Very low density lipoprotein receptor, a negative regulator of the wnt signaling pathway and choroidal neovascularization

Choroidal neovascularization (CNV) in age-related macular degeneration is a leading cause of blindness. Very low density lipoprotein receptor gene knock-out (Vldlr(-/-)) mice have been shown to develop subretinal neovascularization (NV) with an unknown mechanism. The present study showed that in Vldlr(-/-) mice, NV initiated in the choroid and progressed to penetrate the retinal pigment epithelium layer, proliferating in the subretinal space. This phenotype recapitulated what is seen in wet age-related macular degeneration, suggesting that this is a CNV model. The CNV correlated with overexpression of vascular endothelial growth factor in Vldlr(-/-) eyecups and was blocked by a neutralizing antibody against vascular endothelial growth factor receptor-2. The wnt co-receptor LRP5/6 expression was significantly up-regulated in Vldlr(-/-) eyecups compared with that in wild-type mice. Significantly, Vldlr(-/-) mice showed impaired phosphorylation of downstream effectors of the wnt signaling pathway, glycogen synthase kinase-3beta (GSK-3beta), and beta-catenin, concomitant with increased levels of free GSK-3beta and beta-catenin, suggesting an increased activity of the wnt pathway. Down-regulation of VLDLR by small interference RNA resulted in up-regulation of LRP5/6 expression and activation of beta-catenin in cultured endothelial cells. Furthermore, Dickkopf-1, a specific inhibitor of the wnt pathway, effectively decreased vascular endothelial growth factor and beta-catenin levels in the retinal pigment epithelium of Vldlr(-/-) mice and in cells transfected with the VLDLR small interference RNA. These results suggest that VLDLR functions as a negative regulator of CNV, and this function is mediated through the wnt pathway.     

Cloning and embryonic expression of six wnt genes in the medaka (Oryzias latipes) with special reference to expression of wnt5a in the pectoral fin buds

WNTs are secreted signaling molecules which control cell differentiation and proliferation. They are known to play essential roles in various developmental processes. Wnt genes have been identified in a variety of animals, and it has been shown that their amino acid sequences are highly conserved throughout evolution. To investigate the role of wnt genes during fish development from the evolutionary viewpoint, six medaka wnt genes (wnt4, wnt5a, wnt6, wnt7b, wnt8b and wnt8-like) were isolated and their embryonic expression was examined. These wnt genes were expressed in various tissues during embryonic development, and most of their expression patterns were conserved or comparable to those of other vertebrates. Thus, these wnt genes may be useful as molecular markers to investigate development and organogenesis using the medaka. Focus was on wnt5a, which was expressed in the pectoral fin buds, because its expression pattern was particularly comparable to that in tetrapod limbs. Its detailed expression pattern was further examined during pectoral fin bud development. The conservation and diversification of Wnt5a expression through the evolutionary transition from fish fins to tetrapod limbs is discussed.     

Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway

The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.     

The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization.

During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. This work identifies a tissue polarity gene, diego, that comprises six ankyrin repeats and colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. Our data suggest that Frizzled acts through Diego to promote local clustering of Flamingo, and that clustering of Diego and Flamingo in one cell nonautonomously propagates to others.     

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

Regulation of adipocyte differentiation and gene expression-crosstalk between TGFβ and wnt signaling pathways

Obesity results in reduced differentiation potential of adipocytes leading to adipose tissue insulin resistance. Elevated proinflammatory cytokines from adipose tissue in obesity, such as TNFα have been implicated in the reduced adipocyte differentiation. Other mediators of reduced adipocyte differentiation include TGFβ and wnt proteins. Although some overlap exists in the signaling cascades of the wnt and TGFβ pathways it is unknown if TGFβ or wnt proteins reciprocally induce the expression of each other to maximize their biological effects in adipocytes. Therefore, we investigated the possible involvement of TGFβ signaling in wnt induced gene expression and vice versa in 3T3-L1 adipocyte. Effect of TGFβ and Wnt pathways on differentiation was studied in preadipocytes induced to differentiate in the presence of Wnt3a or TGFβ1 and their inhibitors (FZ8-CRD and SB431542, respectively). Regulation of intracellular signaling and gene expression was also studied in mature adipocytes. Our results show that both TGFβ1 and Wnt3a lead to increased accumulation of β-catenin, phosphorylation of AKT and p44/42 MAPK. However, differences were found in the pattern of gene expression induced by the two proteins suggesting that distinct, but complex, signaling pathways are activated by TGFβ and wnt proteins to independently regulate adipocyte function.     

Expression of UNC-5 in the developing Xenopus visual system

Celsr, also called Flamingo (Fmi) genes encode proteins of the cadherin superfamily. Celsr cadherins are seven-pass transmembrane proteins with nine cadherin repeats in the extracellular domain, and an anonymous intracellular C-terminus. The Drosophila Fmi gene regulates epithelial planar cell polarity and dendritic field deployment. The three Flamingo gene orthologs in man and rodents are named, respectively, CELSR1-3 and Celsr1-3. Celsr1 and 2 are expressed during early development, in the brain and epithelia. In this report, we characterized further Celsr genes in the mouse, and examined their developmental pattern of expression. Each Celsr is expressed prominently in the developing brain following a specific pattern, suggesting that they serve distinct functions.     

Role of lbx2 in the noncanonical Wnt signaling pathway for convergence and extension movements and hypaxial myogenesis in zebrafish

It has been suggested that mouse lbx1 is essential for directing hypaxial myogenic precursor cell migration. In zebrafish, the expression of lbx1a, lbx1b, and lbx2 has been observed in pectoral fin buds. It has also been shown that knocking down endogenous lbx2 in zebrafish embryos diminishes myoD expression in the pectoral fin bud. However, downstream lbxs signals remain largely unexplored. Here, we describe a previously unknown function of zebrafish lbx2 (lbx2) during convergent extension (CE) movements. The abrogation of the lbx2 function by two non-overlapping morpholino oligonucleotides (MOs) resulted in the defective convergence and extension movements in morphants during gastrulation. Our transplantation studies further demonstrated that the overexpression of lbx2 autonomously promotes CE movements. Expression of wnt5b is significantly reduced in lbx2 morphants. We have demonstrated that application of the wnt5b MO, a dominant-negative form of disheveled (Dvl) and a chemical inhibitor of Rho-associated kinase Y27632 in zebrafish embryos have effects reminiscent that are of the CE and hypaxial myogenesis defects observed in lbx2 morphants. Moreover, the CE and hypaxial mesoderm defects seen in lbx2 morphants can be rescued by co-injection with wnt5b or RhoA mRNA. However, this reduced level of active RhoA and hypaxial myogenesis defects in the embryos injected with the dominant-negative form of Dvl mRNA cannot be effectively restored by co-injection with lbx2 mRNA. Our results suggest that the key noncanonical Wnt signaling components Wnt5, Dvl, and RhoA are downstream effectors involved in the regulative roles of lbx2 in CE movement and hypaxial myogenesis during zebrafish embryogenesis.     

Wnt signal transduction pathway and apoptosis: a review

The association between the wnt signaling pathway and apoptosis has become more firmly established in the recent scientific literature. Many reports indicate that the wnt signaling pathway regulates apoptosis through a variety of mechanisms.     

The zic1 gene is an activator of Wnt signaling

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, we asked whether Xenopus Zic1 acted through the Wnt pathway. Using Wnt signaling inhibitors, we demonstrate that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated here. Together, our data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates.     

The Eregli Marshes: A new nesting site for the Greater Flamingo,Phoenicopterus ruber,in Turkey

Abstract

35–40 old Flamingo nests were found in the Ere?li Marshes in 1987. Successful breeding probably did not take place. A review of Flamingo breeding in Turkey is given.