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Products for Morphogen Research R&D Systems Tools for Cell Biology Research™ Products for Morphogen Research BMP-4 NEURAL PLATE BMP-7 PROSPECTIVE NEURAL CREST NON-NEURAL ECTODERM Noggin Shh Noggin PRESOMITIC MESODERM NOTOCHORD NON-NEURAL ECTODERM FUTURE FLOOR PLATE BMP-4 Shh Noggin DORSAL AORTA ROOF PLATE Products for Morphogen Research for Products Noggin Wnt-1 Wnt-3a Wnt-4 NT-3 Wnt-6 Wnt-7a EARLY SOMITE Myf5 NEURAL TUBE Pax3 Sim1 BMP-4 INTERMEDIATE MESODERM Shh ShhShh NogginNoggin BMP-4 DORSAL AORTA Ihh NOTOCHORD MORPHOGENS Morphogens are molecules that regulate cell fate during development. Formation of morphogen concentration gradients directs the biological responses of surrounding cells. Graded responses occur as a result of morphogens binding to specific cell surface receptors that subsequently activate intracellular signaling pathways and promote or repress gene expression at specific threshold concentrations. Activation or inactivation of these signaling pathways provides positional information that ultimately determines tissue organization and morphology. Research in model organisms has revealed that morphogens are involved in many aspects of development. For example, morphogens are required in Drosophila for patterning of the dorso-ventral and anterior-posterior axes, segment patterning, and positional signaling in the leg and wing imaginal discs. Proteins belonging to the Wingless/Wnt, Notch, Hedgehog, and TGF-b families have been identified as morphogens that direct a number of these processes. Research in higher organisms has demonstrated that homologues of these same signaling molecules regulate vertebrate axis formation, anterior/posterior polarity during limb development, mesoderm patterning, and numerous other processes that establish an organism’s basic body structure. R&D Systems offers a wide selection of proteins, antibodies, and ELISAs for morphogen-related developmental research. Wnt Family Stress Fibers b-Catenin Merge The molecular name Wnt is derived from Wingless, the Drosophila melanogaster segment polarity gene, and Integrase-1, the vertebrate homologue. Untreated Wnts are a large family of secreted glyco proteins that bind to seven transmembrane G protein- coupled receptors belonging to the Frizzled fami- ly. Frizzled proteins form co-receptor complexes with LRP, ROR, or Ryk molecules. Wnt binding to Frizzled receptors can activate both the canonical Wnt-3a Wnt/b-Catenin signaling pathway and the non- canonical, b-Catenin independent pathways. Wnt signaling regulates multiple developmental processes, including anterior-posterior pattern- ing, patterning of the neural tube and limbs, de- velopment of the reproductive system, somite Wnt-5a development, and formation of the pituitary gland, adrenals, kidneys, and mammary tissues. Demonstration of Wnt-3a and Wnt-5a Bioactivity in Mouse Fibroblasts. Recombinant Mouse Wnt-3a (Catalog # 1324-WN) and Recombinant Human/Mouse Wnt-5a (Catalog # 645-WN) promote stress fiber formation in NIH-3T3 mouse embryonic fibroblast cells, while only Wnt-3a promotes nuclearb -Catenin accumulation. Images courtesy of Dr. Raymond Habas, Robert Wood Johnson School of Medicine. N MW R R 92 66 4000 100 55 43 36 Wnt-3a (High Purity) 90 29 3000 22 80 18 12 70 2000 60 Luciferase Activity Luciferase Proliferation (RFU) Proliferation 50 1000 40 0 0 0.0001 0.001 0.01 0.1 1.0 10 0 0.1 1 10 Wnt-3a (µg/mL) Wnt-11 (µg/mL) Wnt-3a Induces b-Catenin-responsive Transcriptional Activation. b-Catenin- Detection of Wnt-5a Expression in Embryonic Mouse Muscle. Wnt-5a was detected Wnt-11 Induces Proliferation of Rat Small Intestine Epithelial Cells. The IEC-18 rat responsive transcriptional activation was assessed using the TOPflash TCF reporter in in immersion-fixed frozen sections of embryonic mouse muscle (13 d.p.c.) using Rat small intestine epithelial cell line was treated with increasing concentrations of the HEK293T human embryonic kidney cell line following treatment with Recombi- Anti-Human/Mouse Wnt-5a Monoclonal Antibody (Catalog # MAB645). The tissue was Recombinant Mouse Wnt-11 (Catalog # 6179-WN). Cell proliferation was assessed nant Human Wnt-3a (Catalog # 5036-WN; dark green line) or High Purity Recombinant stained using NorthernLights™ 557-conjugated Anti-Rat Secondary Antibody (Catalog using Resazurin (Catalog # AR002). Human Wnt-3a (Catalog # 5036-WNP; light green line). The purity of the High Purity # NL013; red) and nuclei were counterstained with DAPI (blue). Recombinant Human Wnt-3a protein is highlighted in a silver-stained, SDS-PAGE gel loaded with 1 μg of protein/lane under both non-reducing (N) and reducing (R) condi- tions (inset). MW = Molecular weight markers. NorthernLights is a trademark of R&D Systems, Inc. www.RnDSystems.com Products for Wnt Research RECOMBINANT RECOMBINANT RECOMBINANT MOLECULE & NATURAL PROTEINS ANTIBODIES ELISAs MOLECULE & NATURAL PROTEINS ANTIBODIES ELISAs MOLECULE & NATURAL PROTEINS ANTIBODIES ELISAs APC H JNK1/JNK2 H M R R-Spondin 1 H M H M Axin-1 H M R JNK1 M H M R H M R R-Spondin 2 H H Axin-2 H JNK2 H M R H M R R-Spondin 3 H M H M b-Catenin H M R B Ch X H Kremen-1 M H M M R-Spondin 4 H M M Bcl-9 H Kremen-2 H M R H M Ryk H M Bcl9-2 H LRP-1 H sFRP-1 H H CaM Kinase II H M R X LRP-1 Cluster II H H sFRP-2 M M Casein Kinase H M R LRP-1 Cluster IV H sFRP-3 H M H M H 1α,1γ,1δ,1ε LRP-4 H R sFRP-4 H H Casein Kinase 2b H M R LRP-6 H M H M sFRP-5 H Ccd1/DIXDC1 M MESDC2 M H M Shisa-4 H M Dishevelled-1 H MFRP H H M Soggy-1 H M H M M Dishevelled-2 H MKK7 H SOST/Sclerostin H M H M Dishevelled-3 H Myocilin H TAK1 H Dkk-1 H M R H M R H M NeuroD1 H M TCF-7/TCF-1 H Dkk-2 M M Norrin H M H M TCF7L1 H M Dkk-3 H H M H Nucleoredoxin H WIF-1 H H M H Dkk-4 H M H M H PKCα H M R Wnt-1 M Draxin M H M R PKCb1 H R Wnt-2 H Frizzled-1 H M H M PKCb2 H M Wnt-2b M Frizzled-2 M M PKCγ H M R Wnt-3a H M M Frizzled-3 H M PKCδ H Wnt-4 H M H M Frizzled-4 H M H M PKCε H M R Wnt-5a H M H M R Frizzled-5 H H PKCι/λ H M Wnt-5b M M Frizzled-6 H M PKCι/λ/ζ H M R Wnt-6 H Frizzled-7 H M H M PKCθ H M Wnt-7a H H Frizzled-8 H M M Pygopus-1 H M Wnt-7b H Frizzled-9 M Pygopus-2 H Wnt-8a M Frizzled-10 H Renin R H Wnt-8b H M GSK-3α/b H M R H M R ROCK1 H Wnt-9a H GSK-3α H M R H ROCK2 H M R Wnt-9b M H M GSK-3b H H M R H M R ROR1 Receptor H H Wnt-10a M ICAT H Tyrosine Kinase Wnt-10b M JNK H M R H M R RTK-like Orphan H H Wnt-11 H M Receptor 2/ROR2 KEY: H: Human M: Mouse R: Rat B: Bovine Ch: Chicken X: Xenopus Xwnt-8 mRNA 40 R-Spondin 3 (ng/mL) A. B. 0.001 0.01 0.1 1 10 100 DLD-2C2 MCF-7 SK-MEL-28 35 HeLa A431 35000 24000 30 30000 β-Catenin 20000 cells) 6 Luciferase Activity Luciferase 25 25000 20 16000 Xwnt-8 mRNA + Dkk-1 Inhibitor 20000 15 -Catenin (ng/10 -Catenin Luciferase Activity Luciferase C. D. β 12000 15000 10 5 10000 8000 0.001 0.01 0.1 1 10 100 1000 10000 0 R-Spondin 4 (ng/mL) MCF-7 HeLa A431 SK-MEL-28 DLD-2C2 R-Spondin 3 and R-Spondin 4 Induce b-Catenin-responsive Transcriptional Activa- Dkk-1 Antagonizes Wnt Activity during Xenopus Development. A. 16-cell stage Detection of Intracellular b-Catenin using the DuoSet® IC ELISA Development tion. In the presence of 5 ng/mL Recombinant Mouse Wnt-3a (Catalog # 1324-WN), Xenopus embryos were injected with Xenopus Wnt-8 mRNA into a ventral/vegetal blasto- System. The levels of b-Catenin in lysates prepared from the MCF-7 human breast adeno- increasing concentrations of Recombinant Mouse R-Spondin 3 (Catalog # 4120-RS; brown mere. B. A representative Xenopus embryo (stage 35) injected with Xwnt-8 mRNA forms carcinoma, HeLa human cervical epithelial carcinoma, A431 human epithelial carcincoma, line) and Recombinant Mouse R-Spondin 4 (Catalog # 4106-RS; green line) stimulate a secondary axis. C. To counter the effects of Xwnt-8 mRNA, a Wnt antagonist protein, SK-MEL-28 human malignant melanoma, and DLD-2C2 human colorectal adrenocarcino- transcriptional activation of the TOPflash b-Catenin/TCF reporter in the human kidney Recombinant Human Dkk-1 (Catalog # 5439-DK), was injected into the spaces near the ma cell lines were assessed using the Human Total b-Catenin DuoSet IC ELISA (Catalog # cell line, HEK293T. Xwnt-8 mRNA injection site at the 64-cell stage. D. A representative embryo injected with DYC1329; bar graph). The results of the DuoSet IC ELISA are consistent with the relative Xwnt-8 mRNA and then rescued by injection with Recombinant Human Dkk-1 prevents levels of b-Catenin detected by Western blot analysis (inset). the development of a secondary axis. For more information visit our website at www.RnDSystems.com/go/Wnt DuoSet is a registered trademark of R&D Systems, Inc.
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