The effects of extracts of <i>P</i>. <i>virginica</i> on seedling height of four lawn species.

<p>The effects of extracts of <i>P</i>. <i>virginica</i> on seedling height of four lawn species.</p

The effects of extracts of <i>P</i>. <i>virginica</i> on aboveground biomass and root biomass of seedlings of four lawn species.

<p><i>P</i> values (significant level) of aboveground and root biomass were abbreviated as <i>P</i>_A and <i>P</i>_R, respectively.</p

Assay of rVpmaX adhesion and adhesion inhibition to EBL cells visualized by confocal laser scanning microscopy.

<p>Active rVpmaX interacted with fixed EBL cells, and the surplus protein was rinsed away by washing with PBST. The attached protein was immunostained with rabbit anti-rVpmaX antibody and mouse anti-rabbit IgG-FITC. The EBL cell membranes were labeled with 1,19-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and the cell nuclei were counter-labeled with 49,6-diamidino-2-phenylindole (DAPI). (A1–A2) 10 µg rVpmaX adhering to EBL cells. (B) Adhesion inhibition of 10 µg rVpmaX to EBL cells by 10 µl rabbit anti-rVpmaX serum. (C) Adhesion of 20 µg rVpmaX to EBL cells. (D) The adhesion of 20 µg rVpmaX to EBL cells was inhibited by 20 µl rabbit anti-rVpmaX serum. (E) EBL cells without protein added.</p

The effects of extracts of <i>P</i>. <i>virginica</i> on root length of four lawn species.

<p>The effects of extracts of <i>P</i>. <i>virginica</i> on root length of four lawn species.</p

Localization of VpmaX in <i>M. bovis</i> Hubei-1.

<p>Western blot analysis of rVpmaX (lane 1), <i>M. bovis</i> total proteins (lane 2), cell membrane fraction proteins (lane 3), cell soluble cytosolic fraction proteins (lane 4), and bovine serum albumin (lane 5) using rabbit anti-rVpmaX serum and a peroxidase-conjugated secondary antibody.</p

The effects of extracts of <i>P</i>. <i>virginica</i> on seed germination rate of four lawn species.

<p>The effects of extracts of <i>P</i>. <i>virginica</i> on seed germination rate of four lawn species.</p

R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

<div><p>The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, <i>rspo3</i> mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of <i>rspo3</i> promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of <i>rspo3</i> increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of <i>rspo3</i> abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of <i>rspo3</i> results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.</p></div

Gene structure, amino acid sequence, and phylogenetic analysis of zebrafish and other vertebrate Rspo3 orthologs.

<p>(<b>A</b>) Comparison of human, mouse, elephant shark, zebrafish, medaka, fugu, stickleback, and spotted gar <i>RSPO3/rspo3</i> gene structure. Exons are shown as boxes (filled box, protein coding region; open box, UTR). Introns are shown as lines. (<b>B</b>) Amino acid sequence alignment of human, mouse, rat, cow, horse, <i>Xenopus</i>, elephant shark, spotted gar, stickleback, fugu, medaka, and zebrafish RSPO/Rspo3. The identical amino acids are in black and similar amino acids are in grey. Protein domains of zebrafish Rspo3 are shown using black lines, and the domain names are given. (<b>C</b>) Phylogenetic analysis of the RSPO/Rspo family using the Neighbor-Joining (NJ) method. The length of branches represents the genetic distances, and numbers on nodes are bootstrap percentages to indicate the two clades as sisters. The accession numbers are as follows: human RSPO1 NP_001033722.1, mouse Rspo1 NP_619624.2, rat Rspo1 NP_001101450.1, zebrafish Rspo1 NP_001002352.1, human RSPO2 NP_848660.3, mouse Rspo2 NP_766403.1, rat Rspo2 NP_001124047.1, <i>Xenopus</i> Rspo2 NP_001088999.1, human RSPO3 NP_116173.2, mouse Rspo3 NP_082627.3, rat Rspo3 NP_001094460.1, cow Rspo3 NP_001069502.1, horse Rspo3 NP_001103152.1, <i>Xenopus</i> Rspo3 NP_001123245.1, medaka Rspo3 ENSORLP00000007233, fugu Rspo3 ENSTRUP00000009202, stickleback <i>rspo3</i> ENSGACG00000006080, elephant shark Rspo3 SINCAMP00000010032, spotted gar Rspo3 ENSLOCP00000020398, human RSPO4 NP_001025042.2, mouse Rspo4 NP_001035779.1, rat Rspo4 XP_006235323.1. *,Ensembl or GenBank predicted sequence.</p

Rspo3 inhibits the zygotic Wnt/β-catenin signaling in zebrafish embryos.

<p>(<b>A</b>) Rspo3 inhibited Wnt/β-catenin reporter activities. One-cell stage embryos were injected with Topflash reporter DNA together with 600 pg <i>gfp</i> mRNA, 20 pg <i>wnt3a</i> mRNA, 200 pg <i>dkk1</i> mRNA, or 600 pg <i>rspo3</i> mRNA, respectively. Injected embryos were raised to the shield stage and the luciferase activity was measured. Values are means ± S.E. (n = 3). **, ***<i>P</i><0.01 and 0.001 vs. the <i>gfp</i> group. (<b>B</b>) Knockdown of <i>rspo3</i> increased Wnt/β-catenin reporter activity. One-cell stage embryos were injected Topflash reporter DNA together with cMO (16 ng), MO1, or MO2, respectively. Injected embryos were raised to the shield stage and the luciferase activity was measured. (<b>C</b> and <b>D</b>) Knockdown of <i>rspo3</i> increased the expression levels of <i>vent</i> (C) and <i>sp5l</i> (D) mRNA. Embryos were injected with cMO (8 ng), MO1 (4 ng) or MO2 (8 ng) at the one-cell stage, and wild-type embryos were used as control. The <i>vent</i> and <i>sp5l</i> mRNA levels were measured by RT-qPCR at the 90% epiboly stage. Values are means ± S.E. (n = 3). *, **, ***<i>P</i><0.05, 0.01, and 0.001 vs. the cMO group. (<b>E</b>) Rspo3 inhibited Wnt3a activity. Representative views of embryos. The pictures were taken at the 5-somite stage. (<b>F</b>) Quantitative results. The percentages of dorsalized embryos described in (E) were calculated and shown. The total embryo numbers are given at the top. Embryos were injected with 600 pg <i>gfp</i> mRNA, 20 pg <i>wnt3a</i> mRNA, 20 pg <i>wnt3a</i> mRNA plus 200 pg <i>dkk1</i>, or 600 pg <i>rspo3</i> mRNA. (<b>G</b>) Rspo3 inhibited Wnt3a-stimulated Topflash reporter activity. One-cell stage embryos were injected with Topflash DNA together with the indicated mRNA, the injected embryos were raised to the shield stage and luciferase activities were determined. Values are means ± S.E. (n = 3). ***<i>P</i><0.0001, one-way ANOVA test. (<b>H</b>) Rspo3 cannot inhibit <i>β-cateninΔN</i> activity. Representative views of embryos injected with 600 pg <i>gfp</i> mRNA, 50 pg <i>β-cateninΔN</i> mRNA, or 50 pg <i>β-cateninΔN</i> plus 600 pg <i>rspo3</i> mRNA. The pictures were taken at the 5-somite stage. (<b>I, J</b>) Quantitative results (I) and luciferase activity assay (J). Values are means ± S.E. (n = 3). **<i>P</i><0.01, one-way ANOVA test. (<b>K</b>) Knockdown of <i>rspo3</i> decreased the expression of <i>otx2</i> and increased the expression of <i>hoxb1b</i> at the 100% epiboly stage. (<b>L</b>) Overexpression of <i>rspo3</i> increased the expression of <i>otx2</i> and decreased the expression of <i>hoxb1b</i> at the 100% epiboly stage. Dorsal views with anterior to the left are shown. Asterisks indicate the edges of the expression domains (K, L). Scale bars = 200 µm. (<b>M</b>) The percentage of embryos were calculated and shown. The results are from three independent experiments, and the total embryo numbers are given at the top.</p

The averaged soil nitrate-N and ammonium-N (amm-N) (a), and fine root biomass and necromass (b) in 0–10 and 10–20 cm soil in N fertilization and control plots across the study period from May to October, 2010 (Mean ± SE, <i>n</i> = 3).

<p>The biomass and necromass are gained from sequential soil cores.</p