Early development, pattern, and reorganization of the planula nervous system in Aurelia (Cnidaria, Scyphozoa)

We examined the development of the nervous system in Aurelia (Cnidaria, Scyphozoa) from the early planula to the polyp stage using confocal and transmission electron microscopy. Fluorescently labeled anti-FMRFamide, antitaurine, and antityrosinated tubulin antibodies were used to visualize the nervous system. The first detectable FMRFamide-like immunoreactivity occurs in a narrow circumferential belt toward the anterior/aboral end of the ectoderm in the early planula. As the planula matures, the FMRFamide-immunoreactive cells send horizontal processes (i.e., neurites) basally along the longitudinal axis. Neurites extend both anteriorly/aborally and posteriorly/orally, but the preference is for anterior neurite extension, and neurites converge to form a plexus at the aboral/anterior end at the base of the ectoderm. In the mature planula, a subset of cells in the apical organ at the anterior/aboral pole begins to show FMRFamide-like and taurine-like immunoreactivity, suggesting a sensory function of the apical organ. During metamorphosis, FMRFamide-like immunoreactivity diminishes in the ectoderm but begins to occur in the degenerating primary endoderm, indicating that degenerating FMRFamide-immunoreactive neurons are taken up by the primary endoderm. FMRFamide-like expression reappears in the ectoderm of the oral disc and the tentacle anlagen of the growing polyp, indicating metamorphosis-associated restructuring of the nervous system. These observations are discussed in the context of metazoan nervous system evolution.     

Embryonic development and metamorphosis of the scyphozoan <Emphasis Type="Italic">Aurelia</Emphasis>

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of “secondary” gastrulation during early metamorphosis.     

Embryonic and larval development of the sea anemone Haliplanella lineata from Japan

The development of Haliplanella lineata, following fertilization in the laboratory, was studied by light and electron microscopy. Spawned ova were spherical, magenta in color and about 120–150 µm in diameter. Cleavage was holoblastic and radial. Gastrulation occurred by immigration and invagination. Eighteen hours after fertilization, the embryo became a swimming planula larva with an apical organ and ciliary tuft at the aboral end. In the laboratory, planulae lived for about 2 weeks in the swimming state but in no case was there any settlement by larvae in this study. The structural study of planulae concentrated on the development of the aboral ectoderm, because of the functional significance of its cellular organization in larval settlement.     

Post-embryonic larval development and metamorphosis of the hydroidEudendrium racemosum (Cavolini) (Hydrozoa,Cnidaria)

The morphology and histology of the planula larva ofEudendrium racemosum (Cavolini) and its metamorphosis into the primary polyp are described from light microscopic observations. The planula hatches as a differentiated gastrula. During the lecithotrophic larval period, large ectodermal mucous cells, embedded between epitheliomuscular cells, secrete a sticky slime. Two granulated cell types occur in the ectoderm that are interpreted as secretory and sensorynervous cells, but might also be representatives of only one cell type with a multiple function. The entoderm consists of yolk-storing gastrodermal cells, digestive gland cells, interstitial cells, cnidoblasts, and premature cnidocytes. The larva starts metamorphosis by affixing its blunt aboral pole to a substratum. While the planula flattens down, the mucous cells penetrate the mesolamella and migrate through the entoderm into the gastral cavity where they are lysed. Subsequently, interstitial cells, cnidoblasts, and premature cnidocytes migrate in the opposite direction, i.e. from entoderm to ectoderm. Then, the polypoid body organization, comprising head (hydranth), stem and foot, all covered by peridermal secretion, becomes recognisable. An oral constriction divides the hypostomal portion of the gastral cavity from the stomachic portion. Within the hypostomal entoderm, cells containing secretory granules differentiate. Following growth and the multiplication of tentacles, the head periderm disappears. A ring of gland cells differentiates at the hydranth's base. The positioning of cnidae in the tentacle ectoderm, penetration of the mouth opening and the multiplication of digestive gland cells enable the polyp to change from lecithotrophic to planktotrophic nutrition.     

Collar cells in planula and adult tentacle ectoderm of the solitary coral Balanophyllia regia (anthozoa eupsammiidae)

Summary The planula larva of the solitary coral Balanophyllia regia has an ectoderm of flagellate, diplosomal collar cells. The collar of these cells is composed of a ring of microvilli linked with mucus strands. Four types of flagellate gland cells, three types of nematocyst and spirocysts are present in the planula ectoderm. The function of these ectoderm cells is discussed. The mesogloeal muscular and packing tissues of the planula are briefly described. The tentacle of the adult coral, examined for comparison, has an ectoderm of flattened flagellate cells with a shallow collar. Collar cells similar to those of the planula are occasionally found on the tentacle and their function is not known. Independent sensory cells built on a modified collar cell plan with collar of thickened microvilli are common in the tentacle. These are quite separate from the three kinds of tentacular nematocyte. Distended glandular areas occur in the tentacle ectoderm. The flagellate tentacle gastrodermis, muscle and mesogloeal region are briefly described. The evolutionary significance of collar cell ectoderm in a planula is discussed and the occurrence of collar cells throughout the animal kingdom, reviewed.I am most grateful to Paul Tranter of the Plymouth Laboratory for providing material and to Gareth Morgan for assistance with electron microscopy.     

Localization of sensory mechanisms utilized by coral planulae to detect settlement cues

Coral planulae are induced to settle and metamorphose by contact with either crustose coralline algae or marine bacterial biofilms. Larvae of two coral species, Pocillopora damicornis and Montipora capitata, which respond to different metamorphic cues, were utilized to investigate the sensory mechanisms used to detect metamorphic cues. Because the aboral pole of the coral planula is the point of attachment to the substratum, we predicted that it is also the point of detection for cues. To determine where sensory cells for cues are localized along the body, individual larvae were transversely cut into oral and aboral portions at various levels along the oral–aboral axis, and exposed to settlement‐inducing substrata. Aboral ends of M. capitata metamorphosed, while oral ends continued to swim. However, in larvae of P. damicornis, ¾ oral ends (i.e., lacking the aboral pole) were also able to metamorphose, indicating that the cells that detect cues may be distributed along the sides of the body. These cells do not correspond to FMRFamide‐immunoreactive cells that are present throughout the body. Cesium ions induced both aboral and oral ends of larvae of both species to settle, suggesting that oral ends have not lost their capacity to metamorphose, despite lacking sensory cells to detect natural cues. To determine whether sensory cells in larvae of P. damicornis are restricted to one side of the body, swimming behavior over substrata was observed in larvae labeled with diI, a red fluorescent lipophilic membrane stain. The larvae were found to rotate around the oral–aboral axis, with their surface against the substratum, not favoring a particular side for detecting cues. While clarifying the regions of the larval body important for settlement and metamorphosis in coral planulae, we conclude that significant differences between coral species may be due to differences in the distribution of sensory structures in relation to different planular sizes.     

Compartments in Scyphozoa

Polyps of Scyphozoa have a cup-shaped body. At one end is the mouth opening surrounded by tentacles, at the other end is an attachment disc. The body wall consists of two tissue layers, the ectoderm and the endoderm, which are separated by an extracellular matrix, the mesoglea. The polyp's gastric cavity is subdivided by septa running from the apical end to the basal body end. The septa consist of two layers of endoderm and according to biology textbooks the number of septa is four. However, in rare circumstances Aurelia produces polyps with zero, two, six, or eight septa. We found that the number was always even. Therefore we propose that two types of endoderm exist, forming alternating stripes running from the oral body end to the aboral end. The stripes have some properties of developmental compartments. Where cells of different compartments meet, they form a septum. We also propose that the ectoderm is subdivided into compartments. The borders of the ectodermal and endodermal compartments are perpendicular to each other. Tentacles of the polyp and rhopalia (sense organs) of the ephyra (young medusa), respectively, develop at the border between two ectodermal compartments. The number can be even or odd. Rhopalia formation is particularly favored where two ectodermal and two endodermal compartments meet.     

Components of both major axial patterning systems of the Bilateria are differentially expressed along the primary axis of a 'radiate' animal, the anthozoan cnidarian Acropora millepora

Cnidarians are animals with a single (oral/aboral) overt body axis and with origins that nominally predate bilaterality. To better understand the evolution of axial patterning mechanisms, we characterized genes from the coral, Acropora millepora (Class Anthozoa) that are considered to be unambiguous markers of the bilaterian anterior/posterior and dorsal/ventral axes. Homologs of Otx/otd and Emx/ems, definitive anterior markers across the Bilateria, are expressed at opposite ends of the Acropora larva; otxA-Am initially around the blastopore and later preferentially toward the oral end in the ectoderm, and emx-Am predominantly in putative neurons in the aboral half of the planula larva, in a domain overlapping that of cnox-2Am, a Gsh/ind gene. The Acropora homologs of Pax-3/7, NKX2.1/vnd and Msx/msh are expressed in axially restricted and largely non-overlapping patterns in larval ectoderm. In Acropora, components of both the D/V and A/P patterning systems of bilateral animals are therefore expressed in regionally restricted patterns along the single overt body axis of the planula larva, and two 'anterior' markers are expressed at opposite ends of the axis. Thus, although some specific gene functions appear to be conserved between cnidarians and higher animals, no simple relationship exists between axial patterning systems in the two groups.     

Long-wavelength photosensitivity in coral planula larvae

Light influences the swimming behavior and settlement of the planktonic planula larvae of coral, but little is known regarding the photosensory biology of coral at this or any life-history stage. Here we used changes in the electrical activity of coral planula tissue upon light flashes to investigate the photosensitivity of the larvae. Recordings were made from five species: two whose larvae are brooded and contain algal symbionts (Porites astreoides and Agaricia agaricites), and three whose larvae are spawned and lack algal symbionts (Acropora cervicornis, Acropora palmata,and Montastrea faveolata). Photosensitivity originated from the coral larva rather than from, or in addition to, its algal symbionts as species with and without symbionts displayed similar tissue-level electrical responses to light. All species exhibited as much (or more) sensitivity to red stimuli as to blue/green stimuli, which is consistent with a role for long-wavelength visible light in the preference for substrata observed during settlement and in facilitating vertical positioning of larvae in the water column.     

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.     

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.     

The ontogeny of interstitial cells in Pennaria tiarella

The interstitial cells of Pennaria tiarella differentiate exclusively from the central endoderm of the planula. Shortly after their appearance, most of the interstitial cells become cnidoblasts. Subsequently, as the larva transforms into a polyp, both cnidoblasts and interstitial cells migrate from the endoderm, through endoblast and mesoglea, into the ectoderm. It is suggested that some interstitial cells remain in the endoderm and differentiate into the gland and mucous cells of the polyp gastroderm.     

The nature of the adhesion of tentacles to shells during shell-climbing in the sea anemone Calliactis parasitica (Couch)

Phase contrast microscopy and scanning electron microscopy show that during the response of the symbiotic sea anemone Calliactis parasitica (Couch) to shells of Buccinum undatum (L.) three times as many spirocysts as nematocysts are discharged. Observations indicate that spirocysts are responsible for the adhesion of tentacles to shells.Discharge levels are not significantly influenced by the nature of the substratum to which the anemones are attached. The reported observation that fewer tentacles adhere to shells when anemones are settled on shells than when they are fixed on a different substratum is re-interpreted in terms of a new model for the control of spirocyst discharge.     

Prospective fate of early blastomeres in Hydractinia echinata (Cnidaria,Hydrozoa)

Summary Blastomeres of two-cell, four-cell, and eight-cell embryos of Hydractinia echinata were injected with horseradish-peroxidase (HRP) or fluorescein isothiocyanate (FITC)-dextran. The fate of the descendants of the injected blastomeres was followed until the planula larva had developed. The results obtained after HRP or FITC-dextran injection were essentially the same. Blastomeres are equivalent up to the four-cell stage, i.e. half-blastomeres produce half of the ectoderm of the planula larva and quarter-blastomeres give rise to one quarter of the larval ectoderm. During normal embryogenesis, the larval anterior-posterior axis corresponds to the animal-vegetal axis of the zygote. Thus, the labelled areas of larvae consisting of the progeny of injected half or quarter blastomeres normally stretch along the larval anterior-posterior axis. Normally, material giving rise to anterior or posterior larval parts, respectively, is separated at the third cleavage. Irrespective of the type of experiment, the progeny of injected blastomeres always contributed to endoderm formation, i.e. in larvae resulting from injected embryos the endoderm was more or less uniformly labelled. Application of vital stains locally to the exterior of zygotes and following these markers through first and second cleavage, produced evidence that in the vast majority of cases, the second cleavage is meridional. Offprint requests to: A. Schlawny     

Gene structure and larval expression of cnox-2Am from the coral Acropora millepora

We have cloned a Hox-like gene, cnox-2Am, from a staghorn coral, Acropora millepora, an anthozoan cnidarian, and characterised its embryonic and larval expression. cnox-2Am and its orthologs in other cnidarians and Trichoplax most closely resemble the Gsx and, to a lesser extent, Hox 3/4 proteins. Developmental northern blots and in situ hybridisation are consistent in showing that cnox-2Am message appears in the planula larva shortly after the oral/aboral axis is formed following gastrulation. Expression is localised in scattered ectodermal cells with a restricted distribution along the oral/aboral body axis. They are most abundant along the sides of the cylindrical larva, rare in the oral region and absent from the aboral region. These cells, which on morphological grounds we believe to be neurons, are of two types; one tri-or multipolar near the basement membrane and a second extending projections in both directions from a mid-ectodermal nucleus. Anti-RFamide staining reveals neurons with a similar morphology to the cnox-2Am-expressing cells. However, RFamide-expressing neurons are more abundant, especially at the aboral end of the planula, where there is no cnox-2Am expression. The pattern of expression of cnox-2Am resembles that of Gsx orthologs in Drosophila and vertebrates in being expressed in a spatially restricted portion of the nervous system.     

Fine structural studies of the nervous system and the apical organ in the planula larva of the sea anemone Anthopleura elegantissima

The nervous system of the planula larva of Anthopleura elegantissima consists of an apical organ, one type of endodermal receptor cell, two types of ectodermal receptor cells, central neurons and nerve plexus. Both interneural and neuromuscular synapses are found in the nerve plexus. The apical organ is a collection of about 100 long, columnar cells each bearing a long cilium and a collar of about 10 microvilli. The cilia of the apical organ are twisted together to form an apical tuft. The ciliary rootlets of the apical organ cells are extremely long, reaching to the basal processes of the cells adjacent to the mesoglea. All three types of sensory cells are tall and slender in profile and are identified by the presence of one or more of the following features: microtubules, small vesicles, membrane-bound granules and synapses. The interneurons are bipolar cells with somas restricted to the aboral end, adjacent to the apical organ. All synapses observed are polarized or asymmetrical. A diagram including all the elements of the nervous system is presented and the possible functions of the nervous system are discussed in relation to larval behavior.     

Metamorphosis of coeloblastula performed by multipotential larval flagellated cells in the calcareous sponge Leucosolenia laxa

The calcareous sponge Leucosolenia laxa releases free-swimming hollow larvae called coeloblastulae that are the characteristic larvae of the subclass Calcinea. Although the coeloblastula is a major type of sponge larva, our knowledge about its development is scanty. Detailed electron microscopic studies on the metamorphosis of the coeloblastula revealed that the larva consists of four types of cells: flagellated cells, bottle cells, vesicular cells, and free cells in a central cavity. The flagellated cells, the principal cell type of the larva, are arranged in a pseudostratified layer around a large central cavity. The larval flagellated cells characteristically have glutinous granules that are used as internal markers during metamorphosis. After a free-swimming period the larva settles on the substratum, and settlement apparently triggers the initiation of metamorphosis. The larval flagellated cells soon lose their flagellum and begin the process of dedifferentiation. Then the larva becomes a mass of dedifferentiated cells in which many autophagosomes are found. Within 18 h after settlement, the cells at the surface of the cell mass differentiate to pinacocytes. The cells beneath the pinacoderm differentiate to scleroblasts that form triradiate spicules. Finally, the cells of the inner cell mass differentiate to choanocytes and are arranged in a choanoderm that surrounds a newly formed large gastral cavity. We found glutinous granules in these three principal cell types of juvenile sponges, thus indicating the multipotency of the flagellated cells of the coeloblastula.     

Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms

As a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems.