Early development of zooxanthella-containing eggs of the corals Porites cylindrica and Montipora digitata: The endodermal localization of zooxanthellae

We studied the early development of zooxanthellae-containing eggs of the scleractinian corals Porites cylindrica and Montipora digitata to elucidate how zooxanthellae become localized to the endoderm of planulae during the course of development. In both species, zooxanthellae were distributed evenly in the oocytes and delivered almost equally to the blastomeres during cleavage. In P. cylindrica, gastrulation occurred via delamination or ingression, and blastomeres containing zooxanthellae dropped into the blastocoel during gastrulation. Thus, zooxanthellae were restricted to the endodermal cells at the gastrula or early planula stage in P. cylindrica. In M. digitata, gastrulation occurred by a combination of invagination and epiboly to form a somewhat concave gastrula. Zooxanthellae were present in both endodermal and ectodermal cells of early planulae, but they disappeared from the ectoderm as the planulae matured. In our previous study on two species of Pocillopora, we found that zooxanthellae were localized in eggs as well as in embryos, and that blastomeres containing zooxanthellae later dropped into the blastocoel to become restricted to the endoderm (Hirose et al., 2000). The timing and mechanism of zooxanthella localization and types of gastrulation differed among species belonging to the three genera. These results suggest that zooxanthella localization in the embryos reflects the timing of the determination of presumptive endoderm cells and/or specificity of zooxanthellae toward presumptive endoderm cells.     

Catalase characterization and implication in bleaching of a symbiotic sea anemone

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.     

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.     

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.     

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.     

Planulae brooding and acquisition of zooxanthellae inXenia macrospiculata (Cnidaria: Octocorallia)

Brooding in the octocoralXenia macrospiculata is described. Young planulae ofX. macrospiculata were found in brooding pouches located below the anthocodia among the polyps’ cavities. These cavities are connected by and lined with ectoderm. Detached zooxanthellae were present within the brooding pouches, and are most probably acquired later by the planulae. The zooxanthellae enter into ectodermal ameboid cells by phagocytosis, and are then transferred to the endoderm.     

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.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

Scanning electron microscopy (SEM) of the chick embryo primitive streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2--5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the privitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arrangedcells with long. thin processes. Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Phorbol esters alter cell fate during development of sea urchin embryos

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.     

Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development

Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.     

Pattern formation in a pentameral animal: induction of early adult rudiment development in sea urchins

We investigated adult rudiment induction in the direct-developing sea urchin Heliocidaris erythrogramma microsurgically. After removal of the archenteron (which includes presumptive coelomic mesoderm as well as presumptive endoderm) from late gastrulae, larval ectoderm develops properly but obvious rudiments (tube feet, nervous system, and adult skeleton) fail to form, indicating that coelomic mesoderm, endoderm, or both are required for induction of adult development. Recombination of ectoderm and archenteron rescues development. Implanted endoderm alone or left coelom alone each regenerate the full complement of archenteron derivatives; thus, they are uninformative as to the relative inductive potential of the two regions. However, in isolated ectoderm, more limited regeneration gives rise to larvae containing no archenteron derivatives at all, endoderm only, or both endoderm and left coelom. Adult nervous system begins to develop only in the latter, indicating that left coelom is required for the inductive signal. Isolated ectoderm develops a vestibule (the precursor of adult ectoderm) and correctly regulates vestibular expression of the ectodermal territory marker HeET-1, indicating that the early phase of vestibule development occurs autonomously; only later development requires the inductive signal. Another ectodermal marker, HeARS, is regulated properly in the larval ectoderm region, but not in the vestibule. HeARS regulation thus represents an early response to the inducing signal. We compare HeARS expression in H. erythrogramma with that in indirect developers and discuss its implications for modularity in the evolution of developmental mode.