Dicapryloylglycerol and ammonium ions induce metamorphosis of ascidian larvae

Summary Larvae of the tunicate Ciona start metamorphosis between some hours and a few days after hatching. Several substances were found to reduce this time span, such as vital dyes [Cloney (1961) Am Zool 1:67–87; Hirani (1961) Bull Mar Biol Stn Asamushi 11:121–125], heavy-metal ions including copper [see review by Lynch (1961) Am Zool 1:59–66] and the hormone thyroxine [Patricolo et al. (1981) Cell Tissue Res 214:289–301]. This study shows that low concentrations of ammonium ions as well as the second messenger dicapryloylglycerol induce metamorphosis immediately after hatching. On the other hand, when the follicle cells are removed newly hatched Ciona remain larvae for days. Follicle cells are possibly degraded by bacteria, which thereby produce ammonia.     

Presence of thyroid hormones in ascidian larvae and their involvement in metamorphosis

In this study we investigated the presence and localization of thyroxine in Ciona intestinalis larvae and its involvement in metamorphosis. To date, the mechanisms regulating the metamorphosis of ascidians remain largely unknown. In vivo treatment of swimming larvae with exogenous L-thyroxine and thiourea, and in vitro experiments utilizing high performance liquid chromatography, radioimmunoassay, and immunoperoxidase staining demonstrate the presence of thyroxine at the larval stage. This suggests that this hormone may participate in the control of metamorphosis and thus play a different role from that observed in adults.     

Dopamine and serotonin modulate the onset of metamorphosis in the ascidian Phallusia mammillata

Neurotransmitters play an important role in larval metamorphosis in different groups of marine invertebrates. In this work, the role of dopamine and serotonin during metamorphosis of the ascidian Phallusia mammillata larvae was examined. By immunofluorescence experiments, dopamine was localized in some neurons of the central nervous system and in the adhesive papillae of the larvae. Dopamine and serotonin signaling was inhibited by means of antagonists of these neurotransmitters receptors (R(+)-SCH-23390, a D(1) antagonist; clozapine, a D(4) antagonist; WAY-100635, a 5-HT(1A) antagonist) and by sequestering the neurotransmitters with specific antibodies. Moreover, dopamine synthesis was inhibited by exposing 2-cell embryos to alpha-methyl-l-tyrosine. Dopamine depletion, obtained by these different approaches, caused early metamorphosis, while serotonin depletion delayed the onset of metamorphosis. The opposite effects were obtained using agonists of the neurotransmitters: lisuride, a D(2) agonist, inhibited metamorphosis, while DOI hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So, it is possible to suppose that dopamine signaling delayed metamorphosis while serotonin signaling triggers it. We propose a mechanism by which these neurotransmitters may modulate the timing of metamorphosis in larvae.     

Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis

Ascidians are a group of invertebrate chordates that exhibit a biphasic life history, with chordate-specific structures developing during embryogenesis (dorsal neural tube and notochord) and metamorphosis (pharyngeal gill slits and endostyle). Here we characterize the expression of a caudal/Cdx gene homologue, Hec-Cdx, from the ascidian Herdmania curvata. Vertebrate Cdx genes are expressed at gastrulation and in the posterior of the developing neural tube and endoderm. Hec-Cdx expression is initiated at the earliest stages of gastrulation, with peaks in RNA abundance occurring first during neurulation and tailbud extension and then in 3- to 5-day-old juveniles. Hec-Cdx is expressed in a pair of cells in the anterior lip of the blastopore in the late gastrula which form the most posterior portion of the neural plate. During tailbud formation expression is maintained in and solely restricted to these cells. During metamorphosis expression is localized to the intestine of the juvenile. These data, along with data for the H. curvata Otx gene, suggest that the evolution of the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a temporal separation of tissue-specific expression. Received: 10 October 1999 / Accepted: 1 November 1999     

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neogoniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H. curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in N. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Rhythmic contractions of the ampullar epidermis during metamorphosis of the ascidian Molgula occidentalis

Summary The ampullae of Molgula occidentalis are hollow, tubular extensions of the epidermis. They are ensheathed by a secreted tunic. When they grow out shortly after settlement, the ampullae spread the tunic over the substratum to form a firm attachment for the sessile juvenile. A simple squamous epithelium forms the thin ampullar walls. A glandular, simple columnar epithelium forms the distal tip of each ampulla. The glandular cells probably secrete the adhesive that attaches the tunic to the substratum.Repetitive, peristaltic contractions pass from the base to the distal end of each ampulla. Microsurgery, time-lapse cinemicrography and TEM have been used to analyze this phenomenon. The contractions are mediated by a layer of 4–8 nm microfilaments in the base of the ampullar epithelium.Each juvenile has 7–9 ampullae which contract at different frequencies. Isolated ampullae continue to contract normally for several days. Thus each ampulla has an intrinsic rhythm. Microsurgical experiments suggest that there is no specific region within an ampulla with unique pacemaker properties. It is proposed that communication via gap junctions allows the coordination of ampullar cells into a well organized peristaltic wave.     

Hemps, a novel EGF-like protein, plays a central role in ascidian metamorphosis

All chordates share several characteristic features including a dorsal hollow neural tube, a notochord, a pharynx and an endostyle. Unlike other chordate taxa, ascidians have a biphasic life-history with two distinct body plans. During metamorphosis, the larval nerve cord and notochord degenerate and the pharyngeal gill slits and endostyle form. While ascidians, like other marine invertebrates, metamorphose in response to specific environmental cues, it remains unclear how these cues trigger metamorphosis. We have identified a novel gene (Hemps) which encodes a protein with a putative secretion signal sequence and four epidermal growth factor (EGF)-like repeats which is a key regulator of metamorphosis in the ascidian Herdmania curvata. Expression of Hemps increases markedly when the swimming tadpole larva becomes competent to undergo metamorphosis and then during the first 24 hours of metamorphosis. The Hemps protein is localised to the larval papillae and anterior epidermis of the larva in the region known to be required for metamorphosis. When the larva contacts an inductive cue the protein is released, spreading posteriorly and into the tunic as metamorphosis progresses. Metamorphosis is blocked by incubating larvae in anti-Hemps antibodies prior to the addition of the cue. Addition of recombinant Hemps protein to competent larvae induces metamorphosis in a concentration-dependent manner. A subgroup of genes are specifically induced during this process. These results demonstrate that the Hemps protein is a key regulator of ascidian metamorphosis and is distinct from previously described inducers of this process in terrestrial arthropods and aquatic vertebrates.     

ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles

In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. We demonstrate that the MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, we identified the network of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, we show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. These observations lead us to propose a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi.     

The influence of temperature and light on larval pre-settlement metamorphosis: a study of the effects of environmental factors on pre-settlement metamorphosis of the solitary ascidian Styela canopus

This study investigated the effects of two environmental factors, temperature and light, on larval settlement and metamorphosis in the solitary ascidian Styela canopus. The results revealed that larval settlement rates decreased with increasing temperature in the range 12–30°C. We also demonstrated for the first time that pre-settlement metamorphosis of ascidian larvae can occur as a function of temperature. We suggest this could be an adaptation to avoid the greater energetic cost of active larval swimming, presumably resulting from the increasing temperature. They are able to metamorphose into passive drifting post-larvae and to continue planktonic life. This finding has implications for larval dispersal, especially under conditions of ocean warming. In addition, the effect of light intensity on larval settlement and metamorphosis was significantly different between photoperiods of 24 L : 0 D and 12 L : 12 D. These results provide some insight into the complex cues affecting settlement and metamorphosis of ascidian larvae and ascidian distribution in nature.