Gamete interactions in Xenopus laevis: identification of sperm binding glycoproteins in the egg vitelline envelope.

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.

Vg1 and regional specification in vertebrates: a new role for an old molecule.

The Vg1 protein was discovered some ten years ago in a screen for localized maternal RNA molecules involved in early embryonic patterning in the frog Xenopus laevis. The localization of this molecule to the vegetal pole suggested that Vg1 might function as a determinant of embryonic cell fate, and its DNA sequence revealed that it is related to factors involved in induction of the mesoderm. However, it is only in the past year that evidence hinting at the role of Vg1 in early development has emerged. It now seems that although the key component for specifying a vertebrate dorsal axis has been known to us for a decade, cryptic processing pathways have kept its role in this important process hidden from view.

Genomic organization and nucleotide sequence of two distinct histone gene clusters from Xenopus laevis. Identification of novel conserved upstream sequence elements.

We have performed a detailed analysis of the genomic organization and the nucleotide sequence of two distinct Xenopus laevis histone gene clusters totaling approximately 23.5 X 10(3) base-pairs. Each cluster contains at least one copy of each of the five histone genes. However, these genes are present in different arrangements within each cluster and different H1A, H2A and H2B proteins (variants) are encoded by the respective genes of each cluster. Southern blot analysis of genomic X. laevis DNA indicates that each cluster is a member of a distinct family of tandemly repeated histone gene clusters. A comparative analysis of the nucleotide sequences flanking the histone genes within these two clusters has revealed the presence of multiple conserved sequence elements that are specific for each histone gene class and located at preferred upstream positions. Several of these elements correspond to sequences that are known to be required for maximal transcription of the corresponding genes. Most of these sequence elements have not been identified previously, although we find that many of them are present at corresponding locations upstream of histone genes from other organisms. We suggest that the conserved upstream sequence elements may play an important role in the expression of histone genes in vivo.

Ventral mesoderm induction and patterning by bone morphogenetic protein heterodimers in Xenopus embryos.

Bone morphogenetic proteins (BMPs) perform diverse functions in vertebrate development. Here we demonstrate that the heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. We also provide indirect evidence that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. We propose that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development.

Hepatocellular carcinoma presenting as a tumour of the hilar and extrahepatic bile ducts.

A case of hepatocellular carcinoma extending within the large extra- and intrahepatic bile ducts is reported. No primary tumour was found in the liver parenchyma by abdominal ultrasound, spiral computed tomography or magnetic resonance, but transduodenal cholangioscopy showed tumour in the common hepatic ducts and the two main branches. Endoscopic biopsy showed highly differentiated hepatocellular carcinoma. The patient was treated with endoscopic biliary drainage and died at home 7 months after admittance.

[Spontaneous rupture of the kidney with perirenal hemorrhage in undiagnosed polyarteritis nodosa]. / Spontan nyreruptur med perirenal blødning ved ikkeerkendt polyarteritis nodosa.

Spontaneous rupture of the kidney with perirenal haemorrhage is a rare and serious complication of polyarteritis nodosa (PAN), usually requiring urgent surgery. The present case was a 28-year old male, who had been ill for 14 days, with abdominal pain, loss of appetite, nausea and vomiting. The patient was in shock on hospital admission. Explorative laparotomy revealed a massive haemorrhage from the right kidney. The kidney was removed and histological examination of the specimen revealed polyarteritis nodosa. The clinical history of the presented case is similar to previously described cases of kidney rupture in PAN. In the case presented, however, the diagnosis was first established histologically, while the diagnosis in most of the former cases was established on renal angiography, with findings of spindle-formed renal aneurysms.

Xenopus mothers against decapentaplegic is an embryonic ventralizing agent that acts downstream of the BMP-2/4 receptor.

Dorsal-ventral patterning in vertebrate embryos is regulated by members of the TGF-beta family of growth and differentiation factors. In Xenopus the activins and Vg1 are potent dorsal mesoderm inducers while members of the bone morphogenetic protein (BMP) subclass pattern ventral mesoderm and regulate ectodermal cell fates. Receptors for ligands in the TGF-beta superfamily are serine-threonine kinases, but little is known about the components of the signal transduction pathway leading away from these receptors. In Drosophila the decapentaplegic protein (dpp), a homolog of vertebrate BMP-2 and BMP-4, functions in dorsal-ventral axial patterning, and a genetic screen for components involved in signaling by dpp has identified a gene named mothers against decapentaplegic (Mad). Mad encodes a unique, predicted cytoplasmic, protein containing no readily identified functional motifs. This report demonstrates that a gene closely related to Drosophila Mad exists in Xenopus (called XMad) and it exhibits activities consistent with a role in BMP signaling. XMad protein induces ventral mesoderm when overexpressed in isolated animal caps and it ventralizes embryos. Furthermore, XMad rescues phenotypes generated by a signaling-defective, dominant-negative, BMP-2/4 receptor. These results furnish evidence that XMad protein participates in vertebrate embryonic dorsal-ventral patterning by functioning in BMP-2/4 receptor signal transduction.

A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation.

Pattern formation in early embryogenesis is guided by maternal, localized determinants and by inductive interactions between cells. In Xenopus eggs, localized molecules have been identified and some, such as Vg1 and Xwnt-11, can specify cell fates by functioning as inducers or patterning agents. We have used differential screening to identify new Xenopus genes that regulate mesodermal patterning, and we have isolated a new member of the T-box family of transcription factors. This gene, named Brat, is expressed maternally and its transcripts are localized to the vegetal hemisphere of the egg. During early embryonic cleavage, Brat mRNA becomes partitioned primarily within vegetal cells that are fated to form the endoderm. Zygotic expression of Brat begins at the onset of gastrulation within the presumptive mesoderm of the marginal zone. Consistent with its zygotic expression pattern, Brat induces, in a dose-dependent manner, a full spectrum of mesodermal genes that mark tissues across the dorsal-ventral axis, from the blood through the Spemann organizer. Brat also induces endoderm, consistent with its vegetal localization, making Brat a good candidate for a maternal determinant of the endoderm. We tested whether endogenous Brat is required for mesoderm formation by expressing a dominant-negative, transcriptional repressor form of Brat in embryos. This treatment inhibited mesoderm formation and severely disrupted normal development, thereby establishing that Brat plays a critical role in embryonic mesoderm formation and body patterning.

Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4.

We provide a comparative analysis of the expression patterns and ventral mesoderm-inducing properties of Xenopus BMP-2 and BMP-4. Transcripts for BMP-2 and BMP-4 are maternally stored in eggs, and zygotic expression of these genes is uniform in the ectoderm and mesoderm in late blastulae. During gastrulation, BMP-2 is expressed at a low level throughout the ectoderm and marginal zone, but at early neurula stages a patch of dorso-anterior cells displays enhanced expression. In contrast, BMP-4 transcripts are restricted to the ventrolateral marginal zone during gastrulation, and in late gastrula and early neurula BMP-4 is expressed in the epidermis but not the neural plate. At post-neurula stages, BMP-2 and BMP-4 transcripts are associated with a variety of mesodermal structures, including the pharyngeal pouches, heart, blood island, and blastopore. At tailbud stages, BMP-2 and BMP-4 are expressed in neural tissues including the neural tube and brain. In mesoderm induction assays, BMP-2 and BMP-4 induce Xhox3, an early ventral-posterior mesoderm marker, and larval alpha Tl globin, a marker for red blood cells. Induction of red blood cells in response to BMP-4 was demonstrated by staining with a hemoglobin-specific reagent. Little is known about factors that induce hematopoietic lineages in vertebrates, and these results provide evidence linking BMP activity and blood differentiation. Globin induction by BMP-2 and BMP-4 is blocked by co-expression of a dominant-negative activin receptor, suggesting that either endogenous activin signals are required for BMP-mediated induction, or that the truncated activin receptor interferes with signaling by BMP receptors. In assays on marginal zone explants, we demonstrate that BMP-4 respecifies dorsal mesoderm to form ventral mesoderm, consistent with its ability to induce blood and to ventralize embryos. BMP-2, however, does not display such activity. The findings extend and support evidence that BMP-2 and BMP-4 function in ventral mesoderm induction and patterning in Xenopus. Our data furthermore highlight the multiple functions these factors fulfill during early vertebrate embryogenesis.

Processed Vg1 protein is an axial mesoderm inducer in Xenopus.

Vg1 is a TGF beta-related growth factor encoded by a maternal mRNA localized to vegetal blastomeres in Xenopus embryos. Vg1 precursor protein is abundant in vegetal cells, but the processed mature form has not been readily detected and no activity has been demonstrated for the putative Vg1 mature protein. We have engineered a BMP2-Vg1 fusion (BVg1) that promotes formation of mature Vg1 protein in vivo. Injection of BVg1 mRNA induces dorsal mesoderm in animal cap cells, and BVg1 expression in ultraviolet-ventralized embryos fully restores a normal dorsal axis. Blastomeres expressing BVg1 act as a Nieuwkoop center, the region that induces the Spemann organizer. our results lead us to suggest that localized posttranslational processing of Vg1 precursor protein on the future dorsal side of the embryo is a key step in generating dorsal mesoderm and the body axis in Xenopus.

Tbx5 is essential for heart development.

Mutations in the Tbx5 transcription factor cause heart septal defects found in human Holt-Oram Syndrome. The complete extent to which Tbx5 functions in heart development, however, has not been established. Here we show that, in Xenopus embryos, Tbx5 is expressed in the early heart field, posterior to the cardiac homeobox transcription factor, Nkx2.5. During morphogenesis, Tbx5 is expressed throughout the heart tube except the anterior portion, the bulbus cordis. When Tbx5 activity is antagonized with a hormone-inducible, dominant negative version of the protein, the heart fails to develop. These results suggest that, in addition to its function in heart septation, Tbx5 has a more global role in cardiac specification and heart development in vertebrate embryos.

Major transitions in histone gene expression do not occur during development in Xenopus laevis.

In light of the parallels that exist in the structure of histone genes in sea urchins and in the frog, Xenopus laevis, and in the early development of these animals, it has been thought that Xenopus histone gene expression might be subject to the type of developmental regulation observed in sea urchins. We have examined the patterns of histone mRNA accumulation in Xenopus oocytes and embryos by primer extension and S1 nuclease protection techniques. The data demonstrate that histone genes which are active in Xenopus oocytes, and which contribute to large pools of histone mRNA in the absence of DNA replication, are also transcriptionally active in late embryos and in cultured cells. These results suggest that, rather than activating distinct sets of histone genes at different developmental stages, the developing frog embryo reprograms the expression of histone genes active in nondividing oocytes so that their expression becomes coupled to DNA replication subsequently during embryogenesis.

Drosophila 60A gene, another transforming growth factor beta family member, is closely related to human bone morphogenetic proteins.

The 60A gene, a member of the transforming growth factor beta superfamily of signaling proteins, has been identified in Drosophila melanogaster. From its inferred protein sequence we predict the precursor is secreted and processed to release a growth factor-like molecule. The 60A gene is expressed throughout development with peaks of transcription during early embryogenesis, in pupae, and in adult males. The putative 60A protein shows greater sequence similarity to three vertebrate family members (human bone morphogenetic proteins 5, 6, and 7) than to its only Drosophila relative, the protein product of the decapentaplegic (dpp) gene. This observation suggests that the duplication event that gave rise to the two transforming growth factor beta-like proteins in Drosophila predates the divergence of chordates and arthropods.

Systemic coagulation activation and anastomotic leakage after colorectal cancer surgery.

PURPOSE: The aim of the present study was to study whether patients developing anastomotic leakage after colorectal resections for colorectal cancer have laboratory signs of an altered hemostatic balance in the systemic circulation, preoperatively and postoperatively, causing an impaired healing process. METHODS: Patients operated on for colorectal cancer were studied. Seventeen consecutive patients with anastomotic leakage and 17 patients without anastomotic leakage were matched according to age, gender, tumor stage, and localization of tumor. Hemostatic balance was estimated preoperatively and at one, two, and seven days and at three months after surgery by plasma levels of sensitive markers of coagulation activation and fibrinolysis, i.e., prothrombin fragment 1 + 2, thrombin-antithrombin complexes, soluble fibrin, tissue-type plasminogen activator activity, and plasminogen activator inhibitor Type 1. RESULTS: Preoperatively, the hemostatic balance was comparable in patients with and without anastomotic leakage. In the early postoperative period, patients developing anastomotic leakage exhibited signs of systemic coagulation activation, i.e., elevated plasma levels of prothrombin fragment 1 + 2, thrombin-antithrombin complexes, soluble fibrin, and plasminogen activator inhibitor Type 1. The observed coagulation activation appeared before the anastomotic leakage became clinically evident. More patients with anastomotic leakage received perioperative blood transfusions than patients without leakage, despite the fact that duration of surgery and intraoperative blood loss were comparable in the two groups. CONCLUSIONS: Enhanced coagulation activity was observed postoperatively in patients developing anastomotic leakage after colorectal resections for colorectal cancer. Such a hypercoagulable state may contribute to the development of anastomotic leakage by facilitating formation of microthromboses in the perianastomotic area.

Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation.

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

Xenopus laevis sperm-egg adhesion is regulated by modifications in the sperm receptor and the egg vitelline envelope.

The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation.

The TGF-beta superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-beta-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-beta target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the 'common-partner' SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-beta) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-beta signals.

Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation.

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.

Expression of activin mRNA during early development in Xenopus laevis.

Activins are members of the transforming growth factor-beta superfamily, a class of peptide growth factors that can regulate the growth and differentiation of a variety of cell types. In mesoderm induction assays, activins A and B were shown to be very potent inducers and it was only recently demonstrated that they are crucial for initial mesoderm induction in Xenopus embryos. To determine the source of activin protein for initial mesoderm induction and to investigate whether activins may play further roles in embryonic development we have examined the localization of the mRNAs encoding the activin beta A and beta B subunits in Xenopus embryos. Activin beta A and beta B mRNAs are found in the follicle cells surrounding oocytes but not in oocytes themselves or fertilized eggs. During embryogenesis activin mRNA is first detected after the midblastula transition and expression increases as development proceeds. Activin beta B mRNA is homogeneously distributed during blastula and early gastrula stages but restricted to the dorso-anterior region in neurula stage embryos. At the early tailbud stage activin expression becomes confined to the brain, eye analgen, visceral pouches, otic vesicles, and the anterior notochord.