Cyclooxygenase-1 signaling is required for vascular tube formation during development

Prostaglandin endoperoxide synthases (PTGS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze the key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H2 (PGH2). Although COX and prostaglandins have been implicated in a wide variety of physiologic processes, an evaluation of the role of prostaglandins in early mammalian development has been difficult due to the maternal contribution of prostaglandins from the uterus: COX null mouse embryos develop normally during embryogenesis. Here, we verify that inhibition of COX-1 results in zebrafish gastrulation arrest and shows that COX-1 expression becomes restricted to the posterior mesoderm during somitogenesis and to posterior mesoderm organs at pharyngula stage. Inhibition of COX-1 signaling after gastrulation results in defective vascular tube formation and shortened intersomitic vessels in the posterior body region. These defects are rescued completely by PGE(2) treatment or, to a lesser extent, by PGF(2alpha), but not by other prostaglandins, such as PGI(2), TxB(2), or PGD(2). Functional knockdown of COX-1 using antisense morpholino oligonucleotide translation interference also results in posterior vessel defect in addition to enlarged posterior nephric duct, phenocopying the defects caused by inhibition of COX-1 activity. Together, we provide the first evidence that COX-1 signaling is required for development of posterior mesoderm organs, specifically in the vascular tube formation and posterior nephric duct development.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures

The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999     

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of β-catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.     

Wnt/PCP signaling controls intracellular position of MTOCs during gastrulation convergence and extension movements

During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.     

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.     

Circulating proangiogenic molecules PIGF, SDF-1 and sVCAM-1 in patients with systemic lupus erythematosus

Serum concentrations of three angiogenic cytokines: vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1) and placental growth factor (PIGF) and soluble vascular cell adhesion molecule 1 (sVCAM-1), were investigated in the serum of 61 patients with systemic lupus erythematosus (SLE) and 20 healthy subjects. The possible association between serum levels of these proteins and SLE activity, as well as correlation between the concentrations of cytokines were also analysed. All of these factors were detectable in all SLE patients and the healthy control group. The median concentration of VEGF was higher in active SLE (386 pg/mL) than in inactive disease (327 pg/mL) or in the control group (212 pg/mL, p<0.004). The median serum level of SDF-1 was higher in SLE patients (1,814 pg/mL) than in the control group (1,507 pg/mL, p<0.02). The median concentration of PIGF was higher (14 pg/mL) in SLE patients than in the control group (12 pg/mL, p=0.03), and particularly in active disease (17 pg/mL) as compared to the inactive phase (13 pg/mL, p=0.01). The correlations between the levels of cytokines examined and clinical features, laboratory abnormalities and the type of treatment were also analysed. We found a positive correlation between serum concentrations of PIGF and SLE activity according to SLAM score (p=0.33, p=0.13).