GSK3beta/shaggy mediates patterning along the animal-vegetal axis of the sea urchin embryo

In the sea urchin embryo, the animal-vegetal axis is defined before fertilization and different embryonic territories are established along this axis by mechanisms which are largely unknown. Significantly, the boundaries of these territories can be shifted by treatment with various reagents including zinc and lithium. We have isolated and characterized a sea urchin homolog of GSK3beta/shaggy, a lithium-sensitive kinase which is a component of the Wnt pathway and known to be involved in axial patterning in other embryos including Xenopus. The effects of overexpressing the normal and mutant forms of GSK3beta derived either from sea urchin or Xenopus were analyzed by observation of the morphology of 48 hour embryos (pluteus stage) and by monitoring spatial expression of the hatching enzyme (HE) gene, a very early gene whose expression is restricted to an animal domain with a sharp border roughly coinciding with the future ectoderm / endoderm boundary. Inactive forms of GSK3beta predicted to have a dominant-negative activity, vegetalized the embryo and decreased the size of the HE expression domain, apparently by shifting the boundary towards the animal pole. These effects are similar to, but even stronger than, those of lithium. Conversely, overexpression of wild-type GSK3beta animalized the embryo and caused the HE domain to enlarge towards the vegetal pole. Unlike zinc treatment, GSK3beta overexpression thus appeared to provoke a true animalization, through extension of the presumptive ectoderm territory. These results indicate that in sea urchin embryos the level of GSKbeta activity controls the position of the boundary between the presumptive ectoderm and endoderm territories and thus, the relative extent of these tissue layers in late embryos. GSK3beta and probably other downstream components of the Wnt pathway thus mediate patterning both along the primary AV axis of the sea urchin embryo and along the dorsal-ventral axis in Xenopus, suggesting a conserved basis for axial patterning between invertebrate and vertebrate in deuterostomes.     

Na is essential for activation of the inseminated sea urchin egg

The presence of at least 2.5 mM Na during the first several min after insemination is required for the activation of sea urchin eggs. Of those chemical species examined that exist entirely as cations, and which did not activate the unfertilized egg, only Li substitutes for Na. Ammonium and other amines, with pKa values between 9 and 10.8, in the complete absence of Na (a) can induce nuclear activation of unfertilized eggs, and (b) permit the development of the early fertilized egg through the stage that normally requires Na.     

Serine hydroxymethyltransferase is maternally essential in Caenorhabditis elegans

The mel-32 gene in the free living soil nematode Caenorhabditis elegans encodes a serine hydroxymethyltransferase (SHMT) isoform. Seventeen ethylmethanesulfonate (EMS)-induced mutant alleles of mel-32(SHMT) have been generated, each of which causes a recessive maternal effect lethal phenotype. Animals homozygous for the SHMT mutations have no observable mutant phenotype, but their offspring display an embryonic lethal phenotype. The Mel-32 phenotype has been rescued with a transgenic array containing only mel-32(SHMT) genomic DNA. Heteroduplex analysis of the 17 alleles allowed 14 of the mutations to be positioned to small regions. Subsequent sequence analysis has shown that 16 of the alleles alter highly conserved amino acids, while one allele introduces a stop codon that truncates two thirds of the predicted protein. mel-32(SHMT) has a 55-60% identity at the amino acid level with both isoforms of SHMT found in yeast and humans and a 50% identity with the Escherichia coli isoform. The C. elegans mel-32 mutation represents the first case where SHMT has been shown to be an essential gene.     

Bottle cells are required for the initiation of primary invagination in the sea urchin embryo

Invagination of epithelial tissue occurs during gastrulation, neurulation, and organogenesis in many organisms. However, the underlying morphogenetic mechanisms of invagination are not understood. To elucidate these mechanisms, we have analyzed the initial invagination of the vegetal plate in the sea urchin embryo, a process termed primary invagination. At the onset of invagination, a ring of cells with highly constricted apices (bottle cells) encircles a group of two to eight round, central cells. To investigate the morphogenetic role of the bottle cells in the process of primary invagination, we have undertaken a series of laser ablation studies in which different proportions of various cell types were ablated and the effects were recorded using 4-D microscopy. Elimination of a 90 degrees-180 degrees arc of bottle cells markedly retards invagination, but only within the ablated region. Ablation of other cell types does not result in a statistically significant effect on primary invagination. These studies indicate that the number and arrangement of the bottle cells are critical factors for proper initiation of invagination. In addition, we have used the perturbing anti-hyalin antibody mAb183 to show that cell attachment to the hyaline layer is necessary for bottle cell formation and the initiation of primary invagination.     

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.     

BMP4 is essential for lens induction in the mouse embryo

Vertebrate lens development is a classical model system for studying embryonic tissue interactions. Little is known, however, about the molecules mediating such inductive events. Here, we show that Bmp4, which is expressed strongly in the optic vesicle and weakly in the surrounding mesenchyme and surface ectoderm, has crucial roles during lens induction. In Bmp4(tm1) homozygous null mutant embryos, lens induction is absent, but the process can be rescued by exogenous BMP4 protein applied into the optic vesicle in explant cultures. This is associated with rescue of ectodermal expression of Sox2, an early lens placode marker. Substituting the optic vesicle in explant cultures with BMP4-carrying beads, however, does not lead to lens induction, indicating that other factors produced by the optic vesicle are involved. BMP4 appears to regulate expression of a putative downstream gene, Msx2, in the optic vesicle. No change in Pax6 expression is seen in Bmp4(tm1) mutant eyes, and Bmp4 expression appears unaffected in the eyes of homozygous Pax6(Sey-1Neu), suggesting that PAX6 and BMP4 function independently. Based on these results we propose that BMP4 is required for the optic vesicle to manifest its lens-inducing activity, by regulating downstream genes and/or serving as one component of multiple inductive signals.     

The betaL integrin subunit is necessary for gastrulation in sea urchin embryos

Integrins are a family of cell adhesion molecules reported to mediate cellular interactions essential for normal embryonic morphogenesis. Here we describe a beta integrin subunit that is expressed during early embryogenesis in the sea urchin embryo and appears to be necessary for normal development. The deduced amino acid sequence of betaL is similar to vertebrate beta integrin subunits, but is most closely related to the sea urchin betaG subunit. Northern blots show that betaL is expressed at all stages with maximum expression beginning during gastrulation. Immunolocalization and in situ RNA hybridization show that in blastulae betaL is expressed in the blastoderm and by the ring of bottle cells in the vegetal plate during the initial phase of gastrulation. Presumptive secondary mesenchyme cells express high levels of betaL throughout elongation of the archenteron and in the pluteus betaL is expressed by blastocoelar cells, skeletal mesenchyme, and pigment cells. Antibodies and Fab fragments against betaL block spreading of dissociated embryonic cells on RGD (arginine-glycine-aspartate)-containing substrates. Treating embryos with anti-betaL antibodies blocks the initial phase of gastrulation and interferes with the organization of actin filaments. Prior to gastrulation, the antibodies cause thickening of the blastoderm and later in development defects in skeletal patterning result. Probing for antibody in treated embryos indicates that it penetrates the ectoderm to cells within the blastocoel and is actively endocytosed. We propose that betaL forms receptors that bind to RGD-containing ligands and anchors actin filaments. These receptors appear to be essential in several aspects of morphogenesis.     

The 350-kDa sea urchin egg receptor for sperm is localized in the vitelline layer

Previous studies have established by several methods that the 350-kDa egg receptor for sperm is localized on the plasma membrane-vitelline layer complex of the egg of the sea urchin Strongylocentrotus purpuratus. In addition, it has been found that molecules which are cross-reactive with anti-receptor antibody are present in the cortical granules located at the inner face of the plasma membrane. The objective of this study was to define more precisely the locale of the cell surface receptor. We have found that following fertilization, the immunoreactive receptor initially found on the egg surface moved to the fertilization envelope (FE) and then disappeared in parallel with the loss of sperm binding activity. A cross-linked, high-molecular-weight derivative of soybean trypsin inhibitor (hMW-SBTI) which was unable to pass through the elevating FE blocked the loss of both immunoreactivity and the sperm binding activity of the FE, but did not inhibit the vitelline delaminase activity that has been implicated in FE formation. Western blot analysis following SDS-PAGE of the proteins of the FE isolated in the presence of hMW-SBTI and benzamidine revealed the presence of the 350/320-kDa proteins which cross-reacted with anti-receptor antibody. Experiments in which molecules on the surface of unfertilized eggs were labeled with biotin and traced after FE formation revealed that a significant portion of the 350/320-kDa glycoproteins that were incorporated into the FE originated from the cell surface, rather than from the cortical granules. These findings provide strong evidence that in unfertilized eggs the egg receptor for sperm exists as part of the protein complex known as the vitelline layer which serves as a precursor of the FE. Evidence is presented indicating that some of the receptor in the vitelline layer is cryptic and a possible function for this cryptic form of the receptor is proposed.     

An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila

Overexpression of many growth factor receptors, as well as growth factors, has been shown to confer varying degrees of estrogen-independent growth on estrogen receptor (ER) positive breast cancer cells. The proto-oncogene Raf-1 is a key intermediate in the signal transduction pathway of many of these growth factor receptors, and when constitutively activated in fibroblasts is transforming. To examine the effects of Raf-1 kinase activity on the estrogen-dependent growth of human breast cancer cells, ER + MCF-7 breast cancer cells were stably transfected with an expression construct directing the expression of an amino-truncated protein having constitutive kinase activity. Expression of constitutively activated Raf in MCF-7 cells is incompatible with growth in the presence of estrogen; that is, cells down-regulate expression of the transfected Raf. Constitutive Raf activity does allow for growth of the cells in the absence of estrogen, suggesting that activation of growth factor signaling pathways through Raf may confer a selective advantage for growth of breast cancer cells under estrogen-deprived conditions. In addition, the high levels of Raf activity induce apoptosis in cells grown under either condition. This is a novel activity for Raf, and may occur because the levels of the constitutive Raf are extremely high in these cells.     

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.     

Organization of the proximal promoter of the hatching-enzyme gene, the earliest zygotic gene expressed in the sea urchin embryo

The hatching enzyme (HE) gene is the earliest zygotic gene expressed in the sea urchin embryo. To investigate the regulation of the HE gene activity, 5' flanking DNA and the 5' untranslated leader were inserted upstream of reporter genes whose expression was monitored in vivo during development after transfer into eggs. By deletion analysis we showed that no more than 3 kb of flanking sequence are required for correct expression of transgenes. The proximal region of 0.5 kb does not precisely control spatial restriction but drives expression at a nearly maximal level. The proximal promoter was searched extensively for sites of protein-DNA interactions by DNAse protection and gel-shift methods. The 12 sites identified form 3 groups: core promoter; central region; and distal region. The central region bears three sites that contain a direct or inverted CCAAT box. Mutation and deletion analysis showed that, in addition to the core-promoter elements, the two most-distal CCAAT-containing sites are indispensable for promoter activity. These sites bind the same set of proteins, which are abundant in the nuclei of cleavage embryos.     

Surface localization of the sea urchin egg receptor for sperm

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.     

Oral-aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore

Cognitive-behavioral therapy (CBT) has been shown to be a highly effective form of treatment for patients with bulimia nervosa and anorexia nervosa. Issues of satiety disturbances, food restriction, and food choice are central to this form of therapy; however, ingestive behavior research that directly addresses these issues in eating disorder patients has often been overlooked by clinicians. These areas of research are reviewed and the implications of the findings for more effective CBT therapy with anorexics and bulimics are discussed.