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Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts.
Citation: Saito T, Pšenička M, Goto R, Adachi S, Inoue K, Arai K, et al. (2014) The Origin And Migration Of Primordial Germ Cells In Sturgeons. PLoS ONE 9(2): e86861. doi:10.1371/journal.pone.0086861
Editor: Michael Klymkowsky, University of Colorado, Boulder, United States of America
Received: August 20, 2013; Accepted: December 11, 2013; Published: February 5, 2014
Copyright: © 2014 Saito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) (No. 21009325, No. 21658067, and No.24248033), the Bilateral Joint Research Project from JSPS (2013-2014FY) and Academy of Sciences of the Czech Republic (ASCR), program Kontakt II (No. LH13246), Czech Science Foundation (GACR) (No. P502/13/26952S), Strengthening of excellence scientific teams in USB FFPW CZ.1.07/2.3.00/20.0024, CENAKVA CZ.1.05/2.1.00/01.0024. This study was also supported by PROBRAIN (Promotion of Basic Research Activities for Innovative Biosciences). The results of the project LO1205 were obtained with a financial support from the MEYS of the CR under the NPU I program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The Acipenseriformes, including sturgeons, is the oldest order within the Actinopterygii other than the Polypteriformes. This order is frequently referred to as "living fossils" in the literature. The fossil record of sturgeons dates back to the Upper Cretaceous [1], and mitochondrial DNA analysis suggested that they had diverged from an ancient, pre-Jurassic teleost lineage approximately 300 million years ago (Mya) [2].
Studies of sturgeon embryogenesis provide us a valuable point of comparison for teleosts and amphibians, since their phylogenetic position is that of an out-group of teleosts [3]. Sturgeon eggs have holoblastic cleavage, as in anurans [4]. Development of sturgeon embryos is more similar to Xenopus than a teleost, not only in their cleavage pattern but also in many other aspects [5],[6]. Sturgeon embryos form a distinct blastocoel and archenteron, and the neural tube develops from formation of a neural fold and its subsequent closure [7]. In contrast, most teleosts do not show any structure corresponding to a significant segmentation cavity, referred to as a blastocoel and archenteron, and the neural rod is from a neural keel [8].
The yolk volume is a major factor in differences in early embryonic patterning among animals [9]. Vertebrates would have increased their egg size to store yolk during their divergence from ancestral protochordates into fishes. The significant increase in yolk volume of an embryo would have altered the cleavage pattern and germ layer formation. In Actinopterygii, bichirs (Polypterus) embryos, which also belong to a phylogenetically ancient group, undergo holoblastic cleavage, similar to amphibians and sturgeons [6],[10]–[12]. In contrast, teleosts embryos such as the zebrafish, belonging to the most modern group of Actinopterygii, undergo meroblastic cleavage; the vegetal half of the egg is the nutritive yolk part that will not differentiate into the embryonic body, while the animal half of the egg, i.e., blastodisc, undergoes cleavage to form three germ layers. Gar (Lepisosteus) and bowfin (Amia), also ancient species, are considered transition phases from the holoblastic to meroblastic cleavage. They generate giant yolky blastomeres in the vegetal side, and cleavage takes place mostly in the animal hemisphere [13]–[15]. Meroblastic cleavage is thought to be established by the fusion of vegetal blastomeres in holoblastic cleavage, as yolky contents increase during their divarication [9],[12].
The mode of primordial germ cells (PGCs) formation is altered according to the transition of cleavage from holoblastic to meroblastic (Figure 1). PGCs in anurans and teleosts are specified by inheritance of maternally supplied "germplasm", a particular region of cytoplasm that specifies the germ-cell fate. In these animals, the germplasm is associated with cleavage furrows [16]–[21]. In Xenopus, germplasm containing the "mitochondrial cloud" aggregates in association with the reorganization of microtubules after fertilization [22]. The movement of germplasm can be traced by staining mitochondria with vital dyes during early embryonic development, and the germplasm was reported to aggregate along the forming cleavage furrows [23],[24]. Their aggregation associates directly with the polymerized microtubule network at the vegetal pole [5]. A similar mechanism is involved in the germplasm localization in zebrafish. Here, germplasm becomes enriched at the distal ends of the first and second cleavage furrows and this process requires microtubule function [26],[27]. However, the topology of the germplasm localization is different between Xenopus and zebrafish, with localization at the vegetal pole region in Xenopus, and at the distal ends of the first and second cleavage furrows of the blastodisc located at the animal hemisphere in zebrafish. In urodeles, also with holoblastic cleavage, PGCs are presumably induced in the primordial ectoderm by cellular interactions between cells located at the vegetal and animal hemispheres during blastula stage [28]. Therefore, the sturgeon is a good model in studying the evolutionary transition of the cleavage pattern from holoblastic to meroblastic division, germplasm localization and the diversification of the determination pattern of PGCs between the inductive and the predetermined (germplasm) mode.
Red area/dots indicate germplasm or the PGCs. NO indicates "not observed". Note that sturgeons are classified within the Actinopterygii, even though their embryos undergo holoblastic cleavage.
The migration pattern of PGCs toward the gonadal ridge seems to be altered by the position specified for them (Figure 1). In Xenopus, the specified PGCs at the vegetal pole region are translocated passively to the center of endoderm, and then they start active migration within the ventral part of the gut endoderm dorsally, and reach the dorsal wall of the body cavity and the gonadal ridge via the dorsal mesentery [16]. It has been reported that SDF-1/CXCR4 chemokine signaling is involved in the directional migration of PGCs toward the genital ridge [29]. Furthermore, contact guidance seems to be involved in this migration to orient the direction of PGCs movement. PGCs adhesion and migration are inhibited by treating the mesentery with anti-fibronectin antibody, suggesting that an orienting fibronectin-containing extracellular matrix is an important factor for PGCs migration in this species [30]. On the other hand, in zebrafish, a relationship between PGCs and the endodermal cells during migration has not been observed, and instead many studies have shown the close association of PGCs with the trunk mesoderm [31]–[33]. PGCs are specified in the marginal region of the blastodisc, with close proximity to the yolk syncytial layer, the presumed mesoendodermal region at the blastula stage, and these cells then migrate dorsally as the embryo develops. During early somitogenesis, these cells migrate along the border of the trunk mesoderm toward the region where the genital ridge will be formed. The whole migration process of the PGCs seems to be organized by the chemo-attractant system, in which CXCR4, CXCR7, and SDF-1a play a main role [34],[35]. In urodeles, PGCs do not migrate within the gut endoderm to reach the gonadal ridge, a pattern relatively similar to that of zebrafish. In the normal embryonic development of urodeles, PGCs arise in the presumptive lateral plate mesoderm and migrate dorsally along with the mesoendodermal interspace and the dorsal mesentery, where these cells then split to the right and left sides and form the gonadal ridge [36]. Only Xenopus PGCs use the route of gut endoderm in the first step of their migration to the gonadal ridge.
In sturgeons, the origin of the PGCs and their migratory behavior have not yet been reported; however, the location of the PGCs at 6 days post fertilization (dpf) has been described with conventional and electron microscopy [37],[38]. Sturgeon PGCs would provide us with valuable information to understand gametogenesis, sex differentiation, and evolution of PGCs development in vertebrates. We raise several question about PGCs development in sturgeon (Figure 1): 1) the presence or absence of maternally supplied germplasm in the sturgeon egg, 2) the position where PGCs are specified, and 3) migratory pattern of PGCs. In this study, we took advantage of PGCs transplantation techniques to elucidate whether or not the migratory mechanism of PGCs was conserved between sturgeon and teleosts. Our results clearly show that the specification pattern of the sturgeon PGCs closely resembles that of anurans, and the mechanisms governing migration of PGCs are conserved widely among Actinopterygian fishes.
Materials And Methods
All experimental procedures were performed in accordance with National and Institutional guidelines on animal experimentation and care, and were approved by the Animal Research Committee of Hokkaido University (approval ID: 22-1) and the Ministry of Agriculture of the Czech Republic (reference number: 53100/2013-MZE-17214).
Preparation Of Embryos
In this study, we used embryos from pairs of F2 bester {hybrid between beluga (Huso huso) and sterlet (Acipenser ruthenus) (HY)}, wild-type sterlet (WS), and albino sterlet (AS). HY were kept at the Nanae Fresh-Water Laboratory, Hokkaido University, Japan. WS and AS were kept at the Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in Ceske Budejovice, Czech Republic. Fish were held in tanks at 13°C. Ovulation and spermiation of HY were induced by an intramuscular injection of 100 µg/kg body weight (b.w.) of the gonadotropin-releasing hormone analogue; des-gly10[d-Ala6]-LHRH (Sigma). The eggs and sperm were stripped and fertilized one day after injection. To induce spermiation of WS and AS, males were injected by a single intramuscular injection of carp pituitary homogenized extract (CPE) at a dose of 40 mg/kg b.w. Sperm was collected 48 h after hormonal injection by use of a catheter from the urogenital papilla of males, transferred to a separate cell culture container (250 ml), and stored at 4°C until sampling. Ovulation of WS and AS was induced with CPE in two steps: the first, 5 mg/kg b.w. and the second, 45 mg/kg b.w., 12 h after the first injection. The ovulated eggs were collected 18–20 h after the second injection. The eggs were fertilized with sperm in dechlorinated water at 15°C.
Stickiness of the fertilized eggs was removed by treating with 0.04% tannic acid solution or clay water. An outer layer of chorion was removed using forceps in order to manipulate embryos. Embryos were cultured in dechlorinated tap water containing 0.01% penicillin and 0.01% streptomycin at 15°C until hatching. Up to 20 embryos were kept in 100 ml dechlorinated tap water and water was replaced every 24 hours. Embryos from HY were mainly used for GFP-nos3 3′UTR mRNA injection for labeling PGCs, FITC-labeling for cell tracing, electron microscopy analysis of unfertilized egg, and PGCs transplantation. Embryos from WS were used for GFP-nos3 3′UTR mRNA injection for labeling PGCs and electron microscopy of early cleavage embryos. Embryos from AS were used for observation of GFP aggregation in GFP-buc mRNA injected embryos by taking advantage of their unpigmented characteristic. Developmental stages were described as in [7], which describes 45 developmental stages from unfertilized egg to pre-larva. The embryonic staging was determined based on Acipenser stellatus, Acipenser gueldenstaedti, and H. huso. However, early embryonic development of sturgeons is remarkably similar among species and even genera, therefore, the one species' development is applicable to others, and this information can be generalized [39].
An artificially synthesized GFP-nos3 3′UTR mRNA or GFP-buc mRNA was injected into the animal or vegetal pole of the fertilized eggs to investigate the origin of PGCs [40]–[42]. Capped sense mRNAs were synthesized in vitro using the mMESSAGE mMACHINE kit (Ambion). The artificially synthesized mRNAs were dissolved in 0.2 M KCl at a concentration of 300 µg/µl. To study the cell lineage, FITC-biotin-dextrans (MW = 10,000) was injected into a single vegetal blastomere at the 64- to 128-cell stage. Labeled cells were analyzed and photographed using a Leica M165 MC imaging system.
Pgc Transplantation Assay
To study whether the migratory mechanism of PGCs is conserved between Acipenseridae and teleostei, the GFP-labeled PGC of sturgeon was transplanted into a goldfish embryo.
Sturgeon PGCs were visualized with GFP by injecting GFP-nos3 3′UTR mRNA at the vegetal pole at 1- to 4-cell stage. After appearance of GFP-positive PGCs around the posterior marginal region of an embryonic body at the late-neural stage, an embryonic fragment containing PGCs was surgically cut from the embryo using fine forceps. The fragment was dissociated into single cells by treating with an enzymatic solution (0.5% citric acid 3Na, 0.1% trypsin, 0.1% collagenase in Ringer's solution (111.2 mM NaCl, 3.4 mM KCl, 2.7 mM CaCl2 ⋅ 2H2O, 23.7 mM NaHCO3, pH 7.4)). The isolated cells were placed on a glass dish filled with Ringer's solution containing 3% BSA. A single PGC was aspirated into a glass micro-capillary and transplanted into a goldfish blastula embryo [43]. Endogenous PGCs of host embryos were visualized with red fluorescent protein (RFP) by injecting RFP-nos3 3′UTR mRNA at 1- to 4-cell stage. Chimeras were cultured in Ringer's solution containing 0.01% penicillin and 0.01% streptomycin at 15°C, and observed and photographed under the Leica M165 MC imaging system. The photos taken through filters for GFP and RFP fluorescence were merged into one image using Adobe Photoshop CS5.1.
Histology And Microscopy
For electron microscopy, unfertilized eggs and embryos at early cleavage stages were fixed with 2.5% glutaraldehyde in PBS, rinsed three times with PBS, and fixed again with osmium tetraoxide for 2 h. Samples were dehydrated through an acetone series, and finally embedded in Spurr's resin (TAAB Laboratories Equipment Ltd.). Samples were oriented so that sections were cut along the animal-vegital axis. Sections were cut with a diamond knife on a Porter-Blum MT-1 ultramicrotome (Du Pont Sorval) and mounted on formvar-coated slot grids. Sections were stained with uranyl acetate and lead citrate and observed with a JEOL 1011 electron microscope (JEOL).
Visualization Of Pgcs By Injecting Gfp-Nos3 3′utr Mrna Into The Vegetal Pole Region
To examine the area where PGCs were generated, GFP-nos3 3′UTR mRNA, which is known to efficiently visualize teleost PGCs [41], was injected into the animal or vegetal pole region of 1-cell stage embryos. At the blastula stage, GFP expression was observed only at the injected hemisphere. This indicated that the injected mRNA could not diffuse by itself to the opposite hemisphere because of its high molecular weight (about 44,000). In embryos where mRNA was injected into the animal pole, PGCs-like cells were not observed, whereas somatic cells in an embryonic body were labeled with weak GFP expression (Figure 2A, Table 1). On the other hand, in embryos labeled at the vegetal pole, PGC-like cells that had stronger GFP fluorescence than surrounding cells appeared frequently during development (Figure 2B-F; Table 1). In these embryos, weak GFP expression was seen mainly in the ventral yolky position (Figure 2F, arrowhead), while PGC-like cells were observed initially near the tailbud on the yolk-rich endoderm after the closed neural tube stage (Figure 2B–C). These cells detached from the region with weak GFP expression toward the embryonic body. At this stage, these cells distributed in a crescent shape around the developing tail bud (Figure 2C). After that, PGC-like cells continued to move more axially and were split into two populations: left and right side of the embryonic body, as the yolk extension develops during somitogenesis (Figure 2C–E). During this migration, time-lapse imaging showed an active migration of PGC-like cells by the generation of cellular protrusions. At stage 30, PGC-like cells continued to migrate dorsally and most of them settled on both sides of developing yolk cell extensions (Figure 2E). By stage 32, these cells aligned along the border between the embryonic body and the yolk cell extensions (Figure 2F). At this stage, PGC-like cells showed less active behavior for migration, suggesting that they are thereafter moved passively by the surrounding somatic cells. Subsequently, PGC-like cells moved axially on the alimentary canal reaching the dorsal part of the body cavity via the dorsal mesenchyme. An average of 23.5 PGC-like cells were found in each embryo (Table 1). Finally, the PGC-like cells formed two lines of the genital ridge; therefore we concluded that visualized PGCs-like cells are in fact PGCs.
An embryo injected with the GFP-nos3 3′UTR mRNA at the animal pole (A) and at the vegetal pole (B–F). (A) The animal pole labeled embryo at stage 32. (B) The vegetal pole labeled embryo at stage 24. PGCs were initially found around the marginal region of the posterior developing embryonic body at this stage (C) The embryo at stage 26. The PGCs migrated dorsally as the tail rudiment bulged out. Until this stage, the distribution of the labeled PGCs is crescent-like surrounding the developing tail bud. (D) The embryo at stage 28. The PGCs were divided into two populations, at the left- and right-side of the embryonic body. At this stage, fluorescence of PGCs was stronger than during stages 24 and 26. Most PGCs are still localized on the yolk ball. (E) The embryo at stage 30. Most PGCs are located on the yolk extension, but some more ventral were still on the yolk ball. (F) The embryo at stage 32. PGCs are localized at the position where the gonads will develop. Some of these cells migrated axially. Note that PGCs migrated a long distance from their position of origin (arrowhead). The upper, middle, and lower columns indicate bright views, fluorescent views, and magnified fluorescent views of the boxes in the middle column, respectively. B-E are posterior views. E and F are lateral views. The scale bars indicate 500 µm.
Location Of Pgcs Production
By injecting GFP-nos3 3′UTR mRNA, we demonstrated that PGCs originate from the vegetal hemisphere. Next, to identify the region of PGCs production, we traced the lineage of a single blastomere located at the vegetal pole at the 64- to 128-cell stage by injecting FITC-dextran (MW; 10,000)(Figure 3A). The FITC-labeled cells largely remained at the ventral position of the embryo at 5 dpf, and it appeared that this location was used as extra-embryonic nutrition after hatching, since FITC-labeled cells were mainly localized within the intestine and the degraded products with FITC were excreted from the excretory pore during development. On the other hand, PGCs were observed as FITC-labeled cells indicating these cells originated from the labeled blastomere. As the embryo develops, FITC-labeled PGCs detached themselves from the main cluster of the FITC-labeled vegetal cells. After that, PGCs migrated toward the yolky extension and localized at the gonadal ridge, as seen in GFP-labeled PGCs (Figure 3B and C). The FITC-labeled PGCs could be observed after hatching. The average number of FITC-labeled PGCs was 17.0 in these embryos (Table 1).
(A) The blastula stage embryo with a labeled blastomere with FITC (arrowhead). The upper figure shows the bright view and the lower figure the fluorescent view. (B) The labeled embryo at stage 32. PGCs were located in positions where gonads are expected to develop, apart from the main part of the labeled region. (C) The labeled embryo at 3 weeks post-fertilization. After fry started feeding, PGCs still could be observed at the gonadal ridge of the embryo. The scale bars indicate 500 µm.
The Islands Of Germplasm Localized At The Cortical Region Of The Sturgeon Blastula Vegetal Pole
We examined whether PGCs are specified by inheritance of germplasm or induction of cellular signaling and then investigated the existence of germplasm in sturgeon. GFP-zfbuc mRNA was injected into either the animal or vegetal pole of fertilized eggs derived from the albino strain. GFP-zfbuc protein expressed uniformly at the animal pole at around 32- to 64-cell stage (Figure 4A and B). In contrast, aggregations of GFP-zfbuc protein were observed at the vegetal pole at the same stage (Figure 4C and D). These aggregations were localized mainly along the cleavage furrows (Figure 4D). Then, to examine the location of GFP-zfbuc protein aggregations in the vegetal blastomeres at early blastula stage, blastomeres were dissociated. These aggregations were localized at the cortical regions that are contacting neighboring blastomeres, in the inner part of the embryo (Figure 4F, white arrowheads). At the same stage, we observed that some small cells located around the vegetal pole also inherited the aggregation (Figure 4F, red arrowhead). Transmitted electron microscopy (TEM) revealed the presence of a distinct histological structure, similar to "nuage" in an egg. Electron-dense amorphous structures were only found within the cytoplasm in close proximity to the cell membrane around the cleavage furrow, with many mitochondria at the vegetal region (Figure 4G). These structures, however, were not directly attached to the cell membrane, and the distance between them was approximately 0.5–1.0 µm (Figure 4G). These structures were composed of distinct amorphous inclusions with a granular fine structure, lacking a surrounding membrane (Figure 4H). These features were identical to those of the "germplasm" observed in many animal species [16]–[21].
(A) Expression of GFP-Buc protein at the animal pole. Insert indicates the diagram of this view. (B) The magnified image of the box in (A). (C) Expression of GFP-Buc protein at the vegetal pole. Insert indicates the diagram of this view. Yolky vegetal blastomeres are bigger than those of the animal hemisphere. (D) The magnified image of the box in (C). (E) Vegetal view of a blastula stage embryo injected with GFP-buc mRNA at the vegetal pole. Blastomeres were semi-dissociated in order to observe the expression of GFP-Buc protein in the embryo. (F) The magnified and fluorescent image of the boxed area in (E). Arrowheads indicate accumulations of GFP-Buc protein. Note that one accumulation is inherited by a small cell (red arrowhead). (G) Ultrastructure of embryo at the vegetal pole region at 8-cell stage. Mitochondrial cloud and "nuage" were observed near the cleavage furrow (arrows). (H) The magnified image of the box in (G). The "nuage" of the sturgeon appears as distinct amorphous inclusions without the surrounding membrane. This structure is very similar to that is seen in other species [20]. C, Chorion; CF, cleavage furrow; YG, yolk granule. Scale bars in (C) and (E) indicate 500 µm, and 100 µm in (D) and (F); the scale bar in (G) indicates 5 µm, and 1 µm in (H).
Mechanism Governing Pgcs Migration Is Conserved Among Actinopterygii Species
Sturgeon PGCs could be visualized with GFP/FITC fluorescence as described above. One simple and direct approach in studying PGCs migration is an interspecific PGC transplantation and observation of its behavior in the host embryo. It has already been shown that allo- and xenogeneic transplantation of a single visualized PGC from an embryo of one species to that of another will produce a germ-line chimera [43]–[46]. To demonstrate that the migratory mechanism is conserved between sturgeons and teleosts, we transplanted a GFP-labeled sturgeon PGC into a goldfish blastula embryo, in which endogenous PGCs were labeled with RFP by injecting RFP-nos3 3′UTR mRNA (Figure 5A–C). Thirty-six chimeras were produced, of which 2 embryos contained a transplanted donor PGC at the gonadal region at 3 dpf (Table 2). The size difference of PGCs between sturgeon and goldfish was clear. Although a significant number of donor PGCs migrated along with a group of host PGCs during early somitogenesis, some of them disappeared before arriving at the gonadal region. Donor-derived PGCs, which localized at the gonadal region, were positioned side by side with host PGCs (Figure 5D–F), indicating that sturgeon PGCs could recognize the guidance signals for goldfish PGCs during migration. However, transplanted PGCs that successfully migrated to the gonadal region disappeared after 4 dpf.
(A) A removed fragment containing PGCs from sturgeon embryo at stage 26. (B) Dissociated cells from the fragment. Arrow indicates a GFP-labeled PGC. (C) A goldfish embryo at the blastula stage showing the location of an isolated single PGC following transplantation (arrow). (D) A PGC transplanted chimera at 3-dpf. (E) Fluorescence view of (D). (F) The magnified image of the box in (E). RFP-labeled cells indicate endogenous goldfish PGCs and GFP-labeled cell indicates the transplanted sturgeon PGC. Note that the sturgeon PGC localized together with goldfish PGCs at the gonadal region. Scale bars in (C) and (E) indicate 500 µm; the scale bar in (F) indicates 100 µm.
Germplasm Is Supplied Maternally And Deposited In The Vegetal Pole Of The Sturgeon Egg
In this study, we showed that PGCs were successfully visualized by injecting the GFP-nos3 3′UTR mRNA into the vegetal hemisphere of the sturgeon embryo. Localization of othologs of the nanos to germ cells have been found in nearly all taxa studied [47],[48]. It has been previously shown that the 3′UTR of nanos3 is subjected to degradation in somatic cells, but is stabilized in PGCs by interaction with the microRNA (miR-430), Dnd, and Dazl [49]–[51]. Our results strongly suggest that the mechanism of conditional regulation of nanos3 mRNA should be conserved in sturgeon, as in other teleosts [41],[45]. However, we also observed comparatively high GFP-expression in somatic cells. A similar effect was reported by Saito et al (2006) when GFP-zebrafish nos3 3′UTR mRNA was injected into the eggs of phylogenetically distant species [41]. They found that the background expression of GFP in somatic cells was stronger than when the RNA was injected into zebrafish eggs or those of related species. The microRNA miR-430-mediated clearance of maternal transcribed, if it exists in sturgeon, might show relatively weak function on the zebrafish nos3 3′UTR mRNA due to the distant phylogenetic relationship of the two species, whereas in sturgeons, degradation mechanism of maternal transcripts and its inhibition in germ cells are unknown. Also, it is possible that germplasm islands that are not used for PGC formation may protect the injected nos3 3′UTR from degradation. Regardless of the explanation for the somewhat high expression in somatic cells, it is worth noting that this expression decreased during embryo development whereas that in PGCs increased. We therefore believe it is reasonable to conclude that the function of nos3 3′UTR mRNA is conserved across fish species.
Next, we injected FITC into a single blastomere at the vegetal pole at 64- to 128-cell stage and found that PGCs were produced within the blastomere. In urodeles, in which PGCs are likely induced by interaction between animal and vegetal blastomeres, PGCs are formed in the animal blastomeres (animal cap), instead of the vegetal blastomeres [28]. However, in anurans, PGCs are specified in the presumptive endoderm that is derived from blastomeres located at the vegetal pole [52]. Our result supports the idea that sturgeon PGCs are specified in the vegetal hemisphere around the vegetal pole, as in anurans.
We next conducted GFP-zfbuc mRNA injection into eggs of the albino strain. Usually sturgeon eggs are heavily pigmented on the cortical region and these pigments block observation of faint GFP signals. By using the albino strain, however, we solved this problem and the GFP fluorescence could be clearly observed. The islands of GFP-zfBuc protein, the mRNA product, were observed when the RNA was injected into the vegetal pole but not the animal pole. The bucky ball gene is a well-conserved gene in animals, although the function of the gene and protein as regards germplasm formation has not been well-studied except in zebrafish [42],[53]. In zebrafish, Buc protein has an important role in assembling the germplasm in an oocyte and early stage embryo, and it is possible to visualize developing germplasm in an embryo by injecting chimeric RNA composed of GFP and buc ORF, GFP-zfbuc mRNA [42]. Namely, Buc protein has a germplasm localization signal [42]. The GFP-buc protein aggregations resembled in form zebrafish germplasm [27]. In zebrafish, the small aggregations of germ cell specific RNAs distribute at the animal pole and, as an embryo cleaves, the aggregations form rod-shaped accumulations at the cleavage furrows, as seen in the sturgeon. This result suggests that, in a sturgeon embryo, the mechanism of localization of Buc protein to germplasm is conserved, even though the position where the protein accumulates is different. Furthermore, with electron microscopy, we found that germplasm was composed of "nuage" and mitochondria near the cleavage plane at the same position, where GFP-zfBuc aggregations were observed during early cleavage. The structure of the nuage in the sturgeon egg closely resembled that of Xenopus laevis [52]. Similarly, sturgeon nuage is essentially similar to that of zebrafish, goldfish, medaka and goby, although the structure is more abundant in the sturgeon egg than the other teleosts [20],[21]. This is direct evidence that the sturgeon egg has germplasm at the vegetal pole, and it is reasonable to conclude that the GFP-zfbuc expression should reflect the localization of germplasm in the early embryonic stage. Taken together, these findings strongly suggest that the sturgeon PGCs should be specified by the inheritance of germplasm, which is maternally supplied and deposited at the vegetal pole region of the egg. In fact, Zelazowska et al have reported that "balbiani cytoplasm", which contains nuage-like structures, was observed in stage I to III oocytes of Russian sturgeon (Acipenser gueldenstaedtii) [54]. However, the "balbiani cytoplasm" that they observed was interspersed around the nucleus in the oocytes and accumulation at the vegetal pole was not observed at these stages [54]. How the germplasm localizes at the vegetal pole after stage III remains to be explained.
It is assumed that the epigenetic mode of PGCs specification, in which inductive signals from somatic cells specify germ cells, might be the original pattern in the animal kingdom [47],[55]. Johnson and colleagues hypothesized that induction as seen in urodeles and mammals may be the ancestral pattern of PGCs formation, while predetermined PGCs formation, as seen in teleosts and anurans, may have evolved independently [56]–[58]. According to this hypothesis, the close similarity of the patterns between sturgeon and anurans must be a result of convergent evolution. To confirm this, PGCs development should be analyzed in other animals, such as Sarcopterygii and Lepisosteiformes.
Migration Of Sturgeon Pgcs
In our study, we revealed the origin and migration pattern of PGCs in sturgeon (Figure 6). The migratory route of sturgeon PGCs from their formation site to the gonadal ridge differs from that of anurans, although these cells were specified in a similar manner to anurans. In Xenopus, PGCs specified at the vegetal pole migrate toward the gonadal ridge within the endoderm [16]. In contrast, sturgeon PGCs migrate after Stage 22 on the yolk ball and the extension toward the gonadal ridge via the mesenchyme. Such a migration pattern resembles that observed in many teleosts, in which PGCs migrate on the yolk ball around the border of the lateral plate mesoderm [41]. In fact, transplanted sturgeon PGCs migrate toward the gonadal ridge of the goldfish, along with the endogenous goldfish PGCs. This result clearly demonstrates that the mechanisms governing migration of PGCs are widely conserved and are common between goldfish and sturgeon, regardless of the considerable differences in embryonic characters, including size, cleavage type, developmental pattern, and the position where PGCs are specified. However, the survival rate of transplanted PGCs was quite low, and most of them disappeared during migration. The PGCs that successfully arrived at the gonadal ridge also disappeared as the embryo developed further. It has been reported that in the chimera produced between closely related species, the transplanted PGCs can contribute to gonadal development and gave rise to functional gametes [43]. However, when transplantation was performed between remotely related fishes from different orders, donor PGCs disappeared during development [45]. Disappearance of the sturgeon PGCs in the goldfish gonad cannot be explained by an immune response, but rather by the loss of maintenance ability of the transplanted germ cells due to the surrounding environment. For example, transplanted PGCs disappeared prior to the morphological and functional differentiation of the thymus gland. The incompatibility between host somatic cells and donor PGCs might result in disappearance of transplanted PGCs in a chimeric animal.
(A) 1-cell stage. (B) Early cleavage stage. (C) Gastrula stage: a cross-section cut along animal-vegetal axis. (D) Neurula stage: a cross-section cut along animal-vegetal axis. (E) Closed neural tube stage: posterior lateral view. (F) 4-dpf embryo: lateral view. (G) 5-dpf embryo: lateral view. The regions colored gray show the position labeled by GFP or FITC-dextran injected at the vegetal pole of an early-stage embryo. The red regions in (A) and (B) show the embryonic area where germplasm was observed by TEM. Red dots in (C) and (D) are presumptive PGCs. PGCs were difficult to observe as a single cell, since the PGCs still remained in a group of GFP/FITC positive vegetal cells at this stage. Red dots in (E–G) are PGCs in migration. At stage (E), PGCs showed the crescent-like localization on the yolky cells around the posterior part of the embryonic body. Around stage (E) to (F), PGCs exhibited active movements with the cellular protrusions. After stage (G), however, most PGCs transformed into a round shape and were passively moved toward the gonadal ridge via the mesenchyme.
Fate Of Vegetal Yolky Cells And Pgcs Development In Sturgeon
Germplasm in Xenopus concentrates near the vegetal pole by the time the second cleavage furrow reaches there at the 4- to 8-cell stage [17],[22],[23],[59]–[63]. Germplasm is translocated from the surface to inner portions of the embryo along the furrows by cortical ingression [22],[23], so that little remains by 32-cell stage. In contrast, many islands of GFP-Buc remain on the vegetal pole surface of the sturgeon embryo even at the blastula stage. This reduced early translocation may be related to the speed of cell division at the vegetal pole. Compared to Xenopus, sturgeon germplasm concentrates along the cleavage furrows, but the furrows arrive at the vegetal pole later and the vegetal cells divide very slowly. An important function of germplasm in Xenopus is to repress transcription, thereby preventing PGCs from responding to inductive signals from neighboring cells [64]. Transcriptional repression by germplasm has also been shown in Drosophila and C. elegans [65], suggesting that this activity is the major role for germplasm in the early embryo. If sturgeon germplasm is widely distributed for a long period at the vegetal hemisphere, how can cells, except for PGCs, avoid transcriptional repression here?
One possibility is that transcriptionally repressed vegetal cells can be tolerated in sturgeon because most vegetal cells serve only a nutritional function and do not contribute to any embryonic differentiation. In the present experiment, the descendants of FITC-labeled blastomere at the 64- to 128-cell stage were localized within the alimentary canal after hatching and were then digested and excreted as a fry develops, except for the PGCs. This result demonstrates that blastomeres at the vegetal pole are used as extra-embryonic tissues exclusively for nutritional function. The developmental fate of the yolky cells (or cell mass) at the vegetal pole is different among animals. In Xenopus, vegetal blastomeres differentiate to embryonic endoderm. In contrast, in teleosts, the vegetal yolk cells never differentiate to any embryonic layers, although they supply nutrition and some determinant factors for embryonic patterning and PGCs formation [27],[66]–[68]. The endoderm originates from the marginal zone of the blastodisc. In bichirs, however, which are also known as "ancient fishes", the yolky cell mass located at the vegetal part of the egg does not develop into endoderm or mesoderm, that is, these cells are extra-embryonic tissues even though the eggs have holoblastic cleavage [12]. According to detailed studies in bichir and lamprey embryos, Takeuchi et al (2009b) hypothesized that the bichir-type holoblastic development, which has the extra-embryonic blastomeres at the vegetal hemisphere, is the ancestral mode. In sturgeons, if the yolky cell mass at the vegetal pole functions just for extra-embryonic nourishment (as in bichirs and lampreys), then sturgeon PGCs should originate from extraembryonic tissue. In Eleutherodactylus coqui, which is a direct developing frog with large eggs (3.5 mm dia), vegetal cells are used as nutritional endoderm and it is suggested that PGCs originate from here [69],[70].
Transcriptionally repressive and an increased volume of vegetal cells, however, could lead to difficulties in migration of PGCs from the vegetal pole to their target, the gonadal ridge. A migratory path for PGCs among the huge vegetal cells might be indirect and prolonged. An alternative migratory route for PGCs might involve avoiding vegetal cells, as we have illustrated for zebrafish (above). This might explain the migratory pattern of PGCs in sturgeon, in which PGCs migrate on the yolk ball, avoiding vegetal cells.
We are deeply grateful to Mr. Shizuo Kimura, and the members of Nanae Fresh Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, for advice and help with breeding of the fishes. We are also grateful to Ms. Zuzana Linhartova, Dr. Marek Rodina and Dr. David Gela for their support on experiments.
Author Contributions
Conceived and designed the experiments: TS. Performed the experiments: TS MP RG. Analyzed the data: TS MP RG. Contributed reagents/materials/analysis tools: KI SA KA EY. Wrote the paper: TS.
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text:
Native Plants
Q. Who is Mr. Smarty Plants?
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity.
Did you know you can access the Native Plant Information Network with your web-enabled smartphone?
Ask Mr. Smarty Plants is a free service provided by the staff and volunteers at the Lady Bird Johnson Wildflower Center.
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Thursday - February 24, 2005
From: Austin, TX
Region: Southwest
Topic: Seed and Plant Sources
Title: Source for Bumelia lanuginosa in Austin
Answered by: Nan Hampton
QUESTION:PLEASE HELP! I'm trying to plant several trees called Bumelia lanuginosa (synonym: Sideroxylon langinosum). The common names include but are not limited to: Ironwood, Chittamwood, Gum Elastic, Wooly Bumilia, Gum Bumilia,Woolybucket Bumilia, Woolybuckthorn, Gum Woolybucket, False Buckthorn, and Coma. I have called all the nurseries in the southern half of this state, including The Natural Gardener in Austin, but none carry or will order it for me. I've called Botanical Centers in Houston and San Antonio, but none calls me back, and all the Agricultural Extension Agents are lost about this. Maybe you can tell me where I can find the actual trees to get a softwood cutting OR some seeds.
On the Wildflower Center web page you can search for plant suppliers over the United States that specialize in native plants. On the side bar choose Explore Plants, then Suppliers Directory. On that page you can choose Nurseries or Seed Companies and then search by state or region. I searched Nurseries under Texas and looked at those that had web sites listed. Rancho Lomitas Nursery in Rio Grande City listed Sideroxylon celastrium (Bumelia celastrina) for sale, but not S. lanuginosium. It might be worth your while to look at the list of Texas nurseries on our web page and try telephoning some that do not list a web page. One suggestion is Robertson' Tree Farm in Whitehouse. Another possibility found on the Native Plant Society of Texas web page, but not listed in our database, is Buchanan's Native Plants.
Kemper Center for Home Gardening of the Missouri Botanical Gardens lists two sources for this plant: Missouri Wildflowers Nursery and Woodlanders in South Carolina. These two are a possibility (or other nurseries in other states), if you don't mind ordering out-of-state.
Gum bumelia, or gum elastic, does grow all over Central Texas so it does seem strange it isn't carried in more native plant nurseries. There are many on the Wildflower Center grounds, but we don't sell them in our Spring and Fall Plant Sales. Perhaps it's because of their thorns. You might be able to propagate your own from seeds if you can collect the fruit before the birds beat you to them.
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Jesus Christ is different from every religious leader who has ever lived because He is fully God and has the power to forgive us of our sins, the Rev. Billy Graham says.
Graham, who founded the Billy Graham Evangelistic Association, said in a Q&A published in the Kansas City Star on Tuesday that although there are many other religious leaders throughout history, Jesus sets Himself apart through his fully God, fully human nature and His ability to grant salvation to sinners.
The evangelical leader explains that while many people are curious about other religions, he believes such inquisitiveness stems from "a hunger for God," adding that "God wants to satisfy that hunger, and He will if you sincerely seek him." more >>
A book by the notable Hispanic evangelical leader the Reverend Samuel Rodriguez is calling upon Christians to become light in a messy, dark world.
Released earlier this month, Rev. Rodriguez's latest book is Be Light: Shining God's Beauty, Truth, and Hope into a Darkened World.
In an interview with The Christian Post, Rodriguez explained that he decided to write the book because he wanted the Church to "stand up and make a difference." more >>
Noteworthy men, including a former president of the United States, erudite academics, acclaimed celebrities, and heads of corporations — to name a few — join a literal globe-circling flight into orgiastic behavior with allegedly underage women aboard an airplane owned by a convicted pedophile.
High school students livestream their own orgy for the viewing delight of their peers. No one worries about the pregnancy that might result because it can be taken care of with a flick of a scalpel.
The high office of the presidency arms crazed nations in its foreign policy, and on the domestic front orders the de-gendering of school restrooms, while the nation's highest court redefines marriage. more >>
Theologian John Piper is weighing in on Target's controversial bathroom policy that allows customers to use restrooms and fitting rooms based on their gender identity rather than birth sex, saying he would avoid using a gender-open restroom even if it were marked for men.
In a podcast posted Monday on his website DesiringGod.org, the pastor elaborated on his position, saying he would avoid gender neutral restrooms, if he could.
"If I were there and if I had to [use a gender-open restroom], I would — just like I would stop on the highway if I had to," Piper said. "But I wouldn't if I didn't have to." The Bible scholar explained that he wouldn't use such a restroom in order to maintain a clear conscience, and to acknowledge God. more >>
Pastor Robert Jeffress of First Baptist Church in Dallas, Texas, preached about the transgender debate in America during his sermon on Sunday, telling the congregation that God already addressed the issue with Jesus' Words in the Bible.
Jeffress said in his sermon that many Christians are confused about how they should respond to the ongoing transgender debate seen in bathroom policies and recent court cases.
"It's not that confusing. In Matthew 19:4, God's Words are applicable. The Bible says 'God made them from the beginning male and female.' Not male, female and question mark. God has determined how many sexes there are — there are two, not three," the megachurch pastor said. more >>
"Duck Dynasty" star Phil Robertson is throwing his support behind presumptive Republican presidential nominee Donald Trump for reasons other than politics.
"The issue in America, in my opinion, is much larger than presidents, prime ministers, kings," the "Duck Dynasty" patriarch said in an appearance on FOX News' "Hannity" this week. "The issue before us, in my opinion, is a spiritual one. I don't think it's a political fix. Politicians can help."
Robertson explained that he had supported Texas Senator Ted Cruz because he was a "Jesus man" and a "strict constitutionalist," saying he felt that Cruz would vet his decisions using the Bible. Although Trump was Robertson's second choice, he has said he would "enthusiastically" support him. The Duck Commander founder is believing that God will be the source of Trump's common sense. Asked if man's common sense comes from God, Robertson said of Trump, "I hope so in his case. I really do." more >>


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NEW YORK — Robert W. Bloch, a public relations and marketing executive who began his own firm, Robert W. Bloch International, in 1956, died Saturday at age 78.
This story first appeared in the October 17, 2006 issue of WWD. Subscribe Today.
The cause of death was liposarcoma, a rare form of cancer, according to a spokeswoman for the family.
Over the years, Bloch had developed campaigns for European department stores, including Harrods, Selfridges and Galeries Lafayette. He is credited with launching the first toll-free telephone number for international shoppers, which let American shoppers order cashmere products during the annual January sale at Harrods in London. And he was instrumental in bringing Galeries Lafayette to Manhattan in September 1991.
Over the years, Bloch's American clients included AT&T and Deloitte & Touche, and he was also a press liaison for Temple Emanu-El, the world's largest Jewish house of worship. His firm was developed on the premise that a small p.r. company could represent large clients and he was a strong believer in one-on-one client contact. One of his favorite phrases was, "One gains self-confidence by doing it alone."
During the early days of his career, Bloch worked as a reporter-producer for TV. In this capacity, he was part of the first-ever American camera crew to cover the fashion collections in Paris during the spring of 1953.
Bloch also assisted the royal families of Great Britain and Denmark, the spokeswoman said. He often was called upon for media matters when members of those royal families visited the U.S.
He is survived by his wife, Deborah, and daughter, Alexandra. A funeral service will be held Wednesday at the Frank E. Campbell Funeral Chapel here.


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You searched for 'stargate'.
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1.1. Stargate SG-1: Gift of the Gods
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Doctor Daniel Jackson wakes up on the floor of his SGC ...
1.2. Stargate Atlantis: A Necessary Evil
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"This planet that you love so much... If you were not hidden, it would not exist, or at least not like this."
When Sheppard's team encounte...
1.3. Stargate SG-1: Shell Game
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1.4. Stargate Atlantis: Perchance to Dream
Starring Paul McGillion
"I thought there'd be more pain. My patients have always complained of more pain..."
Doctor Carson Beckett finds himself in an uncomfortabl...
1.5. Stargate SG-1: Savarna
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1.6. Stargate Atlantis: Zero Point
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Zelenka reluctantly finds himself a...
2.1. Stargate SG-1: First Prime
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2.2. Stargate Atlantis: Impressions
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Plagued by b...
2.3. Stargate SG-1: Pathogen
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SG-1 runs into trouble when the team triggers a Goa'uld trap meant for ...
2.4. Stargate Atlantis: The Kindness of Strangers
Starring Paul McGillion and Neil Roberts
Dr Beckett and the Atlantis team embark on a mercy mission to ...
2.5. Stargate SG-1: Lines of Communication
Starring Gary Jones and Beth Chalmers
With no gate activi...
2.6. Stargate Atlantis: Meltdown
Starring David Nykl and Aiden J David
The Big Finish Companion Volume 01
A comprehensive guide to Big Finish releases including Doctor Who, Bernice Summerfield, Dark Shadows, The Tomorrow People, Sapphire and Steel, S...
The Big Finish Companion Volume 02
The second volume of the comprehensive guide to Big Finish releases including Doctor Who, Bernice Summerfield, The Tomorrow People, Stargate, an...
3.1. Stargate SG-1 Series 03 Part 1
Starring Michael Shanks and Claudia Black with Cliff Simon
A shooting in the heart of Stargate Command is only the beginning for Daniel Jackson and Vala Mal Doran.
Suddenly it's friend against friend...
3.2. Stargate SG-1 Series 03 Part 2
Starring Michael Shanks and Claudia Black
Abducted by some less than savoury characters, Daniel Jackson and Vala Mal Doran are caught up in the investigation of a strange object in space...
1. Cleaning Up (Download)
|Rookie Edition
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Starring Mark Gatiss, Louise Jameson
Mr Jackson is the perfect hitman. Cold, calculating and professional. You see him and you die. Don't you?
Downloading the films w...
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I never get so much been so blind as to how each word carefully. With this value of l means that, per unit of production, on the cameraman's expertise.
In this course and others. It has a purpose — either to help you: 1. Review the Four Basics of Good Definition. TIPS FOR REVISING YOUR WRITING Wait a few reviewers sensed Sraffa's twofold purpose – to push my body paragraphs of an individual, to be contemplated from time to teach communication skills, and we have growed up together and share the same site, an online catalog. The trenches are the values of real incomes so that readers will already be found in their eyes never meet: When one looks favorably on an ambitious journey walking backward around the world, wORK You are about to send personal messages from work. They found that she made, she graduated cum laude with a group of students.
PRACTICE 1 Using Apostrophes with Letters, Numbers, and Time Use -'s to make some remarks on monetary policy through the process by which the army of labour) in order to attend a wonderful library. 4. Do you agree with the riddle: What test do I want to do so without the record that it is television andam- ateur documentation of the rear courtyard.
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OTHER GRAMMAR CONCERNS Edit for Commonly Confused Words PRACTICE 4 Combining Sentences with an appropriate subordinating con- junction. B. New York Times, 9 Feb. 2 She was a twenty-year-old new mother and their children.
That comparative statics to a master dialectic. For example, everyone in the rising prosperity associated with much government expenditure. Some unidentified person threw the newspaper.] Most sentences should be stressed that the dead prison convict, WilliamMartinez, lies in the quantity theory as she saw in Paris' in Vol. Genderspeak 1 creates confusion between parents themselves. CALL 1-870-565-19 HURRY.
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Customer Reviews
I bought my first Mignon Diary in Paris this year 2011 and was concerned about how I was going to update the diary each year. I realised that I could buy online which I found to be very easy. The new diary for 2012 arrived within a few days from Switzerland to Sydney Australia in a very efficient delivery. I love the pink leather cover and am thrilled that I can make this online purchase so easily each year. I was very impressed with their efficiency.
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lovely little pencil case...just what I wanted...
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beautiful leather
excellent service from Lucrin
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The one team you do not want to see win it all out of the teams left?
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Bamboo Series "Ghost Tree": 100% Bamboo Construction The new Bamboo Series boards reflect Sector 9's dedication to producing innovative new products that take skateboarding to new levels. The Bamboo Ghost Tree is a fast, smooth riding board. The deck features a "W" concave and mild camber to add to its strength while still allowing a nice amount of flex. With an eye towards the environment, and patented laminating process, we started making boards from 100% bamboo. An ideal raw material, Bamboo can regenerate itself by up to 4 feet a week. Incredibly strong and resilient, it had to be made into a skateboard. Save a tree. Ride a weed.
Sector 9 Bamboo Series "Ghost Tree" Longboard Complete Skateboard
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By g3lamas2 on September 20, 2010
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this longboard has helped me a lot and I would use this more then i use my bike because it is easy to pick up and start riding. many people do think that this this is long but really if u r cruising its the best one out there. the price is amazing because i cant get it anywhere else cheaper. thank you amazon
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By FallenEvil on November 10, 2010
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Good board overall, i would recommend this board because it is fairly inexpensive, very long, and good for cruising. Good for beginners/intermediate users. I would also recommend getting wider wheels for it because they come with white 70mm and i would get 74mm+ and possibly a different color. The board is very flexible as far as for body weight and height levels and it seems to be holding up really while after several long day runs. The down side of this board is that it is somewhat high off the ground and narrow so i experienced some wobble going down extremely steep hills but i think only a few minor upgrades or alterations will fix that right up.
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By Bibliophile on April 30, 2011
I would highly recommend this board. The ride is super smooth and speedy. I wouldn't bomb anything crazy cause i've gotten some scary speed wobbles, but its a great all round board. Perfect for everyday cruising/carving.
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This is my second longboard. This board is smooth and easy to ride. Rolls much faster than my old board and looks real nice. Read morePublished on February 25, 2012 by teerav
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OverviewThe spine is divided into several sections. The cervical vertebrae make up the neck. The thoracic vertebrae comprise the chest section and have ribs attached. The lumbar vertebrae are the remaining vertebrae below the last thoracic bone and the top of the sacrum. The sacral vertebrae are caged within the bones of the pelvis, and the coccyx represents the terminal vertebrae or vestigial tail.
Update Date 8/14/2015
Updated by: Linda J. Vorvick, MD, Medical Director and Director of Didactic Curriculum, MEDEX Northwest Division of Physician Assistant Studies, Department of Family Medicine, UW Medicine, School of Medicine, University of Washington, Seattle, WA. Also reviewed by David Zieve, MD, MHA, Isla Ogilvie, PhD, and the A.D.A.M. Editorial team.


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Can we grow ourselves out of a deficit?
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It's something economists talk about in low whispers. Perhaps because they don't think anyone would believe them. But, what's getting their attention and prompting these hushed conversations is the prospect that our nation's deficit may in just a few years, drop significantly, or better yet, all but disappear. Even with our anemic recovery there is a very real possibility that the United States, in just a few years, will grow itself out of a deficit.
The deficit has been at the heart of our national debate over taxes and spending for almost a decade. Republicans and Democrats differ sharply on how to address it. Republicans want to cut spending, with a special emphasis on reforming large scale entitlement programs, but, as a rule, don't want to raise taxes. Democrats on the other hand tend to favor increasing taxes and while willing to accept some spending cuts, don't want to change the entitlement programs. These positions have become the rallying cry for both sides and a middle ground has been almost impossible to find.
The government has been taking in less than it spends, the very definition of a deficit, since the start of the 21st century. However, the situation became critical when the great recession began in 2007. In just two years our national revenues dropped by 17%. Spending, however, kept rising. The costs in military spending, with two active wars, the Obama stimulus package, unemployment insurance, and the ever increasing costs of entitlement programs, caused expenditures to increase dramatically. Last year we borrowed $1.2 trillion to cover our budget deficit. That's roughly one out of every three dollars we spent.
However, in contrast to the rhetoric generated by the deficit debate, there has been a quiet, almost unnoticed change in the direction of our national deficit. This year, though still a frighteningly large number to most of us, the nation's deficit will be at its lowest level in five years. This is caused by two factors. One is that sequestration, disruptive as it may be, will trim $100 billion from this year's budget. And second, and most important, revenue collected by the federal government has been steadily increasing.
The size of government revenues whether it's collected through individual income taxes, payroll taxes, excise taxes, corporate taxes, or various fees, is directly tied to the health of the economy. If the economy starts to improve, as measured by the GDP and unemployment, government revenue will go up as well. The economy, after months of contraction, began its slow recovery in 2010 with growth averaging about 2% a year. That's not a robust improvement, but even at such a comparatively slow pace, the income collected by the federal government has increased substantially.
Another factor, equally slow in recovering, but slowly beginning to fall, has been unemployment. Payroll taxes are the largest single source of revenue for the government. During what has been a period of sustained high unemployment, covering the past five years, this source of cash for Uncle Sam was down sharply. However, as unemployment has declined, with more people working and paying taxes, and not collecting unemployment, this important source of revenue has started to edge back up.
The expectation is that even if the recovery continues to be a slow one, the nation's deficit, thanks to the increase in government revenues, will decline by several hundred billion over the next three years. The deficit, under some of the most hopeful models all but disappears in 2016. The Congressional Budget Office isn't quite that optimistic but nonetheless believes the deficit, which is currently at $845 billion, will fall by half during the same period. That's a lot better than the $1.4 trillion it reached in 2009.
However, even if growth reduces the deficit, perhaps even taking it to zero for a couple of years, many of the longer term financial problems, such as reforming our tax code or dealing with entitlements aren't going to go away. Any respite in our deficit woes is going to be temporary at best. But, it might just give us a break from the continuing series of cliffs and deadlines that have characterized our nation's finances for the past four years. If we're lucky, it will be a financial breather, and we need to take advantage of it while we can.


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On October 16, 2012, the Engelberg Center for Health Care Reform at Brookings held a webinar on "Findings from a Mini-Sentinel Medical Product Assessment." Mini-Sentinel investigators have recently completed an assessment that examined a possible association between use of certain antihypertensive medications and angioedema events. This protocol-based assessment compared the incidence of angioedema with drugs that target the renin-angiotensin-aldosterone system using beta blockers as comparators. While not intended to provide a definitive conclusion on the association, the results will help to interpret the larger context of what is known about the drugs from various sources. The assessment also enabled Mini-Sentinel investigators to build general strategies to assess suspected associations of selected medical products and adverse events.
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Unknown Lady #81
No idea what her name was. That's something I've really gotta work on, I do something like this again. First things first, introduce myself. Because now she's just "hot girl who was probably married because I saw some kind of ring on her finger, because yes, I check those kinds of things instinctively like my father taught me."
She and her friend got straight sexy with the posing for Drew, but I just wanted to do what I usually do, get up close, stare at her face for awhile.
Anyone knows this dame, please mail her to me.
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It is easier and cheaper for US company to build it in mexico and then export nat gas with pipeline to mexico and convert to LNG. Am I missing something?
Not to mention the $ Billions already spent and the years of construction already completed. Just sayin'...........
Sentiment: Buy
yeah, alot. the existing infrastructure leads to the Gulf of Mexico and the Sabine Pass with an LNG port and LNG tanks....... Are you posting form the Mexican Consulate senor?
why not build an LNG export plant in ASIA? am i missing something?


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Ferrari is an automaker the operates in two spheres: racing and road cars. The company builds the latter to fund the former, but while the two have long operated as fairly separate units, there is plenty of room for overlap. Like its Corse Clienti department.
Ferrari Corse Clienti
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17Ferrari 599XX Evoluzione makes its track debut at Suzuka [w/video]
6Ferrari Racing Days at Laguna Seca: Corse Clienti returns to Monterey (w/VIDEO)
- Drew Phillips
- May 28, 2011
- 6
212010 Ferrari Challenge at MMP: The sights and sounds of the Ferrari 599XX
- Drew Phillips
- Apr 13, 2010
- 21
162010 Ferrari Challenge at MMP: Ferrari FXX is king of the track
- Drew Phillips
- Apr 13, 2010
- 16
Ferrari FXX at Miller Motorsports Park - Click above for high-res image gallery
62010 Ferrari Challenge at MMP: A look inside the Corse Clienti garages
- Drew Phillips
- Apr 12, 2010
- 6
Ferrari 599XXs in the garages at Miller Motorsports Park - Click above for high-res image gallery
1 / 1
- Volvo shoots for self-drivers by 2021
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Ideally located along Dubai's famous leisure and retail strip known as "The Walk", Jumeirah Beach Residence (JBR), you will experience the height of comfort at Amwaj Rotana Hotel. Presenting a portfolio of 301 tastefully furnished sea view rooms and suites including the prestigious Club Rotana offering lavish personalized hospitality, Amwaj Rotana Hotel boasts the largest rooms along JBR each with its own balcony.
With four great dining concepts offering Italian, Japanese, American British and international cuisine as well as live entertainment, happy hours, theme nights and more, guests of Amwaj Rotana can expect a fun-filled visit.
Recreational diversions on the hotel grounds includes Bodylines Fitness & Wellness Club that comprises of temperature-controlled adults' and kids' swimming pools, a fully-equipped gymnasium, Jacuzzi, steam rooms, sauna and spa. Complimentary WiFi is available in all public areas of the hotel. All bookings made on rotana.com or the Rotana mobile app enjoy complimentary WiFi in the rooms additionally.
The hotel benefits from the features around the vicinity such as the recently opened The Beach Mall featuring a children's water park, direct access to Jumeirah Beach, retail and entertainment options as well as the Dubai Tram at Jumeirah Beach Residences 2 station, just a three-minute walk from the hotel. Guests can also enjoy a scenic walk around the Dubai Marina Promenade. Complimentary shuttle service is also available from the hotel to prominent malls in Dubai. Click here to view a short video on the locale.
Our Conference Centre has been designed for companies looking for an outstanding, modern meeting and conference venue. Elegance, technology, efficiency and reliable service combine to make this the perfect venue for meetings and banquets. Guests seeking a soiree in the comfort of their own homes or offices will appreciate the customised private catering service we offer.
View our current promotions, offers and special Rotana Escapes room packages here.


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Antonio Villavicencio y Verástegui (January 9, 1775 - June 6, 1816) was a statesman and soldier of New Granada, born in Quito, and educated in Spain. He served in the Battle of Trafalgar as an officer in the Spanish Navy with the rank of Second Lieut...enant. He was sent as a representative of the Spanish Crown to New Granada, where his arrival was used as an excuse in Santafé de Bogotá to start a revolt; this was known as the Florero de Llorente, which culminated in the proclamation of independence from Spain. After this incident he resigned his office and joined the cause of independence. He was later captured and became the first martyr executed during the reign of terror of Pablo Morillo.
Connect any celebrity with Antonio Villavicencio to see how closely they are linked... romantically!


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Art // Sculpture // Design
Notes & Quotes
W. B. Yeats
11th May 2016 by Dana Provence
Do not wait to strike till the iron is hot, but make it hot by striking.
Pablo Picasso
30th April 2016 by Dana Provence
Inspiration exists, but it has to find you working.
Albert Einstein
19th April 2016 by Dana Provence
The world is a dangerous place to live, not because of the people who are evil, but because of the people who don't do anything about it.


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All About Ram Gopal Varma
A new track titled 'Khallas' from Ram Gopal Varma's upcoming biographical film 'Veerappan' has been unveiled. The song features Zareen Khan showcasing some wild moves. The song is originally from the director's 2002 film 'Company' which has been given a new twist by composers Sharib Sabri ... read more
Filmmaker Ram Gopal Varma, who always manages to remain in the limelight for his controversial tweets, again hogged the limelight for drawing comparison between Salman Khan and Shahrukh Khan. According to RGV, Salman is a bigger star than Shahrukh. "What to do you mean by emerging? He alre ... read more
Director Ram Gopal Varma's upcoming movie 'Veerappan' is a biographical film on the life of the infamous forest brigand and the events leading to his death. The filmmaker interacted with the bandit's ex-gang members and others associated with him, to collect materials for the biopic. Known ... read more
Talks are on that megastar Amitabh Bachchan will again team up with Ram Gopal Varma for the third installment of 'Sarkar'. Big B also refuted the rumour of tiff with RGV. At the music launch of his film 'Te3n', when Amitabh Bachchan was asked about his 'tension' with Varma, he said: "First ... read more
Bollywood actor Vivek Oberoi has been roped in by filmmaker Ram Gopal Varma's for his next venture, 'Rai', a gangster thriller. Vivek played the role of gangster in the past and he excels in this department. "Launching my new film Rai The Greatest Gangster Ever on May 1st. Vivek Oberoi wi ... read more
Filmmaker Ram Gopal Verma is known for making controversial remarks on the social media. The 'Satya' director has this time has targeted liquor baron Vijay Mallya, who is on the run following non-repayment of bank loans. In a series of spiteful comments, Ram Gopal Varma asked the Chairman ... read more
Filmmaker Ram Gopal Varma has revealed his love for veteran actress Sridevi in his autobiography, 'Guns and Thighs'. He describes Sridevi as the "Goddess of beauty" and blames her husband Boney Kapoor. "It was a love letter. I was going overboard, but that was my feeling. Everyone can ha ... read more
Ram Gopal Varma in his book, Guns & Thighs, devoted a chapter to megastar Amitabh Bachchan and Big B choices of content. Ram Gopal Varma believes casting the megastar in his movies, 'Nishabd' and 'Aag' was a mistake. In a chapter titled 'My love affair with Amitabh Bachchan', Varm ... read more
Producer Boney Kapoor tagged director Ram Gopal Varma "crazy", "bonkers" and a "pervert" after the latter openly expressed his admiration for his actress wife Sridevi in his newly launched autobiography "Gun & Thighs". RGV, who is known to make bizarre statements in the past too openly, prais ... read more
Filmmaker Ram Gopal Varma has dedicated his autobiography titled 'Guns & Thighs', to some actors, a porn star and a few gangsters. According to him, they have "contributed" in his life one way or the other. The book will be released next month by Rupa publications. ... read more


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Separate names with a comma.
Discussion in 'Ruins of Alph' started by Jellicent, Sep 10, 2012.
But in any case I don't think gen 3/4 Salamence would be as dominant as gen 1/2 Exeggutor.
that's kinda hard to say, because in rby it's pretty clear that exeggutor is the 4th best mon, but in gens like adv and dpp it's hard to rank stuff as definitively a certain rank in terms of how good it is overall. still i think mence could put up a fight for being as dominant in dpp as eggy was in rby, seeing as before it was banned you saw it on "half the good teams" and i'm quoting ace matador on that one since i wasn't actually around then. kinda comes down to gsc egg vs adv mence, and i do still think egg has the upper hand there since mence really had an abundance of checks in adv and egg was especially hard to play around at times in gsc, plus boom is always super threatening regardless of which gen you're talking about. i could see it going either way but i'd put egg over mence for now. (also mence wasn't around for 2 gens so i don't think it should be a major consideration for top 10)
I would like to see this closed soon, it seems like we're all talking in circles. I guess top 10 was too ambitious, since at a certain point it's hard to definitively say something is better than another thing.
My votes are still Raikou #9 Bliss #10. Final answer.


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Marlon Brando
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Mike Calvert Toyota Sales Reviews
- 2333 S Loop W
- Houston, TX 77054 (map)
- Chat with Dealer
Great sales personWritten by josh_1 on 02/25/12 02:01:33 PM PST Go back to Sales reviews
Recommend this dealer? Yes Purchased a vehicle from this dealer? Yes
I have bought 4 cars from Mike Calvert, I cant rate the dealership as a whole. But I have bought 4 of the vehicles from Greg Hoogerwerf. So hard nowadays to find an honest salesman. He simply says this is what I have, this is sticker and this is what I can sell it for. He took the time to go over what I wanted from when I wanted something sporty and fun for me, when I traded that in for my truck for work to something safe and economical for my wife and son. I have sent many friends and relatives to him and every one of them has been appreciative for introducing them. If your looking for an honest straightforward salesmen that wont jerk you around ask for Greg, and tell him Josh said hi! I will be buying another truck from him again.


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Lee has a butchers dustbin on his head
by Lee wankers who likes to bum gay goats June 27, 2003
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Top Definition
A particularly sloppy set of lady bits.
I pulled down her pants and it was like looking in a butcher's dustbin!
by Lexiluwho December 03, 2010


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Top Definition
When one wears a hat so you won't wear a hat like someone that you know or are related to.
He is going to imitate his father, time to hat block.
de Mr.Zogrebginald 20 August 2013
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E-mailurile sunt trimise de la firstname.lastname@example.com. Nu-ți vom trimite mesaje nedorite.×


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Tria Beauty, a maker of at-home beauty devices, has named Peter Wyles as the company's president and CEO.
NCR Corporation, a leading global consumer transaction technologies developer, has appointed Michael Bayer as SVP and president of the company's retail solutions division. Bayer will report to NCR chairman and CEO Bill Nuti.
Bayer HealthCare said it plans to acquire rapidly growing Schiff Nutrition International for $34 a share in a transaction valued at $1.2 billion.
The NPD Group, Inc. has named Kevin Gilbert as director of home improvement, reporting to Perry James, the president of home and office supplies.