Ecological regulation of development: induction of marine invertebrate metamorphosis

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding--lecithotrophic--larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.     

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.     

The impact of waterborne cues from conspecifics and other species on the larvae of Halichondria panacea Pallas, 1766 (Porifera: Demospongiae)

The impacts of different concentrations of the excretory-secretory products (ESPs) of the solitary ascidian Styela rustica (Linnaeus, 1767) and the sponge Halichondria panacea (Pallas, 1766) on the settlement, metamorphosis, and mortality rates of H. panacea larvae were studied in a laboratory experiment. At high concentrations, substances released into the environment by the ascidian S. rustica exerted a negative impact on the metamorphosis rate of sponge larvae. The exposure to moderate or high concentrations of ESPs from conspecific adults led to high mortality of larval sponges; however, low conspecific ESP concentrations markedly stimulated metamorphosis; larval mortality was low. Apparently, different concentrations of the same ESPs can have effects with a different strength and focus. This should be taken into account in the study of chemically mediated interactions between aquatic organisms.     

Characterization and expression of calmodulin gene during larval settlement and metamorphosis of the polychaete Hydroides elegans

The polychaete Hydroides elegans (Serpulidae, Lophotrochozoa) is a problematic marine fouling organism in most tropical and subtropical coastal environment. Competent larvae of H. elegans undergo the transition from the swimming larval stage to the sessile juvenile stage with substantial morphological, physiological, and behavior changes. This transition is often referred to as larval settlement and metamorphosis. In this study, we examined the possible involvement of calmodulin (CaM) - a multifunctional calcium metabolism regulator, in the larval settlement and metamorphosis of H. elegans. A full-length CaM cDNA was successfully cloned from H. elegans (He-CaM) and it contained an open reading frame of 450 bp, encoding 149 amino acid residues. It was highly expressed in 12h post-metamorphic juveniles, and remained high in adults. In situ hybridization conducted in competent larvae and juveniles revealed that He-CaM gene was continuously expressed in the putative growth zones, branchial rudiments, and collar region, suggesting that He-CaM might be involved in tissue differentiation and development. Our subsequent bioassay revealed that the CaM inhibitor W7 could effectively inhibit larval settlement and metamorphosis, and cause some morphological defects of unsettled larvae. In conclusion, our results revealed that CaM has important functions in the larval settlement and metamorphosis of H. elegans.     

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     

Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis

The swimming larvae of the chordate ascidians possess a dorsal hollowed central nervous system (CNS), which is homologous to that of vertebrates. Despite the homology, the ascidian CNS consists of a countable number of cells. The simple nervous system of ascidians provides an excellent experimental system to study the developmental mechanisms of the chordate nervous system. The neural fate of the cells consisting of the ascidian CNS is determined in both autonomous and non-autonomous fashion during the cleavage stage. The ascidian neural plate performs the morphogenetic movement of neural tube closure that resembles that in vertebrate neural tube formation. Following neurulation, the CNS is separated into five distinct regions, whose homology with the regions of vertebrate CNS has been discussed. Following their larval stage, ascidians undergo a metamorphosis and become sessile adults. The metamorphosis is completed quickly, and therefore the metamorphosis of ascidians is a good experimental system to observe the reorganization of the CNS during metamorphosis. A recent study has shown that the major parts of the larval CNS remain after the metamorphosis to form the adult CNS. In contrast to such a conserved manner of CNS reorganization, most larval neurons disappear during metamorphosis. The larval glial cells in the CNS are the major source for the formation of the adult CNS, and some of the glial cells produce adult neurons.     

Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10(-7)-10(-2) M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) < 9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10(-5) M and IBMX at 10(-6)-10(-4) M induced 41-83% larval settlement and ACH at 10(-7)-10(-5) M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemical, most of the larvae settled within 24 h. Compounds at concentrations of 10(-3)-10(-2) M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10(-6)-10(-4) M and L-DOPA at 10(-5) M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

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.     

The biology of coral metamorphosis: molecular responses of larvae to inducers of settlement and metamorphosis

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring > 8000 Acropora unigenes were used to follow gene expression changes during the 12 h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common—an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.     

Induction of metamorphosis with potassium ions requires development of competence and an anterior signalling centre in the ascidian Herdmania momus

 Increased K⁺ concentration in seawater induces metamorphosis in the ascidian Herdmania momus. Larvae cultivated at 24°C exhibit highest rates of metamorphosis when treated with 40 mM KCl-elevated seawater at 21°C. At 24°C, H. momus larvae develop competence to respond to KCl-seawater and initiate metamorphosis approximately 3 h after hatching. Larval trunks and tails separated from the anterior papillae region, but maintained in a common tunic at a distance of greater than 60 μm, do not undergo metamorphosis when treated with KCl-seawater; normal muscle degradation does not occur in separated tails while ampullae develop from papillae-containing anterior fragments. Normal programmed degradation of myofibrils occurs when posterior fragments are fused to papillae-containing anterior fragments. These data indicate that H. momus settlement and metamorphosis only occurs when larvae have attained competence, and suggest that an anterior signalling centre is stimulated to release a factor that induces metamorphosis. Received: 15 May 1996 / Accepted: 19 September 1996     

GABA is an inhibitory neurotransmitter in the neural circuit regulating metamorphosis in a marine snail

The marine mud snail, Tritia (=Ilyanassa) obsoleta, displays a biphasic life cycle. During the initial phase of early development, embryos hatch from benthic egg capsules to become weakly swimming veliger larvae. In the second phase, adult T. obsoleta are facultative carnivores and major agents of community disturbance. Metamorphosis is the irreversible developmental event that links these two life history stages. When physiologically competent, larvae can respond to appropriate environmental cues by settling onto their mudflat habitat and transforming themselves into miniature adult snails. Two neurotransmitters—serotonin and nitric oxide—have opposing effects on the metamorphic process in this species. In multiple other species of gastropod and bivalve molluscs, a third neurotransmitter, the classically inhibitory compound γ‐aminobutyric acid (GABA), can induce settlement or metamorphosis upon external application to competent larvae. In this situation, GABA is presumed to mimic the action of ligands from the juvenile environment that bind to larval chemosensory receptors and activate the metamorphic pathway. Results of our experiments contradict this commonly reported action of GABA on molluscan larvae. External application of GABA to competent larvae of T. obsoleta elicited no response, but instead attenuated the action of serotonin (5‐HT), a metamorphic inducer. Our investigations into the responses of larval T. obsoleta to multiple GABAergic reagents support our hypothesis that GABA functions internally as a neurotransmitter in the pathway that controls the initiation of metamorphosis. Our results also suggest that GABA acts directly on or downstream from serotonergic neurons to regulate the metamorphosis‐inducing effects of this neurotransmitter. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 736–753, 2018     

Isolation and characterization of genes that are expressed during Ciona intestinalis metamorphosis

In ascidians, the events of metamorphosis transform the non-feeding, mobile tadpole larva into a filter-feeding, fixed juvenile, and the process involves rearrangements of cells, two organs and physiological changes. Differential screening was used to isolate two genes that are not expressed in swimming larvae but are expressed immediately after the initiation of metamorphosis in Ciona intestinalis. One of the genes, Ci-meta1, encodes a polypeptide with a putative secretion signal sequence, 6 epidermal growth factor (EGF)-like repeats and 13 calcium-binding EGF-like repeats. The gene begins to be expressed immediately after the beginning of metamorphosis in the adhesive organ and is likely to be associated with the signal response for metamorphosis. Another gene named Ci-meta2 encodes a protein with a putative secretion signal and three thrombospondin type-1 repeats. Ci-meta2 gene expression begins at the larval stage and is upregulated in the metamorphosing juveniles. Ci-meta2 expression is found in three regions; the adhesive organ which is also associated with settlement, the neck region between the trunk and the tail of the larva which is associated with tail resorption, and dorsal regions of the trunk which correspond to the location of the siphon primordium. This gene may be involved in the dynamic arrangement of cells during ascidian metamorphosis.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.     

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.