nerve extends along the dorsal midline of the tail and another extends along the ventral midline. Each nerve is composed of 50–70 naked axons lying in a groove in the base of the epidermis, and each projects to the visceral ganglion. The cell bodies of the caudal sensory neurons occur in pairs within the epidermis, and are situated along the courses of the nerves. A single cilium arises from an invagination in the soma of each neuron, passes through the inner cuticular layer of the tunic and enters a tail fin formed by the outer cuticular layer. We propose that these cells are mechanoreceptors. The caudal sensory system is similar in representative species of ten families of ascidians.Abbreviations a axial complex of the tail - ac accessory centriole - ax axon - bb basal body - bl basal lamina - c cilium - cep common epidermal cells - cs ciliary sheath - dcv dense-cored vesicles - dsn dorsal sensory nerve - ec ependymal cells - ep epidermis - gj gap junction - h hemocoel - hc hemocoelic chamber - icl inner cuticular layer of the tunic - m caudal muscle - nc dorsal nerve cord - ncl neurocoel - no notochord - ocl outer cuticular layer of the tunic - sc sensory cell - sn sensory nerve - sv sensory vesicle - vg visceral ganglion - vsn ventral sensory nerve     

Ovulation and egg segregation in the tunic of a colonial ascidian,Diplosoma listerianum (Tunicata,Ascidiacea)

Summary The process of egg segregation in the tunic of the ovoviviparous ascidian Diplosoma listerianum was studied by light and electron microscopy. One egg at a time was seen to mature in each zooid. The eggs had large yolk and grew on the ovary wall enveloped in four layers: (1) outer follicle cells (OFC), long and rich in RER (rough endoplasmic reticulum) and with dense granules in the Golgi region; (2) flat inner follicle cells (IFC); (3) a loosely fibrillar vitelline coat (VC); (4) test cells encased on the egg surface. The growing egg protrudes from the ovary wall and presses on the contiguous epidermis. Granulocytes enter the space between the epidermis and the egg and insinuate cytoplasmic protrusions, disrupting the continuity of the OFC layer. At ovulation, OFC and IFC are discharged and form a post-ovulatory follicle (corpus luteum). The epidermis shrinks and closes, possibly by activation of microfilaments, causing the egg to be completely surrounded by the tunic. In the zooid, the wound caused by the passage of the egg is repaired both by contraction of the epidermis and by phagocytic activity. Altered spermatozoans are found in phagocytosing cells in the lumen of the ovary. These are presumably remnants of those which entered to fertilize the egg before segregation.     

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea) 2: demonstration of proteins

In a previous paper, cellulose fibres were demonstrated in the larval, the metamorphosing, and the juvenile tunics. In this paper we used cytochemical methods and X-ray microanalysis to obtain additional information on tunic morphogenesis in Halocynthia papillosa. The chemical composition of the tunic evolves with its structural complexity. The larval and juvenile fibres are shown to be structurally and chemically different. While neither proteins nor glycosaminoglycans seem to be associated with the larval fibres, the juvenile fibres consist of a cellulose core wrapped in a sheath of tannophilic proteins. Patches of glycosaminoglycans line their longitudinal axes. In the course of metamorphosis, the cuticle undergoes profound modifications in regions of spine morphogenesis. Granular material that was previously called fibro-granular material (Lübbering et al., 1993) is essential to the formation of cuticular plates and spines. During metamorphosis, this material accumulates in epidermal granules and is discharged into the tunic. It crosses the fundamental layer of the tunic and reaches the cuticle. Our results strongly suggest that this material consists of proteins rich in cysteine and hydrophobic amino acids.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.