Screening assays for hedgehog agonists and antagonists

Abstract

The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here hedgehog-related genes, comprise morphogenic signals produced by embryonic patterning centers, and are involved in the formation of ordered spatial arrangements of differentiated tissues in vertebrates. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

1 - 62 . (canceled) 
     
     
         63 . The isolated vertebrate hedgehog polypeptide of  claim 64 , wherein said polypeptide is recombinantly produced. 
     
     
         64 . An isolated vertebrate hedgehog polypeptide, wherein said polypeptide binds to a naturally-occurring patched protein. 
     
     
         65 . A pharmaceutical composition comprising a vertebrate hedgehog polypeptide that binds to a naturally-occurring patched protein formulated in a pharmaceutically acceptable carrier. 
     
     
         66 . The pharmaceutical composition of  claim 65 , wherein said vertebrate hedgehog polypeptide is recombinantly produced. 
     
     
         67 . An isolated nucleic acid comprising a nucleotide sequence encoding a vertebrate hedgehog polypeptide, wherein the polypeptide binds to a naturally-occurring patched receptor. 
     
     
         68 . The nucleic acid of  claim 67 , further comprising
 at least one transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.   
     
     
         69 . An expression vector, which replicates in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of  claim 68 . 
     
     
         70 . A host cell transfected with the expression vector of  claim 69  and expressing said polypeptide. 
     
     
         71 . A method of producing a recombinant hedgehog polypeptide comprising culturing the cell of  claim 70  in a cell culture medium to express said hedgehog polypeptide, and expressing and isolating said polypeptide from said cell culture. 
     
     
         72 . A method for promoting one or more of growth, differentiation, or survival of a neuronal cell, comprising contacting said cell with an amount of a hedgehog polypeptide effective to promote one or more of growth, differentiation, or survival of said neuronal cell, wherein said hedgehog polypeptide binds a naturally occurring patched receptor. 
     
     
         73 . The method of  claim 74 , wherein said mammalian cell is a mammalian neuronal cell. 
     
     
         74 . A method for promoting one or more of growth, differentiation, or survival of a mammalian cell responsive to hedgehog induction, comprising contacting the cell with an amount of a hedgehog polypeptide effective to promote, relative to the cell in the absence of hedgehog treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell, wherein said hedgehog polypeptide binds a naturally occurring patched receptor. 
     
     
         75 . An isolated antibody that binds to a vertebrate hedgehog polypeptide. 
     
     
         76 . The isolated antibody of  claim 75 , wherein the antibody binds to a mammalian hedgehog polypeptide. 
     
     
         77 . The isolated antibody of  claim 76 , wherein the antibody binds to a mammalian Sonic hedgehog polypeptide. 
     
     
         78 - 79 . (canceled) 
     
     
         80 . An antibody preparation comprising an antibody that specifically binds to a vertebrate hedgehog polypeptide. 
     
     
         81 . The antibody preparation of  claim 80 , wherein said hedgehog polypeptide is a mammalian Sonic hedgehog polypeptide. 
     
     
         82 - 83 . (canceled) 
     
     
         84 . The antibody preparation of  claim 81 , wherein said polypeptide is a Sonic hedgehog polypeptide. 
     
     
         85 . A method of detecting hedgehog expression in a cell comprising measuring hedgehog mRNA expression or hedgehog protein expression in said cell. 
     
     
         86 - 87 . (canceled) 
     
     
         88 . A method for promoting hedgehog signaling in a cell, comprising administering to the cell an amount of a hedgehog agonist effective to promote hedgehog signaling. 
     
     
         89 . A method for inhibiting hedgehog signaling in a cell, comprising administering to the cell an amount of a hedgehog antagonist effective to inhibit hedgehog signaling. 
     
     
         90 . The method of  claim 88 , wherein the hedgehog agonist is a hedgehog polypeptide. 
     
     
         91 . The method of  claim 89 , wherein the hedgehog antagonist is an antibody that binds a vertebrate hedgehog polypeptide. 
     
     
         92 . A method for inhibiting one or more of growth, differentiation, or survival of a mammalian cell responsive to hedgehog induction, comprising contacting the cell with an amount of a hedgehog antagonist effective to inhibit, relative to the cell in the absence of treatment with a hedgehog antagonist, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. 
     
     
         93 . The method of  claim 92 , wherein the hedgehog antagonist is an antibody that binds a vertebrate hedgehog polypeptide. 
     
     
         94 . A method for inhibiting one or more of growth, differentiation, or survival of a cell, comprising contacting said cell with an amount of a hedgehog antagonist effective to inhibit one or more of growth, differentiation, or survival of said neuronal cell. 
     
     
         95 . The method of  claim 94 , wherein the hedgehog antagonist is an antibody that binds a vertebrate hedgehog polypeptide. 
     
     
         96 . (canceled) 
     
     
         97 . The method of  claim 94 , wherein said cell is a neuronal cell. 
     
     
         98 . The method of  claim 94 , wherein said cell is a mammalian cell and wherein said antagonist is an antibody preparation comprising an antibody that specifically binds to a mammalian hedgehog polypeptide.