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.     

The multi-level regulation of clownfish metamorphosis by thyroid hormones

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Induction of metamorphosis in the sand dollar Peronella japonica by thyroid hormones

The larva of the sand dollar Peronella japonica lacks a mouth and gut, and undergoes metamorphosis into a juvenile sand dollar without feeding. In the present study, it was found that thyroid hormones accelerate the metamorphosis of P. japonica larvae. The contents of thyroid hormones in larvae increased gradually during development. Thiourea and potassium perchlorate, inhibitors of thyroid hormone synthesis, delayed larval metamorphosis and simultaneously repressed an increase in the content of thyroxine in the larval body. These results suggest that the P. japonica larva has a system for synthesis of thyroid hormones that act as factors for inducing metamorphosis.     

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis

Abstract. Metamorphosis in the South African clawed frog, Xenopus laevis , is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T₄) and triiodothyronine (T₃). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH.
These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH.
Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.     

Comparative analysis of thyroxine distribution in ascidian larvae

The ascidian endostyle is a mucus-secreting pharyngeal organ, it has iodine-concentrating activity and the biosynthesis of thyroid hormones has been well documented. According to our recent findings, ascidians possess thyroid hormones, which are localized in mesenchymal cells. We have studied the presence and localization of l-thyroxine (T₄) in Ascidia malaca (Traustedt), Ascidiella aspersa (Müller), Phallusia mamillata (Cuvier) and Ciona intestinalis (Linnaeus) larvae and its involvement in metamorphosis. In vivo treatment of swimming larvae with exogenous T₄ and thiourea (a thyroid hormone synthesis inhibitor), demonstrate the presence of T4 during larval development. These results were confirmed by in vitro experiments utilizing dot blotting, radioimmunoassay and immunoperoxidase staining. The hormone was localized in mesenchymal cells of all four ascidians, spread out in the body cavity, under the adhesive papillae and around the intestine. The presence of TH in mesenchymal cells could be related to blood cells, musculature and heart tissue differentiation. The results suggest that this hormone could be involved in the control of metamorphosis.     

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.     

Nervous network in larvae of the ascidian Ciona intestinalis

 With the use of the monoclonal antibody UA301, which specifically recognizes the nervous system in ascidian larvae, the neuronal connections of the peripheral and central nervous systems in the ascidian Ciona intestinalis were observed. Three types of peripheral nervous system neurons were found: two located in the larval trunk and the other in the larval tail. These neurons were epidermal and their axons extended to the central nervous system and connected with the visceral ganglion directly or indirectly. The most rostral system (rostral trunk epidermal neurons, RTEN) was distributed bilateral-symmetrically. In addition, presumptive papillar neurons in palps were found which might be related to the RTEN. Another neuron group (apical trunk epidermal neurons, ATEN) was located in the apical part of the trunk. The caudal peripheral nervous system (caudal epidermal neurons, CEN) was located at the dorsal and ventral midline of the caudal epidermis. In the larval central nervous system, two major axon bundles were observed: one was of a photoreceptor complex and the other was connected with RTEN. These axon bundles joined in the posterior sensory vesicle, ran posteriorly through the visceral ganglion and branched into two caudal nerves which ran along the lateral walls of the caudal nerve tube. In addition, some immunopositive cells existed in the most proximal part of the caudal nerve tube and may be motoneurons. Received: 8 September 1997 / Accepted: 14 December 1997     

Role of thyroid hormones and their receptors in peripheral nerve regeneration.

After peripheral nerve injury in adult mammals, reestablishment of functional connections depends on several parameters including neurotrophic factors, the extracellular matrix, and hormones. However, little is known about the contribution of hormones to peripheral nerve regeneration. Thyroid hormones, which are required for the development and maturation of the central nervous system, are also important for the development of peripheral nerves. The action of triiodothyronine (T3) on responsive cells is mediated through nuclear thyroid hormone receptors (TRs) which modulate the expression of specific genes in target cells. Thus, to study the effect of T3, it is first necessary to know whether the target tissues possess TRs. The fact that sciatic nerve cells possess functional TRs suggests that these cells can respond to T3 and, as a consequence, that thyroid hormone may be involved in peripheral nerve regeneration. The silicone nerve guide model provides an excellent system to study the action of local administration of T3. Evidence from such studies demonstrate that animals treated locally with T3 at the level of transection have more complete regeneration of sciatic nerve and better functional recovery. Among the possible regulatory mechanisms by which T3 enhances peripheral nerve regeneration is rapid action on both axotomized neurons and Schwann cells which, in turn, produce a lasting and stimulatory effect on peripheral nerve regeneration. It is probable that T3 up- or down-regulates gene expression of one or more growth factors, extracellular matrix, or cell adhesion molecules, all of which stimulate peripheral nerve regeneration. This could explain the greater effect of T3 on nerve regeneration compared with the effect of any one growth factor or adhesion molecule.     

Preventing ascidian fouling in aquaculture: screening selected allelochemicals for anti-metamorphic properties in ascidian larvae

Fouling by ascidians causes major stock losses and disrupts production in marine aquaculture, especially bivalve aquaculture. Currently, no cost effective solution exists despite the testing of many prospective control techniques. This study examined a range of allelochemicals suspected to inhibit metamorphosis in marine larvae. Five allelochemicals were screened in a larval metamorphosis bioassay using Ciona savignyi Herdman to determine their potential as a remedy for ascidian fouling in bivalve aquaculture. Three of the compounds tested inhibited ascidian larval metamorphosis and increased mortality at low concentrations. These were radicicol (99% inhibition of metamorphosis [IC??], 0.8 μg ml?1; 99% lethal concentration [LC??], 2.5 μg ml?1; 99% lethal time [LT??], 7.0 days), polygodial (IC??, 0.003 μg ml?1; LC??, 0.9 μg ml?1; LT??, 6.4 days), and ubiquinone-10 (IC??, 3.2 μg cm?2; LC??, 14.5 μg cm?2; LT??, 5.6 days; expressed as μg cm?2 due to insolubility in water and ethanol). While spermidine significantly affected metamorphosis and mortality of C. savignyi, the effect was insufficient to achieve inhibition in 99% of larvae over the 7-day timeframe of the assay. Muscimol did not affect metamorphosis or mortality at the concentrations tested. The present study demonstrates that radicicol, polygodial and ubiquinone-10 have potential for future development in antifoulant formulations targeted towards the inhibition of metamorphosis in ascidian larvae, while spermidine and muscimol appear unsuitable.     

Texture preferences of ascidian tadpole larvae during settlement

Ascidian tadpole larvae settle on hard surfaces and undergo metamorphosis into sessile adults. To test whether tadpoles evaluate the texture of surfaces they settle upon, we presented tadpoles with surfaces that were divided into halves; each half had one of four different textures: smooth, fine sandpaper, coarse sandpaper, and sandblasted. In all cases, twice as many individuals settled on one side over the other, but this was not consistently the smooth side or the rough side. More tadpoles settled on a smooth surface than one scoured by sandpaper, but more tadpoles settled on a sandblasted surface than smooth one. This indicates tadpoles are capable of finer tactile discrimination than merely detecting a hard surface, and supports the hypothesis that ascidian tadpoles have mechanoreceptive sensory neurons.     

Photorefractoriness in European starlings: associated hypothalamic changes and the involvement of thyroid hormones and prolactin

Responsivity to photostimulation in previously photorefractory European starlings is caused by subjection to short daylengths and is characterized by a marked activation of the hypothalamus in terms of synthesis of gonadotropin releasing hormone. This active hypothalamic state is amplified for a time by a subsequent exposure to long days but is soon completely reversed as the birds become photorefractory again. This latter effect of long photoperiods and the concurrent secretion of prolactin are dependent on the presence of thyroid hormones. Conceivably, prolactin causes photorefractoriness by inhibition at a hypothalamic level.     

Neurula rotation determines left-right asymmetry in ascidian tadpole larvae

Tadpole larvae of the ascidian Halocynthia roretzi show morphological left-right asymmetry. The tail invariably bends towards the left side within the vitelline membrane. The structure of the larval brain is remarkably asymmetric. nodal, a conserved gene that shows left-sided expression, is also expressed on the left side in H. roretzi but in the epidermis unlike in vertebrates. We show that nodal signaling at the late neurula stage is required for stereotypic morphological left-right asymmetry at later stages. We uncover a novel mechanism to break embryonic symmetry, in which rotation of whole embryos provides the initial cue for left-sided expression of nodal. Two hours prior to the onset of nodal expression, the neurula embryo rotates along the anterior-posterior axis in a counterclockwise direction when seen in posterior view, and then this rotation stops when the left side of the embryo is oriented downwards. It is likely that epidermis monocilia, which appear at the neurula rotation stage, generate the driving force for the rotation. When the embryo lies on the left side, protrusion of the neural fold physically prevents it from rotating further. Experiments in which neurula rotation is perturbed by various means, including centrifugation and sandwiching between glass, indicate that contact of the left epidermis with the vitelline membrane as a consequence of neurula rotation promotes nodal expression in the left epidermis. We suggest that chemical, and not mechanical, signals from the vitelline membrane promote nodal expression. Neurula rotation is also conserved in other ascidian species.