Selective Degradation of Specific Components of Fertilization Coat and Differentiation of Hatching Gland Cells during the Two Phase Hatching of Bufo japonicus Embryos

The embryonic hatching process in the toad, Bufo japonicus , consists of two phases: rupture of the outer jelly strings at stage 20 (neural tube) and an escape from the inner jelly layers and fertilization coat (FC) of individual embryos at stage 23 (tailbud). SDS-PAGE analyses of FCs revealed that, of the eight major protein bands, two components with 58 K and 62 K in molecular weight gradually decreased from stage 18–19 on and totally disappeared at stage 22. When the FCs were treated with a hatching medium prepared by culturing denuded prehatching embryos, both 58 K and 62 K components of the FCs were solubilized, and in the solubilized materials 18 K and 31 K components appeared. Electron microscopy showed that a meshwork of filament bundles present in the FCs before stage 17 became dissociated at stage 19–20, and completely disappeared at stage 23, just before the hatching of embryos. Hatching gland cells (HGCs), an epidermal cell with numerous secretory granules, were first identified at stage 19, and underwent active secretion of the granules during stage 19–23. These results indicate that the hydrolytic degradation of 58K and 62 K components in FCs effected by the hatching enzyme constitutes the basic mechanism of embryonic hatching during both the first and second phases.     

Structures of Xenopus Embryonic Epidermal Lectin Reveal a Conserved Mechanism of Microbial Glycan Recognition

Intelectins (X-type lectins), broadly distributed throughout chordates, have been implicated in innate immunity. Xenopus laevis embryonic epidermal lectin (XEEL), an intelectin secreted into environmental water by the X. laevis embryo, is postulated to function as a defense against microbes. XEEL is homologous (64% identical) to human intelectin-1 (hIntL-1), which is also implicated in innate immune defense. We showed previously that hIntL-1 binds microbial glycans bearing exocyclic vicinal diol groups. It is unknown whether XEEL has the same ligand specificity. Also unclear is whether XEEL and hIntL-1 have similar quaternary structures, as XEEL lacks the corresponding cysteine residues in hIntL-1 that stabilize the disulfide-linked trimer. These observations prompted us to further characterize XEEL. We found that hIntL-1 and XEEL have similar structural features. Even without the corresponding intermolecular disulfide bonds present in hIntL-1, the carbohydrate recognition domain of XEEL (XEEL_CRD) forms a stable trimer in solution. The structure of XEEL_CRD in complex with d-glycerol-1-phosphate, a residue present in microbe-specific glycans, indicated that the exocyclic vicinal diol coordinates to a protein-bound calcium ion. This ligand-binding mode is conserved between XEEL and hIntL-1. The domain architecture of full-length XEEL is reminiscent of a barbell, with two sets of three glycan-binding sites oriented in opposite directions. This orientation is consistent with our observation that XEEL can promote the agglutination of specific serotypes of Streptococcus pneumoniae. These data support a role for XEEL in innate immunity, and they highlight structural and functional conservation of X-type lectins among chordates.     

Expression pattern of BXR suggests a role for benzoate ligand-mediated signalling in hatching gland function

The Xenopus laevis nuclear receptor BXR has recently been shown to be activated by a class of endogenous benzoate metabolites, indicating the presence of a novel and unsuspected benzoate ligand-dependent signalling pathway. The receptor is expressed ubiquitously in blastula and gastrula stage embryos, and its expression declines during neurula stages. In order to examine further this novel vertebrate signalling system, we have examined the expression of the BXR gene in tailbud stage embryos and adults. We show here that in Xenopus tailbud stage embryos expression is restricted to the hatching gland, suggesting a role in hatching gland function. Neither BXR nor a BXR-VP16 fusion is sufficient to specify hatching gland in neurally-induced tissue. In adults, BXR expression is abundant in the brain and gonads. This expression pattern in adults is distinct from any of the putative mammalian homologues. A nuclear receptor that mediates benzoate signalling has yet to be found in mammals.     

Expression of epidermis-specific antigens during embryogenesis of the ascidian, Halocynthia roretzi

We have produced two monoclonal antibodies (Epi-1 and Epi-2) which specifically recognize epidermal cells and their derivative, the larval tunic, of developing embryos of the ascidian Halocynthia roretzi. The antigens, examined by indirect immunofluorescence staining, first appear at the early tailbud stage and are present until at least the swimming larval stage. There were distinct and separate puromycin and actinomycin D sensitivity periods for each antigen. Aphidicolin, a specific inhibitor of DNA synthesis, prevented the appearance of each antigen when embryos were exposed to the drug continuously from cleavage stages. These results suggest that the antigens are synthesized during embryogenesis by developing epidermal cells and that several rounds of DNA replication are required for the antigen expression. Early cleavage stage embryos, including fertilized but unsegmented eggs, in which cytokinesis had been blocked with cytochalasin B expressed the antigens, and blastomeres exhibiting the antigens were always of the epidermis lineage. In partial embryos produced by four separated blastomere pairs of the 8-cell embryos, the expression of antigens was seen only in those developed from the animal blastomere pairs, which are progenitors of epidermal cells. These observations indicate that differentiation of epidermal cells in ascidian embryos takes place in a typical "mosaic" fashion.     

Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis

Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

卤虫(Artemia salina)孵化腺细胞分化时相的免疫细胞化学研究(简报)

动物孵化酶(hatching enzyme,HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type ofcells)。完全分化的HGC内充满了低电子密度的酶原颗粒(孵化酶原颗粒),在鱼胚中的分布因物种而异。在大多数鱼中,HGC分布在胚体的外表面和/或卵黄囊中,一般为外胚层来源。如在虹蹲鱼HGC分布在胚体的前表面、卵黄囊、咽部、鳃的内表面及外表面,属于外胚层来源。而日本鳉鱼HGC     

卤虫（Artemia salina）孵化腺细胞分化时相的免疫细胞化学研究（简报）

动物孵化酶(hatching enzyme HE)是早期胚胎在特定发育阶段由孵化腺细胞产生和分泌的,在动物早期胚胎孵化中具有关键性作用^[4]。孵化腺细胞(hatching gland cell,HGC)一般为单细胞腺体,是从胚胎发育到特定阶段(孵化前)出现、至胚胎孵出后的特定时期消失的一时性细胞(transient type of     

Isolation of cDNA Clones for Epidermis-Specific Genes of the Ascidian Embryo

The present investigation was conducted to isolate cDNA clones that correspond to epidermis-specific genes of the ascidian embryo. When cleavage of fertilized eggs of Halocynthia roretzi is blocked by treatment with cytochalasin B and the arrested eggs are reared as one-celled embryos for about 30 hr, they develop features of differentiation of the epidermis only. Translation in vitro of poly(A)⁺ RNA from cleavage-arrested embryos and analysis of the products by two-dimensional gel electrophoresis revealed several predominant polypeptides that were not detected in a similar analysis of fertilized eggs, suggesting the appearance of epidermis-specific mRNAs in cleavage-arrested embryos. A cDNA library was constructed from arrested one-celled embryos. Differential screening of the library with a total cDNA probe from cleavage-arrested embryos and with a similar probe from fertilized eggs yielded eight different cDNA clones specific for the cleavage-arrested embryos. Northern blot analysis revealed that the mRNAs that corresponded to these cDNAs were present in normal tailbud embryos. In addition, in situ hybridization of whole-mount specimens showed that the mRNAs were restricted to the epidermal cells of tailbud embryos.     

Characterization, fate, and function of hamster cortical granule components

Little is known about the composition and function of mammalian cortical granules. In this study, lectins were used as tools to: (1) estimate the number and molecular weight of glycoconjugates in hamster cortical granules and show what sugars are associated with each glycoconjugate; (2) identify cortical granule components that remain associated with the oolemma, cortical granule envelope, and/or zona pellucida of fertilized oocytes and preimplantation embryos; and (3) examine the role of cortical granule glycoconjugates in preimplantation embryogenesis. Microscopic examination of unfertilized oocytes revealed that the lectins PNA, DBA, WGA, RCA(120), Con A, and LCA bound to hamster cortical granules. Moreover, LCA and Con A labeled the zona pellucida, cortical granule envelope, and plasma membrane of fertilized and artificially activated oocytes and two and eight cell embryos. Lectin blots of unfertilized oocytes had at least 12 glycoconjugates that were recognized by one or more lectins. Nine of these glycoconjugates are found in the cortical granule envelope and/or are associated with the zona pellucida and plasma membrane following fertilization. In vivo functional studies showed that the binding of Con A to one or more mannosylated cortical granule components inhibited blastomere cleavage in two-cell embryos. Our data show that hamster cortical granules contain approximately 12 glycoconjugates of which nine remain associated extracellularly with the fertilized oocyte after the cortical reaction and that one or more play a role in regulating cleavage divisions.     

Micromorphological studies of adventitious bud formation on Picea abies embryos treated with cytokinin

Embryos of Picea abies (L.) Karst were pulse-treated with water or cytokinin for 2 h and then cultured on medium lacking cytokinin. Adventitious buds developed on cytokinin-treated embryos, but not on water-treated embryos. The general appearance and the surface morphology were similar on water and BA (benzyladenine)-treated embryos after 3 days. The epidermal cells were elongating after 6 days on water-treated embryos, while they were dividing on cytokinin-treated embryos. Furthermore, the cells surrounding the stomata had started to proliferate on BA-treated embryos. This was the first micromorphological sign of bud initiation. During the second week prominent meristemoids developed from these cells. A stoma was observed on the top of each meristemoid. The variation in developmental pattern of meristemoids among different embryos as well as within each embryo was small. However, during the subsequent development of bud primordia and buds, the morphological variation was significant. The meristemoids continued to develop into cone-shaped bud primordia, which successively changed shape during the transition to adventitious buds. The epidermal cells divided and the epidermis did not rupture during the formation of adventitious bud primordia. The epidermis was identified as the protoderm of the bud primordium.     

Loss of test cells leads to the formation of new tunic surface cells and abnormal metamorphosis in larvae of Ciona intestinalis (Chordata, Ascidiacea)

The larvae of the ascidian Ciona intestinalis from which the chorion with the test cells and follicle cells were removed developed normally without the test cells until the early tailbud stage. A number of round-shaped cells morphologically similar to the test cells but with different lectin affinities and autofluorescence, then appeared on the neck region of the demembranated embryos. The new cells had three different types: round, particulate, and granular, and these cells increased in number after the late tailbud stage. The morphology of the adhesive papillae, tunic layers and epidermis of the demembranated larvae was similar to that of control larvae; however, the affinity to lectins was different in the swimming period. Control larvae attached to the substratum after the swimming period, resorbed the tail completely and underwent rotation of the visceral organs. Conversely, rotation occurred before completion of tail resorption in the demembranated larvae. Furthermore, the metamorphic events progressed more slowly in the demembranated larvae. These results suggest that the test cells play important roles in normal development and morphogenesis of ascidian larvae. Received: 4 December 1998 / Accepted: 9 April 1999     

Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis.

Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.     

Par6b Regulates the Dynamics of Apicobasal Polarity during Development of the Stratified Xenopus Epidermis

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the “basolateral” state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasticity of hatching in amphibians: evolution, trade-offs, cues and mechanisms

Many species of frogs and salamanders, in at least 12 families, alter their timing of hatching in response to conditions affecting mortality of eggs or larvae. Some terrestrially laid or stranded embryos wait to hatch until they are submerged in water. Some embryos laid above water accelerate hatching if the eggs are dehydrating; others hatch early if flooded. Embryos can hatch early in response to predators and pathogens of eggs or delay hatching in response to predators of larvae; some species do both. The phylogenetic pattern of environmentally cued hatching suggests that similar responses have evolved convergently in multiple amphibian lineages. The use of similar cues, including hypoxia and physical disturbance, in multiple contexts suggests potential shared mechanisms underlying the capacity of embryos to respond to environmental conditions. Shifts in the timing of hatching often have clear benefits, but we know less about the trade-offs that favor plasticity, the mechanisms that enable it, and its evolutionary history. Some potentially important types of cued hatching, such as those involving embryo-parent interactions, are relatively unexplored. I discuss promising directions for research and the opportunities that the hatching of amphibians offers for integrative studies of the mechanisms, ecology and evolution of a critical transition between life-history stages.     

Larval Tunic and the Function of the Test Cells in Ascidians

Abstract In normal ascidian development, cuticular fins begin to form at the late tailbud stage and are fully formed at hatching. When one or several neurulae were manually demembranated (follicle cells, vitelline coat and test cells removed) and cultured in seawater they failed to form caudal fins. Fins were normal when the follicle cells alone were removed. The shape of the fins was normal when demembranation was delayed to the late tailbud stage. Does demembranation cause the loss of an essential factor produced by the embryos themselves or do the test cells provide a factor for fin morphogenesis? Demembranated neurulae of Ascidia callosa were cultured in groups ranging in size from 2 to 80 in 1 ml volumes of seawater. The mean lengths of the caudal fins increased with group size. In larger groups, some embryos developed fins that were normal in shape and as long as undemembranated controls. Results were similar with Corella inflata. These experiments suggest that a diffusible substance from the embryos facilitates fin morphogenesis and that test cells are not required. Test cells deposit ‘ornaments’ on the tunic in some species. In other species no ornaments are produced. Ten families are compared. It is proposed that the test cells make the tunic hydrophilic.     

Taking the plunge: California Grunion embryos emerge rapidly with environmentally cued hatching

The process of hatching has been well studied in some model species of teleosts: the medaka Oryzias latipes, the mummichog Fundulus heteroclitus, and the zebrafish Danio rerio. These models are compared to the California Grunion, Leuresthes tenuis that has some unique features of reproduction related to tidal synchrony of spawning and environmentally cued hatching (ECH). During oviposition at spring tides, this marine teleost spawns out of water to bury its clutches on sandy beaches in the high intertidal zone. After embryos of L. tenuis reach hatching competence, hatching can be triggered at any time. Incubation above the water line inhibits hatching until ECH is triggered by rising tides during the following lunar phase, and hatching occurs within a few seconds. We review the embryo's response to environmental cues at hatching and the effects of the surrounding medium on the chorionase and chorion for this form of ECH. Leuresthes tenuis shares some similarities as well as some important differences with the model species. Comparison of hatching across teleostean taxa indicates great variability in stage at hatching and in duration of incubation that suggest hatching plasticity in response to environmental cues may be more widespread than currently appreciated.     

Differential structure and activity between human and mouse intelectin-1: human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer

Human intelectin-1 (hITLN-1) is a 120-kDa lectin recognizing galactofuranosyl residues found in cell walls of various microorganisms but not in mammalian tissues. Although mouse intelectin-1 (mITLN-1) has been identified previously, its biochemical properties and functional characteristics have not been studied. Therefore, we have compared structures and saccharide-binding specificities of hITLN-1 and mITLN-1 using recombinant proteins produced by mammalian cells. Recombinant hITLN-1 is a trimer, disulfide-linked through Cys-31 and Cys-48, and N-glycosylated at Asn-163. Despite 84.9% amino acid identity to hITLN-1, recombinant and intestinal mITLN-1 are unglycosylated 30-kDa monomers. Recombinant hITLN-1, as well as recombinant and intestinal mITLN-1 were purified by Ca(2+)-dependent adsorption to galactose-Sepharose. In competitive binding studies, hITLN-1 was eluted from galactose-Sepharose by 100 mM 2-deoxygalactose, a galactofuranosyl disaccharide, d-xylose, and both d- and l-ribose. In contrast, mITLN-1 was partially eluted by the galactofuranosyl disaccharide, and only minimally by the other saccharides indicating that the two intelectins have different saccharide-binding specificities. When the N- and C-terminal regions of hITLN-1 were replaced, respectively, with those of mITLN-1, galactose-Sepharose binding was associated with the C-terminal regions. Finally, hITLN-1 binding to galactose-Sepharose was not affected by the substitution of the Cys residues in the N-terminal region that are necessary for oligomer formation, nor was it affected by the removal of the N-linked oligosaccharide at Asn-163. Although both hITLN-1 and mITLN-1 recognize galactofuranosyl residues, our comparative studies, taken together, demonstrate that these intelectins have different quaternary structures and saccharide-binding specificities.