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.     

C- and H-Tyrosine incorporation into Ascidia ceratodes tunichrome in vivo

Radiolabeled tyrosine is incorporated into newly biosynthesized tunichrome of Ascidia ceratodes at a rate comparable to previous findings with phenylalanine. ³H-tyrosine incorporation indicates that formation of the catechol and pyrogallol moieties occurs via the NIH shift pathway. Radiolabel is found in many compartments other than solely tunichrome (tunic > whole body ? blood plasma ? tunichrome). The amount of label in the tunic, whole bodies and blood plasma declines over the 14 days following exposure to ³H-tyrosine, whereas the radiolabel in the tunichrome continues to increase. These results are consistent with two alternative hypotheses: tunichromes may be formed either by sequential hydroxylation of di- and tri-peptide precursors, or by selective cleavage of a larger DOPA- or TOPA-containing compound. In either event, hydroxylation may precede formation of tunichrome's carbon backbone.     

Ascidian larval tunic: Extraembryonic structures influence morphogenesis

Summary The larval tunic of Corella inflata is composed of two cuticular layers, extracellular filaments and ground substance. It lies outside the epidermis and most of it is known to be produced by the epidermis. The dorsal, ventral and caudal fins are specialized parts of the tunic that are essential for larval locomotion. The following hypothesis was tested: Morphogenesis of the larval fins is dependent upon the presence of extraembryonic structures (test cells, chorion or follicle cells) before completion of the late tail bud stage of development. We tested this by dechorionating embryos of Corella inflata and Ascidia paratropa. The operation removes all extraembryonic structures. It was performed mainly on neurula, early tail-bud and late tail-bud stages.Fin formation is inhibited when neurulae are dechorionated but not when late tail-bud or older embryonic stages are dechorionated. Dechorionated neurulae produce all of the major components of the tunic (cuticular layers, filaments and ground substance) but they are unable to form functional fins. At the time of dechorionation, in all experiments, the embryos had no fins.Removal of the follicle cells does not inhibit fin formation. The test cells are known to secrete granular ornaments that attach to the surface of the tunic. The fibrous, acellular chorion may serve to contain the test cells and their products or products of the embryo that are not firmly attached. The test cells may induce or control the morphogenesis of the larval fins in ascidians before the late tail-bud stage of development. We suggest ways of testing this hypothesis and an alternative hypothesis.     

Oxidative transformation of a tunichrome model compound provides new insight into the crosslinking and defense reaction of tunichromes

Tunichromes, small oligopeptides with dehydrodopa units isolated from the blood cells of ascidians, have been implicated in the defense reactions, metal binding, wound repair, or tunic formation. Their instability and high reactivity has severely hampered the assessment of their biological role. Experiments conducted with the model compound, 1,2-dehydro-N-acetyldopamine, indicated that the instability of tunichromes is due to this basic structure. Exposure of this catecholamine derivative to even mild alkaline condition such as pH 7.5 causes rapid nonenzymatic oxidation. High performance liquid chromatography and mass spectrometry studies confirmed the production of dimeric and other oligomeric products in the reaction mixture. The nonenzymatic reaction seemed to proceed through the intermediary formation of semiquinone free radical and superoxide anion. Ultraviolet and visible spectral studies associated with the oxidation of tunichromes isolated from Ascidia nigra by tyrosinase indicated the probable formation of oligomeric tunichrome products. Attempts to monitor the polymerization reaction by mass spectrometry ended in vain. Tunichrome also exhibited instability in mild alkaline conditions generating superoxide anions. Based on these studies, a possible role for oxidative transformation of tunichrome in defense reaction, tunic formation and wound healing is proposed.     

Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.     

More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus neurula embryos

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation.     

Embryonic and larval development of a cold adapted Antarctic ascidian

Ova of the Antarctic ascidian Cnemidocarpa verrucosa were mature at 240–245 μm. At 0 to −1.5°C, embryos hatched as swimming tadpoles at 8 days from fertilization, which is close to the ages at which some Antarctic echinoderm and nemertean embryos hatch as blastulae. Comparisons of Antarctic and temperate ascidian larvae suggest that the ascidian’s development rate is affected by low environmental temperatures to about the same extent as embryos and larvae of an echinoid, nemertean, and calanoid copepods. The ascidian’s tadpoles were bright orange and large, >2 mm in length including tunic and >1.5 mm in length without tunic. The large and brightly colored tadpoles were conspicuous when swimming, which supports the hypothesis that larvae of C. verrucosa are chemically defended against predators. Metamorphosed juveniles were found in cultures within 16 days from fertilization, when some unsettled tadpoles still moved but were less active. The potential pelagic period may therefore be 16 or more days with 8 days as an unhatched embryo and up to 8 or more days as a tadpole. The resting metabolic rate of tadpole larvae was 15 pmol O₂ h⁻¹ individual⁻¹ which is equivalent to larval respiration rates in Antarctic echinoderms. A low resting metabolic rate suggests a potential mechanism for the extended larval lifespan in C. verrucosa.     

Quantitative aspects of RNA synthesis in normal and lithium-treated embryos ofXenopus laevis

Summary The treatment ofXenopus early embryos with lithium chloride produces exogastruale — embryos which fail to gastrulate normally and in which the rates of cell division are reduced. In the present study estimations of incorporations of (5-³H) uridine and the specific activities of the 5-ribonucleotide precursor pools showed that exogastrulae have higher rates of RNA synthesis per cell than control neurulae. Sub-cellular fractionations showed that a greater proportion of labelled RNA was retained in the nuclei of exogastrulae than of neurulae, while neurulae showed a greater incorporation into polysomes.     

Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.     

Ontogeny of the appendicularian <Emphasis Type="Italic">Oikopleura dioica</Emphasis> (Tunicata,Chordata) reveals characters similar to ascidian larvae with sessile adults

Appendicularians have always occupied a central role in considerations of tunicate and chordate evolution. Two hypotheses have been proposed – one holds that appendicularia represents the sister taxon to the remaining tunicates, the other suggests that appendicularians were derived from an ascidian-like ancestor. In the present study I report results from electron microscopic investigation of larval tunicates including the first electron microscopic investigation of the tail of the early ontogenetic appendicularian “Streckform” and discuss their phylogenetic implications. The early “Streckform” of Oikopleura dioica Fol, 1872 is invested with an extracellular covering that consists of an inner electron-light layer and an electron-dense outermost layer. In addition, the extracellular covering forms fin blades. Because these traits are shown to be similar to the tunic of different ascidian larvae, the extracellular covering in early appendicularian embryos is suggested to be homologous to the larval tunic of ascidian larvae. Overall, the tail of early developmental stages of appendicularians consists of a mosaic of apomorphic and plesiomorphic features. The straight, continuous endodermal strand was inherited from a common chordate ancestor whereas the finlets of larvae, consisting of extracellular material, were inherited from a common tunicate ancestor. The horizontal orientation of the tail as a whole was inherited from the last common ancestor of appendicularians and aplousobranch ascidians, and the discovered floating extension at the posterior tip of the tail is unique to the holoplanktonic Oikopleura dioica. These findings support the hypothesis that Appendicularia is derived from a sessile, ascidian-like ancestor.     

Isolation, properties and structural studies on a compound from tunicate blood cells that may be involved in vanadium accumulation.

A novel compound, for which the trivial name tunichrome is proposed, was isolated from the vanadium-rich blood cells of the tunicate Ascidia niga. Preliminary structural studies suggest a molecular weight of about 390, the presence of conjugated vinyl groups, and an acidic group, possibly carboxyl, with an apparent pKa of 3.0. Elements C, H, N and O comprise 98.4% of the sample weight, the number of atoms per mol of tunichrome being 14.1, 22.2, 1.5 and 10.6 respectively, which indicates some heterogeneity in the sample. Tunichrome readily reduces Fe(III) and V(V). In an initial fast step, 2 mol of V(V) are reduced, or 4 mol of Fe(III)-phenanthroline per mol of tunichrome; in a further slow reaction, another 9 mol of Fe(III)-phenanthroline or Fe(III)-bipyridine are reduced. The initial reaction is first-order with respect to tunichrome and Fe(III). Above pH 3.5, tunichrome is rapidly hydrolysed, 13 mol of OH- being consumed per mol of tunichrome. The hydrolysis involves polymerization and loss of the characteristic absorption peak at 325 nm. It is suggested that the presence of tunichrome may be linked to vanadium accumulation by the blood cells. The mechanism involves entry of vanadate via an anionic channel into vacuoles of the blood cells, where it is reduced to V(IV) or V(III), both of which, being cationic, cannot escape from the vacuole.     

Early embryo orientation by direction of the cardinal point in natural clutches of two ranidae species inhabiting geographically distant regions

The azimuth (the least angle with the north-south direction) of the first cleavage furrow and anteroposterior axes of neurula was measured on projections of photographs of natural clutches. The azimuth distribution of the craniocaudal axis of Rana dalmatina neurulae in clutches from southern Italy and the first cleavage furrows of R. arvalis embryos in clutches from central Russia proved identical. Both craniocaudal axes and first cleavage furrows were mostly oriented from west to east. The azimuth distribution of the craniocaudal axis of R. arvalis neurulae in clutches subjected to repeated cold shock proved to be random. The causes and mechanisms of predominant orientation of the embryos in natural clutches of frogs are discussed. We propose that magnetic sensitivity is acquired by cytoskeleton elements, most likely microtubules, during reorganization in the course of normal development or due to experimental influences.     

SCANNING ELECTRON MICROSCOPICAL APPEARANCE OF CELLS FROM XENOPUS LAEVIS EMBRYOS OF DIFFERENT STAGES

The scanning electron microscopical appearances of cells isolated from different regions of Xenopus laevis embryos of different stages, and cultured in vitro have been compared. Blastula inner ectoderm cells initially show filopodia, then become flattened onto the substrate and then form pseudopodia. Blastula outer ectoderm cells are initially similar, but do not form pseudopodia. Most of the ectoderm cells from gastrulae and neurulae are featureless. Endoderm cells from blastulae do not initially form filopodia, but later form pseudopodia. Most of the endoderm cells from gastrulae and neurulae show neither filopodia nor pseudopodia, but in the gastrula some elongated, cylindrical cells are observed. Thus cells change their appearance after the three hour culture period; cells from different regions of embryos of the same stage show different appearances in vitro ; and cells from equivalent regions of embryos of different stages show different behaviours in vitro.     

Functional gametes derived from explants of single blastomeres containing the "germ plasm" in Xenopus laevis: a genetic marker study

Single blastomeres containing the "germ plasm" were isolated from 32-cell embryos of Xenopus albino (ap/ap) or wild type and cultured in vitro until the corresponding normal control embryos reached the neurula stage. The resulting explants from albinos were implanted into wild-type host neurulae and vice versa. The formation of functional gametes, eggs or sperm, of donor type was tested when the operated host embryos had reached sexual maturity. The color of the eggs laid by the experimental females and the presence or absence of melanophores in the epidermis and of pigment granules in the eyes of hatched larvae from matings of the experimental males with albino females made possible the identification of donor-type gametes. Twelve males and 12 females of the wild-type hosts, and 16 males and 14 females of the albino hosts survived. Six animals produced donor-type eggs or sperm, most of them being germ line chimeras. This shows that functional gametes can develop from explants derived from single blastomeres containing the "germ plasm."     

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     

Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

A METHOD FOR THE REMOVAL OF THE JELLY AND VITELLINE MEMBRANE FROM THE EMBRYOS OF XENOPUS LAEVIS

A simple procedure is described for removing the jelly and vitelline membrane of Xenopus laevis embryos. The method is based on the observation that incubation of the embryos in the mixed solution of trypsin and sodium thioglycolate at pH 8.0 causes effective dissolution of these structures. This solution is equally effective in this respect on the embryos at different developmental stages. Normal development is obtained from all of the denuded neurulae and from many of the denuded earlier embryos. Some chemical properties of the jelly and the vitelline membrane of Xenopus laevis are discussed based upon these observations.     

Early embryo orientation with respect to the cardinal points in natural clutches of two ranidae species inhabiting geographically distant regions

The azimuth (the least angle with the north-south direction) of the first cleavage furrow and anteroposterior axes of neurula was measured on projections of photographs of natural clutches. The azimuth distribution of the craniocaudal axis ofRana dalmatina neurulae in clutches from southern Italy and the first cleavage furrows ofR. arvalis embryos in clutches from central Russia proved identical. Both craniocaudal axes and first cleavage furrows were mostly oriented from west to east. The azimuth distribution of the craniocaudal axis ofR. arvalis neurulae in clutches subjected to repeated cold shock proved to be random. The causes and mechanisms of predominant orientation of the embryos in natural clutches of frogs are discussed. We propose that magnetic sensitivity is acquired by cytoskeleton elements, most likely microtubules, during reorganization in the course of normal development or due to experimental influences.     

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.