WO2004108930A1 - Genes participating in fgf signal transduction - Google Patents

Genes participating in fgf signal transduction Download PDF

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Publication number
WO2004108930A1
WO2004108930A1 PCT/JP2004/007910 JP2004007910W WO2004108930A1 WO 2004108930 A1 WO2004108930 A1 WO 2004108930A1 JP 2004007910 W JP2004007910 W JP 2004007910W WO 2004108930 A1 WO2004108930 A1 WO 2004108930A1
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gene
fgf
seq
minutes
amino acid
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PCT/JP2004/007910
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French (fr)
Japanese (ja)
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Hitoshi Okamoto
Yoshikazu Hirate
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Riken
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]

Definitions

  • the present invention relates to a novel gene involved in FGF signaling.
  • the central nervous system and the cerebellum in the central nervous system are thought to be formed inductively by the organizer activity of the midbrain hindbrain boundary. It has been reported that several signal molecules (WntK FGF8) and transcription factors (Islet3, 0tx2, Gbx2, Pax2 / 5/8, Engl / 2/3) are involved in the formation of the midbrain-hindbrain boundary. (See Non-Patent Documents 1 to 11).
  • the caudal limit of Otx2 expression positions the isthmic organizer.Nature 401, 164-168. Millet, S., Campbell, K., Epstein, DJ, Losos, K., Harris, E. and Joyner, AL, 1999.A role for Gbx2 in repression of Otx2 and positioning the mid / hindbrain organizer.Nature 401, 161-164.
  • Engrailed-1 mutant mice an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.Development 120, 2065-2075.
  • an object of the present invention is to provide a novel gene involved in FGF signal transduction, which enables analysis of the formation mechanism of the midbrain tectum and cerebellum at the midbrain hindbrain boundary. Disclosure of the invention
  • the present inventors have conducted intensive studies, succeeded in identifying a novel gene family involved in FGF signaling, and completed the present invention.
  • the present invention includes the following.
  • a gene involved in FGF signal transmission that encodes the following protein (a) or (b):
  • a gene involved in FGF signal transmission comprising the polynucleotide of the following (a) or (b):
  • (6) a step of allowing a lead compound to act on the transgenic cell according to (4) or the transgene according to (5): E: a nick animal;
  • FIG. 1 shows the results of examining the homology between human cnpyl and zebrafish cnpyl.
  • FIG. 2 is a diagram showing the results of examining homology between human cnpy2 and zebrafish cnpy2.
  • FIG. 3 is a diagram showing the results of examining the homology between human cnpy3 and zebrafish cnpy3.
  • FIG. 4 is a diagram showing the result of examining the homology between human ciipy4 and zebrafish cnpy4.
  • ⁇ Figure 5 is a micrograph showing the observation of the expression of the cnpyl, cnpyS and cnpy4 genes in zebrafish embryos.
  • FIG. 6 is a micrograph showing the expression of the cnpyl, cnpy3, and cnpy4 genes in zebrafish embryos.
  • FIG. 7 is a micrograph showing the expression of the cnpy2, cnpy3, and cnpy4 genes in mouse embryos.
  • FIG. 8 is a micrograph of a normal embryo and a knockdown embryo, which observed morphological abnormalities around ⁇ B.
  • FIG. 9 is a micrograph of the observed morphological abnormalities of the otocyst in the normal embryo and the knockdown embryo.
  • FIG. 10 is a photomicrograph showing that normal zephrafish embryos and cnpyl-M0 knockdown embryos were embedded with FGF8b beads, and the induction of engrailed gene expression was observed.
  • FIG. 11 is an electrophoresis photograph in which activated ERK was detected by Western blot in npyl-M0 knockdown embryos and cnpy3 / 4-M0 knockdown embryos.
  • Figure 12 is a photomicrograph of the immunohistochemical method used to detect which part of the zebrafish embryo had lost activated ERK.
  • zebrafish Diorerio
  • Islet-3 Kikuchi, Y., Segawa, ⁇ ., Tokumoto,., Tsubokawa, ⁇ ., Hott, ⁇ ., Uyemura, ⁇ . And Okamoto, ⁇ ., 1997.
  • a new gene group whose expression was controlled by Ocular and cerebellar defects in zebrafis induced by overexpression of the LIM domains of the islet 3 LIM / homeodomain protein. Neuron 18, 369-382.
  • Islet-3 is a LIM / homeodomain transcription factor that controls the expression of a signal from the midbrain that promotes the development of the midbrain-hindbrain boundary (hereinafter referred to as thigh B). Then, the present inventors have identified a novel gene family that is under expression control by Islet-3 and is involved in FGF signal transduction. This new gene family will be referred to as the canopy gene family, and each gene belonging to the family will be named with the word rcnpyj.
  • the cnpy gene family derived from zebrafish has four types of cnpyl to 4 genes.
  • the nucleotide sequence containing the coding region of the cnpyl gene is shown in SEQ ID NO: 1, and the amino acid sequence of cnpyl encoded by the cnpyl gene is shown in SEQ ID NO: 2.
  • SEQ ID NO: 3 shows the partial amino acid sequence of cnpy2 encoded by the cnpy2 gene.
  • the nucleotide sequence containing the coding region of the cnpy3 gene is shown in SEQ ID NO: 4, and the cnpy3 gene encodes Is shown in SEQ ID NO: 5.
  • the nucleotide sequence including the coding region of the cnpy4 gene is shown in SEQ ID NO: 6, and the amino acid sequence of cnpy4 encoded by the cnpy4 gene is shown in SEQ ID NO: 7.
  • cnpyl based on the amino acid sequence of cnpyl (SEQ ID NO: 2), a homology search was performed on the human genome database published by the National Center for Biotechnology Information (NCBI) to obtain human-derived amino acids.
  • NCBI National Center for Biotechnology Information
  • the cnpy gene family has been identified. There are four human cnpy gene families, including human cnpy 1-4 genes.
  • the nucleotide sequence containing the coding region of the human cnpyl gene is shown in SEQ ID NO: 8
  • amino acid sequence of human cnpyl encoded by the human cnpyl gene is shown in SEQ ID NO: 9.
  • the nucleotide sequence containing the coding region of the human ciipy2 gene is shown in SEQ ID NO: 10, and the amino acid sequence of human cnpy2 encoded by the human cnpy2 gene is shown in SEQ ID NO: 11.
  • the nucleotide sequence containing the coding region of the human cnpy3 gene is shown in SEQ ID NO: 12, and the amino acid sequence of human cnpy3 encoded by the human cnpy3 gene is shown in SEQ ID NO: 13.
  • the nucleotide sequence containing the coding region of the human cnpy4 gene is shown in SEQ ID NO: 14, and the amino acid sequence of human cnpy4 encoded by the human cnpy4 gene is shown in SEQ ID NO: 15.
  • FIG. 1 shows the results of measuring the homology between cnpyl derived from zebrafish and cnpyl derived from human. This alignment was prepared by the Lipman-Pearson method (default parameter setting) of Geiietyx-Mac, verlO (Genetics).
  • FIG. 2 shows the results of measuring the homology of ciipy2 (partial amino acid sequence) derived from zebrafish and cnpy2 derived from human.
  • FIG. 3 shows the results of measuring the homology between cnpy3 derived from zebrafish and cnpy3 derived from human.
  • FIG. 1 shows the results of measuring the homology between cnpyl derived from zebrafish and cnpyl derived from human. This alignment was prepared by the Lipman-Pearson method (default parameter setting) of Geiietyx-Mac, verlO (Genetics).
  • FIG. 2 shows the results of measuring the homology of ciipy2 (partial
  • FIGS. 1 to 4 show the results of measuring the homology between cnpy4 derived from zebrafish and cnpy4 derived from human.
  • the cnpy gene family derived from zebrafish and the cnpy gene family derived from human show high homology, and it is considered that genes belonging to both families have the same function.
  • mouse-derived cnpy gene family by performing a homology search on the mouse genome database published at NCBI based on the amino acid sequence of cnpyl (SEQ ID NO: 2). it can. Also, it is not limited to zebrafish, human, and mouse, but includes, for example, By searching databases such as ascidians, sea urchins, Drosophila and nematodes, the cnpy gene family in various metazoans can be identified.
  • SEQ ID NO: 16 shows the partial amino acid sequence of mouse cnpyl encoded by the mouse-derived mouse cnpyl gene.
  • the nucleotide sequence containing the coding region of mouse cnpy2 gene is shown in SEQ ID NO: 17, and the amino acid sequence of mouse cnpy2 encoded by mouse cnpy2 gene is shown in SEQ ID NO: 18.
  • the nucleotide sequence containing the coding region of mouse cnpy3 gene is shown in SEQ ID NO: 19, and the amino acid sequence of mouse cnpy3 encoded by mouse cnpy3 gene is shown in SEQ ID NO: 20.
  • the nucleotide sequence containing the coding region of mouse cnpy4 gene is shown in SEQ ID NO: 21, and the amino acid sequence of mouse cnpy4 encoded by mouse cnpy2 gene is shown in SEQ ID NO: 22.
  • the gene involved in FGF signaling means a cnpy gene included in all of these cnpy gene families.
  • the genes involved in FGF signaling according to the present invention include 2, 3, 5, 7,
  • One or more amino acids are substituted, deleted, or added in any of the amino acid sequences shown in 9, 11, 13, 13, 15, 16, 18, 20, and 22.
  • a gene encoding a protein having a function related to FGF signal transduction (hereinafter referred to as “mutant protein”). That is, the gene involved in FGF signal transduction according to the present invention includes a gene encoding a mutant protein.
  • mutant proteins include, for example, SEQ ID NOs: 1, 4, 6, 8,
  • It can be prepared by deleting, substituting or adding one or more bases in the base sequences shown in 10, 12, 14, 17, 19 and 21.
  • a conventionally known method can be appropriately used without any particular limitation.
  • site-directed mutagenesis methods can be used to replace certain bases.
  • a site-directed mutagenesis method for example, a site-directed mutagenesis method of T. Kunkel (Kunkel, TA Proc. Nati. Acad.
  • a mutagenesis kit using site-directed mutagenesis for example, Mutan-K (Takara Shuzo) or Mutan-G (Takara Shuzo) or the like, Mutations can also be introduced using the Takara Shuzo LA PCR in vitro Mutagenes is series kit.
  • the gene involved in the FGF signaling is any one of the nucleotide sequences represented by SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 14, 17, 19 and 21. It contains a nucleotide sequence that hybridizes under stringent conditions to a complementary nucleotide sequence, and contains a polynucleotide encoding a protein having a function involved in FGF signaling. Hybridizing under stringent conditions means that under stringent conditions, so-called specific hybrids are formed and non-specific hybrids are not formed.
  • nucleic acids having high homology that is, DNAs having homology of 60% or more, preferably 80% or more hybridize with each other, and if the homology is lower than that, it means that they do not hybridize.
  • the stringent condition means, for example, a condition at a sodium concentration of 15 to 900 mM and a temperature of 37 to 70 ° C, preferably 68 ° C.
  • FGF signaling means a signal cascade that proceeds when FGF binds to a receptor.
  • Involved in FGF signal transduction means the role of constituting the above signal cascade and the function of activating the above signal cascade. Therefore, “genes involved in FGF signal transduction” means genes included in a part of the signal cascade or genes having a function of activating the signal cascade. Whether a gene is involved in FGF signal transduction is determined by the presence or absence of expression of a gene downstream of the signal cascade in a cell or transgenic animal in which the gene has been knocked out, and a part of the signal cascade.
  • the transgenic animal is not particularly limited, and transgenic zebrafish, transgenic mouse and the like can be used.
  • the genes downstream of the signal cascade include the englled genes (Ekker, M.,
  • ERKs a family of protein-serine / threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF.Cell 65, 663-675.
  • the presence or absence of the expression of the gene downstream of the above signal cascade can be confirmed according to a standard method, for example, by Northern hybridization, western plotting, in situ hybridization, RT-PCR, etc. can do.
  • Whether or not to activate the protein that constitutes a part of the above-mentioned signal strength scape can be confirmed according to a standard method.For example, it can be confirmed by a method such as Western blotting or immunohistochemistry. it can.
  • the gene involved in FGF signal transduction it becomes possible to analyze the formation mechanism of the mesencephalic tectum and cerebellum at the midbrain hindbrain boundary.
  • hypoplasia of the midbrain optic tectum and malformation of the midbrain hindbrain boundary were observed. It can be said that it is involved in the formation of the brain tectum and the midbrain hindbrain boundary.
  • transgenic animals that inhibited translation from the transcript of the cnpy3 gene, hypoplasia of the midbrain tectum, malformation of the midbrain hindbrain border, malformation and development of otoliths Since degeneration of the lens is observed in the late stage, it can be said that the cnpy3 gene is involved in the formation of the midbrain tectum, the midbrain hindbrain boundary, and the otic vesicle.
  • the FGF signal is deeply involved in the formation of various organs such as the inner ear, blood vessels, limb buds, teeth, etc., as well as in canceration of cells.
  • the discovery of extracellular factors that can regulate the activity of this signal is expected to open up a wide range of applications as regulators of organ formation and cell growth. Therefore, by using a transgenic animal in which the function of a gene involved in FGF signal transduction according to the present invention is deficient, it is possible to identify a molecule that works in association with the FGF signal at the midbrain-hindbrain boundary. It is possible to identify novel molecules that are linked to the regulation of the function of FGF, which is deeply involved in organ formation and cell growth control.
  • FIGS. 5 and 6 The results of observing the expression of the cnpyh cnpy3 and cnpy4 genes in zebrafish embryos are shown in FIGS. 5 and 6.
  • the first column from the left shows the expression of the cnpyl gene
  • the second column shows the expression of the cnpy3 gene
  • the third column shows the expression of the cnpy4 gene
  • the first row from the top shows the expression at the 8-cell stage.
  • One zebrafish embryo, the second line is a zebrafish embryo in the dome stage (blastula stage), and the third line is a zebrafish embryo in the 30% epiboly stage (early gastrulation stage).
  • the first column from the left shows the expression of the cnpyl gene
  • the second column shows the expression of the cnpy3 gene
  • the third column shows the expression of the cnpy4 gene.
  • the second line is a zebrafish embryo at the tail bud embryo stage
  • the third line is a zebrafish embryo 18 hours after fertilization
  • the fourth line is a 25 zebrafish embryo. A zebrafish embryo in time.
  • the cnpyl gene is weakly expressed at the primary level, and after fertilization.
  • mice ICR
  • Embryos were excised from the mice (ICR) on day 10. 5 of gestation, washed with PBS, and fixed with 4% paraformaldehyde / PBS overnight. 75, 50, 25% ethanol after dehydration with ethanol
  • Rehydration was performed with the / PBSTw series. Treat with 10 mg / ml protein inase K / PBSTw for 5 to 15 minutes, wash twice with 2 mg / ml glycine / PBSTw for 5 minutes, and wash twice with PBSTw for 5 minutes, then 0.2% glutaraldehyde / 4% paraformaldehyde / The cells were fixed with PBSTw for 20 minutes. After washing twice with PBSTw for 5 minutes, treat with 70 for 1 hour, cool completely in ice water, and add 6%
  • NTMT 0.1 M sodium chloride, 0.1 M Tris-HCl, pH 9.5, 50 mM magnesium chloride
  • FIG. 7 shows the results of observation of the mouse embryo at day 10.5 after fertilization.
  • FIG. 7 shows the results of observing the expression of the cnpy2 gene, the cnpy3 gene, and the cnpy4 gene in order from the left.
  • Fig. 7 Expression of cnpy2 gene, cnpy3 gene and cnpy4 gene was observed throughout the mouse embryo, but strong expression was observed particularly in the limb bud and tail.
  • cnpy M0 knockdown embryos embryos in which the cnpyl gene was knocked down by the same method (called cnpy M0 knockdown embryos; knockdown embryos of other cnpy gene families are also referred to), cnpy3_M0 knockdown embryos, cnpy3 / 4 -M0 knockdown embryos and cnpy4-M0 knockdown embryos were prepared and morphological abnormalities were observed.
  • each antisense morpholino oligo is as follows.
  • antisense morpholino oligos were prepared using lx Danieau buf fer (58 mM sodium chloride, 0.7 mM potassium chloride, 0.5 mM magnesium sulfate, 0.6 mM calcium nitrate, 5.0 mM HEPES, pH 7.0. 6). Antisense morpholino oligos were microinjected into zebrafish embryos at the 1-2 cell stage.
  • FIG. 4 is a photograph taken from the side around ⁇ ⁇ B in a down embryo.
  • FIG. 9 is a photograph of oticles (indicated by arrows) in normal zebrafish embryos, cnpy3-M0 knockdown embryos, and cnpy3 / 4-M0 knockdown embryos, from left to right.
  • Example 2 suggested that the cnpy gene family is involved in FGF signal transduction. Therefore, in this example, the relationship between the cnpy gene family and FGF signal was examined.
  • Recombinant mouse FGF-8b (R & D system) was dissolved in PBS, and heparin, insolubi 1 i zed on acrylic beads (Sigma), which had been washed with PBS beforehand, was added at a concentration of 0.5 mg / ml. Incubate at 4 ° C overnight.
  • FGF-soaked beads are embedded in normal embryos or cnpyl-M0 knockdown embryos 15 hours after fertilization, and 1/3 diluted Ringer solution (39 mM sodium chloride, 0.97 mM potassium chloride, 1.8 mM chloride) After incubation in calcium, 1.7 mM Hepes, pH 7.2) for 3 hours, fix with 4% paraformaldehyde Then, the expression of the engrailed gene was examined by in situ hybridization. The results are shown in FIG. As can be seen from FIG. 10, in the cnpyl-MO knockdown embryo, the degree of ectopic expression of the engrailed gene was significantly reduced when FGF8b beads were embedded. In normal zebrafish embryos (normal embryos), it is found that ectopic expression of the engrailed gene is increased by embedding FGF8b beads. These results revealed that the cnpyl gene is essential for FGF signal transmission.
  • FGF signals are transmitted by FGF binding to the receptor and activating a signal cascade downstream of the receptor.
  • Activation of ERK is an indicator of FGF signal (Gotoli, Y. and Nishida, E., 1996.Signals for mesoderm induction. Roles of fibroblast growth factor (FGF) / mitogen-activated protein kinase (MAPK) pathway. Biochim Biopys Acta 1288, F 7.). Therefore, activated ERK was detected by Western plot in cnpyl-MO knockdown embryos and cnpy3 / 4-M0 knockdown embryos. Normal zebrafish embryos (normal embryos) and fgf3-M0 knockdown embryos were used as controls.
  • the sequence of igi3-M0 is CATTGTGGCATGGCGGGATGTCGGC (SEQ ID NO: 26). After fertilization
  • a novel gene involved in FGF signaling can be provided.
  • the formation mechanism of the midbrain tectum and cerebellum by the midbrain hindbrain boundary and the formation mechanism of various organs such as the inner ear, blood vessels, limb buds, and teeth And analysis of cell carcinogenesis mechanism.
  • SEQ ID NOS: 23 to 26 are synthetic DNAs.

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Abstract

It is intended to provide novel genes participating in FGF signal transduction by which the mechanisms of forming mesencephalic tectum in the mesencephalon/metenchephalon border, cerebellum, etc. can be analyzed. (1) A gene participating in FGF signal transduction and encoding the following protein (a) or (b). (a) A protein containing one of the amino acid sequences represented by SEQ ID NOS:2, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20 and 22. (b) A protein comprising an amino acid sequence derived from one of the amino acids represented by SEQ ID NOS:2, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20 and 22 by substitution, deletion or addition of one or more amino acids and having a function of participating in FGF signal transduction.

Description

明 細 書  Specification
FGFシグナル伝達に関与する遺伝子 技術分野 Genes involved in FGF signaling
本発明は、 FGFシグナル伝達に関与する新規な遺伝子に関する。 背景技術  The present invention relates to a novel gene involved in FGF signaling. Background art
中枢神経系のうち中脳視蓋部と小脳は中脳後脳境界部の持つオーガナイザー活 性によって誘導的に形成されると考えられている。 中脳後脳境界部の形成にはい くつかのシグナル分子 (WntK FGF8)や転写因子 (Islet3、 0tx2、 Gbx2、 Pax2/5/8, Engl/2/3) が関与していることが報告されている （非特許文献 1〜1 1参照）。  The central nervous system and the cerebellum in the central nervous system are thought to be formed inductively by the organizer activity of the midbrain hindbrain boundary. It has been reported that several signal molecules (WntK FGF8) and transcription factors (Islet3, 0tx2, Gbx2, Pax2 / 5/8, Engl / 2/3) are involved in the formation of the midbrain-hindbrain boundary. (See Non-Patent Documents 1 to 11).
しかしながら、 これらの既知遺伝子の働きだけでは中脳後脳境界部による中脳 視蓋部及び小脳の形成を説明することはできない。  However, the function of these known genes alone cannot explain the formation of the midbrain tectum and cerebellum by the midbrain hindbrain boundary.
非特許文献 1  Non-patent document 1
McMahon, A. P. and Bradley, A. , 1990. The Wnt-1 (int-1) pro to - oncogene is required for development of a large region of the mouse brain. Cell 62, 1073-1085.  McMahon, A. P. and Bradley, A., 1990.The Wnt-1 (int-1) pro to-oncogene is required for development of a large region of the mouse brain.Cell 62, 1073-1085.
非特許文献 2  Non-patent document 2
McMahon, A. P. , Joyner, A. L. , Bradley, A. and McMahon, LA., 1992. The midbrain-hindbrain phenotype of nt-1-/Wnt-1- mice results from stepwise deletion of engrailed - expressing cells by 9.5 days postcoitum. Cell 69, 581-595.  McMahon, AP, Joyner, AL, Bradley, A. and McMahon, LA., 1992.The midbrain-hindbrain phenotype of nt-1- / Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69, 581-595.
非特許文献 3  Non-patent document 3
Crossley, P.H. , Martinez, S. and Martin, G. I. , 1996. Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66-68.  Crossley, P.H., Martinez, S. and Martin, G.I., 1996.Midbrain development induced by FGF8 in the chick embryo.Nature 380, 66-68.
非特許文献 4  Non-patent document 4
Kikuchi, Y. , Segawa, Η. , Tolomoto, Μ. , Tsubokawa, Τ. , Hotta, Υ. , Kikuchi, Y., Segawa, Η., Tolomoto, Μ., Tsubokawa, Τ., Hotta, Υ.,
Uyemura, Κ. and Okamoto, Η. , 1997. Ocular and cerebellar defects in zebraf ish induced by overexpression of the LIM domains of the islet - 3 LIM/homeodomain protein. Neuron 18, 369-382. Uyemura, Κ. And Okamoto, Η., 1997.Ocular and cerebellar defects in zebraf ish induced by overexpression of the LIM domains of the islet-3 LIM / homeodomain protein.Neuron 18, 369-382.
非特許文献 5  Non-patent document 5
Broccoli, V. , Boncinelli, E. and furst, 1 , 1999. The caudal limit of Otx2 expression positions the isthmic organizer. Nature 401, 164-168. Millet, S.， Campbell, K. , Epstein, D. J. , Losos, K. , Harris, E. and Joyner, A. L. , 1999. A role for Gbx2 in repression of Otx2 and positioning the mid/hindbrain organizer. Nature 401, 161-164.  Broccoli, V., Boncinelli, E. and furst, 1, 1999.The caudal limit of Otx2 expression positions the isthmic organizer.Nature 401, 164-168. Millet, S., Campbell, K., Epstein, DJ, Losos, K., Harris, E. and Joyner, AL, 1999.A role for Gbx2 in repression of Otx2 and positioning the mid / hindbrain organizer.Nature 401, 161-164.
非特許文献 6  Non-patent document 6
Pfef fer, P. L. , Gerster, T. , Lun, K. , Brand, M. and Busslinger, M. , 1998. Characterization of three novel members of the zebraf ish Pax2/5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function. Development 125, 3063-3074.  Pfef fer, PL, Gerster, T., Lun, K., Brand, M. and Busslinger, M., 1998.Characterization of three novel members of the zebraf ish Pax2 / 5/8 family: dependency of Pax5 and Pax8 expression on the Pax2.1 (noi) function.Development 125, 3063-3074.
非特許文献 7  Non-patent document 7
Lun, K. and Brand, M. , 1998. A series of no isthmus (noi) alleles of the zebraf ish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary. Development 125, 3049-3062.  Lun, K. and Brand, M., 1998.A series of no isthmus (noi) alleles of the zebraf ish pax2.1 gene reveals multiple signaling events in development of the midbrain-hindbrain boundary.Development 125, 3049-3062.
非特許文献 8  Non-patent document 8
Fjose, A.， Njolstad, P. R. , Nornes, S.， Molven, A. and Krauss, S.， 1992. Structure and early embryonic expression of the zebraf ish engrailed 2 gene. Mec . Dev. 39, 51-62.  Fjose, A., Njolstad, P.R., Nornes, S., Molven, A. and Krauss, S., 1992. Structure and early embryonic expression of the zebraf ish engrailed 2 gene.Mec. Dev. 39, 51-62.
非特許文献 9  Non-patent document 9
Ekker, M. , Wegner, J. , Akimenko, M. A. and Westerf ield, M.， 1992. Coodinate embryonic expression oi three zebraf ish engrailed genes. Development 116, 1001-1010.  Ekker, M., Wegner, J., Akimenko, M.A. and Westerfield, M., 1992.Coodinate embryonic expression oi three zebraf ish engrailed genes.Development 116, 1001-1010.
非特許文献 10  Non-patent document 10
Wurst, W. , Auerbach, A. B. and Joyner, A. L. , 1994. Multiple developmental defects in Engrailed - 1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development 120, 2065-2075. Wurst, W., Auerbach, AB and Joyner, AL, 1994.Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.Development 120, 2065-2075.
非特許文献 1 1  Non-patent document 1 1
Joyner, A丄， Skarnes, W. C. and Rossant, J. , 1989. Production of a mutation in mouse En- 2 gene by homologous recombination in embryonic stem cells. Nature 338, 153-156.  Joyner, A 丄, Skarnes, W.C. and Rossant, J., 1989.Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells.Nature 338, 153-156.
そこで、 本発明は、 中脳後脳境界部による中脳視蓋部及び小脳の形成メカニズ ム等の解析を可能とする、 FGF シグナル伝達に関与する新規な遺伝子を提供する ことを目的とする。 発明の開示  Therefore, an object of the present invention is to provide a novel gene involved in FGF signal transduction, which enables analysis of the formation mechanism of the midbrain tectum and cerebellum at the midbrain hindbrain boundary. Disclosure of the invention
上述した目的を達成するために、 本発明者らが鋭意検討し、 FGF シグナル伝達 に関与する新規な遺伝子ファミリーを同定することに成功し、 本発明を完成する に至った。  In order to achieve the above-mentioned object, the present inventors have conducted intensive studies, succeeded in identifying a novel gene family involved in FGF signaling, and completed the present invention.
本発明は以下を包含する。  The present invention includes the following.
(1) 以下の （a) 又は （b) のタンパク質をコードする、 FGF シグナル伝 達に関与する遺伝子。  (1) A gene involved in FGF signal transmission that encodes the following protein (a) or (b):
( a ) 配列番号 2、 3、 5、 7、 9、 1 1、 1 3、 1 5、 1 6、 1 8、 2 0及び 2 2に表すアミノ酸配列のうちいずれか 1のアミノ酸配列を含むタンパク質 (a) a protein comprising the amino acid sequence of any one of the amino acid sequences represented by SEQ ID NOs: 2, 3, 5, 7, 9, 11, 11, 13, 15, 16, 18, 20, and 22;
( b ) 配列番号 2、 3、 5、 7、 9、 1 1、 1 3、 1 5、 1 6、 1 8、 2 0及び 2 2に表すアミノ酸配列のうちいずれか 1のアミノ酸配列において、 1又は複数 個のアミノ酸が置換、 欠失又は付加されたアミノ酸配列からなり、 FGF シグナル 伝達に関与する機能を有するタンパク質。 (b) In the amino acid sequence of any one of the amino acid sequences represented by SEQ ID NOs: 2, 3, 5, 7, 9, 11, 11, 13, 15, 16, 18, 20, and 22, 1 Or a protein comprising an amino acid sequence in which a plurality of amino acids have been substituted, deleted or added, and having a function involved in FGF signal transduction.
(2) 以下の （a) 又は （b) のポリヌクレオチドからなる FGFシグナル伝 達に関与する遺伝子。  (2) A gene involved in FGF signal transmission, comprising the polynucleotide of the following (a) or (b):
(a) 配列番号 1、 4、 6、 8、 1 0、 1 2、 1 4、 1 7、 1 9及び 2 1に表す 塩基配列のうちいずれか 1の塩基配列を含むポリヌクレオチド  (a) a polynucleotide comprising the nucleotide sequence of any one of the nucleotide sequences represented by SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 14, 17, 19, and 21
( b ) 配列番号 1、 4、 6、 8、 1 0、 1 2、 1 4、 1 7、 1 9及び 2 1に表す 塩基配列のうちいずれか 1の塩基配列に相補的な塩基配列に対して、 ストリンジ ェントな条件下でハイブリダィズする塩基配列を含み、 FGF シグナル伝達に関与 する機能を有するタンパク質をコードするポリヌクレオチド (b) a base sequence complementary to any one of the base sequences represented by SEQ ID NOs: 1, 4, 6, 8, 10, 12, 12, 14, 17, 19 and 21; Contains nucleotide sequences that hybridize under stringent conditions and is involved in FGF signaling. Encoding a protein having a function to perform
( 3 ) 上記 （1 ) 又は （2 ) 記載の FGFシグナル伝達に関与する遺伝子を有 するベクター。  (3) A vector having the gene involved in FGF signal transduction according to the above (1) or (2).
( 4 ) 上記 （1 ) 又は （2 ) 記載の FGFシグナル伝達に関与する遺伝子の機 能を欠損させた遺伝子導入細胞。  (4) A transgenic cell in which the function of the gene involved in FGF signal transduction according to (1) or (2) is deficient.
( 5 ) 上記 （1 ) 又は （2 ) 記載の FGFシグナル伝達に関与する遺伝子の機 能を欠損させたトランスジエニック動物。  (5) A transgenic animal lacking the function of the gene involved in FGF signal transduction according to the above (1) or (2).
( 6 ) 上記 （4 ) 記載の遺伝子導入細胞又は上記 （5 ) 記載のトランスジ： E: ニック動物に対してリード化合物を作用させる工程と、  (6) a step of allowing a lead compound to act on the transgenic cell according to (4) or the transgene according to (5): E: a nick animal;
中脳 ·後脳境界部において FGFシグナルと関連して働くリード化合物を同定す る工程とを含むスクリーニング方法。  Identifying a lead compound that works in association with the FGF signal at the midbrain / hindbrain boundary.
本明細書は本願の優先権の基礎である日本国特許出願 2003-161005号の明細書 および/または図面に記載される内容を包含する。 図面の簡単な説明  This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2003-161005, which is a priority document of the present application. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 ヒト cnpylとゼブラフィッシュ cnpylとの相同性を検討した結果を示 す図である。  FIG. 1 shows the results of examining the homology between human cnpyl and zebrafish cnpyl.
図 2は、 ヒト cnpy2とゼブラフィッシュ cnpy2との相同性を検討した結果を示 す図である。  FIG. 2 is a diagram showing the results of examining homology between human cnpy2 and zebrafish cnpy2.
図 3は、 ヒト cnpy3とゼブラフィッシュ cnpy3との相同性を検討した結果を示 す図である。  FIG. 3 is a diagram showing the results of examining the homology between human cnpy3 and zebrafish cnpy3.
図 4は、 ヒト ciipy4とゼブラフィッシュ cnpy4との相同性を検討した結果を示 す図である。 · 図 5は、 cnpyl、 cnpyS及ぴ cnpy4遺伝子のゼブラフィッシュ胚における発現を 観察した顕微鏡写真である。  FIG. 4 is a diagram showing the result of examining the homology between human ciipy4 and zebrafish cnpy4. · Figure 5 is a micrograph showing the observation of the expression of the cnpyl, cnpyS and cnpy4 genes in zebrafish embryos.
図 6は、 cnpyl、 cnpy3及び cnpy4遺伝子のゼブラフィッシュ胚における発現を 観察した顕微鏡写真である。  FIG. 6 is a micrograph showing the expression of the cnpyl, cnpy3, and cnpy4 genes in zebrafish embryos.
図 7は、 cnpy2遺伝子、 cnpy3遺伝子及び cnpy4遺伝子のマウス胚における発現 を観察した顕微鏡写真である。 図 8は、 正常胚及びノックダウン胚における丽 B周辺の形態異常を観察した顕 微鏡写真である。 FIG. 7 is a micrograph showing the expression of the cnpy2, cnpy3, and cnpy4 genes in mouse embryos. FIG. 8 is a micrograph of a normal embryo and a knockdown embryo, which observed morphological abnormalities around 丽 B.
図 9は、 正常胚及びノックダウン胚における耳胞の形態異常を観察した顕微鏡 写真である。  FIG. 9 is a micrograph of the observed morphological abnormalities of the otocyst in the normal embryo and the knockdown embryo.
図 1 0は、 正常なゼプラフィッシュ胚及び cnpyl- M0 ノックダウン胚に FGF8b ビーズを埋め込み、 engrailed遺伝子の発現誘導を観察した顕微鏡写真である。 図 1 1は、 npyl- M0ノックダウン胚及び cnpy3/4 - M0ノックダウン胚において活 性化 ERKをウエスタンブロットによって検出した電気泳動写真である。  FIG. 10 is a photomicrograph showing that normal zephrafish embryos and cnpyl-M0 knockdown embryos were embedded with FGF8b beads, and the induction of engrailed gene expression was observed. FIG. 11 is an electrophoresis photograph in which activated ERK was detected by Western blot in npyl-M0 knockdown embryos and cnpy3 / 4-M0 knockdown embryos.
図 1 2は、 免疫組織化学法によって、 ゼブラフィッシュ胚のどの部分で活性化 ERKが消失したのかを検出した顕微鏡写真である。 発明を実施するための形態  Figure 12 is a photomicrograph of the immunohistochemical method used to detect which part of the zebrafish embryo had lost activated ERK. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明者は、先ず、 モデル動物としてゼブラフィッシュ （Daniorerio) Islet - 3 (Kikuchi, Y. , Segawa, Η. , Tokumoto, . , Tsubokawa, Τ. , Hott , Υ. , Uyemura, Κ. and Okamoto, Η. , 1997. Ocular and cerebellar defects in zebrafis induced by overexpression of the LIM domains of the islet 3 LIM/homeodomain protein. Neuron 18, 369-382.) によって発現制御を受ける新規遺伝子群の探索を行った。 ここで、 Islet- 3は、 中脳 ·後脳境界部 （以下、 腿 Bと呼ぶ） の発生を促進する中 脳からのシグナルの発現を制御する LIM/ホメォドメイン型の転写因子である。 そして、 本発明者は、 Islet- 3による発現制御を受け、 且つ FGFシグナル伝達 に関与する新規な遺伝子ファミリ一を同定した。この新規な遺伝子ファミリ一を、 canopy 遺伝子フアミリーと呼ぶこととし、 当該ファミリーに属する各遺伝子を rcnpyj なる文言を付して命名する。  First, the present inventors have proposed that zebrafish (Daniorerio) Islet-3 (Kikuchi, Y., Segawa, Η., Tokumoto,., Tsubokawa, Τ., Hott, Υ., Uyemura, Κ. And Okamoto, Η., 1997. A new gene group whose expression was controlled by Ocular and cerebellar defects in zebrafis induced by overexpression of the LIM domains of the islet 3 LIM / homeodomain protein. Neuron 18, 369-382.) Was searched. Here, Islet-3 is a LIM / homeodomain transcription factor that controls the expression of a signal from the midbrain that promotes the development of the midbrain-hindbrain boundary (hereinafter referred to as thigh B). Then, the present inventors have identified a novel gene family that is under expression control by Islet-3 and is involved in FGF signal transduction. This new gene family will be referred to as the canopy gene family, and each gene belonging to the family will be named with the word rcnpyj.
ゼブラフィッシュ由来の cnpy遺伝子ファミリ一は、 cnpyl〜4遺伝子の 4種類 存在している。 cnpyl 遺伝子のコード領域を含む塩基配列を配列番号 1に示し、 cnpyl遺伝子がコードする cnpylのアミノ酸配列を配列番号 2に示す。 cnpy2遺伝 子がコードする cnpy2の部分アミノ酸配列を配列番号 3に示す。 cnpy3遺伝子の コード領域を含む塩基配列を配列番号 4に示し、 cnpy3遺伝子がコードする cnpy3 のアミノ酸配列を配列番号 5に示す。 cnpy4 遺伝子のコード領域を含む塩基配列 を配列番号 6に示し、 cnpy4遺伝子がコードする cnpy4のアミノ酸配列を配列番 号 7に示す。 The cnpy gene family derived from zebrafish has four types of cnpyl to 4 genes. The nucleotide sequence containing the coding region of the cnpyl gene is shown in SEQ ID NO: 1, and the amino acid sequence of cnpyl encoded by the cnpyl gene is shown in SEQ ID NO: 2. SEQ ID NO: 3 shows the partial amino acid sequence of cnpy2 encoded by the cnpy2 gene. The nucleotide sequence containing the coding region of the cnpy3 gene is shown in SEQ ID NO: 4, and the cnpy3 gene encodes Is shown in SEQ ID NO: 5. The nucleotide sequence including the coding region of the cnpy4 gene is shown in SEQ ID NO: 6, and the amino acid sequence of cnpy4 encoded by the cnpy4 gene is shown in SEQ ID NO: 7.
また、 cnpylのアミノ酸配列 （配列番号 2 ) に基づいて、 Na t i onal Cen ter fo r B iotechnology Informat ion (NCBI)で公開されているヒトゲノムデータベースに 対して相同性検索を行うことによって、ヒト由来の cnpy遺伝子ファミリーを同定 した。 ヒト由来の cnpy遺伝子ファミリ一は、 ヒ卜 cnpy 1〜4遺伝子の 4種類存在 している。 ヒト cnpyl遺伝子のコード領域を含む塩基配列を配列番号 8に示し、 ヒ卜 cnpyl遺伝子がコ一ドするヒト cnpylのアミノ酸配列を配列番号 9に示す。 ヒト ciipy2遺伝子のコード領域を含む塩基配列を配列番号 1 0に示し、ヒト cnpy2 遺伝子がコードするヒト cnpy2のアミノ酸配列を配列番号 1 1に示す。ヒ卜 cnpy 3 遺伝子のコード領域を含む塩基配列を配列番号 1 2に示し、 ヒト cnpy3遺伝子が コードするヒト cnpy3のアミノ酸配列を配列番号 1 3に示す。 ヒト cnpy4遺伝子 のコード領域を含む塩基配列を配列番号 1 4に示し、 ヒト cnpy4遺伝子がコード するヒ卜 cnpy4のアミノ酸配列を配列番号 1 5に示す。  In addition, based on the amino acid sequence of cnpyl (SEQ ID NO: 2), a homology search was performed on the human genome database published by the National Center for Biotechnology Information (NCBI) to obtain human-derived amino acids. The cnpy gene family has been identified. There are four human cnpy gene families, including human cnpy 1-4 genes. The nucleotide sequence containing the coding region of the human cnpyl gene is shown in SEQ ID NO: 8, and the amino acid sequence of human cnpyl encoded by the human cnpyl gene is shown in SEQ ID NO: 9. The nucleotide sequence containing the coding region of the human ciipy2 gene is shown in SEQ ID NO: 10, and the amino acid sequence of human cnpy2 encoded by the human cnpy2 gene is shown in SEQ ID NO: 11. The nucleotide sequence containing the coding region of the human cnpy3 gene is shown in SEQ ID NO: 12, and the amino acid sequence of human cnpy3 encoded by the human cnpy3 gene is shown in SEQ ID NO: 13. The nucleotide sequence containing the coding region of the human cnpy4 gene is shown in SEQ ID NO: 14, and the amino acid sequence of human cnpy4 encoded by the human cnpy4 gene is shown in SEQ ID NO: 15.
ゼブラフィッシュ由来の cnpylとヒト由来の cnpylとについて相同性を測定し た結果を図 1に示す。 このァライメントは、 Geiietyx- Mac, verlO (ゼネテイクス） の Lipman-Pearson法 （デフォルトパラメータ設定） で作製した。 同様に、 ゼブ ラフィッシュ由来の ciipy2 (部分アミノ酸配列） とヒト由来の cnpy2 とについて 相同性を測定した結果を図 2に示す。 ゼブラフィッシュ由来の cnpy3とヒ卜由来 の cnpy3とについて相同性を測定した結果を図 3に示す。 ゼブラフィッシュ由来 の cnpy4とヒト由来の cnpy4とについて相同性を測定した結果を図 4に示す。 図 1乃至 4に示すように、ゼブラフィッシュ由来の cnpy遺伝子フアミリーとヒ ト由来の cnpy遺伝子ファミリーとは、高い相同性を示すため、両ファミリーに属 する遺伝子は同機能を有することが考えられる。  Fig. 1 shows the results of measuring the homology between cnpyl derived from zebrafish and cnpyl derived from human. This alignment was prepared by the Lipman-Pearson method (default parameter setting) of Geiietyx-Mac, verlO (Genetics). Similarly, FIG. 2 shows the results of measuring the homology of ciipy2 (partial amino acid sequence) derived from zebrafish and cnpy2 derived from human. Fig. 3 shows the results of measuring the homology between cnpy3 derived from zebrafish and cnpy3 derived from human. FIG. 4 shows the results of measuring the homology between cnpy4 derived from zebrafish and cnpy4 derived from human. As shown in FIGS. 1 to 4, the cnpy gene family derived from zebrafish and the cnpy gene family derived from human show high homology, and it is considered that genes belonging to both families have the same function.
また、 cnpylのアミノ酸配列 （配列番号 2 ) に基づいて、 NCBIで公開されてい るマウスゲノムデータべ一スに対して相同性検索を行うことによって、 マウス由 来の cnpy遺伝子ファミリーを同定することもできる。 また、 ゼブラフィッシュ、 ヒト、 マウスに限定されず、 例えば、 ゥシ、 ブ夕、 ラット、 ニヮトリ、 力エル、 ホヤ、ゥニ、ショウジヨウバエ、線虫等のデータベースを検索することによって、 各種後生動物における cnpy遺伝子ファミリーを同定することができる。 It is also possible to identify the mouse-derived cnpy gene family by performing a homology search on the mouse genome database published at NCBI based on the amino acid sequence of cnpyl (SEQ ID NO: 2). it can. Also, it is not limited to zebrafish, human, and mouse, but includes, for example, By searching databases such as ascidians, sea urchins, Drosophila and nematodes, the cnpy gene family in various metazoans can be identified.
マウス由来のマウス cnpyl遺伝子がコードするマウス cnpylの部分アミノ酸配 列を配列番号 1 6に示す。 マウス cnpy2遺伝子のコード領域を含む塩基配列を配 列番号 1 7に示し、 マウス cnpy2遺伝子がコードするマウス cnpy2のアミノ酸配 列を配列番号 1 8に示す。 マウス cnpy3遺伝子のコード領域を含む塩基配列を配 列番号 1 9に示し、 マウス cnpy3遺伝子がコードするマウス cnpy3のアミノ酸配 列を配列番号 2 0に示す。 マウス cnpy4遺伝子のコード領域を含む塩基配列を配 列番号 2 1に示し、 マウス cnpy2遺伝子がコードするマウス cnpy4のアミノ酸配 列を配列番号 2 2に示す。  SEQ ID NO: 16 shows the partial amino acid sequence of mouse cnpyl encoded by the mouse-derived mouse cnpyl gene. The nucleotide sequence containing the coding region of mouse cnpy2 gene is shown in SEQ ID NO: 17, and the amino acid sequence of mouse cnpy2 encoded by mouse cnpy2 gene is shown in SEQ ID NO: 18. The nucleotide sequence containing the coding region of mouse cnpy3 gene is shown in SEQ ID NO: 19, and the amino acid sequence of mouse cnpy3 encoded by mouse cnpy3 gene is shown in SEQ ID NO: 20. The nucleotide sequence containing the coding region of mouse cnpy4 gene is shown in SEQ ID NO: 21, and the amino acid sequence of mouse cnpy4 encoded by mouse cnpy2 gene is shown in SEQ ID NO: 22.
本発明に係る FGFシグナル伝達に関与する遺伝子は、これら全ての cnpy遺伝子 ファミリーに含まれる cnpy遺伝子を意味する。  The gene involved in FGF signaling according to the present invention means a cnpy gene included in all of these cnpy gene families.
また、 本発明に係る FGFシグナル伝達に関与する遺伝子は、 2、 3、 5、 7、 The genes involved in FGF signaling according to the present invention include 2, 3, 5, 7,
9、 1 1、 1 3 ,、 1 5、 1 6、 1 8、 2 0及び 2 2に表すアミノ酸配列のうちい ずれかのアミノ酸配列において、 1又は複数個のアミノ酸が置換、 欠失又は付加 されたアミノ酸配列からなり、 FGF シグナル伝達に関与する機能を有するタンパ ク質（以下、「変異型タンパク質」と総称する）をコードする遺伝子を含んでいる。 すなわち、 本発明に係る FGFシグナル伝達に関与する遺伝子は、 変異型タンパク 質をコードする遺伝子を含んでいる。 One or more amino acids are substituted, deleted, or added in any of the amino acid sequences shown in 9, 11, 13, 13, 15, 16, 18, 20, and 22. And a gene encoding a protein having a function related to FGF signal transduction (hereinafter referred to as “mutant protein”). That is, the gene involved in FGF signal transduction according to the present invention includes a gene encoding a mutant protein.
変異型タンパク質をコードする遺伝子は、 例えば、 配列番号 1、 4、 6、 8、 Genes encoding mutant proteins include, for example, SEQ ID NOs: 1, 4, 6, 8,
1 0、 1 2、 1 4、 1 7、 1 9及び 2 1に示す塩基配列における 1以上の塩基を 欠失、 置換又は付加することによって作製することができる。 1以上の塩基を欠 失、 置換又は付加する際には、 従来公知の手法を特に限定することなく、 適宜使 用することができる。 例えば、 部位特異的突然変異誘発方法を使用して、 所定の 塩基を置換することができる。 部位特異的突然変異誘発方法としては、 例えば T. クンケル（Kunkel)の部位特異的変異導入法（Kunkel, T. A. Proc. Nat i. Acad.It can be prepared by deleting, substituting or adding one or more bases in the base sequences shown in 10, 12, 14, 17, 19 and 21. When one or more bases are deleted, substituted or added, a conventionally known method can be appropriately used without any particular limitation. For example, site-directed mutagenesis methods can be used to replace certain bases. As a site-directed mutagenesis method, for example, a site-directed mutagenesis method of T. Kunkel (Kunkel, TA Proc. Nati. Acad.
Sc i. U. S. A. 82, 488-492 (1985) )、 Gapped dupl ex法等が挙げられる。 ま た、 通常のクンケル法で使用する 1〜2 本の改変用オリゴヌクレオチドを、 最高Sc. U. S. A. 82, 488-492 (1985)), and the Gapped duplex method. In addition, one or two modification oligonucleotides used in the usual Kunkel method are
16 本のオリゴヌクレオチドを同時に用いて多部位での効率よい置換を行う改良 法なども採用することができる。 本発明では、 部位特異的突然変異誘発法を利用 した変異導入用キット （例えば Mu t an- K (宝酒造社製）や Mu t an- G (宝酒造社製））な どを用いて、 あるいは、 宝酒造社製の LA PCR in vi t ro Mut agenes i s シリーズキ ットを用いて変異を導入することもできる。 Improvement of efficient multi-site substitution using 16 oligonucleotides simultaneously A law can also be adopted. In the present invention, a mutagenesis kit using site-directed mutagenesis (for example, Mutan-K (Takara Shuzo) or Mutan-G (Takara Shuzo)) or the like, Mutations can also be introduced using the Takara Shuzo LA PCR in vitro Mutagenes is series kit.
さらに、本発明に係る FGFシグナル伝達に関与する遺伝子は、配列番号 1、 4、 6、 8、 1 0、 1 2、 1 4、 1 7、 1 9及び 2 1に表すいずれかの塩基配列の相 補的な塩基配列に対して、 ストリンジェントな条件下でハイブリダィズする塩基 配列を含み、 FGF シグナル伝達に関与する機能を有するタンパク質をコードする ポリヌクレオチドを含んでいる。 ストリンジェントな条件下でハイブリダイズす るとは、 ストリンジェントな条件下において、 いわゆる特異的なハイブリッドが 形成され、 非特異的なハイブリッドが形成されないことを意味する。 例えば、 相 同性が高い核酸同士、すなわち 60%以上、好ましくは 80%以上の相同性を有する DNA 同士がハイプリダイズし、 それより相同性が低い場合はハイブリダィズしな いことを意味する。 ストリンジェン卜な条件とは、 例えば、 ナトリウム濃度が 15 〜900mMであり、 温度が 37〜70°C、 好ましくは 68°Cでの条件を意味する。  Furthermore, the gene involved in the FGF signaling according to the present invention is any one of the nucleotide sequences represented by SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 14, 17, 19 and 21. It contains a nucleotide sequence that hybridizes under stringent conditions to a complementary nucleotide sequence, and contains a polynucleotide encoding a protein having a function involved in FGF signaling. Hybridizing under stringent conditions means that under stringent conditions, so-called specific hybrids are formed and non-specific hybrids are not formed. For example, nucleic acids having high homology, that is, DNAs having homology of 60% or more, preferably 80% or more hybridize with each other, and if the homology is lower than that, it means that they do not hybridize. The stringent condition means, for example, a condition at a sodium concentration of 15 to 900 mM and a temperature of 37 to 70 ° C, preferably 68 ° C.
ここで、 「FGFシグナル伝達」 とは、 FGFが受容体に結合することにより進行す るシグナルカスケードを意味する。 「FGFシグナル伝達に関与する」 とは、 上記シ グナルカスケ一ドを構成する役割及び上記シグナルカスケードを活性化する機能 を意味する。 従って、 「FGFシグナル伝達に関与する遺伝子」 とは、 当該シグナル カスケードの一部に含まれる遺伝子や、 当該シグナルカスケ一ドを活性化する機 能を有する遺伝子を意味する。 ある遺伝子が FGFシグナル伝達に関与するか否か は、 当該遺伝子をノックアウトした細胞又はトランスジエニック動物において、 上記シグナルカスケ一ドの下流にある遺伝子の発現の有無や、 上記シグナルカス ケードの一部を構成するタンパク質を活性化するか否かにより確認できる。 トラ ンスジエニック動物としては、 特に限定されないが、 トランスジエニック ·ゼプ ラフィッシュやトランスジエニック ·マウス等を用いることができる。 上記シグ ナルカスケードの下流にある遺伝子としては、 engrai l ed 遺伝子 （Ekker, M. , Here, “FGF signaling” means a signal cascade that proceeds when FGF binds to a receptor. “Involved in FGF signal transduction” means the role of constituting the above signal cascade and the function of activating the above signal cascade. Therefore, “genes involved in FGF signal transduction” means genes included in a part of the signal cascade or genes having a function of activating the signal cascade. Whether a gene is involved in FGF signal transduction is determined by the presence or absence of expression of a gene downstream of the signal cascade in a cell or transgenic animal in which the gene has been knocked out, and a part of the signal cascade. Can be confirmed by activating or not activating the protein constituting The transgenic animal is not particularly limited, and transgenic zebrafish, transgenic mouse and the like can be used. The genes downstream of the signal cascade include the englled genes (Ekker, M.,
Wegner, J. , Akimenko, Μ. A. and Wes terf ie ld, M. , 1992. Coodinate embryonic express ion of three zebraf ish engrai led genes. Deve lopment 116, 1001-1010. )、 sei遺伝子 (Furt auer, M. , Lin, W.， Ang, S. L.， T isse, B. and T isse, C. , 2002. Sef is a feedback-induced antagonist of as/MAPK-inediated FGF signalling. Nat Cell Biol 4, 170-4.； Tsang, M. , Friesel, R. , Kudoh, T. and Dawid, I.B.， 2002. Identification of Sef, a novel modulator of FGF signalling, Nat Cell Biol 4, 165- 9. )、 sprouty2遺伝子（Chambers, D. , Medhurst, A. D. , Walsh, F. S. , Price, J. and Mason, I., 2000. Differential display of genes expressed at the midbrain - hindbrain junction identifies sprouty2： an FGF8-inducibl e member of a family of intracellular FGF ant gonists. Mol. Cell. Neurosci. 15, 22-35.) を挙げることができる。 また、 シグナルカスケードの一部を構成し て活性化されるタンパク質としては、 E K (Boulton, T. G. ' Nye, S. H. , Robbins, D. J. , Ip, N. Y. , Radziejewska, E.， Morgenbesser, S. D. , DePinho, A., Panayotatos, Ν·， Cobb, M. H. and Yancopoulos, G. D.， 1991. ERKs： a family of protein - serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675. ) を挙げ ることができる。 Wegner, J., Akimenko, Μ. A. and Wes terf ie ld, M., 1992.Coodinate embryonic express ion of three zebraf ish engrai led genes.Deve lopment 116, 1001-1010.), sei gene (Furt auer, M., Lin, W., Ang, SL, T isse, B. and T isse, C., 2002. Sef is a feedback-induced antagonist of as / MAPK-inediated FGF signaling. Nat Cell Biol 4, 170-4 .; Tsang, M., Friesel, R., Kudoh, T. and Dawid, IB, 2002. Identification of Sef, a novel modulator of FGF signaling, Nat Cell Biol 4, 165-9.) Sprouty2 gene (Chambers, D., Medhurst, AD, Walsh, FS, Price, J. and Mason, I., 2000. Differential display of genes expressed at the midbrain-hindbrain junction identifies sprouty2: an FGF8-inducibl e member Neurosci. 15, 22-35.) a family of intracellular FGF ant gonists. EK (Boulton, TG 'Nye, SH, Robbins, DJ, Ip, NY, Radziejewska, E., Morgenbesser, SD, DePinho, A. et al. , Panayotatos, Ν, Cobb, MH and Yancopoulos, GD, 1991. ERKs: a family of protein-serine / threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF.Cell 65, 663-675.) Can be
上記シグナルカスケードの下流にある遺伝子の発現の有無は、 定法に従って確 認することができ、 例えば、 ノーザンハイプリダイゼイシヨン、 ウェスタンプロ ッティング、 insitu ハイブリダィゼイシヨン、 RT- PCR等の手法で確認すること ができる。  The presence or absence of the expression of the gene downstream of the above signal cascade can be confirmed according to a standard method, for example, by Northern hybridization, western plotting, in situ hybridization, RT-PCR, etc. can do.
また、 上記シグナル力スケードの一部を構成する夕ンパク質を活性化するか否 かは、 定法に従って確認することができ、 例えば、 ウェスタンブロッテイング、 免疫組織化学法等の手法で確認することができる。  Whether or not to activate the protein that constitutes a part of the above-mentioned signal strength scape can be confirmed according to a standard method.For example, it can be confirmed by a method such as Western blotting or immunohistochemistry. it can.
本発明に係る FGFシグナル伝達に関与する遺伝子によれば、 中脳後脳境界部に よる中脳視蓋部及び小脳の形成メカニズム等の解析が可能となる。 特に、 cnpyl 遺伝子の転写産物からの翻訳を阻害したトランスジエニック動物においては、 中 脳視蓋部の低形成及び中脳後脳境界部の形成異常が観察されることから、 cnpyl 遺伝子は、中脳視蓋部及び中脳後脳境界部の形成に関与しているといえる。また、 cnpy3 遺伝子の転写産物からの翻訳を阻害したトランスジエニック動物において は、 中脳視蓋部の低形成、 中脳後脳境界部の形成異常、 耳石の形成異常及び発生 後期におけるレンズの変性が観察されることから、 cnpy3遺伝子は、中脳視蓋部、 中脳後脳境界部及び耳胞の形成に関与しているといえる。 According to the gene involved in FGF signal transduction according to the present invention, it becomes possible to analyze the formation mechanism of the mesencephalic tectum and cerebellum at the midbrain hindbrain boundary. In particular, in transgenic animals that inhibited the translation of cnpyl gene from transcripts, hypoplasia of the midbrain optic tectum and malformation of the midbrain hindbrain boundary were observed. It can be said that it is involved in the formation of the brain tectum and the midbrain hindbrain boundary. In transgenic animals that inhibited translation from the transcript of the cnpy3 gene, hypoplasia of the midbrain tectum, malformation of the midbrain hindbrain border, malformation and development of otoliths Since degeneration of the lens is observed in the late stage, it can be said that the cnpy3 gene is involved in the formation of the midbrain tectum, the midbrain hindbrain boundary, and the otic vesicle.
また、 FGF シグナルは、 中脳後脳境界部の形成以外にも、 内耳、 血管、 肢芽、 歯牙、 等の様々な器官の形成や、 細胞の癌化などに深く関わっている。 このシグ ナルの活性を調節できる細胞外因子を発見できれば、 器官形成や細胞の増殖の制 御因子として広い応用の道が開けると期待される。したがって、本発明に係る FGF シグナル伝達に関与する遺伝子の機能を欠損させたトランスジエニック動物を用 いれば、 中脳 ·後脳境界部において: FGFシグナルと関連して働く分子を同定する ことが可能となり、 器官形成や細胞の増殖制御に深く関わる FGFの働きの制御に 繋がる新規分子を同定することができる。  In addition to the formation of the midbrain-hindbrain boundary, the FGF signal is deeply involved in the formation of various organs such as the inner ear, blood vessels, limb buds, teeth, etc., as well as in canceration of cells. The discovery of extracellular factors that can regulate the activity of this signal is expected to open up a wide range of applications as regulators of organ formation and cell growth. Therefore, by using a transgenic animal in which the function of a gene involved in FGF signal transduction according to the present invention is deficient, it is possible to identify a molecule that works in association with the FGF signal at the midbrain-hindbrain boundary. It is possible to identify novel molecules that are linked to the regulation of the function of FGF, which is deeply involved in organ formation and cell growth control.
すなわち、 本発明に係る FGFシグナル伝達に関与する遺伝子を導入した遺伝子 導入細胞又は本発明に係る FGFシグナル伝達に関与する遺伝子を導入した卜ラン スジエニック動物に対してリード化合物を作用させる工程と、 中脳 ·後脳境界部 において FGFシグナルと関連して働くリード化合物を同定する工程とを含むスク リーニング方法を提供することができる。  That is, a step of causing a lead compound to act on a transgenic cell into which a gene involved in FGF signaling according to the present invention has been introduced or on a transgenic animal into which a gene involved in FGF signaling according to the present invention has been introduced. Identifying a lead compound that works in association with the FGF signal at the brain-hindbrain boundary.
以下、 実施例を用いて本発明をより詳細に説明するが、 本発明の技術的範囲は これら実施例に限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.
〔実施例 1〕 cnpy遺伝子ファミリーの発現解析  [Example 1] Expression analysis of cnpy gene family
本例では、先ず、ゼブラフィッシュ胚における cnpy遺伝子ファミリーの発現を 観察した。  In this example, first, the expression of the cnpy gene family in zebrafish embryos was observed.
ゼブラフィッシュ胚を 4%パラホルムアルデヒド/ PBS (0. 8% NaC l, 0. 02¾ KC1, 0. 02 M P04, pH7. 3)で 4 °Cで一晩固定し、 PBSTw (PBS+0. 1% Tween - 20)で 5 分間Zebrafish embryos 4% paraformaldehyde / PBS (0. 8% NaC l , 0. 02¾ KC1, 0. 02 M P0 4, pH7. 3) were fixed at 4 ° C overnight with, PBSTw (PBS + 0. 1% Tween-20) for 5 minutes
4回洗う。 胚をメタノールに置換し- 20GCで 30分以上おいた後、 75， 50, 25% メ タノ一ル /PBSTwで順次置換する。 PBSTwで 5分間 2回リンスした後、 受精後 24 時間以降の胚については Prote inase K (10 mg/ml in PBSTw)で室温で 3〜 10分間 消化。 2 mg/nil グリシン/ PBSTwでリンスした後、 4% パラホルムアルデヒド/ PBSTw で 20分間固定する。 PBSTwで 5分間 5回洗い、 HM (50% ホルムアミド、 5x SSC (0. 75 M塩化ナトリウム， 75 mM クェン酸ナトリウム， pH7. 0) , 50 mg/ml へパリン， 0. 1¾Wash 4 times. After replacing the embryo with methanol and leaving it at -20 GC for 30 minutes or more, replace it with 75, 50, 25% methanol / PBSTw sequentially. After rinsing twice with PBSTw for 5 minutes, embryos 24 hours after fertilization are digested with protein inase K (10 mg / ml in PBSTw) at room temperature for 3 to 10 minutes. After rinsing with 2 mg / nil glycine / PBSTw, fix with 4% paraformaldehyde / PBSTw for 20 minutes. Wash 5 times for 5 minutes with PBSTw, HM (50% formamide, 5x SSC (0.75 M sodium chloride, 75 mM sodium citrate, pH 7.0), 50 mg / ml heparin, 0.1¾
Tween- 20， 5 mg/ml toml a RNA)を加え、 65 °Cで 1〜8時間プレハイブリダィゼィ シヨンを行う。 80 °Cで 10分間の熱変性を行ったディゴキシジェニンラベルされ た cnpyl, cnpy3, cnpy4 全長に対するアンチセンスリポプローブ/ Hb4 を加え、 65 t:でー晚ハイブリダィゼイシヨンを行う。 その後、 胚を 50% ホルムアミ ド /2xSSCTw (0. 3 M塩化ナトリゥム， 30 mM クェン酸ナトリゥム、 0. 1% Tween-20) で 30分間 2回、 2xSSTwで 15分間、 0. 2xSSCTwで 30分間 2回いずれも 65 でで洗 浄を行う。 2% B locking reagent (Roche) /MBT (0. 1 Mマレイン酸、 0. 15 M塩化ナ トリゥム、 0. 1% Tween-20)を加え室温で 1時間ブロッキングを行い、 AP標識抗 DI G 抗体（Roche)を PBSTwで 5000倍希釈し、室温で 2時間あるいは 4 。(：で一晚ィンキ ュペートする。 PBSTwで 20分間 6回洗浄した後、 SB (100 塩化ナトリウム、 50 mM 塩化マグネシウム、 100 mM Tr i s-HC l, H 9. 5, 0. 1% Tween20)で 5分間 回置換し、 SS (NBT/BCIP (Roche) , SB)で発色を行う。 その後、 PBSTwで 5分間 2 回洗浄し、 100% エタノールで 10分間脱色を行う。 50% エタノール/ PBSTwで 5分 間、 PBSTwで 5分間 2回洗浄した後、 25, 50, 75%グリセロール/ PBSTwで各 5分間 置換し、 75% グリセロール/ PBSTwでマウントし検鏡する。 Tween-20, 5 mg / ml toml a RNA) and prehybridization at 65 ° C for 1-8 hours Do the chillon. Add digoxigenin-labeled cnpyl, cnpy3, cnpy4 antisense lipoprobe / Hb4, which has been heat denatured at 80 ° C for 10 minutes, and perform hybridization at 65 t :. The embryos were then washed twice with 50% formamide / 2xSSCTw (0.3 M sodium chloride, 30 mM sodium citrate, 0.1% Tween-20) for 30 minutes twice, 2xSSTw for 15 minutes, and 0.2xSSCTw for 30 minutes 2 Wash at 65 each time. Add 2% B locking reagent (Roche) / MBT (0.1 M maleic acid, 0.15 M sodium chloride, 0.1% Tween-20), block for 1 hour at room temperature, and AP-labeled anti-DIG antibody (Roche) diluted 5000 fold with PBSTw, 2 hours or 4 at room temperature. After washing with PBSTw 6 times for 20 minutes, SB (100 sodium chloride, 50 mM magnesium chloride, 100 mM Tris-HCl, H9.5, 0.1% Tween20) 5 times with, and develop color with SS (NBT / BCIP (Roche), SB), then wash twice with PBSTw for 5 minutes, and decolorize with 100% ethanol for 10 minutes 50% ethanol / PBSTw After washing twice for 5 minutes with PBSTw for 5 minutes, replace with 25, 50, and 75% glycerol / PBSTw for 5 minutes each, mount with 75% glycerol / PBSTw, and observe with a microscope.
cnpyh cnpy3及び cnpy4遺伝子のゼブラフィッシュ胚における発現を観察した 結果を図 5及び図 6に示す。なお、図 5は、左から 1列目が cnpyl遺伝子の発現、 2列目が cnpy3遺伝子の発現、 3列目が cnpy4遺伝子の発現を示しており、 上か ら 1行目が 8細胞期にあるゼブラフィッシュ胚であり、 2行目がドーム期 （胞胚 期） にあるゼブラフィッシュ胚であり、 3行目が 30% ep iboly期 （初期原腸胚期） にあるゼブラフィッシュ胚である。  The results of observing the expression of the cnpyh cnpy3 and cnpy4 genes in zebrafish embryos are shown in FIGS. 5 and 6. In FIG. 5, the first column from the left shows the expression of the cnpyl gene, the second column shows the expression of the cnpy3 gene, the third column shows the expression of the cnpy4 gene, and the first row from the top shows the expression at the 8-cell stage. One zebrafish embryo, the second line is a zebrafish embryo in the dome stage (blastula stage), and the third line is a zebrafish embryo in the 30% epiboly stage (early gastrulation stage).
図 6は、 左から 1列目が cnpyl遺伝子の発現、 2列目が cnpy3遺伝子の発現、 3列目が cnpy4遺伝子の発現を示しており、 上から 1行目が胚盾期 （中期原腸胚 期） にあるゼブラフィッシュ胚であり、 2行目が尾芽胚期にあるゼブラフイツシ ュ胚であり、 3行目が受精後 18時間にあるゼブラフィッシュ胚であり、 4行目が 受精後 25時間にあるゼブラフィッシュ胚である。  In Fig. 6, the first column from the left shows the expression of the cnpyl gene, the second column shows the expression of the cnpy3 gene, and the third column shows the expression of the cnpy4 gene. The second line is a zebrafish embryo at the tail bud embryo stage, the third line is a zebrafish embryo 18 hours after fertilization, and the fourth line is a 25 zebrafish embryo. A zebrafish embryo in time.
図 5及び図 6から、 cnpyl 遺伝子は、 マ夕一ナルな発現が弱く見られ、 受精後 From Fig. 5 and Fig. 6, the cnpyl gene is weakly expressed at the primary level, and after fertilization.
12時間頃から孵化腺原基での発現が見られるようになり、 受精後 14時間以降は 丽 B において特異的に発現が見られることが判った。 また、 図 5及び図 6から、 cnpy3遺伝子及ぴ cnpy4遺伝子は、 マターナルな発現が強く見られ、 胚性発現は 胚全体に弱く見られることが判つた。 From around 12 hours, expression in the hatching gland primordium was observed, and it was found that expression was specifically observed in 丽 B after 14 hours from fertilization. Also, from FIGS. 5 and 6, the cnpy3 gene and the cnpy4 gene show strong maternal expression, and It was found that it was weakly observed throughout the embryo.
次に、 本例では、 マウス胚における cnpy遺伝子ファミリーの発現を観察した。 妊娠 10. 5 日目のマウス（ICR)から胚を摘出し、 PBSで洗浄後、 4%パラホルムァ ルデヒド /PBS で一晩固定した。 エタノールで脱水後、 75, 50, 25%エタノール Next, in this example, the expression of the cnpy gene family in mouse embryos was observed. Embryos were excised from the mice (ICR) on day 10. 5 of gestation, washed with PBS, and fixed with 4% paraformaldehyde / PBS overnight. 75, 50, 25% ethanol after dehydration with ethanol
/PBSTwシリーズで再水和を行った。 10 mg/ml Prote inase K/PBSTwで 5〜 15分間 処理し、 2 mg/mlグリシン/ PBSTwで 5分間、 PBSTwで 5分間 2回洗浄し、 0. 2% グ ルタルアルデヒド /4% パラホルムアルデヒド/ PBSTw で 20 分間固定した。 PBSTw で 5分間 2回洗浄した後、 70 でで 1時間処理した後氷水中で完全に冷やし、 6%Rehydration was performed with the / PBSTw series. Treat with 10 mg / ml protein inase K / PBSTw for 5 to 15 minutes, wash twice with 2 mg / ml glycine / PBSTw for 5 minutes, and wash twice with PBSTw for 5 minutes, then 0.2% glutaraldehyde / 4% paraformaldehyde / The cells were fixed with PBSTw for 20 minutes. After washing twice with PBSTw for 5 minutes, treat with 70 for 1 hour, cool completely in ice water, and add 6%
H202/PBSTwを加え室温で 1時間置く。 PBSTwで 5分間 3回洗浄した後、 70 °Cで保 温しておいたプレハイブリダィゼイシヨン溶液 （50% ホルムアミド、 5x SSC, HAdding H 2 0 2 / PBSTw placed at room temperature for 1 hour. After washing three times with PBSTw for 5 minutes, prehybridization solution (50% formamide, 5x SSC, H
4. 5, 50 mg/ml torul a RNA, 1% SDS, 50 mg/mlへパリン） を加え、 70 で 5分 間置いた後、 新しいプレハイブリダィゼイシヨン溶液を加え、 1時間以上インキ ュべ一トする。 80 °Cで 10分間の熱変性を行ったディゴキシジェニンラベルされ た cnpy2, cnpy3, cnpy4全長に対するアンチセンスリボプローブ/プレハイブリダ ィゼィション溶液を加え、 70 °Cで揺らしながらー晚ハイブリダイゼィシヨンを行 う。 Solut ionl (50%ホルムアミド、 5xSSC, pH4. 5, 1% SDS)を加え 70 で 30分 間 3回洗浄を行う。 Solut ionl ： Solut ion2 (5xSSC, pH4. 5, 0. 1% Tween- 20)を加 え 70 で 10分間洗浄を行う。 Solut ion を加え室温で 5分間 3回洗浄する。4. Add 5,50 mg / ml torul a RNA, 1% SDS, 50 mg / ml heparin), leave at 70 for 5 minutes, add new prehybridization solution, and incubate for 1 hour or more. Check. Add the antisense riboprobe / prehybridization solution to the digpygenin-labeled cnpy2, cnpy3, and cnpy4 full-length denatured at 80 ° C for 10 minutes, and perform the hybridization while shaking at 70 ° C. U. Add Solutionl (50% formamide, 5xSSC, pH4.5, 1% SDS) and wash 3 times at 70 for 30 minutes. Solut ionl: Wash with Solut ion2 (5xSSC, pH 4.5, 0.1% Tween-20) at 70 for 10 minutes. Add Solution and wash 3 times for 5 minutes at room temperature.
Solut ion2を加え室温で 30分間洗浄する。 Solut ion3 (50¾ ホルムアミド， 2x SSC, pH4. 5, 1% SDS)を加え 70 °Cで 5分間 回洗浄を行う。 Solut ion3を加え 70 ででAdd Solution2 and wash at room temperature for 30 minutes. Add Solution3 (50¾ formamide, 2x SSC, pH 4.5, 1% SDS) and wash 5 times at 70 ° C. Add Solut ion3 and add 70
30 分間 3 回洗浄を行う。 Solut ion ： TBSTw (150 mM塩化ナトリウム、 100 mMWash 3 times for 30 minutes. Solut ion: TBSTw (150 mM sodium chloride, 100 mM
Tri s-HCl, pH7. 5, 0. 1% Tween-20) =1 : 1を加え 70 °Cで 10分間インキュベートす る。 TBSTwを加え、 室温で 5分間 5回インキュベートする。 2 Blocking reagen tAdd Tris-HCl, pH 7.5, 0.1% Tween-20) = 1: 1 and incubate at 70 ° C for 10 minutes. Add TBSTw and incubate 5 times for 5 minutes at room temperature. 2 Blocking reagen t
(Roche) /TBST で 60分間ブロッキングを行う。 AP標識抗 DI G抗体（Roche)を TBSTw で 5000倍希釈し、 4 °Cで一晩インキュベートする。 TBSTwを加え、 室温で 5分間(Roche) Perform blocking with / TBST for 60 minutes. Dilute AP-labeled anti-DIG antibody (Roche) 5000-fold with TBSTw and incubate at 4 ° C overnight. Add TBSTw, 5 minutes at room temperature
4回インキュベートする。 TBSTwを加え、 室温で 1時間 6回インキュベートする。Incubate 4 times. Add TBSTw and incubate 6 times for 1 hour at room temperature.
NTMT (0. 1 M塩化ナトリウム、 0. 1 M Tris-HCl, pH9. 5, 50 mM塩化マグネシウム、NTMT (0.1 M sodium chloride, 0.1 M Tris-HCl, pH 9.5, 50 mM magnesium chloride,
1% Tween-20) を加え室温で 5 分間 2 回イ ンキュベー トする。 発色液Add 1% Tween-20) and incubate twice for 5 minutes at room temperature. Coloring solution
(NBT/BCIP/NTMT)を加え室温で遮光して 1〜5時間発色を行う。 PBSTwを加え室温 で 5分間 5回洗浄する。 100% エタノールで 10分間脱色を行う。 50% エタノール /PBSTwで 5分間、 PBSTwで 5分間 2回洗浄した後、 25, 50, 75%グリセロール/ PBSTw で各 5分間置換し、 75% グリセロール/ PBSTwでマウントし検鏡する。 Add (NBT / BCIP / NTMT) and shade at room temperature for 1-5 hours. Add PBSTw and room temperature 5 times for 5 minutes each. Decolorize with 100% ethanol for 10 minutes. After washing twice with 50% ethanol / PBSTw for 5 minutes and with PBSTw twice for 5 minutes, replace with 25, 50, and 75% glycerol / PBSTw for 5 minutes each, mount with 75% glycerol / PBSTw, and observe with a microscope.
受精後 10. 5日目のマウス胚を観察した結果を図 7に示す。なお、図 7において、 左から順に cnpy2遺伝子、 cnpy3遺伝子及び cnpy4遺伝子の発現を観察した結果 である。 図 7力ゝら、 cnpy2遺伝子、 cnpy3遺伝子及び cnpy4遺伝子ともにマウス胚 全体に亘つて発現が見られたが、 特に肢芽及び尾部における強い発現を観察する ことができる。  FIG. 7 shows the results of observation of the mouse embryo at day 10.5 after fertilization. FIG. 7 shows the results of observing the expression of the cnpy2 gene, the cnpy3 gene, and the cnpy4 gene in order from the left. Fig. 7 Expression of cnpy2 gene, cnpy3 gene and cnpy4 gene was observed throughout the mouse embryo, but strong expression was observed particularly in the limb bud and tail.
〔実施例 2〕 アンチセンスモルフォリノオリゴによるノックダウン実験  [Example 2] Knockdown experiment using antisense morpholino oligo
ゼブラフィッシュにおいて、 転写産物からタンパク質への翻訳をアンチセンス モルフォリノオリゴ (M0)の顕微注入により効果的に阻害できる （ノックダウン） ことが知られている。 そこで、 本例では、 cnpyl 遺伝子を同手法でノックダウン した胚 （cnpy卜 M0ノックダウン胚と呼ぶ。 その他の cnpy遺伝子ファミリーのノ ックダウン胚も同様に称する。）、 cnpy3_M0ノックダウン胚、 cnpy3/4 - M0ノックダ ゥン胚及び cnpy4-M0ノックダウン胚を作製し、 形態異常を観察した。  It is known that in zebrafish, translation of a transcript into a protein can be effectively inhibited (knockdown) by microinjection of antisense morpholino oligo (M0). Therefore, in this example, embryos in which the cnpyl gene was knocked down by the same method (called cnpy M0 knockdown embryos; knockdown embryos of other cnpy gene families are also referred to), cnpy3_M0 knockdown embryos, cnpy3 / 4 -M0 knockdown embryos and cnpy4-M0 knockdown embryos were prepared and morphological abnormalities were observed.
各アンチセンスモルフオリノオリゴの配列は以下の通りである。  The sequence of each antisense morpholino oligo is as follows.
cnpyl-MO； ATGGTGACATGCTGGTCTCCTGAG (配列番号 2 3 ) cnpyl-MO; ATGGTGACATGCTGGTCTCCTGAG (SEQ ID NO: 23)
cnpy3-M0； TTAGAAC GAGC AAT ACTAAAAC C C C (配列番号 2 4 ) cnpy3-M0; TTAGAAC GAGC AAT ACTAAAAC C C C (SEQ ID NO: 24)
cnpy4-M0； GTAAACATTTCCATACTGACTACAA (配列番号 2 5 ) cnpy4-M0; GTAAACATTTCCATACTGACTACAA (SEQ ID NO: 25)
これらアンチセンスモルフォリノオリゴは、 lx Danieau buf fer (58 mM塩化ナ トリウム、 0. 7 mM塩化カリウム、 0. 5 mM硫酸マグネシウム、 0. 6 mM硝酸カルシ ゥム、 5. 0 mM HEPES, pH7. 6)にそれぞれ溶解した。 1〜2細胞期のゼブラフイツシ ュ胚にアンチセンスモルフォリノオリゴを顕微注入した。  These antisense morpholino oligos were prepared using lx Danieau buf fer (58 mM sodium chloride, 0.7 mM potassium chloride, 0.5 mM magnesium sulfate, 0.6 mM calcium nitrate, 5.0 mM HEPES, pH 7.0. 6). Antisense morpholino oligos were microinjected into zebrafish embryos at the 1-2 cell stage.
結果を図 8及び図 9に示す。図 8において、 （a)は正常なゼブラフィッシュ胚に おける MHB周辺を上方から撮影した写真であり、（b)は正常なゼブラフィッシュ胚 における MHB周辺を側方から撮影した写真であり、 （c)は cnpy卜 M0ノックダウン 胚における MHB周辺を上方から撮影した写真であり、 （d)は cnpyl- M0ノックダウ ン胚における MHB周辺を側方から撮影した写真であり、 （e)は cnpy3- M0ノックダ ゥン胚における MHB周辺を上方から撮影した写真であり、 （ί)は cnpy3-M0ノック ダウン胚における丽 B周辺を側方から撮影した写真である。 図 9において、 左か ら順に、 正常なゼブラフィッシュ胚、 cnpy3 - M0 ノックダウン胚及び cnpy3/4- M0 ノックダウン胚における耳胞 （矢印で示す） を撮影した写真である。 The results are shown in FIGS. In FIG. 8, (a) is a photograph taken from above of the vicinity of MHB in a normal zebrafish embryo, (b) is a photograph taken from the side of MHB in a normal zebrafish embryo, and (c) ) Is a photograph taken from above of the vicinity of MHB in cnpy M0 knockdown embryo, (d) is a photograph taken from the side of MHB in cnpyl-M0 knockdown embryo, and (e) is a photograph taken from the side. This is a photograph taken from above of the vicinity of MHB in the knockdown embryo. (Ί) shows the cnpy3-M0 knockout. 4 is a photograph taken from the side around 周 辺 B in a down embryo. FIG. 9 is a photograph of oticles (indicated by arrows) in normal zebrafish embryos, cnpy3-M0 knockdown embryos, and cnpy3 / 4-M0 knockdown embryos, from left to right.
図 8及び図 9の結果から、 各ノックダウン胚において以下の形態異常が観察さ れた。 cnpy卜腸ノックダウン胚では中脳視蓋部の低形成及び MHBの形成異常が観 察された。 cnpy3-M0ノックダウン胚では軽度の耳胞及び耳石の形成異常が観察さ れた。 cnpy3/4- M0ノックダウン胚においては重度の耳胞及び耳石の形成異常が見 られた。 また、 cnpy4 - M0 単独では異常はほとんど見られない （図示せず） ので、 cnpy4遺伝子は耳胞及び耳石の形成において cnpy3遺伝子と協調的に働いている と考えられた。 MHB形成及び耳胞の形成において FGFシグナルが重要な役割を担 つていることが知られているので、 本例によって cnpy遺伝子ファミリ一は FGF のシグナル伝達に関与していることが示唆された。  From the results of FIGS. 8 and 9, the following morphological abnormalities were observed in each knockdown embryo. In cnpy intestinal knockdown embryos, hypoplasia of the midbrain tectum and abnormal formation of MHB were observed. In cnpy3-M0 knockdown embryos, mild dysplasia of otocysts and otoliths was observed. In cnpy3 / 4-M0 knockdown embryos, severe otocysts and otolith dysplasia were observed. Since cnpy4-M0 alone showed almost no abnormalities (not shown), it was considered that the cnpy4 gene cooperated with the cnpy3 gene in the formation of otocysts and otoliths. Since it is known that FGF signal plays an important role in MHB formation and otocyst formation, this example suggested that the cnpy gene family 1 is involved in FGF signal transduction.
〔実施例 3〕 繊維芽細胞増殖因子 （FGF) シグナルへの関与  [Example 3] Involvement in fibroblast growth factor (FGF) signal
実施例 2により cnpy遺伝子ファミリ一が FGFシグナル伝達に関与することが示 唆されたため、 本例では、 cnpy遺伝子ファミリーと FGFシグナルとの関連につい て検討した。  Example 2 suggested that the cnpy gene family is involved in FGF signal transduction. Therefore, in this example, the relationship between the cnpy gene family and FGF signal was examined.
<FGFビーズ実験〉 <FGF beads experiment>
間脳及び中脳付近に FGFSb に浸したビーズを埋め込むことによって異所的に engrai led 遺伝子の発現が誘導されることが知られている （Cross ley, P. H. , Mart inez, S. and Mart in, G. R. , 1996. Midbrain deve lopment induced by FGF8 in the chick embryo. Nature 380, 66-68. )。 そこで、 正常なゼブラフィッシュ 胚及び cnpyl- MOノックダウン胚に FGF8bビーズを埋め込み、 engrai led遺伝子の 発現誘導を確認した。  It is known that the engrai led gene expression is ectopically induced by implanting beads soaked in FGFSb near the diencephalon and midbrain (Crossley, PH, Mart inez, S. and Mart in, GR, 1996. Midbrain deve lopment induced by FGF8 in the chick embryo. Nature 380, 66-68.). Therefore, FGF8b beads were embedded in normal zebrafish embryos and cnpyl-MO knockdown embryos, and the induction of expression of the engrai led gene was confirmed.
Recombinant mouse FGF - 8b (R&D sys tems)を PBSに溶解し、 あらかじめ PBSで 洗浄しておいた heparin, insolubi 1 i zed on acryl ic beads (S igma)を加え、 0. 5 mg/mlの濃度で 4 °Cで一晩インキュベートする。 受精後 1 5時間の正常胚あるい は cnpyl- M0ノックダウン胚に FGFを浸したビーズを埋め込み、 1/3希釈 Ringer 液 （39 mM塩化ナトリウム、 0. 97 mM塩化カリウム、 1. 8 mM塩化カルシウム、 1. 7 mM Hepes, pH7. 2)中で 3時間インキュベート後、 4%パラホルムアルデヒドで固定 し、 in situハイブリダイゼィションによって engrailed遺伝子の発現を調べた。 結果を図 1 0に示す。 図 1 0から判るように、 cnpyl-MOノックダウン胚におい ては、 FGF8bビーズを埋め込んだところ、 engrailed遺伝子の異所的な発現の程度 が大幅に減少した。なお、正常なゼブラフィッシュ胚（正常胚）においては、 FGF8b ビーズを埋め込むことによって、 engrailed 遺伝子の異所的な発現が増加してい ることが判る。 この結果から、 cnpyl遺伝子は、 FGFシグナルの伝達に必須である ことが明らかとなった。 Recombinant mouse FGF-8b (R & D system) was dissolved in PBS, and heparin, insolubi 1 i zed on acrylic beads (Sigma), which had been washed with PBS beforehand, was added at a concentration of 0.5 mg / ml. Incubate at 4 ° C overnight. FGF-soaked beads are embedded in normal embryos or cnpyl-M0 knockdown embryos 15 hours after fertilization, and 1/3 diluted Ringer solution (39 mM sodium chloride, 0.97 mM potassium chloride, 1.8 mM chloride) After incubation in calcium, 1.7 mM Hepes, pH 7.2) for 3 hours, fix with 4% paraformaldehyde Then, the expression of the engrailed gene was examined by in situ hybridization. The results are shown in FIG. As can be seen from FIG. 10, in the cnpyl-MO knockdown embryo, the degree of ectopic expression of the engrailed gene was significantly reduced when FGF8b beads were embedded. In normal zebrafish embryos (normal embryos), it is found that ectopic expression of the engrailed gene is increased by embedding FGF8b beads. These results revealed that the cnpyl gene is essential for FGF signal transmission.
く活性化 ERKのウェスタンプロットによる検出〉 Detection of activated ERK by Western plot>
FGFのシグナルは FGFが受容体に結合しその下流にあるシグナルカスケ一ドを 活性化することによって伝わる。 ERKの活性化は FGFシグナルの指標となる（Gotoli, Y. and Nishida, E.， 1996. Signals for mesoderm induction. Roles of f ibroblas t growth factor (FGF) /mitogen- activated protein kinase (MAPK) pathway. Biochim Biop ys Acta 1288, F卜 7. )。 そこで、 cnpyl- MO ノックダウン胚及び cnpy3/4-M0ノックダウン胚において活性化 ERKをウエスタンプロットによつて検 出した。 なお、 正常なゼブラフィッシュ胚 （正常胚） 及び fgf3- M0ノックダウン 胚をコントロールとした。  FGF signals are transmitted by FGF binding to the receptor and activating a signal cascade downstream of the receptor. Activation of ERK is an indicator of FGF signal (Gotoli, Y. and Nishida, E., 1996.Signals for mesoderm induction. Roles of fibroblast growth factor (FGF) / mitogen-activated protein kinase (MAPK) pathway. Biochim Biopys Acta 1288, F 7.). Therefore, activated ERK was detected by Western plot in cnpyl-MO knockdown embryos and cnpy3 / 4-M0 knockdown embryos. Normal zebrafish embryos (normal embryos) and fgf3-M0 knockdown embryos were used as controls.
igi3- M0の配列は CATTGTGGCATGGCGGGATGTCGGC (配列番号 26)である。 受精後 The sequence of igi3-M0 is CATTGTGGCATGGCGGGATGTCGGC (SEQ ID NO: 26). After fertilization
18 時間の正常胚、 igi3-M0 ノックダウン胚、 cnpy3/4- M0 ノックダウン胚、 及び cnpyl- M0ノックダウン胚の卵膜と卵黄を取り除く。 胚を Sa即 le buffer (125 mMRemove 18-hour normal, igi3-M0 knockdown, cnpy3 / 4-M0 knockdown, and cnpyl-M0 knockdown embryos egg membrane and yolk. Embryos are transferred to Sa-le buffer (125 mM
Tris-HCl, pH6.8, 2% SDS, 10%グリセ□ール、 50 mg/mlブロモフエノールブルー） で溶解し、 95 °C、 5分間処理する。 SDS- PAGEゲルに 1レーンあたり 3匹分のサン プルをアプライし、 電気泳動を行う。 泳動後、 エレクトロブロッテイングにより タンパク質を PVDF メンブレンに転写する。 5%スキムミルク/ TBSTw (TBS, 0.05%Dissolve in Tris-HCl, pH6.8, 2% SDS, 10% glycerol, 50 mg / ml bromophenol blue) and treat at 95 ° C for 5 minutes. Apply 3 samples per lane to the SDS-PAGE gel and perform electrophoresis. After electrophoresis, transfer the protein to a PVDF membrane by electroblotting. 5% skim milk / TBSTw (TBS, 0.05%
Tween- 20)で一晩 4 ででブロッキングを行う。 活性化 ERKを検出するためには 1% スキムミルク/ TBSTWで 1:10000希釈した anti-diphosphorylated ERK (sigma)を 加え、 全 ERK を検出するためには 1%スキムミルク/ TBSTW で 1:10000 希釈した anti-ERKl (Santa Cruz)を加え、 37 °Cで 1時間インキュベートする。 TBSTwで 10 分間 3 回洗浄する。 活性化 ERK を検出するためには 1%スキムミルク/ TBSTW でPerform blocking with Tween-20) overnight at 4. To detect activated ERK, add anti-diphosphorylated ERK (sigma) diluted 1: 10,000 in 1% skim milk / TBSTW, and to detect total ERK, anti-diphosphorylated ERK diluted 1: 10000 in 1% skim milk / TBSTW -Add ERKl (Santa Cruz) and incubate at 37 ° C for 1 hour. Wash 3 times for 10 minutes each with TBSTw. 1% skim milk / TBSTW to detect activated ERK
1:10000希釈した ant i- mouse IgG, HRP conjugated (Santa Cruz)を加え、 全 ERK を検出するためには 1%スキムミルク/ TBSTWで 1:20000希釈した anti- rabbit IgG, HRP conjugated (Santa Cruz)を加え、 37 °Cで 1時間インキュベートする。 TBSTw で 10分間 3回洗浄する。 Western Lightning Cherai luminescence Reagent Plus (Perkin Elmer)を用いシグナルを検出する。 1: 10,000 diluted anti-mouse IgG, HRP conjugated (Santa Cruz) To detect, add anti-rabbit IgG, HRP conjugated (Santa Cruz) diluted 1: 20000 with 1% skim milk / TBSTW and incubate at 37 ° C for 1 hour. Wash 3 times with TBSTw for 10 minutes. The signal is detected using Western Lightning Cherai luminescence Reagent Plus (Perkin Elmer).
結果を図 1 1に示す。 図 1 1から判るように、 cnpy卜 M0 ノックダウン胚及び cnpy3/4-M0ノックダウン胚においては、 igi3-M0ノックダウン胚と同様に、 活性 化 ERKがほぼ消失していることが分かった。 この結果からも、 cnpyl遺伝子は FGF シグナルの伝達に必須であることが明らかどなった。 また、 cnpy3 遺伝子及び cnpy4遺伝子は、 FGFシグナルの伝達に必須であることが明らかとなった。  The results are shown in FIG. As can be seen from FIG. 11, the activated ERK was almost eliminated in the cnpy M0 knockdown embryo and the cnpy3 / 4-M0 knockdown embryo, as in the igi3-M0 knockdown embryo. These results also revealed that the cnpyl gene is essential for FGF signal transduction. In addition, it was revealed that the cnpy3 gene and the cnpy4 gene were essential for FGF signal transmission.
〈免疫組織化学による活性化 ERKの検出〉 <Detection of activated ERK by immunohistochemistry>
ウェスタンブロットにより活性化 ERKの消失が確認されたので、 免疫組織化学 によつて、ゼブラフィッシュ胚のどの部分で活性化 ERKが消失したのかを調べた。 受精後 18時間の正常胚あるいは fgi3-M0, cnpyl - MO, cnpy3_M0各ノックダウン 胚を 4%パラホルムアルデヒドで 4 °Cでー晚固定する。 PBSTwで 5分間 4回洗浄す る。 100%メタノールで脱水し、 - 20 °Cに 30分以上おく。 75， 50, 25%メタノール /PBSTwで各 5分間おく。 PBSTwで 5分間 2回洗浄する。 2% Blocking reagent/MBT を加え、 80 °C、 20分間及び、室温で 1時間インキュベートする。 PBSTwで 1:10000 希釈した anti- diphosphorylated ERKを加え、 4 °Cでー晚インキュベートする。 PBSTwで 20分間 6 回洗浄を行う。 PBSTwで 1:1000希釈した ant i- mouse IgG, Biotin- conjugated (sigma)を加え 4 °Cでー晚インキュベートする。 PBSTwで 20 分間 6回洗浄を行う。 ABC co即 lex (Vector Laboratory)を加え室温で 2時間ィン キュベー卜する。 PBSTwで 20分間 6回洗浄を行う。 0. l M Tris-ΗΠ, pH8.2で 5 分間 2回置換する。 MB/0.1 M Tris- HC1, pH8.2を加え 20分おき、 1:1000希釈し た過酸化水素水を適量加え発色を行う。 PBSTwで 5分間 2回洗浄する。 25, 50, 75% グリセ口一ル /PBSTwで各 5分間置換し、 75% グリセロール/ PBSTwでマウントし検 鏡する。  Since the disappearance of activated ERK was confirmed by Western blot, immunohistochemistry was used to examine which parts of the zebrafish embryo had lost activated ERK. 18 hours after fertilization, fix normal embryos or fgi3-M0, cnpyl-MO, and cnpy3_M0 knockdown embryos at 4 ° C with 4% paraformaldehyde. Wash 4 times for 5 minutes with PBSTw. Dehydrate with 100% methanol and keep at -20 ° C for more than 30 minutes. Incubate in 75, 50, 25% methanol / PBSTw for 5 minutes each. Wash twice with PBSTw for 5 minutes. Add 2% Blocking reagent / MBT and incubate at 80 ° C for 20 minutes and at room temperature for 1 hour. Add anti-diphosphorylated ERK diluted 1: 10000 with PBSTw and incubate at 4 ° C. Wash 6 times with PBSTw for 20 minutes. Add anti-mouse IgG and Biotin-conjugated (sigma) diluted 1: 1000 with PBSTw and incubate at 4 ° C. Wash 6 times with PBSTw for 20 minutes. Add ABC co Immediate lex (Vector Laboratory) and incubate at room temperature for 2 hours. Wash 6 times with PBSTw for 20 minutes. 0. Displace twice with lM Tris-II, pH 8.2 for 5 minutes. Add MB / 0.1 M Tris-HC1, pH 8.2 and add an appropriate amount of 1: 1000 diluted hydrogen peroxide solution every 20 minutes to develop color. Wash twice with PBSTw for 5 minutes. Replace with 25, 50, 75% glycerol / PBSTw for 5 minutes each, mount with 75% glycerol / PBSTw, and observe.
結果を図 12に示す。 図 1 2から判るように、 正常胚においては、 活性化 ERK は終脳及び匪 B に多くみられたが、 cnpyl- M0 ノックダウン胚あるいは cnpy3 - M0 ノックダウン胚においてはこれらの領域での活性化 ERKが消失していた。 この結 果からも、 cnpyl遺伝子及び cnpy3遺伝子は、 FGFシグナルの伝達に必須であるこ とが明らかとなった。 The results are shown in FIG. As can be seen from Fig. 12, activated ERK was found abundantly in telencephalon and maraudal B in normal embryos, but was active in these regions in cnpyl-M0 knockdown embryos or cnpy3-M0 knockdown embryos. ERK had disappeared. This result The results also revealed that the cnpyl gene and cnpy3 gene were essential for FGF signal transmission.
本明細書で引用した全ての刊行物、 特許および特許出願をそのまま参考として 本明細書にとり入れるものとする。 産業上の利用の可能性  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential
以上、 詳細に説明したように、 本発明により、 FGF シグナル伝達に関与する新 規な遺伝子を提供することができる。 本発明に係る遺伝子を使用することによつ て、 中脳後脳境界部による中脳視蓋部及び小脳の形成メカニズム並びに、 内耳、 血管、 肢芽、 歯牙、 等の様々な器官の形成メカニズムや、 細胞の癌化メカニズム 等の解析が可能となる。 配列表フリーテキスト  As described above in detail, according to the present invention, a novel gene involved in FGF signaling can be provided. By using the gene according to the present invention, the formation mechanism of the midbrain tectum and cerebellum by the midbrain hindbrain boundary, and the formation mechanism of various organs such as the inner ear, blood vessels, limb buds, and teeth And analysis of cell carcinogenesis mechanism. Sequence listing free text
配列番号 23〜26は合成 DNAである。  SEQ ID NOS: 23 to 26 are synthetic DNAs.

Claims

請求の範囲 The scope of the claims
1. 以下の （a) 又は （b) のタンパク質をコードする、 FGF シグナル伝達 に関与する遺伝子。 1. A gene involved in FGF signaling that encodes the following protein (a) or (b):
(a) 配列番号 2、 3、 5、 7、 9、 1 1、 13、 15、 16、 18、 20及び 22に表すアミノ酸配列のうちいずれか 1のアミノ酸配列を含むタンパク質 (a) a protein comprising the amino acid sequence of any one of the amino acid sequences represented by SEQ ID NOs: 2, 3, 5, 7, 9, 11, 11, 13, 15, 16, 18, 20, and 22
(b) 配列番号 2、 3、 5、 7、 9、 11、 13、 15、 16、 18、 20及び 22に表すアミノ酸配列のうちいずれか 1のアミノ酸配列において、 1又は複数 個のアミノ酸が置換、 欠失又は付加されたアミノ酸配列からなり、 FGF シグナル 伝達に関与する機能を有するタンパク質。 (b) In the amino acid sequence of any one of the amino acid sequences represented by SEQ ID NOs: 2, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, and 22, one or more amino acids are substituted. A protein comprising a deleted or added amino acid sequence and having a function related to FGF signal transduction.
2. 以下の （a) 又は （b) のポリヌクレオチドからなる FGFシグナル伝達 に関与する遺伝子。  2. A gene involved in FGF signal transduction, comprising the polynucleotide of (a) or (b) below.
( a ) 配列番号 1、 4、 6、 8、 10、 12、 14、 17、 19及び 21に表す 塩基配列のうちいずれか 1の塩基配列を含むポリヌクレオチド  (a) a polynucleotide comprising any one of the nucleotide sequences shown in SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 17, 19 and 21
( b ) 配列番号 1、 4、 6、 8、 10、 12、 14、 17、 19及び 21に表す 塩基配列のうちいずれか 1の塩基配列に相補的な塩基配列に対して、 ストリンジ ェントな条件下でハイブリダィズする塩基配列を含み、 FGF シグナル伝達に関与 する機能を有するタンパク質をコードするポリヌクレオチド  (b) Stringent conditions for a nucleotide sequence complementary to any one of the nucleotide sequences shown in SEQ ID NOs: 1, 4, 6, 8, 10, 12, 14, 17, 19 and 21 A polynucleotide comprising a nucleotide sequence that hybridizes below and encoding a protein having a function involved in FGF signaling
3. 請求項 1又は 2記載の FGFシグナル伝達に関与する遺伝子を有するべク ター。  3. A vector having the gene involved in FGF signaling according to claim 1 or 2.
4. 請求項 1又は 2記載の FGFシグナル伝達に関与する遺伝子の機能を欠損 させた遺伝子導入細胞。  4. A transgenic cell in which the function of the gene involved in FGF signaling according to claim 1 or 2 is deficient.
5. 請求項 1又は 2記載の FGFシグナル伝達に関与する遺伝子の機能を欠損 させたトランスジエニック動物。  5. A transgenic animal in which the function of the gene involved in FGF signaling according to claim 1 or 2 is deficient.
6. 請求項 4記載の遺伝子導入細胞又は請求項 5記載のトランスジエニック 動物に対してリード化合物を作用させる工程と、  6. a step of causing a lead compound to act on the transgenic cell according to claim 4 or the transgenic animal according to claim 5,
中脳 ·後脳境界部において FGFシグナルと関連して働くリード化合物を同定す る工程とを含むスクリーニング方法。  Identifying a lead compound that works in association with the FGF signal at the midbrain / hindbrain boundary.
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