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(12) United States Patent (10) Patent No.: US 7,060,450 B1 Tabin Et Al

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(12) United States Patent (10) Patent No.: US 7,060,450 B1 Tabin Et Al USOO706045OB1 (12) United States Patent (10) Patent No.: US 7,060,450 B1 Tabin et al. (45) Date of Patent: Jun. 13, 2006 (54) SCREENING ASSAYS FORAGONSTS AND OTHER PUBLICATIONS ANTAGONSTS OF THE HEDGEHOG Niswander, L., S. Jeffrey. G.R. Martin, and C. Tickle. A SIGNALING PATHWAY positive feedback loop coordinates growth and patterning in the vertebrate limb. Oct. 13, 1994. Nature. vol. 371. pp. (75) Inventors: Clifford Tabin, Cambridge, MA (US); 609-612. Valeria Marigo, Milan (IT); Philip W. Forbes, A.J. Y. Nakano, A.M. Taylor, and P.W.Ingham. Ingham, Summertown (GB); Andrew Genetic analysis of hedgehog signalling in Drosophila P. McMahon, Lexington, MA (US) embryo. 1993. Development Supplement. 115-124.* (73) Assignees: President and Fellows of Harvard Bowie et al., “Deciphering the Message in the Protein College, Cambridge, MA (US); Sequences: Tolerance to Amino Acid Substitutions”, Sci Imperial Cancer Research Technology ence 247: 1306-1310. 1990.* Ltd., London (GB) Creighton, T.E. Proteins Structures and Molecular Prin ciples, W.H. Freeman and Company: New York, N.Y. pp. (*) Notice: Subject to any disclaimer, the term of this 223-227. 1984. patent is extended or adjusted under 35 Dingermanse, M.A. et al., “The expression of liver-specific U.S.C. 154(b) by 877 days. genes with rat embryonic hepatocytes in a discontinuous process', Differentiation 56: 153-162. 1994.* (21) Appl. No.: 09/711,724 Lerner, R.A., Antibodies of predetermined specificity in biology and medicine, Adv. Immunol. 36: 1-44. 1984.* (22) Filed: Nov. 13, 2000 Zardoya et al., “Evolution and orthology of hedgehog genes, Trends in Genetics. 12(12): 496-497. 1996.* Related U.S. Application Data Anderson, R. et al., “Maintenance of ZPA signaling in (63) Continuation of application No. 08/674.509, filed on cultured mouse limb bud cells', Devel. 117:142-1433 Jul. 2, 1996, now Pat. No. 6,261,786, which is a (1993). continuation-in-part of application No. 08/460,900, Angier, N. "Biologists find key genes that shape patterning filed on Jun. 5, 1995, now Pat. No. 6,165,747, which of embryos, New York Times, Jan. 11, 1994, C-1. is a continuation-in-part of application No. 08/462, Basler, KI., and G. Struhl, “Compartment boundaries and 386, filed on Jun. 5, 1995, now abandoned, which is the control of Drosophila limb pattern by hedgehog pro a continuation-in-part of application No. 08/435,093, tein', Nature 368:208--214 (1994). filed on May 4, 1995, now abandoned, which is a Basler, K. et al., “Control of Cell Pattern in the Neural Tube: continuation-in-part of application No. 08/356,060, Regulation of Cell Differentiation by dorsalin-1, a Novel filed on Dec. 14, 1994, now Pat. No. 5,844,079, TGFB Family Member, Cell 73: 687-702 (May 21, 1993). which is a continuation-in-part of application No. Bass, S. et al., “Hormone phage: An Enrichment Method for 08/176.427, filed on Dec. 30, 1993, now Pat. No. Variant Proteins with Altered Binding Properties”. Proteins: Structure, Function, and Genetics 8:309-314 (1990). 5,789,543. Bejsovec, A. and E. Wieschaus, "Segment polarity gene (51) Int. C. interactoins modulate epidermal patterning in Drosophila GOIN 33/566 (2006.01) embryos', Devel. 119:501-517 (1993). CI2N 5/10 (2006.01) BienZ, M., "Homeotic genes and positional signalling in the CI2N 15/63 (2006.01) Drosophila viscera ', TIG 10:22-26 (Jan. 1994). CI2N I/2 (2006.01) CI2N I/17 (2006.01) (Continued) (52) U.S. Cl. ....................... 435/7.21: 435/7.1; 435/7.2: Primary Examiner Robert S. Landsman 435/69.1: 435/325; 435/348; 435/320.1; 435/252.3: Assistant Examiner—Zachary C. Howard 435/254.2:536/23.1536/23.5; 530/350 (74) Attorney, Agent, or Firm Fish & Neave IP Group of (58) Field of Classification Search ................. 435/7.1, Ropes & Gray LLP 435/7.2, 7.21, 69.1, 325, 348,320.1, 248, 435/252.3, 254.2: 536/23.1, 23.5; 530/350 (57) ABSTRACT See application file for complete search history. (56) References Cited The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here U.S. PATENT DOCUMENTS hedgehog-related genes, comprise morphogenic signals pro duced by embryonic patterning centers, and are involved in 5,223,408 A 6, 1993 Goddel et al. ............. 435/69.3 the formation of ordered spatial arrangements of differenti 5,585,087 A 12/1996 Lustig et al. ................ 424.9.2 ated tissues in vertebrates. The present invention makes 5,837,538 A * 11/1998 Scott et al. ..... 435/325 available compositions and methods that can be utilized, for 6,027,882 A 2/2000 Scott et al. .................... 435/6 example to generate and/or maintain an array of different FOREIGN PATENT DOCUMENTS vertebrate tissue both in vitro and in vivo. WO WO 90,02809 3, 1990 WO WO 92.15679 9, 1992 29 Claims, 24 Drawing Sheets US 7,060,450 B1 Page 2 OTHER PUBLICATIONS Fallon, J.F. et al., “FGF-2: Apical ectodermal ridge growth Bitgood, M. and McMahon, A., “Hedgehod and Bmp Genes signal for chick limb development, Science 264: 104 are Coexpressed at Many Diverse Sites of Cell-Cell Interna 107(1994). tion in the Mouse Embryo”, Dev. Biol. 172 (1):126-138 Fan, C. et al., "Long-range Sclerotome induction by Sonic (1995). hedgehod: Direct role of the amino-terminal cleavage prod Blair, S.S., “Hedgehog digs up an old Friend’, Nature uct and modulation by the cyclic AMP signaling pathway'. 373:656-657 (Feb. 23, 1995). Cell 81: 457-465 (May 5, 1995). Brand-Saberi, B. et al., “The ventralizing effect of the Fietz, M. et al., The hedgehog gene family in Drosophila and notochord on somite differentiation in chick embryos, Anat. vertebrate development, Develop. Supp: 43-51 (1994). Embryol. 188:239-245 (1993). 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Gérard, M. et al., “Structure and activity of regulatory Chang et al., Products, genetic linkage and limb pattening elements involved in the activation of the Hoxd-11 gene activity of a murine hedgehog gene, Development during late gastrulation’, EMBO.J. 12:3539-3550 (1993). 120:3339-3353, Nov. 1994. Gurdon, J.B., “The Generation of diversity and pattern in Charité, J. et al., “Ectopic Expression of Hoxb-8 Causes animal development, Cell 68:185-199 (1992). Duplication of the ZPA in the Forelimb and Homeotic Gustin, K. et al., “Characterization of the role of individual Transformation of Axial Structures, Cell 78:589-601 protein binding motifs within the hepatitis B virus enhancer (1994). Coffman et al., “Xotch, the Xenopus homolog of Drosophila 1 on X promoter activity using linker scanning mutagenies. notch”, Science 249:1438-1441 (1990). Virolgy 193:653-660 (1993). Concordet, J. and Ingham, P., “Developmental biology. Hall, T., et al., “A potential catalytic site revealed by the Patterning goes sonic', Nature 375(6529):279-280 (May 1.7-A crystal structure of the amino-terminal signalling 1995). domain of Sonic hedgehog, Nature 378 (6553):212 Currie et al., “Induction of a specific muscle cell type by a 216(Nov. 1995). hedgehod-like protein in zebrafish', Nature 383:452-455 Halpern, M.E., et al., “Induction of muscle pioneers and (1996). floor plate is distinguished by the Zebrasish no tail muta Curry et al., “Sequence analysis reveals homology between tion, Cell 75:99-111 (1993). two proteins of the flagellar radial spoke', Mol. Cell. Biol. Hamburger, V. and H.L. Hamilton, “A series of normal 12:3967-3977 (1992). stages in the development of the chick embryo, J. Morph. Davidson, E.H., “How embryos work: a comparative view 88:49-92 (1951). of diverse modes of cell fate specification', Devel. 108:365 Hammerschmidt, M. et al., “The world according to hedge 389 (1990). hog, TIG 13(1):14-21 (1997). Davis, A.P. and M.R. Capecchi. “Axial homeosis and Haramis, A. et al., “The limb deformity mutation disrupts appendicular skeleton defects in mice with a targeted dis the SHH/FGF-4 feedback loop and regulation of 5-HoxD ruption of hoxd-1”, Devel. 120:2187-2198 (1994). genes during limb pattern formation, Devel. 121 (12:4161 Dickinson W., “Molecules and morphology: Where's the 4170 (Dec. 1995). homology’, TIG II, (4): 119-120 (1995). Hardy, A., et al., “Gene expression, polarising activity and Dollé, P. et al., “Coordinate expression of the murine Hox-5 complex homoeobox-containing genes during limb pattern skeletal patterning in reaggregated hind limb mesenchyme'. formation', Nature 342:767-772 (1989). Devel. 121 (12):4329-4337 (Dec. 1995). Echelard, Y, et al., “Sonic Hedgehog, a member of a family Hatta, K. et al., “The cyclops mutation blocks specification of putative signaling molecules, is implicated in the regu of the floor plate of the Zebrafish central nervous system’, lation of CNS polarity”, Cell 75:1417-1430 (1993).
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