Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
  • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
  • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• Wnt genes of vertebraten and invertebraten encode a large family of secreted, cystein rich proteins that play key roles as intercellular signaling molecules in a wide variety of biological processes (for an extensive review see (Wodarz and Nusse 1998).
• the first Wnt gene, mouse WnM was discovered as a proto-oncogene activated by integration of mouse mammary tumor virus in mammary tumors (Nusse and Varmus 1982). Consequently, the involvement of the Wg/Wnt pathway in cancer has been largely studied. With the identification of the Drosophila polarity gene wingless as a wnt-1 homologue (Cabrera, Alonso et al.
• the secreted Wnt protein binds to Frizzle cell surface receptors and activates the cytoplasmic protein Dishevelled (Dsh). Dsh then transmits the signal to a complex of several proteins, including the protein kinase Shaggy(Sgg)/GSK3, the scaffold protein Axin and ⁇ -Catenin, the vertebrate homologue of Armadillo.
• ⁇ -Catenin is targeted for degradation after being phosphorylated by Sgg.
• ⁇ -Catenin escape from degradation and accumulate into the cytoplasm.
• Free cytoplasmic ⁇ -Catenin translocates to the nucleus by a still obscure mechanism, and modulates gene transcription through binding the Tcf/Lef family of transcription factors (Grosschedl R 1999). Mutations in ⁇ -catenin, APC, and Axin have been found in several human c cancers, suggesting that constitutive activation of canonical Wnt pathway contributes to human carcinogenesis (Uthoff SM, Eichenberger MR, McAuliffe TL, Hamilton G and Galandiuk S. (2001). MoI. Carcinog., 31, 56-62.
• Wnt signaling pathways that signal independently of ⁇ -catenin, which may signal through calcium flux, c-Jun NH 2 -terminal kinase, and G proteins. These pathways might get activated in parallel to the canonical, bCatenin dependent pathway in tumors characterized by Wnt ligand upregulation (Huguet EL, McMahon JA, McMahon AP, Bicknell R and Harris AL. (1994).
• Wnt signalling In addition to its role in cancerogenesis, Wnt signalling also plays a role in skeletogenesis, bone formation and fracture repair (Hartmann (2000), Holmen (2005)). For instance, upregulation of Wnt-proteins have been shown to correlate with pathobiology of rheumatoid arthritis and osteoarthritis (Sen et al. (2000), Nakamura (2005), Holmen (2005)).
• the invention relates to a method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of:
• a derivate as stated under d) is a the nucleic acid molecule according to a) or b) or a fragment according to c) with an arbitrary molecule attached to it, said candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified nucleic acid molecule or fragment, respectively.
• Percent (%) homologous with respect to the following (poly)peptide sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequences with SEQ ID. Nos 4, 5, 6 and 8 after aligning the sequence and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative amino acid substitution as part of the sequence identity.
• the % identity values used herein can be generated by WU-BLAST-2, which was obtained from (Tatusova TA 1999). WU-BLAST-2 uses several search parameters, most of which are set to the default values.
• percent (%) homologous with respect to the mentioned nucleic acid sequences with the SEQ ID. Nos 1, 2, 3, and 7 is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the said nucleic acid sequences.
• identity values used herein can be generated using BLAST module of WU-BLAST-2 set to the default parameters.
• the (poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 encoded by nucleic acid molecules with the SEQ ID. Nos 1, 2, 3 and 7, respectively, are representatives of a novel family of proteins present in invertebrate, for example in Drosophila, leeches, slugs, snails and earthworms, and in vertebrate organisms, for example in mammals including humans, apes, monkeys, dogs, cats, rabbits, goats, pigs, hamsters, cows, horses, sheep, mice and rats.
• the (poly)peptides with the SEQ ID. Nos 4, 5, 6 and 8 are hereinafter referred to as 3L3 or WLS (wntless) proteins.
• the (poly)peptide with the SEQ ID. No 4 is the 3L3 protein of Drosophila (3L3-PA), as well as the (poly)peptide with the SEQ ID. No 8 (3L3-PB).
• the (poly)peptides with the SEQ ID. Nos 5 and 6 are the 3L3 proteins of caenorhabditis (C.) elegans and human, respectively, which are structural and functional homologous of Drosophila 3L3.
• the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 52 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 55 % homologous, more preferably at least 60 % homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 % homologous.
• the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 75 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 80 % homologous, more preferably at least 85 % homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 % homologous.
• the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 88 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.
• the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 93 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 94 % homologous, more preferably at least 95 % homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 % homologous.
• the nucleic acid molecule, the fragment or the derivate as stated under step e) is at least 98 % homologous to the nucleic acid molecule according to a) or b), to the fragment according to c) or to the derivate according to d), respectively, preferably at least 99 % homologous.
• the invention further relates to a method for screening for a substance that inhibits or enhances the secretion of a protein of the Wnt-family comprising the steps of: a) bringing a candidate substance into contact with a (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide; or b) bringing a candidate substance into contact with a (poly)peptide fragment of the (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 under conditions that permit binding of said substance to said (poly)peptide fragment, said fragment comprises the part of the (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8 which effects Wnt-protein secretion; or c) bringing a candidate substance into contact with a derivate of the
• polypeptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8 and a Wnt protein.
• a derivate as stated under c) is a (poly)peptide with the amino acid sequence of SEQ ID. Nos 4, 5, 6 or 8, or a (poly)peptide fragment according to b) with an arbitrary molecule attached to the N- or C-terminal part or to a side chain of an amino acid, and a candidate substance showing an affinity to said derivate which is at the most 50% increased or decreased compared to the affinity between the candidate substance and the corresponding unmodified (poly)peptide or (poly)peptide fragment, respectively.
• the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 52 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 55 % homologous, more preferably at least 60 % homologous, even more preferably at least 65 % homologous, yet even more preferably at least 70 % homologous.
• the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 75 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 80 % homologous, more preferably at least 85 % homologous, even more preferably at least 86 % homologous, yet even more preferably at least 87 % homologous.
• the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 88 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 89 % homologous, more preferably at least 90 % homologous, even more preferably at least 91 % homologous, yet even more preferably at least 92 % homologous.
• the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 93 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 94 % homologous, more preferably at least 95 % homologous, even more preferably at least 96 % homologous, yet even more preferably at least 97 % homologous.
• the (poly)peptide, the (poly)peptide fragment or the derivate as stated under step d) is at least 98 % homologous to the (poly)peptide according to a), to the (poly)peptide fragment according to b) or to the derivate according to c), respectively, preferably at least 99 % homologous.
• the invention relates to an antibody which specifically binds to the (poly)peptide according to step a), to the (poly)peptide fragment according step b), to the derivate according to step c) or to the (poly)peptide, the (poly)peptide fragment or the derivate according to step d), or to a (poly)peptide domain of the (poly)peptide with the amino acid sequence of SEQ ID. No 4, 5, 6 or 8, preferably to the (poly)peptide domain which is involved in Wnt-protein secretion or which binds to a Wnt-protein.
• the invention relates to a siRNA with a target sequence being a fragment of the nucleic acid molecule coding for a (poly)peptide with the amino acid sequences of SEQ ID. Nos 4, 5, 6 or 8, preferably with a target sequence of SEQ ID Nos 9 or 10.
• the invention relates to the use of said antibody or said siRNA, the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) as a drug.
• the invention relates also to the use of said antibody or said siRNA, the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) for the preparation of a medicament for the treatment of Wnt-signaling related disorders, preferably cancer, bone or joint disorders or developmental disorders.
• Wnt-signaling related disorders preferably cancer, bone or joint disorders or developmental disorders.
• Preferred cancer types are Wnt-dependent cancer types, preferably colorectal cancer, lung cancer, nasopharyngeal carcinoma, preferably Wnt-2 dependent nasopharyngeal carcinoma, small intestinal adenocarcinoma, fundic gland polyps (gastric), gastric carcinoma, gastric (intestinal-like), gastric adenoma (without associated adenocarcinoma), gastrointestinal carcinoid tumor, esophageal adenocarcinoma, juvenile nasopharyngeal angiofibromas, melanoma, pilamatricomas, lung adenocarcinomas, ovarian carcinoma, uterine cervix, uterine endometrial, breast fibromatoses, prostate, thyroid carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma associated with hepatitis C, medulloblastoma, desmoid tumor, Wilm's tumor (kidney), pancreatic (
• Preferred types of bone or joint disorders are osteoarthritis or rheumatoid arthritis, respectively.
• the invention relates to a pharmaceutical composition comprising said antibody or said siRNA.
• the invention relates to an assay for studying diseases induced by an altered Wnt secretion or for drug screening comprising the use of an organism, for example a vertebrate or an invertebrate organism, preferably selected from the group comprising Drosophila, mice, rats, rabbits, chicken, frogs, pigs, sheep, worms, for example C. elegans, and fishes, for example zebrafish, or a cell line systems, preferably human cell line systems, said organisms or cell line system, respectively, showing increased or reduced or no expression of 3L3 or express a mutated 3L3 (poly)peptide in at least one tissue or organ.
• an organism for example a vertebrate or an invertebrate organism, preferably selected from the group comprising Drosophila, mice, rats, rabbits, chicken, frogs, pigs, sheep, worms, for example C. elegans, and fishes, for example zebrafish, or a cell line systems, preferably
• said organisms express the 3L3 gene comprising at least one of the nucleic acid molecules shown in SEQ. ID Nos 1 to 3 or 7 as a transgene.
• said 3L3 gene comprises a mutation selected from the group consisting of deletions, point mutations, insertions and inversions.
• Still another aspect of the invention is a method for the modification of the Wnt secretion of a cell comprising the following step:
• said substance is selected from said antibody or said siRNA, the nucleic acid molecule according to step a) or b), the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), or the (poly)peptide according to step a), the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d).
• the invention relates further to the use of the nucleic acid molecule according to step a) or b) of the screening method, the fragment according to step c), the derivate according to step d) or the nucleic acid molecule, the fragment or the derivate according to step e), or said antibody or said siRNA to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells in vivo and in vitro.
• the invention relates to the use of the (poly)peptide according to step a) of said screening method, the (poly)peptide fragment according to step b), the derivate according to step c) or the (poly)peptide, the (poly)peptide fragment or the derivate according to step d) to regulate the Wnt-signaling pathway in cells, preferably to regulate cell fate determination or to control the development of stem cells in vivo and in vitro.
• Fig. la-c The CustalW protein alignment shows the high homology between the 3L3 protein of Drosophila, C. elegans and human.
• the putative signal sequence is indicated by a dashed line above the sequence and the putative transmembrane domains are underlined (TMHMM prediction).
• Fig. 2 A) Normal adult pharates as control.
• Fig. 3 A) Distal less-LacZ expression in a wild type disc.
• Fig. 4 A) Wg protein expression in a wild type disc.
• Fig. 5 TOP Flash Luciferase assay.
• Wnt3A-V5 and siRNA_h3L3 cotransfected cells show no induction of the Wnt signaling dependent Luciferase reporter gene as the mock control, where empty vector (pcDNA3) was cotransfected with siRNA_GFP.
• empty vector pcDNA3
• Wnt3A-V5 and siRNA_GFP cotransfected cells showed a
• Fig. 6 Co-Immunoprecipitation, IP: ⁇ -HA, blot: ⁇ -V5.
• h3L3-HA is interacting with Wnt3A-V5 (Lane 1).
• Negative controls no interaction could be observed between CD2-HA and Wnt3A-V5 (Lane 2) and no band could be detected in EGFP transfected cells (Lane 3).
• Fig. 7 siRNA against the C. elegans homologue of 3L3.
• siRNA against 3L3 leads to an abnormal arrangement of Oocytes within the gonad (B) and sometimes to a defect in distal tip cell migration (C).
• Fig. 8 TOP Flash Renilla Luciferase assay. WLS RNAi showed a clear down-regulation of Wg secretion compared to GFP RNAi.
• Fig. 9 Secretion of Wnt3a is impaired upon knock-down of hWLS
• Fig. 10 B-I) Cell surface stainings of HEK-293T cells.
• Wnt3a-V5 cannot reach the cell surface of cells co-transfected with sihWLS (B) whereas cotransfection of siGFP leads to detectable levels of Wnt3a-V5 at the cell surface (C).
• Wnt3aC77A-V5 is neither detectable at the surface of cells treated with sihWLS (D) nor siGFP (E).
• F-I HA-CD2 is detected on the cell surface (F,G) while CD2-HA is not (H,I); CD2-HA can be readily detected by conventional staining (not shown). Surface levels of HA-CD2 are not affected by sihWLS
• FIG. 11 RNAi against the C. elegans WLS ortholog phenocopies the ABar spindle orientation defect of mom-2 and mom-3 mutant embryos Embryos are shown anterior to the left and ventral up.
• A Wild-type embryo, the ABar spindle (yellow) is oriented perpendicular to the spindle of ABpr (blue), i.e. perpendicular to the optical section shown (indicated by the yellow dot).
• B B
• RNAi(mom-2) injected animals the two spindles are in parallel orientation.
• C,D In mom-3(or78) mutant embryos (C) as well as in RNAi(R06B9.6) embryos (D) the ABar and ABpr spindles are also in parallel orientation.
• Fig. 12 Secretion of Wnt3a is impaired upon knock-down of hWLS
• the Wnt signaling cascade is essential for the development of both vertebrates and invertebrates, and has been implicated in tumorigenesis.
• the Drosophila Wnt genes are one of the best characterized within the Wnt-protein family, which includes more than hundred genes.
• Wg is required for formation of parasegment boundaries and for maintenance of engrailed (en) expression in adjacent cells.
• the epidermis of embryo defective in Wg function shows only a rudimentary segmentation, which is reflected in an abnormal cuticle pattern. While the ventral cuticle of wild type larvae displays denticle belts alternating with naked regions, the cuticle of Wg mutant larvae is completely covered with denticles.
• Wg controls dorso-ventral positional information.
• Wg patters the future leg by the induction of ventral fate (Struhl and Basler 1993).
• the ventral half of the leg develops into a mirror image of the dorsal side (Baker 1988). Accordingly, reduced Wg activity leads to the transformation of wing to notal tissue, hence the name of the gene (Sharma and Chopra 1976).
• Wg suppresses ommatidial differentiation in favor of head cuticle development, and is involved in establishing the dorso-ventral axis across the eye field (Heberlein, Borod et al. 1998).
• CG6210 encodes for a protein which consists of a signal sequence at the N-terminus and 7 putative transmembrane domains.
• the other suppressor su20.54 has a point mutation that leads to an amino acid residue exchange from Prolin to Serin in the first transmembrane domain.
• Drosophila embryos are progressively subdivided into reiterated segments by the localized activities of several genes.
• Wingless (Wg) and Hedgehog (Hh) are the most important genes of the so-called segment polarity gene group, which eventually divides the A/P axis into 14- 15 stripes prefiguring the future segments.
• Wg or Hh function leads to a disruption of the formation of the repeating exoskeletal structures on the ventral side of the embryo, which is the most obvious phenotypic outcome of the activity of the segment polarity genes.
• Genetic experiments called “germline clones” allow the inventors to generate Drosophila mutant embryos, which completely lack the protein of interest (in the present case 3L3) and then to study the segmentation pattern. In particular "germline clones" are necessary to get rid of the maternal component, which is often present in embryo for many genes.
• Wg plays a pivotal role also in the development of adult appendages of Drosophila, such as antennae, legs, and wings. Therefore inventors studied the phenotype of 3L3 mutant adult pharates, which represent flies just before hatching. They observed that all the appendages, whose development depends on Wg function, are abnormal (Fig. 2 A to C). In particular aristae of antennae are missing, legs are shorter and show segmentation defects, and in 5% of the cases a so-called "wing-to-notum" transformations was observed, which are pathognomonic for a Wg loss of function. These findings give evidence for the conclusion that 3L3 is a positive component in the Wg/Wnt signaling pathway.
• imaginal cells which are organized in sack-like epithelia called "imaginal discs".
• the wing imaginal disc comprises around 20 cells when it is formed during embryonic development and proliferates during the three larval instars to generate a disc of ca. 75'00O cells at the end of the larval life.
• Wingless behaves as a morphogen, whose spatial concentration varies and to which cells respond differently at different threshold concentrations.
• target genes i.e. genes, which are activated by Wg.
• target genes i.e. genes, which are activated by Wg.
• Two kinds of target genes are distinguished in the wing imaginal discs.
• long- range target genes such as Distalless
• target genes which need a high level of the morphogen for their expression (so called “short-range target genes” (such as Senseless).
• h3L3 human homologue of 3L3 (h3L3) is involved in Wnt signaling a Wnt reporter gene assay (TOP-flash) was performed.
• TOP-flash a Wnt reporter gene assay
• This is a luciferase based assay with 5 TCF binding sites in front of the luciferase gene.
• the activity of the Wnt signaling pathway can be measured by the luminescence of the luciferase. From preceding discussed results it was known that 3L3 is important in Drosophilas Wnt producing cells.
• one batch of human 293T cells was transfected with mWnt3A- V5 and siRNA against h3L3, another batch of cells was transfected with the TOP-flash reporter construct.
• WLS wntless mutant cells in Drosophila wing discs it was observed an accumulation of Wg in the Wg producing cells.
• Kc cells we developed an assay to measure the secretion of Wg into the culture medium.
• the Renilla luciferase gene was fused to the N- terminus of wg and transfected into Kc cells.
• the cells were treated with dsRNA of WLS or GFP.
• WLS RNAi showed a clear down-regulation of Wg secretion compared to GFP RNAi (Fig. 8).
• siRNAhWLS-A two different siRNAs (siRNAhWLS-A, SiRNAhWLS-B) were validated by RT-PCR for their effectiveness to knock-down the expression of the endogenous hWLS gene in HEK-293T cells. Independent transfection of both resulted in an 85% decrease of hWLS transcripts (not shown; SiRNAhWLS-B was mostly used and is henceforth referred to as sihWLS, but siRNAhWLS-A showed the same effects). Treatment of responder cells with sihWLS, or treatment of producer cells with siRNA against GFP (siGFP) did not appreciably affect the outcome of the Wnt signalling assay.
• siRNAhWLS-A siRNAhWLS-A
• MOM-3 has been shown to be involved in many different, canonical and non-canonical Wnt pathways such as early blastomere polarization (Rocheleau, Thorpe), VPC specification (Eisenmann and Kim), Q neuroblast migration (Harris). These processes depend on three different Wnt's: MOM-2 (Rochelau, Thorpe), LIN-44 (Jiang and Sternberg) and EGL-20 (Harris).
• Non-canonical Wnt signaling controls orientation of EMS division (the EMS cell is the precursor of the future endoderm and mesoderm precursor cells) and the fate of endoderm. Loss of non-canonical Wnt signaling leads to the loss of endoderm and the development of more mesoderm. Several components of this non-canonical Wnt signaling have been described. One of them, mom-3
• RNAi against c3L3 led to typical loss of canonical and non-canonical Wnt phenotypes such as defects in distal tip cell migration and embryonic lethality. Together, these results show that c3L3 is responsible for the mom-3 phenotype and that RNAi against c3L3 mimics a loss of mom-3 phenotype.
• Wnt signaling in C.elegans is involved in many different developmental steps.
• the earliest step known is the MOM-2 dependent orientation of mitotic spindles and division planes in the four to eight cell embryo. Since mom-3 worms are mutant in the wntless orthologue
• R06B9.6 we injected dsRNA of R06B9.6 into the gonads of wt hermaphrodites. As expected all embryos analyzed injected with dsRNA of R06B9.6, or carrying a mutant allele of mom-2 or mom-3 showed a parallel orientation of the Abar spindle to the ABpr spindle. In contrast, wt embryos had the two spindles oriented perpendicularly (Fig. 11).
• 3L3 which is encoded by the gene CG6210.
• 3L3 plays a role in the Wg/Wnt signaling pathway, and in particular in the Wg/Wnt secretion pathway of the Wnt proteins. Further it has been shown that 3L3 physically interacts to a Wnt-protein. Therefore 3L3 proteins are very promising targets for developing drugs which positively or negatively regulate the Wnt pathway by regulating the secretion of the Wnt ligands.
• the sev-wg transgene which ectopically expresses Wg in the eye, leads to a rough eye phenotype that served as a phenotypic marker (Brunner et al., 1997) in the screen.
• Males carrying an FRT80 on the left arm and the sev-wg on the right arm of the 3 rd chromosome were feeded for 12 hours with 2ImM Ethyl methan sulfonate (EMS). 24 hours after the application of EMS the males were crossed to females encoding an eyeless-flp recombinase on the X chromosome and carrying an FRT80 M w+ on the 3 rd chromosome.
• Male offspring with the ey-flp and both FRT chromosomes were screened for a suppression of the rough eye phenotype. Complementation analysis between different alleles led to the complementation group 3L3 consisting of two alleles.
• TMHMM TOP predicted one N-terminal signal sequence and 7 putative transmembrane domains for CG6210-PB.
• Germline clones It is an experimental procedure used to study the phenotype of embryo of Drosophila melanogaster ⁇ r ⁇ the complete absence of the protein of interest.
• Imaginal discs are hollow sacs of cells that make adult structures during metamorphosis. They arise as pockets in the embryonic ectoderm and grow inside the body cavity until the larva becomes a pupa, at which point they turn inside out (“evaginate”) to form the body wall and appendages.
• Mutant clones in the wing imaginal discs represent groups of cells, which are mutant for the protein of interest. In Drosophila melanogaster this is achieved by mitotic recombination, using the yeast-specific recombinase flippase (flp) driven by the heat-shock promoter (hs-
• Heat-shock at 37.0 degrees for 60 minutes was made 24-72 hours AEF, and then larvae were dissected 4 days later.
• 3L3 heterozygous mutant flies carrying the transgene wg-GAL4 (the transcriptional activator GAL4 is then expressed only in the cells, which express also Wg) were crossed with flies, which were also 3L3 heterozygous mutant and carried the trangene UAS-3L3 (UAS binding sites for the GAL4 protein activate the transcription of 3L3.
• the protocol used to see extracellular Wg differs from the usual protocol in the fact that the staining with the primary antibody is performed in PBS at 4 degrees (in order to block endocytosis) for 30-60 minutes, and that fixation is done after the primary antibody in PBS, with 4% formaldehyde at 4 degrees for 20-30 minutes (Strigini and Cohen, 2000).
• Cells were transfected by the Calcium-Phosphate method. 16 hours after transfection cells were washed and mixed. 24 hours after mixing, the cells were lysed with IxPLB from Promega and analyzed.
• Cells were transfected by the Calcium-Phosphate method. 36 hours after transfection, cells were lysed with RIPA buffer for 1 hour 30 minutes at 4 0 C. The lysate was centrifuged for 30 minutes at 4°C and then incubated with Protein G Sepharose beads and rabbit ⁇ -HA overnight at 4°C. Beads were washed 4x with TBS and then Proteins were eluted with SDS- Loading buffer from the beads at 95 0 C for 10 minutes. Western blotting was performed with the mouse ⁇ -V5 antibody.
• the mWnt3A cDNA sequence was cloned into pcDNA3 followed by a V5-tag and a His-tag.
• the h3L3 cDNA was cloned into pcDNA3 followed by a HA-tag.
• the siRNAs against 3L3 and GFP were generated by Qiagen.
• CD2 was cloned with a C-terminal HA-tag into pcDNA3.
• Socket cells mediate spicule morphogenesis in Caenorhabditis elegans males. Dev Biol 211, 88-99.

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