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. 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