Characterization of superoxide dismutases in anoxia- and hyperoxia-tolerant symbiotic cnidarians

Many cnidarians, such as sea anemones, contain photosynthetic symbiotic dinoflagellates called zooxanthellae. During a light/dark cycle, the intratentacular O(2) state changes in minutes from hypoxia to hyperoxia (3-fold normoxia). To understand the origin of the high tolerance to these unusual oxic conditions, we have characterized superoxide dismutases (SODs) from the three cellular compartments (ectoderm, endoderm and zooxanthellae) of the Mediterranean sea anemone Anemonia viridis. The lowest SOD activity was found in ectodermal cells while endodermal cells and zooxanthellae showed a higher SOD activity. Two, seven and six SOD activity bands were identified on native PAGE in ectoderm, endoderm and zooxanthellae, respectively. A CuZnSOD was identified in both ectodermal and endodermal tissues. MnSODs were detected in all compartments with two different subcellular localizations. One band displays a classical mitochondrial localization, the three others being extramitochondrial. FeSODs present in zooxanthellae also appeared in endodermal host tissue. The isoelectric points of all SODs were distributed between 4 and 5. For comparative study, a similar analysis was performed on the whole homogenate of a scleractinian coral Stylophora pistillata. These results are discussed in the context of tolerance to hyperoxia and to the transition from anoxia to hyperoxia.     

Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

An ultrastructural study of larval settlement in the sea anemone Urticina crassicornis (Cnidaria,Actiniaria)

Laboratory-reared larvae of the sea anemone Urticina (= Tealia) crassicornis have been examined by electron microscopy prior to and following settlement on algal substrata. At 18 days postfertilization, the free-swimming planula larva measures about 600 μm long. A stomodaeal invagination occurs at the narrow end of the larva and connects with a solid mass of endoderm in the core region. The endoderm possesses septa with well-developed myonemes and is situated subjacent to a thin sheet of mesoglea. The uniformly ciliated ectoderm that constitutes the outer layer of the larva contains: (1) spirocysts, (2) nematocysts, (3) mucus, (4) three types of membrane-bound granules, (5) a basiepithelial nerve plexus, and (6) a few nongranular cells that may represent sensory neurons. Within several minutes after the introduction of the algal substratum, the planula characteristically directs its broadened aboral end toward the alga and secretes a refractile sheet of material. As the aboral end attaches to the substratum, the larva becomes noticeably shorter along its oral-aboral axis, presumably owing to the contractions of myonemes that are located within the endodermal septa. All three types of granules and the ectodermal mucoid substances are exocytosed during settlement, but spirocysts and nematocysts characteristically remain undischarged. Ovoid, PAS⁺ granules are believed to be at least partly responsible for adhesion, since these granules are concentrated at the aboral end prior to settlement and are somewhat similar in ultrastructure to putative viscid granules produced by other species. Contrary to a previous report based on light microscopy, no discrete sensory organ is evident in serial sections of the aboral ectoderm. The ability of planulae to detect suitable substrata appears to depend instead on sparsely distributed sensory cells that occur throughout the larval ectoderm.     

Communication compartments in the ectoderm of embryos of Patella vulgata

Summary Patterns of gap junctional communication in the ectoderm of embryos of Patella vulgata have been studied by intracellular injection of the fluorescent dye Lucifer Yellow, and by analysis of its subsequent spread to adjacent cells (dye-coupling). We found that dye-coupling became progressively restricted to different domains of the ectoderm, forming communication compartments. These communication compartments are characterized by their high coupling abilities within the compartment, and reduction of coupling across their boundaries. During development, the pretrochal (anterior) ectoderm becomes subdivided into two communication compartments, the apical organ and the anlage of the head ectoderm. The posttrochal (posterior) ectoderm becomes subdivided into different communication compartments in two successive phases. Firstly, in the 15-h embryo the dorsal and ventral domains of the ectoderm form separate communication compartments. A dorso-ventral communication boundary restricts the passage of dye between the two domains. Secondly, in the 24-h embryo dye-coupling becomes further compartmentalized in both the dorsal and ventral domains. These compartments correspond to the anlagen of different ectodermal structures. In order to study whether any level of coupling persists between the ectodermal compartments we injected currents through a microelectrode inserted into one cell of one compartment and monitored its spread by means of a second microelectrode inserted into one cell of another compartment (electrical coupling). Despite the absence of dye-coupling, electrical coupling between the ectodermal dye-coupling compartments was detected, which suggests that some level of communication is maintained between compartments. Our results demonstrate that within the ectoderm layer of Patella vulgata the transfer of dyes becomes progressively restricted to communication compartments and, concomitantly with the specification of the different ectodermal anlagen, these compartments become subdivided into smaller communication compartments.     

Drosophila endoderm development requires a novel homeobox gene which is a target of Wingless and Dpp signalling.

We have identified and cloned a novel type of homeobox gene that is composed of two homeodomains and is expressed in the Drosophila endoderm. Mutant analysis reveals that its activity is required at the foregut/midgut boundary for the development of the proventriculus. This organ regulates food passage from the foregut into the midgut and forms by the infolding of ectoderm and endoderm-derived tissues. The endodermal outer wall structure of the proventriculus is collapsed in the mutants leading to a failure of the ectodermal part to invaginate and build a functional multilayered organ. Lack-of-function and gain-of-function experiments show that the expression of this homeobox gene in the proventriculus endoderm is induced in response to Wingless activity emanating from the ectoderm/endoderm boundary whereas its expression in the central midgut is controlled by Dpp and Wingless signalling emanating from the overlying visceral mesoderm.     

Quantitative analysis of epithelial cell aggregation in the simple metazoan Hydra reveals a switch from homotypic to heterotypic cell interactions

Hydra, a member of the diploblastic phylum Cnidaria, exhibits the most basic type of organized metazoan tissues. Two unicellular sheets of polarized epithelial cells - ectoderm and endoderm - form a double layer throughout the body column. The double layer can be reestablished from single-cell suspensions by tissue-specific cell-sorting processes. However, the underlying pattern of interactions between ectodermal and endodermal epithelial cells responsible for double-layer formation is unclear. By analyzing cell interactions in a quantitative adhesion assay using mechanically dissociated Hydra epithelial cells, we show that aggregation proceeds in two steps. First, homotypic interactions within ectodermal epithelial cells (ecto-ecto) and within endodermal epithelial cells (endo-endo) form homotypic cell clusters. Second, at an aggregate size of about ten epithelial cells/cluster, ectodermal and endodermal clusters start to form heterotypic aggregates. Homotypic ecto-ecto interactions are inhibited by a polyclonal anti-Hydra membrane antiserum, and under these conditions homotypic endo-endo interactions do not proceed beyond a size of about ten epithelial cells/cluster. These data suggest that homotypic cell clusters reduce their initial homotypic affinity and acquire a new heterotypic affinity. A link between cell adhesion and cell signaling in early Hydra aggregates is discussed.     

Generation of bilateral symmetry in Anthozoa: a model

Polyps of Anthozoa usually display bilateral symmetry with respect to their mouth opening, to their pharynx, and in particular to the arrangement of their mesenteries. Mesenteries, which are endodermal folds running from the apical to the basal end of the body, subdivide the gastric cavity into pouches. They form in a bilateral symmetric sequence. In this article I propose that early in polyp development the endoderm subdivides successively into three different types of compartments. A mesentery forms at the border between compartments. Two of the compartments are homologous to those of Scyphozoa. They form by mutual activation of cell states that locally exclude each other. The third compartment leads to siphonoglyph formation and is an evolutionary innovation of the Anthozoa. The mechanism that controls the number and spatial arrangement of the third type of compartment changes the radial symmetry into a bilateral one and occasionally into a different one. The dynamics of its formation indicate an activator-inhibitor mechanism. Computer models are provided that reproduce decision steps in the generation of the mesenteries.     

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.     

BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning

BMPRIA is a receptor for bone morphogenetic proteins with high affinity for BMP2 and BMP4. Mouse embryos lacking Bmpr1a fail to gastrulate, complicating studies on the requirements for BMP signaling in germ layer development. Recent work shows that BMP4 produced in extraembryonic tissues initiates gastrulation. Here we use a conditional allele of Bmpr1a to remove BMPRIA only in the epiblast, which gives rise to all embryonic tissues. Resulting embryos are mosaics composed primarily of cells homozygous null for Bmpr1a, interspersed with heterozygous cells. Although mesoderm and endoderm do not form in Bmpr1a null embryos, these tissues are present in the mosaics and are populated with mutant cells. Thus, BMPRIA signaling in the epiblast does not restrict cells to or from any of the germ layers. Cells lacking Bmpr1a also contribute to surface ectoderm; however, from the hindbrain forward, little surface ectoderm forms and the forebrain is enlarged and convoluted. Prechordal plate, early definitive endoderm, and anterior visceral endoderm appear to be expanded, likely due to defective morphogenesis. These data suggest that the enlarged forebrain is caused in part by increased exposure of the ectoderm to signaling sources that promote anterior neural fate. Our results reveal critical roles for BMP signaling in endodermal morphogenesis and ectodermal patterning.     

GATA factors as key regulatory molecules in the development of Drosophila endoderm

Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent ( srp , dGATAb , ABF ), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe , and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans , in which a pair of GATA genes, end-1/3 , specifies endodermal fate, and a downstream pair of GATA genes, elt-2/7 , activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes.     

Scanning electron microscopy of the muscle system of Hydra magnipapillata

The fine structure of the ectodermal and endodermal muscle layers of Hydra magnipapillata has been analyzed by scanning electron microscopy after hydrolytic removal of the mesoglea with NaOH and subsequent exposure of the basal and lateral aspects of the layers by mechanical dissection. The ectodermal muscle layer consists of fibrous processes of epithelial cells extending longitudinally to the body axis, whereas the endodermal muscle layer comprises cells with hexagonal bases and several strands of myonemes oriented circularly. In each layer, the muscular elements tightly interdigitate, extending a continuous muscle sheet along the mesoglea. The ectodermal and endodermal muscle sheets communicate with each other via foliate microprojections penetrating the mesoglea. On the lateral aspect of the ectodermal epithelium, spiny nerve fibers run along the upper surface of the muscle processes. The spines are often attached to muscle processes, suggesting that the former monitor muscle contraction. Nerve fibers occasionally come into contact with the mesoglea through narrow gaps between the muscle processes. In the hypostomal ectoderm, a small spindle-shaped cell, probably sensory in nature, extends an apical cilium and a long basal process.     

Ectoderm,endoderm, and the evolution of heterodont dentitions

Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3β, α‐fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth‐forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals. genesis 48:382–389, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

The POU domain protein spg (pou2/Oct4) is essential for endoderm formation in cooperation with the HMG domain protein casanova

The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.     

Expression of the cell surface-associated glycoprotein, fibronectin, in the early mouse embryo.

The expression of the cell surface-associated glycoprotein fibronectin was studied by indirect immunofluorescence in the early stages of mouse embryogenesis. Fibronectin was not detectable in early preimplantation embryos. Trace amounts of the protein were first found between the cells of the inner cell mass of late blastocysts. In implanted early egg cylinders, fibronectin was deposited between the ectoderm and endoderm of the inner cell mass and in the nascent Reichert's membrane. With development, the visceral and the parietal endoderm cells became positive for the protein, but no fibronectin was detected in ectoderm cells. During segregation of mesoderm from ectoderm, fibronectin appeared in mesoderm cells and as a band between the two germ layers. In the developing amnion and chorion, the protein was localized between the ectodermal and mesodermal cell layers. The results indicate that fibronectin is an early differentiation market for the stage of endoderm formation in the inner cell mass of the mouse blastocyst. It is also a marker of mesoderm appearance and seems to be associated with the accumulating extracellular matrix material in the developing embryo.     

Development and evolution of chordate cartilage

Deuterostomes are a monophyletic group of animals containing vertebrates, lancelets, tunicates, hemichordates, echinoderms, and xenoturbellids. Four out of these six extant groups-vertebrates, lancelets, tunicates, and hemichordates-have pharyngeal gill slits. All groups of deuterostome animals that have pharyngeal gill slits also have a pharyngeal skeleton supporting the pharyngeal openings, except tunicates. We previously found that pharyngeal cartilage in hemichordates and cephalochordates contains a fibrillar collagen protein similar to vertebrate type II collagen, but unlike vertebrate cartilage, the invertebrate deuterostome cartilages are acellular. We found SoxE and fibrillar collagen expression in the pharyngeal endodermal cells adjacent to where the cartilages form. These same endodermal epithelial cells also express Pax1/9, a marker of pharyngeal endoderm in vertebrates, lancelets, tunicates, and hemichordates. In situ experiments with a cephalochordate fibrillar collagen also showed expression in pharyngeal endoderm, as well as the ectoderm and the mesodermal coelomic pouches lining the gill bars. These results indicate that the pharyngeal endodermal cells are responsible for secretion of the cartilage in hemichordates, whereas in lancelets, all the pharyngeal cells surrounding the gill bars, ectodermal, endodermal, and mesodermal may be responsible for cartilage formation. We propose that endoderm secretion was primarily the ancestral mode of making pharyngeal cartilages in deuterostomes. Later the evolutionary origin of neural crest allowed co-option of the gene network for the secretion of pharyngeal cartilage matrix in the new migratory neural crest cell populations found in vertebrates.     

Effects of Inducers on Inner and Outer Gastrula Ectoderm Layers of Xenopus laevis

Abstract. Gastrula ectoderm, isolated from Xenopus laevis , was cultured in Holtfreter solution or modified Leibovitz medium (L-15) by the sandwich-method with or without inducer. The ectoderm (SD cell layers) consists of two cell sheets, representing a superficial (S) and a deep (D) layer. In the L-15 medium rather than in Holtfreter solution, the two cell layers separate out into distinct cell masses. This difference in cell affinity under certain experimental conditions could indicate that the deep layer contains endodermal cells. However, an endodermal character of the deep layer can be ruled out by induction experiments with vegetalizing factor or dorsal blastopore lip as inducers. Under the influence of vegetalizing factor the outer as well as the inner ectoderm layer differentiated into mesodermal derivatives such as notochord and somites. The results of the experiments with dorsal blastopore lip as inducer indicate that both inner and outer ectoderm layers are responsive to the neural stimulus. The lower neural competence of the outer ectoderm layer observed by several authors in normogenesis is discussed with regard to the hypothesis about short distance diffusion of the neuralizing factor and/or close cell-to-cell contact between inducing tissue and ectodermal target cells.     

Clonal analysis of early mammalian development

Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.