Blastula Embryos Research Articles

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper ( Epinephelus coioides)

Follicle consists of an oocyte and a lot of surrounding follicular cells, and significant interactions exist between the oocyte and the somatic cells. In this study, a novel cDNA has been screened from a subtractive cDNA library between tail bud embryos and blastula embryos in the protogynous hermaphrodite orange-spotted grouper ( Epinephelus coioides). Its full-length cDNA is 821 bp, and has an ORF of 414 bp for encoding a peptide of 137 aa, which shows 38%, 37%, 33%, and 33% homology with 4 putative proteins screened from zebrafish ( Danio rerio). Conserved domain search in NCBI reveals a single C2 domain existing in the C2 domain superfamily proteins, and has only 7 β strands in comparison with 8 β strands of C2 domains in other C2 domain superfamily proteins. Artificial sex reversal, RT-PCR analysis and Western blot detection demonstrated ovary-specific expression of the C2 domain factor, and therefore the novel gene was designated as E. coioides ovary-specific C2 domain factor, EcOC2 factor. Moreover, predominant expression of EcOC2 factor was further revealed in grouper mature ovary, and its strong immunofluorescence signals were located between granulosa cells and oocyte zona radiata in grouper mature follicles. The data indicate that the novel EcOC2 factor might be a main component that associates between granulosa cells and the oocyte during oocyte maturation, and might play significant roles in regulating oocyte maturation and ovulation. Further studies on its developmental behaviour and physiological functions will elucidate the interactions between oocyte and the surrounding somatic cells and the underlying molecular mechanisms.

Identification of a Spindlin homolog in gibel carp ( Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp ( Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin ( CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and β-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with β-tubulin and other spindle proteins during oocyte maturation and egg fertilization.

Activation of the mouse Oct4 promoter in medaka embryonic stem cells and its use for ablation of spontaneous differentiation

The determination and maintenance of the cell fate is ultimately due to differential gene activity. In the mouse, expression of the transcription factor Oct4 is high in totipotent inner cell mass, germ cells and undifferentiated embryonic stem (ES) cells, but dramatically reduced or extinct upon differentiation. Here, we show that medaka blastula embryos and cells of the ES cell line MES1 are able to activate the Oct4 promoter. Ectopic expression of a fusion gene for beta-galactosidase and neomycin resistance from the Oct4 promoter conferred resistance to G418. G418 selection led to a homogenous population of undifferentiated ES cells which were able to undergo induced or directed differentiation into various cell types including neuron-like cells and melanocytes. Furthermore, GFP-labeled GOF18geo-MES1 cells after differentiation ablation were able to contribute to a wide variety of organ systems derived from all the three germ layers. Most importantly, we show that drug ablation of differentiation on the basis of Oct4 promoter is a useful tool to improve ES cell cultivation and chimera formation: MES1 cells after differentiation ablation appeared to be better donors than the parental MES1 line, as the permissive number of input donor cells increases from 100 to 200, resulting in an enhanced degree of chimerism. Taken together, some transcription factors and cis-acting regulatory sequences controlling totipotency-specific gene expression appear to be conserved between mammals and fish, and medaka ES cells offer an in vitro system for characterizing the expression of totipotency-specific genes such as putative Oct4 homologs from fish.

VegT activation of the early zygotic gene Xnr5 requires lifting of Tcf-mediated repression in the Xenopus blastula

Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-β factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localised throughout the vegetal pole, and β-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, we have defined a minimal promoter, which mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional β-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that β-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a β-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. We propose a model in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of β-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of β-catenin/XTcf-3.

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development.

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.

Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka, Oryzias latipes.

In order to investigate the function of gonadal somatic cells in the sex differentiation of germ cells, we produced chimera fish containing both male (XY) and female (XX) cells by means of cell transplantation between blastula embryos in the medaka, Oryzias latipes. Sexually mature chimera fish were obtained from all combinations of recipient and donor genotypes. Most chimeras developed according to the genetic sex of the recipients, whose cells are thought to be dominant in the gonads of chimeras. However, among XX/XY (recipient/donor) chimeras, we obtained three males that differentiated into the donor's sex. Genotyping of their progeny and of strain-specific DNA fragments in their testes showed that, although two of them produced progeny from only XX spermatogenic cells, their testes all contained XY cells. That is, in the two XX/XY chimeras, germ cells consisted of XX cells but testicular somatic cells contained both XX and XY cells, suggesting that the XY somatic cells induced sex reversal of the XX germ cells and the XX somatic cells. The histological examination of developing gonads of XX/XY chimera fry showed that XY donor cells affect the early sex differentiation of germ cells. These results suggest that XY somatic cells start to differentiate into male cells depending on their sex chromosome composition, and that, in the environment produced by XY somatic cells in the medaka, germ cells differentiate into male cells regardless of their sex chromosome composition.

Functional characterization of Ets-binding sites in the sea urchin embryo: three base pair conversions redirect expression from mesoderm to ectoderm and endoderm

Because of the limited knowledge of target genes for the ets family of transcription factors, it is yet unclear how specificity of biological function among different members is achieved in this class of proteins. In the present study, we compared two Ets-binding sites in two differentially expressed genes of the sea urchin embryo. The first gene examined is the cytoskeletal actin CyIIa, which is transiently expressed in skeletogenic and secondary mesenchyme and in its terminal and permanent phase in the gut. The second one encodes the hatching enzyme gene of Strongylocentrotus purpuratus, and is regulated cell-autonomously and asymmetrically along the maternally determined animal–vegetal axis. The Ets sites within the regulatory regions of these two genes interact and form different binding complexes with proteins present in the nuclei of mesenchyme blastula embryos. We also demonstrated that the DNA binding specificity of the CyIIa Ets-binding site can be converted to the other type of Ets site, as in the hatching enzyme promoter, by changing only three base pairs near the Ets core sequence. Switching of these three base pairs near the central GGA trinucleotide motif characteristic of all Ets-binding targets was also sufficient to redirect expression of a reporter gene construct containing a heterologous basal promoter from mesenchyme to non-mesenchyme cell type in transgenic sea urchin embryos. These observations suggest that binding affinity of ets transcription factors plays an important role in determining cell type-specific gene expression.

Beta-catenin, MAPK and Smad signaling during early Xenopus development.

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. In this study, we have systematically analyzed and compared the signaling pattern of four major pathways by localization of the activated key components beta-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). We have determined semi-quantitatively the distribution of these components at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages, by immunofluorescence on serial cryosections. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, we have manipulated maternal beta-catenin signaling and determined the effect on the other pathways in the blastula embryo. We found that the patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on beta-catenin at this stage.

Single-cell internalization during zebrafish gastrulation

During gastrulation, germ layers are formed as prospective mesodermal and endodermal cells internalize and come to underlie the ectoderm [1–9]. Despite the pivotal role of gastrulation in animal development, the cellular interactions underlying this process are poorly understood. In zebrafish, mesoderm and endoderm formation requires the Nodal signals Cyclops and Squint and their cofactor One-eyed pinhead (Oep) [10–14]. We found that marginal cells in maternal-zygotic oep (MZoep) mutants do not internalize during gastrulation and acquire neural and tail fates at the expense of head and trunk mesendoderm. The lack of internalization in MZoep embryos and the cell-autonomous requirement for oep in Nodal signaling enabled us to test whether internalization can be achieved by individual cells or whether it depends on interactions within a group of cells. We found that individual MZoep mutant cells transplanted to the margin of wild-type blastula embryos initially internalize with their neighbors but are unable to contribute to the mesendoderm. In the reciprocal experiment, single wild-type cells transplanted to the margin of MZoep mutant embryos autonomously internalize and can express the mesendodermal markers axial/foxA2 and sox17. These results suggest that internalization and mesendoderm formation in zebrafish can be attained autonomously by single cells.

Xoom is maternally stored and functions as a transmembrane protein for gastrulation movement in Xenopus embryos.

Xoom has been identified as a novel gene that plays an important role in gastrulation of Xenopus laevis embryo. Although Xoom is actively transcribed during oogenesis, distribution and function of its translation product have not yet been clarified. In the present study, the polyclonal antibody raised against Xoom was generated to investigate a behavior of Xoom protein. Anti-Xoom antibodies revealed that there are two forms of Xoom protein in Xenopus embryos: (i) a 45 kDa soluble cytoplasmic form; and (ii) a 44 kDa membrane-associated form. Two forms of Xoom protein were ubiquitously detected from unfertilized egg to tadpole stage, with a qualitative peak during blastula and gastrula stages. Immunohistochemical examination showed that Xoom protein is maternally stored in the animal subcortical layer and divided into presumptive ectodermal cells during cleavage stages. Enzymatic digestion of membrane protein and immunologic detection of Xoom showed that Xoom exists as a membrane-associated protein. To examine a function of Xoom protein, anti-Xoom antibodies were injected into blastocoele of stage 7 blastula embryo. Anti-Xoom antibodies caused gastrulation defect in a dose- dependent manner. These results suggest that maternally prepared Xoom protein is involved in gastrulation movement on ectodermal cells.

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum.

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.

Expression of GFP in Nuclear Transplants Generated by Transplantation of Embryonic Cell Nuclei from GFP-Transgenic Fish into Nonenucleated Eggs of Medaka,Oryzias latipes

In order to investigate whether foreign genes can be used as genetic markers of donor nuclei in fish nuclear transplantation, expression of the GFP gene derived from donor nuclei was examined in nuclear transplants in medaka (Oryzias latipes). Embryonic nuclei were obtained from blastula embryos produced by crossing of transgenic fish of the wild-type strain heterozygous for the GFP gene with nontransgenic ones or by mutual crossing between transgenic fish. The GFP gene was driven by the promoter of the medaka elongation factor gene, EF-1alpha-A, which is known to induce GFP expression in many tissues except for the muscle in the transgenic fish. The nuclei were transplanted into nonenucleated unfertilized eggs of the orange-red strain. Adult nuclear transplants were successfully obtained at the rate of about 2% of the operated eggs. They were triploid and had no reproductive potential. The GFP gene was expressed in embryos, fry, and adults of nuclear transplants in a pattern similar to that in the transgenic fish. These results indicate that GFP is useful as a foreign genetic marker of donor nuclei in fish nuclear transplantation.

Mesoderm induction in Xenopus. Oocyte expression system and animal cap assay.

AbstractVertebrate mesoderm forms a diversity of tissues, including notochord, somitic muscle, pronephros, mesenchyme, and blood. In the amphibian Xenopus laevis, mesoderm arises in the equatorial region (marginal zone) of the blastula embryo in response to inducing signals produced by vegetal pole cells (1). This model of Xenopus mesoderm induction is derived from tissue recombination experiments in which vegetal pole tissue (prospective endoderm) was cocultured with animal pole tissue (prospective ectoderm) and resulted in the conversion of animal pole tissue into mesoderm (2). Therefore, vegetal pole cells of the cleavage embryo secrete factors that can redirect animal pole cells from ectodermal fates to mesodermal fates. These studies identified the source of mesoderm-inducing signals (vegetal pole) and established the animal pole explant as a responsive tissue useful for identifying mesoderm-inducing factors (3–5).KeywordsBody Wall MuscleAnimal PoleVitelline MembraneVegetal PoleMesoderm InductionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Temperature dependence of membrane lipid composition in early blastula embryos of Lytechinus pictus: selective sorting of phospholipids into nascent plasma membranes.

Lytechinus pictus eggs were fertilized and incubated at 10, 16, and 23 degrees C until the early blastula stage of embryonic development. The phospholipid composition of the embryos and control unfertilized eggs remain identical and unchanged as incubating temperatures are varied; thus, neither incubating temperature, fertilization nor membrane assembly affect their total phospholipid composition. This result agrees with metabolic studies by others, using only a single incubation temperature, and indicates that embryonic development to the early blastula stage occurs with little, if any, de novo phospholipid biosynthesis. However, as in all poikilotherms, the phospholipid composition of the nascent plasma membranes varies with the incubation temperature. Thus, until the blastula stage of embryonic development, the lipids of these newly formed plasma membranes are derived from lipid pools within the embryo whose phospholipid composition is static. The variation of plasma membrane composition is primarily reflected in an increase in the phosphatidylethanolamine (PE): phosphatidylcholine (PC) ratio as incubating temperatures decrease; this is achieved by an exchange of PE for PC. Several mechanisms are considered for the specificity of the selective sorting and assembly of these phospholipids into the nascent plasma membranes.

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor-derived gametes.

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES-like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma-irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor-derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells.

A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: evidence of counter-adhesive activity in vivo.

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+-binding, counter-adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration-dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C-terminal, Ca2+-binding, EF-hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration-dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60-100 ng peptide 4.2 or 0.25-0.4 microg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso-anterior markers at the neural and tail-bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counter-adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.

Microinjection into zebrafish embryos.

Microinjection remains the most popular and effective of the methods to introduce DNA, RNA, and proteins into fertilized zebrafish eggs. The method is simple and reliable. A microinjection pipet is filled with the DNA or RNA solution and attached to an apparatus that forces the solution out of the pipet with air pressure. A small amount of solution is then expelled into the cytoplasm of the embryo before withdrawing the pipet, and the injected embryos are incubated to develop further. Once inside the cells, the foreign DNA or RNA is transcribed and/or translated within the developing embryos and the functional roles of their protein products can be evaluated by morphological, physiological or molecular changes. Thus, microinjection has been widely used for generating transgenic fish (, , ), analyzing gene function by overexpression of DNA or RNA (, , ) and mapping cell fate in early blastula embryos (,).

Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number.

Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis. This is reflected by the fact that the ES cell technology to date could only be fully developed in mice. In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism. Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation. The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts. Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution. During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells. Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells. Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka.

A protein of the basal lamina of the sea urchin embryo.

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus. The protein has been named Pl-200K or Hp-200K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.

THE OLIGOMERIC INTEGRITY OF TOPOSOME IS ESSENTIAL FOR ITS MORPHOGENETIC FUNCTION

Sea urchin embryos are uniquely suitable for the study of morphogenetic cell interactions. Efforts to identify the molecules responsible for morphogenetic cell adhesion led to the isolation of a 22S glycoprotein complex fromParacentrotus lividussea urchin embryo, that has been called toposome. The biological activity of toposome in mediating cellular adhesion has been fully documented. Its function in determining positional guidance during the development of the sea urchin embryo has been proposed. Here studies on the molecular structure of toposome are reported showing that, under non-reducing conditions, it is resolved in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in a major band with an apparent molecular weight of 260 kDa, a doublet of 180–160kDa and a lower band of 80kDa. Digestion with EndoH endoglycosidase reduced the molecular sizes of the bands of 10%, 20% and 40%, respectively. In order to establish if the oligomeric integrity of toposome was essential for its function, the biological activity of each subunit on cells dissociated from sea urchin blastula embryos was tested. The resulting swimming embryoids were lacking skeleton, while reaggregating cells supplemented with native toposome developed into pluteus-like structures with skeletal elements.