Patent Application: US-201113030703-A

Abstract:
the present invention is directed to isolated polypeptides and antibodies suitable for producing therapeutic preparations , methods , and kits relating to bone deposition . one objective of the present invention is to provide compositions that improve bone deposition . yet another objective of the present invention is to provide methods and compositions to be utilized in diagnosing bone dysregulation . the therapeutic compositions and methods of the present invention are related to the regulation of wise , sost , and closely related sequences . in particular , the nucleic acid sequences and polypeptides include wise and sost as well as a family of molecules that express a cysteine knot polypeptide .

Description:
the present invention is directed to compositions and methods that promote bone deposition in vertebrates . in particular , the present invention is directed to compositions and methods that antagonize the interaction between sost and wise proteins with their natural receptors particularly lrp5 and lrp 6 . for example , any peptide of at least 20 , preferably 25 , 30 , 35 , 40 , 50 or more amino acids encoded by seq id no : 1 , 3 , 5 , 7 , 9 , 11 , 13 , 19 , 83 , 84 , 88 , 89 , 90 , 92 , 94 , 96 , 97 , 99 , 100 , 102 , 104 , 106 , 107 or 108 , or any fragment of any sequence thereof , may be used to raise antibodies suitable for antagonizing the interaction between sost and wise proteins with their natural receptors . preferably the immunogen selected is encoded by full length seq id no : 1 , 3 , 5 , 7 , 9 , 11 , 13 , 19 , 83 , 84 , 88 , 89 , 90 , 92 , 94 , 96 , 97 , 99 , 100 , 102 , 104 , 106 , 107 or 108 , preferably full length seq id no 1 or 3 . alternatively the proteins and peptides of seq id no : 2 , 4 , 6 , 8 , 10 , 12 , 14 - 18 , 20 , 85 - 87 , 91 , 93 , 95 , 98 , 101 , 103 , 105 , 109 - 140 or 202 - 214 may be used to raise antagonists of the present invention . preferable sequences in this regard are seq id no : 113 , 119 and most preferably 214 . a further embodiment are blocking peptides that antagonize the interaction between sost and wise proteins with their natural receptors . these include seq id no 21 - 82 . we have isolated the novel wnt inhibitor ; wise , that affects craniofacial anterior - posterior patterning , whose biochemical function we want to address . wise is a secreted molecule with a genomic structure containing two exons ( 200 and 400 bp ) and a large 2 . 5 kb intron ( fig1 a , 1 b ). the second exon encodes a cystein knot motif , which bears some homology to known dan -, and ccn - family members ( fig1 d , 1 e ). wise is mapped to human chromosome 7p21 . 1 , which is linked to the hoxa cluster by 10 . 6 mb ( fig1 c ). the four mammalian hox clusters are thought to have evolved from a single cluster , as in drosophila , we therefore searched other clusters for a possible wise family member . we found that both hoxb and hoxc clusters had an orf that was examined further . the hoxc cluster orf , at 4 mb upstream shares homology to the ccn family . the hoxb cluster contained an ore at 5 mb upstream . the hoxb orf encodes a known gene , sost ( fig1 c ). sost was positionally cloned from a familial mutation affecting bone density . sost and wise both share the same gene structure , and produce a secreted protein whose second exon ( 70 % homologous ) encodes a cystein knot ( fig1 b ). unlike the known cystein knot motifs from dan - ( cerberus , dan , gremlin , caronte ) or ccn - ( nov , ctgf , cyr61 ) family members , wise and sost cystein knots contain 8 cysteins instead of 9 ( fig1 d ). other molecules , mucin2 and vwf have cystein knots containing 10 cysteins , but are arranged in a manner similar to both the ccn - and dan - family ( fig1 d and 1e ). dan and ccn cystein knots share about 50 % homology to those of wise and sost ( fig1 e ). in addition to the cystein knot domain , ccn proteins also encode for insulin binding , von willderbrand ( bmp antagonist - like domain ), and tsp1 domains . however , the dan family appears to only encode for a cystein knot domain . other genes that encode a cystein knot domain include slits , vwf , mucins , and ndp ( fig1 d and 1e ). dan - family members are able to bind to and inhibit bmp proteins , and only cerberus has been shown , in addition , to bind and inhibit wnt activity . the cystein knots of sost and wise ( also termed ectodin and usag - 1 ) are very similar to that of the dan - family , thus not surprisingly that both , sost and wise , also function by binding to and inhibiting bmps ; where sost binds and inhibits bmp6 strongly and bmp7 weakly ( fig2 b ). whereas , wise inhibits the activity of ( strongest to weakest ) bmp7 ; bmp2 ; bmp4 ; then bmp6 ( fig2 a ). yet , wise appears to bind bmp2 the strongest , then bmp7 . the actions of wise as a bmp2 inhibitor is unclear as xenopus noggin ( bmp4 & amp ; 2 inhibitor ) injected animal cap assays tell a different story . wise is able to induce en2 at a distance in xenopus noggin animal cap assays , through the activation of wnt genes ( data not shown ). we wanted to test if wise injections could induce the neural gene ncam , like the bmp inhibitor noggin ( fig2 f ). we found that wise , alone , was unable to activate the expression of ncam , and thus unable to inhibit bmp4 and 2 like the known bmp inhibitor noggin . therefore , wise may function in vivo preferentially by binding to and inhibiting bmp7 , instead of bmp2 and 4 . we then asked if sost and wise could be redundant by looking to see if sost could also induce en2 , like wise . xenopus embryos were either injected with noggin and / or with sost or wise . we found that neither wise nor sost could induce en2 without the addition of noggin . however , wise and noggin injected animal caps induced en2 expression ; however sost and noggin injected caps did not ( fig2 f ). this unexpected finding leads us to examine these two genes more closely . despite their similarity to the dan - family , the cystein knots of wise and sost appear to be most similar to that of the ccn - family ( fig1 d , 1 e ). ccn - family members , cyr61 and ctgf , are known inhibitors of both wnt and bmp pathways . interestingly , dan - family member cerberus appears to bind wnt proteins directly , whereas ctgf binds the wnt co - receptor lrp1 and lrp6 , and it is unknown as to how cyr61 inhibits the pathways . itasaki et al . ( 2003 ) showed that the cystein knot of wise functioned to inhibit the wnt pathway by binding lrp6 . we were curious if sost could function in a similar fashion . sost rna was either microinjected alone or in combination with other factors into xenopus embryos and dorsal marginal zones were assayed for early immediate wnt response genes , siamois and xnr3 ( fig2 g ). we found that , like wise , sost was able to inhibit the action of wnt on siamois and xnr3 ( fig2 g ). this wnt inhibition by sost was found to be working upstream from beta - catenin ( fig2 g ). like wise , sost was also able to rescue secondary axis formation by wnt ( fig2 c , 2 d , 2 e ). however , unlike wise , sost was unable to completely restore a normal axis ( fig2 e ). the inhibition of wnt activity by wise is from an interaction with the first two egf / ywtd propeller repeats found in the amino - terminal of lrp6 . we tested if sost acted in a similar fashion to inhibit the wnt pathway and found that like wise , sost was able to bind to lrp6 , and lrp5 ( fig2 h ). interestingly , sost is unable to bind to the human hbm g171v mutation ( fig2 h ). molecular dissection of sost revealed putative lrp binding sites located in the first arm of the cystein knot ( fig2 i and fig1 b ). in addition , upon destruction of the cystein knot , m8s , sost was unable to bind to lrp6 ( fig2 i ). in conclusion , we find that sost binds to and inhibits the activity of bmp6 strongly , and bmp7 weakly , whereas wise binds to and inhibits bmp7 strongly , and possibly bmp2 more weakly . in addition to bmp modulation , wise and sost bind lrp - 5 and - 6 to also modulate the wnt pathway ( fig2 i ). the wise mutant mice are viable and appear to develop an undulated retina ( fig3 d ) similar to that seen in patients with norrie - disease . 15 the retina of wise null mice have less optic nerve fibers ( fig3 f , 3 g ), however the optic nerve itself appears normal in diameter and trajectory ( fig3 h , 3 i ). additionally , they have an increased thickness of rods and cones layer ( fig3 f , 3 g ). neurofilament staining reveals a loss of horizontal cells within the inner nuclear bipolar cell layer ( fig3 j , 3 k ), which suggests that neighboring photoreceptors would be unable to communicate . retinal ganglion cell marker , pax6 , stains elongated neuronal cell bodies of the inner nuclear layer ( fig3 l ). in wise mutants , they appear rounded instead of elongated ( fig3 m ). wise protein is found in the inner plexiform layer , ganglion cell layer , and in the rod and cone layer of a 2 . 5 month mouse retina ( fig3 n ). unlike wise , sost is found in the tissues adjacent to the neuroepithelium of the diencephalon at e18dpc ( data not shown ). development of the teeth in wise mutants also shows abnormalities ; the incisors need weekly clipping from weaning onwards and the maxillary incisors are supernumerary ( fig4 k ). the molars display abnormal patterning with or without supernumerary buds ( fig4 m , 4 o ). the most severe phenotype is seen in the 1295v / ev mouse where three molars are often found in reverse orientation and fusion of m1 and m2 ( fig4 o ). a less severe phenotype is seen in the c57bl6 mouse , which displays supernumerary m1 molars ( fig4 m ). the maxillary molars in both strains develop severe fusion of all molars ( fig4 k ). the sost mutation does not present a tooth phenotype , probably because sost is expressed in the polarized odontoblasts ( which later gives rise to the periodontal ligament ) and the surrounding osteoblasts ( fig4 b , 4 e , 4 h ). wise , on the other hand , is expressed in the inner enamel epithelium and dental follicle surrounding the tooth bud , as well as in the maxillary incisors ( fig4 c , 4 f , 4 i ). thus , sost and wise are expressed in complementary cell types and thus result with a different tooth phenotype . one cell type that both genes appear to affect in a similar fashion is the bone . wise mutants have an increased alkaline phosphatase positive hypertrophic chondrocyte layer ( fig5 i , 5 j ) which results in an increase in cartilage matrix and bone deposition in the metaphysis plate ( fig5 g , 5 h ). both sost and wise are expressed in hypertrophic chondrocytes , osteoblasts and bone lining cells , and sost is also expressed in osteocytes ( fig5 a , 5 b , 5 e , 5 f , 5 n ). however , sost expression in early bone development ( e14 . 5dpc ) is restricted to the bone lining cells and not osteoblasts ( fig5 b ). this suggests that early bone deposition by osteoblasts is modulated by wise . then during bone remodeling ( 4 months ), once the bone lining cells re - differentiate into osteoblasts , it is sost that would modulate osteoblasts bone deposition instead of wise , as wise is no longer expressed ( fig5 e , 6 i ). in concurrence , wise mutant mice have increased bone density during early prenatal bone development ( under 4 months ), and cease to have an affect once bone - modeling starts ( 4 months ; fig5 o , 6 i ). sost mutations also leads to increased bone density , however this could be during later bone remodeling stages as wise is absent and can not compensate for its loss after 4 months ( fig5 e , 5 f , 6 i ). thus , wise functions to affect bone density before bone remodeling occurs by an increase in hypertrophic chondrocytes and osteoblasts ( fig5 a , 5 g , 5 j ). in addition to increased chondrocytes in the growth plate of 4 month wise mutants , we also observed thickened and extra phalanges ( astericks ; yellow circle , fig6 a , 6 b , 6 e , 6 f ). the vertebra and long bones revealed very slight increases in cortical thickness , mostly evident in the long bones ( fig6 c , 6 d , 6 g , 6 h ). consequently , we have found a new wise family member , sost . both wise and sost are linked to a hox cluster further supporting hox cluster duplication hypotheses . sost functions like wise to inhibit both bmp and wnt pathways , however , unlike wise , is unable to induce en2 expression . the inability to induce en2 is very similar to other cystein knot family members , like ctgf and nov . the phenotypes we observe in wise mutants are also similar to that of sost and lrp mutations , with some exceptions , ie . teeth . interestingly , itasaki et al . ( 2003 ) demonstrated that wise inhibits the wnt pathway by binding to an area encompassing the first two egf / ywtd propeller domains of lrp6 . yet , lrp6 null mice are lethal and do not resemble phenotypes seen in wise mutants . the autosomal recessive disorder that causes low peak bone mass , osteoporosis - pseudoglioma syndrome ( oppg ), has been shown to be due to an inactivation or deletion of lrp5 , and an autosomal dominant point mutation in lrp5 , g171v , results in a high bone mass disorder . furthermore . houston and wylie ( 2002 ) and gong et al . ( 2001 ) have shown that lrp5 is expressed in osteoblasts and in the retinal cell layer . therefore , the bone density phenotypes wise and sost are complementary to those in lrp5 mutants . however , the wise tooth phenotype is not seen in lrp5 or lrp6 mutants . interestingly , the runx2 null mouse develops supernumerary tooth buds , similar to those in wise . runx2 has been shown to be an important ossification selector gene for deciding between the osteoblast or chondrocyte lineage . previous studies have reported normal runx2 expression in an lrp5 null background . kato et al . ( 2002 ) concluded that lrp5 must affect bone density independently of runx2 . however , runx2 acts to regulate transcription of sost ( probably wise too ; fig6 k ), and we now report that sost / wise act to regulate bone deposition through binding to lrp5 / 6 . therefore , only one pathway exists for osteoblast differentiation - proliferation involving runx2 and lrp5 . runx2 acts upstream to regulate transcription of sost / wise , which in turn bind to lrp5 / 6 to regulate bone deposition ( fig6 k ). runx2 appears to function during hypertrophic chondrocytes differentiation . wise in turn acts to induce proliferation of the hypertrophic chondrocyte layer , which leads to an increase in cartilage matrix deposition and ultimately increased bone deposition ( fig6 i ). as disclosed herein , proteins , peptides and nucleic acids of the present invention may be isolated from natural sources , prepared synthetically or recombinantly , or any combination of the same . methods for isolating peptides and nucleic acids of the present invention are well known in the art . generally any purification protocol suitable for isolating nucleic acids or proteins can be used . for example , affinity purification as discussed below in the context of antibody isolation can be used in a more general sense to isolate any peptide or protein . nucleic acids can be purified using agarose gel electrophoresis , as is known in the art . column chromatography techniques , precipitation protocols and other methods for separating proteins and / or nucleic acids may also be used . ( see , e . g ., scopes , protein purification : principles and practice ( 1982 ); u . s . pat . no . 4 , 673 , 641 ; ausubel et al ., supra ; and sambrook et al ., supra ; and leonard et al ., j . biol . chem . 265 : 10373 - 10382 ( 1990 ). for example , peptides may be produced synthetically using solid phase techniques such as described in “ solid phase peptide synthesis ” by g . barany and r . b . merrifield in peptides , vol . 2 , edited by e . gross and j . meienhoffer , academic press , new york , n . y ., pp . 100 - 118 ( 1980 ). similarly , nucleic acids can also be synthesized using the solid phase techniques , such as those described in beaucage , s . l ., & amp ; iyer , r . p . ( 1992 ) advances in the synthesis of oligonucleotides by the phosphoramidite approach . tetrahedron . 48 , 2223 - 2311 ; and matthes et al ., embo j ., 3 : 801 - 805 ( 1984 ). modifications of peptides of the present invention with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo . stability can be assayed in a number of ways . for instance , peptidases and various biological media , such as human plasma and serum , have been used to test stability . see , e . g ., verhoef et al ., eur . j . drug metab pharmacokin . 11 : 291 - 302 ( 1986 ). half life of the peptides of the present invention is conveniently determined using a 25 % human serum ( v / v ) assay . the protocol is generally as follows . pooled human serum ( type ab , non - heat inactivated ) is delipidated by centrifugation before use . the serum is then diluted to 25 % with rpmi tissue culture media and used to test peptide stability . at predetermined time intervals a small amount of reaction solution is removed and added to either 6 % aqueous trichloracetic acid or ethanol . the cloudy reaction sample is cooled ( 4 ° c .) for 15 minutes and then spun to pellet the precipitated serum proteins . the presence of the peptides is then determined by reversed - phase hplc using stability - specific chromatography conditions . other useful peptide modifications known in the art include glycosylation and acetylation . in the case of nucleic acids , existing sequences can be modified using recombinant dna techniques well known in the art . for example , single base alterations can be made using site - directed mutagenesis techniques , such as those described in adelman et al ., dna , 2 : 183 , ( 1983 ). alternatively , nucleic acids can be amplified using pcr techniques or expression in suitable hosts ( cf . sambrook et al ., molecular cloning : a laboratory manual , 1989 , cold spring harbor laboratory , new york , usa ). peptides and proteins may be expressed using recombinant techniques well known in the art , e . g ., by transforming suitable host cells with recombinant dna constructs as described in morrison , j . bact ., 132 : 349 - 351 ( 1977 ); and clark - curtiss & amp ; curtiss , methods in enzymology , 101 : 347 - 362 ( wu et al ., eds , 1983 ). peptides and nucleic acids of the present invention may also be available commercially , or may be produced commercially , given the structural and / or functional properties of the molecules desired . the present invention also contemplates agents that antagonize binding of sost and / or wise to its native receptor ( s ) (“ sost agonist ”). sost agonosts include small organic molecules including a peptidomimetic , which is an organic molecule that mimics the structure of a peptide ; or a peptoid such as a vinylogous peptoid . additional nonpeptide , small organic molecule sost agonists useful in a method of the invention can be identified by screening assays as described herein . preferred embodiments of the present invention include sost agonists that are preferably sost antibodies , wise antibodies or lrp antibodies , although the invention also contemplates inhibitory peptides and small molecular inhibitors as described above . antibodies of the invention are preferably chimeric , more preferably humanized antibodies , ideally monoclonal antibodies preferably raised against murine proteins , most preferably murine sost . methods for producing such antibodies are discussed immediately below . sost antagonist antibodies , including anti - sost antibodies , may be raised using as an immunogen , such as a substantially purified full length protein , such as murine sost , but may also be a sost , wise or lrp protein of human , mouse or other mammalian origin . the immunogenmay be prepared from natural sources or produced recombinantly , or a peptide portion of a protein , which can include a portion of the cystiene knot domain , for example , a synthetic peptide . a non - immunogenic peptide may be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin ( bsa ) or keyhole limpet hemocyanin ( klh ), or by expressing the peptide portion as a fusion protein . various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described , for example , by harlow and lane ( supra , 1988 ). particularly useful antibodies for performing methods of the invention are monoclonal antibodies that that specifically bind to lrp molecules , wise or , most preferably , sost . such antibodies are particularly useful where they bind sost with at least an order of magnitude greater affinity than they bind another protein . methods for creating chimeric antibodies , including humanized antibodies , is discussed in greater detail below . methods for producing both monoclonal and polyclonal antibodies from identified proteins or peptides are well known in the art . in order to prepare recombinant chimeric and humanized antibodies that may function as sost antagonists of the present invention , the nucleic acid encoding non - human antibodies must first be isolated . this is typically done by immunizing an animal , for example a mouse , with prepared α5β1 integrin or an antigenic peptide derived therefrom . typically mice are immunized twice intraperitoneally with approximately 50 micrograms of protein antibody per mouse . sera from immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by elisa with the expressed polypeptide . for immunohistology , active antibodies of the present invention can be identified using a biotin - conjugated anti - mouse immunoglobulin followed by avidin - peroxidase and a chromogenic peroxidase substrate . preparations of such reagents are commercially available ; for example , from zymad corp ., san francisco , calif . mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production . positive supernatants of such hybridomas can be identified using the assays common to those of skill in the art , for example , western blot analysis . the nucleic acids encoding the desired antibody chains can then be isolated by , for example , using hybridoma mrna or splenic mrna as a template for pcr amplification of the heavy and light chain genes [ huse , et al ., science 246 : 1276 ( 1989 )]. nucleic acids for producing both antibodies and intrabodies can be derived from murine monoclonal hybridomas using this technique [ richardson j . h ., et al ., proc natl acad sci usa 92 : 3137 - 3141 ( 1995 ); biocca s ., et al ., biochem and biophys res comm , 197 : 422 - 427 ( 1993 ) mhashilkar , a , m ., et al ., embo j 14 : 1542 - 1551 ( 1995 )]. these hybridomas provide a reliable source of well - characterized reagents for the construction of antibodies and are particularly useful once their epitope reactivity and affinity has been characterized . isolation of nucleic acids from isolated cells is discussed further in clackson , t ., et al ., nature 352 : 624 - 628 ( 1991 ) ( spleen ) and portolano , s ., et al ., supra ; barbas , c . f ., et al ., supra ; marks , j . d ., et al ., supra ; barbas , c . f ., et al ., proc natl acad sci usa 88 : 7978 - 7982 ( 1991 ) ( human peripheral blood lymphocytes ). humanized antibodies optimally include at least a portion of an immunoglobulin constant region ( fc ), typically that of a human immunoglobulin [ jones et al ., nature , 321 : 522 - 525 ( 1986 ); riechmann et al ., nature , 332 : 323 - 329 ( 1988 ); and presta , curr . op . struct . biol ., 2 : 593 - 596 ( 1992 )]. a number of methods have been described to produce recombinant antibodies , both chimeric and humanized . controlled rearrangement of antibody domains joined through protein disulfide bonds to form chimeric antibodies may be utilized ( konieczny et al ., haematologia , 14 ( 1 ): 95 - 99 , 1981 ). recombinant dna technology can also be used to construct gene fusions between dna sequences encoding mouse antibody variable light and heavy chain domains and human antibody light and heavy chain constant domains ( morrison et al ., proc . natl . acad . sci . usa , 81 ( 21 ): 6851 - 6855 , 1984 .). dna sequences encoding the antigen binding portions or complementarity determining regions ( cdr &# 39 ; s ) of murine monoclonal antibodies may be grafted by molecular means into the dna sequences encoding the frameworks of human antibody heavy and light chains ( jones et al ., nature , 321 ( 6069 ): 522 - 525 , 1986 . ; riechmann et al ., nature , 332 ( 6162 ): 323 - 327 , 1988 .). the expressed recombinant products are called “ reshaped ” or humanized antibodies , and comprise the framework of a human antibody light or heavy chain and the antigen recognition portions , cdr &# 39 ; s , of a murine monoclonal antibody . other methods for producing humanized antibodies are described in u . s . pat . nos . 5 , 693 , 762 ; 5 , 693 , 761 ; 5 , 585 , 089 ; 5 , 639 , 641 ; 5 , 565 , 332 ; 5 , 733 , 743 ; 5 , 750 , 078 ; 5 , 502 , 167 ; 5 , 705 , 154 ; 5 , 770 , 403 ; 5 , 698 , 417 ; 5 , 693 , 493 ; 5 , 558 , 864 ; 4 , 935 , 496 ; 4 , 816 , 567 ; and 5 , 530 , 101 , each incorporated herein by reference . techniques described for the production of single chain antibodies ( u . s . pat . no . 4 , 946 , 778 ) can be adapted to produce single chain humanized antibodies to α5β1 integrin . affinity purification of an antibody pool or sera provides a practitioner with a more uniform reagent . methods for enriching antibody granulation inhibitors using antibody affinity matrices to form an affinity column are well known in the art and available commercially ( antibodyshop , c / o statens serum institut , artillerivej 5 , bldg . p2 , dk - 2300 copenhagen 5 ). briefly , an antibody affinity matrix is attached to an affinity support ( see e . g . ; cnbr sepharose ( r ), pharmacia biotech ). a mixture comprising antibodies is then passed over the affinity matrix , to which the antibodies bind . bound antibodies are released by techniques common to those familiar with the art , yielding a concentrated antibody pool . the enriched antibody pool can then be used for further immunological studies , some of which are described herein by way of example . a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis , by combining a number of chemical “ building blocks ” such as reagents . for example , a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks ( amino acids ) in every possible way for a given compound length ( i . e ., the number of amino acids in a polypeptide compound ). millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks . preparation and screening of combinatorial chemical libraries is well known to those of skill in the art . such combinatorial chemical libraries include , but are not limited to , peptide libraries ( see , e . g ., u . s . pat . no . 5 , 010 , 175 , furka , int . j . pept . prot . res . 37 : 487 - 493 ( 1991 ) and houghton et al ., nature 354 : 84 - 88 ( 1991 )). other chemistries for generating chemical diversity libraries can also be used . such chemistries include , but are not limited to : peptides ( e . g ., pct publication no . wo 91 / 19735 ), encoded peptides ( e . g ., pct publication wo 93 / 20242 ), random bio - oligomers ( e . g ., pct publication no . wo 92 / 00091 ), benzodiazepines ( e . g ., u . s . pat . no . 5 , 288 , 514 ), diversomers such as hydantoins , benzodiazepines and dipeptides ( hobbs et al ., proc . nat . acad . sci . usa 90 : 6909 - 6913 ( 1993 )), vinylogous polypeptides ( hagihara et al ., j . amer . chem . soc . 114 : 6568 ( 1992 )), nonpeptidal peptidomimetics with glucose scaffolding ( hirschmann et al ., j . amer . chem . soc . 114 : 9217 - 9218 ( 1992 )), analogous organic syntheses of small compound libraries ( chen et al ., j . amer . chem . soc . 116 : 2661 ( 1994 )), oligocarbamates ( cho et al ., science 261 : 1303 ( 1993 )), and / or peptidyl phosphonates ( campbell et al ., j . org . chem . 59 : 658 ( 1994 )), nucleic acid libraries ( see ausubel , berger and sambrook , all supra ), peptide nucleic acid libraries ( see , e . g ., u . s . pat . no . 5 , 539 , 083 ), antibody libraries ( see , e . g ., vaughn et al ., nature biotechnology , 14 ( 3 ): 309 - 314 ( 1996 ) and pct / us96 / 10287 ), carbohydrate libraries ( see , e . g ., liang et al ., science , 274 : 1520 - 1522 ( 1996 ) and u . s . pat . no . 5 , 593 , 853 ), small organic molecule libraries ( see , e . g ., benzodiazepines , baum c & amp ; en , january 18 , page 33 ( 1993 ); isoprenoids , u . s . pat . no . 5 , 569 , 588 ; thiazolidinones and metathiazanones , u . s . pat . no . 5 , 549 , 974 ; pyrrolidines , u . s . pat . nos . 5 , 525 , 735 and 5 , 519 , 134 ; morpholino compounds , u . s . pat . no . 5 , 506 , 337 ; benzodiazepines , 5 , 288 , 514 , and the like ). another approach uses recombinant bacteriophage to produce large libraries . using the “ phage method ” ( scott and smith , science 249 : 386 - 390 , 1990 ; cwirla , et al , proc . natl . acad . sci ., 87 : 6378 - 6382 , 1990 ; devlin et al ., science , 49 : 404 - 406 , 1990 ), very large libraries can be constructed ( 10 6 - 10 8 chemical entities ). a second approach uses primarily chemical methods , of which the geysen method ( geysen et al ., molecular immunology 23 : 709 - 715 , 1986 ; geysen et al . j . immunologic method 102 : 259 - 274 , 1987 ; and the method of fodor et al . ( science 251 : 767 - 773 , 1991 ) are examples . furka et al . ( 14th international congress of biochemistry , volume # 5 , abstract fr : 013 , 1988 ; furka , int . j . peptide protein res . 37 : 487 - 493 , 1991 ), houghton ( u . s . pat . no . 4 , 631 , 211 , issued december 1986 ) and rutter et al . ( u . s . pat . no . 5 , 010 , 175 , issued apr . 23 , 1991 ) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists . devices for the preparation of combinatorial libraries are commercially available ( see , e . g ., 357 mps , 390 mps , advanced chem tech , louisville ky ., symphony , rainin , woburn , mass ., 433a applied biosystems , foster city , calif ., 9050 plus , millipore , bedford , mass .). in addition , numerous combinatorial libraries are themselves commercially available ( see , e . g ., comgenex , princeton , n . j ., tripos , inc ., st . louis , mo ., 3d pharmaceuticals , exton , pa ., martek biosciences , columbia , md ., etc .). the present invention provides methods for identifying diagnostic and therapeutic sost antagonists . several exemplary methods for identifying such antagonists are described herein , including cell - based and in vitro techniques . a general method of identifying sost antagonists involves evaluating the effects of antagonist candidates on bone deposition under controlled conditions . preferably bone deposition is determined using x - ray techniques on live animals . preferred animals include rodents , more preferred are primates . hand or paw bones are particularly useful subjects for such study . briefly , the test animal is treated with a predetermined dose of a sost antagonist candidate . a control animal is treated with a control solution , preferably a non - irritating buffer solution or other carrier . when the sost antagonist candidate is delivered in a carrier , the control solution is ideally the carrier absent the sost antagonist candidate . multiple doses of the sost antagonist candidate may be applied to the test animal , preferably following a predetermined schedule of dosing . the dosing schedule may be over a period of days , more preferably over a period of weeks . once the dosing schedule has been completed , both test and control animals are examined to determine the level of bone deposition present . this may be accomplished by any suitable method , but is preferably performed on live animals using x - ray equipment . methods for x - ray examination of bones in animals are well known in the art . a sost antagonist candidate suitable for use as a sost antagonist is identified by noting significant bone deposition in the test animal when compared to the control animal . ideally bone deposition in the test bone ( s ) of the test animal should be at least 10 %, more preferably 20 %, most preferably 30 % or 40 % or more pone deposition than is present in the same bones of the control animal . where necessary , levels of bone deposition may be calculated by determining the volume of bone deposition present in each animal . calculations may be performed by constructing a 3 - dimensional image of the bone deposition and calculating the volume from the image with the aid of e . g ., computed axial tomography . in an exemplary embodiment , intravenous injection of a sost antagonist candidate , for example a monoclonal antibody described herein , may be made into a test animal , with a control animal receiving an equal volume of control solution without the sost antagonist candidate . identical dosing should be done on a weekly basis for four weeks . suitable dosage will depend on the nature of the particular sost antagonist candidate being tested . by way of example , in dosing it should be noted that systemic injection , either intravenously , subcutaneously or intramuscularly , may also be used . for systemic injection of a sost antagonist candidate or a sost antagonist , dosage should be about 5 mg / kg , preferably more preferably about 15 mg / kg , advantageously about 50 mg / kg , more advantageously about 100 mg / kg , acceptably about 200 mg / kg . dosing performed by nebulized inhalation , eye drops , or oral ingestion should be at an amount sufficient to produce blood levels of the sost antagonist candidate similar to those reached using systemic injection . the amount of sost antagonist candidate that must be delivered by nebulized inhalation , eye drops , or oral ingestion to attain these levels is dependent upon the nature of the inhibitor used and can be determined by routine experimentation . it is expected that , for systemic injection of the monoclonal antibody sost antagonist candidates described herein , therapeutic levels of the antibody may be detected in the blood one week after delivery of a 15 mg / kg dose . while the methods noted above can be used to identify any type of sost antagonist , they are best suited for screening sost antagonist candidates that are suspected as being sost antagonists , usually through some relationship to known sost antagonists ( e . g ., by belonging to the same chemical family or sharing some other structural or functional feature with a known sost antagonist .) moreover , novel sost antagonists may be identified using a process known as computer , or molecular modeling , as discussed below . computer modeling technology allows visualization of the three - dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule . the three - dimensional construct typically depends on data from x - ray crystallographic analyses or nmr imaging of the selected molecule . the molecular dynamics require force field data . the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity . prediction of what the molecule - compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers , usually coupled with user - friendly , menu - driven interfaces between the molecular design program and the user . an example of the molecular modelling system described generally above consists of the charmm and quanta programs , polygen corporation , waltham , mass . charmm performs the energy minimization and molecular dynamics functions . quanta performs the construction , graphic modelling and analysis of molecular structure . quanta allows interactive construction , modification , visualization , and analysis of the behavior of molecules with each other . a number of articles review computer modeling of drugs interactive with specific proteins , such as rotivinen , et . al ., acta pharmaceutica fennica 97 , 159 - 166 ( 1988 ); ripka , new scientist 54 - 57 ( jun . 16 , 1988 ); mckinaly and rossmann , annu . rev . pharmacol . toxiciol . 29 , 111 - 122 ( 1989 ); perry and davies , osar : quantitative structure - activity relationships in drug design pp . 189 - 193 ( alan r . liss , inc . 1989 ); lewis and dean , proc . r . soc . land . 236 , 125 - 140 and 141 - 162 ( 1989 ); and , with respect to a model receptor for nucleic acid components , askew , et al ., j . am . chem . soc . 111 , 1082 - 1090 ( 1989 ). askew et al . constructed a new molecular shape which permitted both hydrogen bonding and aromatic stacking forces to act simultaneously . askew et al . used kemp &# 39 ; s triacid ( kemp et al ., j . org . chem . 46 : 5140 - 5143 ( 1981 )) in which a u - shaped ( diaxial ) relationship exists between any two carboxyl functions . conversion of the triacid to the imide acid chloride gave an acylating agent that could be attached via amide or ester linkages to practically any available aromatic surface . the resulting structure featured an aromatic plane that could be roughly parallel to that of the atoms in the imide function ; hydrogen bonding and stacking forces converged from perpendicular directions to provide a microenvironment complimentary to adenine derivatives . other computer programs that screen and graphically depict chemicals are available from companies such as biodesign , inc ., pasadena , calif ., allelix , inc , mississauga , ontario , canada , and hypercube , inc ., cambridge , ontario . although these are primarily designed for application to drugs specific to particular proteins , they can be adapted to design of drugs specific to regions of rna , once that region is identified . whether identified from existing sost antagonists or from molecular modelling techniques , sost antagonists generally must be modified further to enhance their therapeutic usefulness . this is typically done by creating large libraries of compounds related to the sost antagonist , or compounds synthesized randomly , based around a core structure . in order to efficiently screen large and / or diverse libraries of sost antagonist candidates , a high throughput screening method is necessary to at least decrease the number of candidate compounds to be screened using the assays described above . high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds ( potential modulator or ligand compounds ). such “ combinatorial chemical libraries ” or “ candidate libraries ” are then screened in one or more assays , as described below , to identify those library members ( particular chemical species or subclasses ) that are able to promote bone deposition . the compounds thus identified can serve as conventional “ lead compounds ” or can themselves be used as potential or actual therapeutics . candidate compounds of the library can be any small chemical compound , or a biological entity , such as a protein , sugar , nucleic acid or lipid , as described previously . typically , test compounds will be small chemical molecules and peptides . the assays discussed below are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays , which are typically run in parallel ( e . g ., in microtiter formats on microtiter plates or similar formats , as depicted in fig5 , in robotic assays ). it will be appreciated that there are many suppliers of chemical compounds , including sigma ( st . louis , mo . ), aldrich ( st . louis , mo . ), sigma - aldrich ( st . louis , mo . ), fluka chemika - biochemica analytika ( buchs switzerland ) and the like . accordingly , the present invention provides methods for high throughput screening of granulation inhibitor candidates . the initial steps of these methods allow for the efficient and rapid identification of combinatorial library members that have a high probability of being sost antagonists . these initial steps take advantage of the observation that sost antagonists are also lrp or sost binding agents . any method that determines the ability of a member of the library , termed a binding candidate , to specifically bind to sost , wise or an lrp protein is suitable for this initial high throughput screening . for example , competitive and non - competitive elisa - type assays known to one of ordinary skill in the art may be utilized . binding candidates that are found to bind sost , wise or an lrp protein with acceptable specificity , e . g ., with a k a for sost , wise or an lrp protein of at least about 10 5 mol − 1 , 10 6 mol − 1 or greater , preferably 10 7 mol − 1 or greater , more preferably 10 8 mol − 1 or greater , and most preferably 10 9 mol − 1 or greater , are sost antagonist candidates and are screened further , as described above , to determine their ability to promote bone deposition . a number of well - known robotic systems have been developed for solution phase chemistries . these systems include automated workstations like the automated synthesis apparatus developed by takeda chemical industries , ltd . ( osaka , japan ) and many robotic systems utilizing robotic arms ( zymate ii , zymark corporation , hopkinton , mass . ; orca , hewlettpackard , palo alto , calif . ), which mimic the manual synthetic operations performed by a chemist . any of the above devices are suitable for use with the present invention . the nature and implementation of modifications to these devices ( if any ) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art . in addition , numerous combinatorial libraries are themselves commercially available ( see , e . g ., comgenex , princeton , n . j ., asinex , moscow , ru , tripos , inc ., st . louis , mo ., chemstar , ltd , moscow , ru , 3d pharmaceuticals , exton , pa ., martek biosciences , columbia , md ., etc .). individuals to be treated using methods of the present invention may be any individual suffering from bone loss , such as a sufferer of osteoporosis or simply an individual recovering from a broken limb . such an individual is a vertebrate such as a mammal , including a dog , cat , horse , cow , or goat ; a bird ; or any other animal , particularly a commercially important animal or a domesticated animal , more particularly a human being . methods of the present invention are suitable for use on any individual suffering bone loss as a result of injury or disease . some embodiments of the methods described herein are particularly suited for treatment of osteoporosis . in therapeutic use sost antagonists generally will be in the form of a pharmaceutical composition containing the antagonist and a pharmaceutically acceptable carrier . pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other buffers or solvents or vehicles such as glycols , glycerol , oils such as olive oil or injectable organic esters . the selection of a pharmaceutically acceptable carrier will depend , in part , on the chemical nature of the sost antagonist , for example , whether the sost antagonist is an antibody , a peptide or a nonpeptide , small organic molecule . a pharmaceutically acceptable carrier may include physiologically acceptable compounds that act , for example , to stabilize the sost antagonist or increase its absorption , or other excipients as desired . physiologically acceptable compounds include , for example , carbohydrates , such as glucose , sucrose or dextrans , antioxidants , such as ascorbic acid or glutathione , chelating agents , low molecular weight proteins or other stabilizers or excipients . one skilled in the art would know that the choice of a pharmaceutically acceptable carrier , including a physiologically acceptable compound , depends , for example , on the route of administration of the sost antagonist and on its particular physio - chemical characteristics . the methods of the present invention include application of sost antagonists in cocktails including other medicaments , for example , antibiotics , fungicides , and anti - inflammatory agents . alternatively , the methods may comprise sequential dosing of an afflicted individual with a sost antagonist and one or more additional medicaments to optimize a treatment regime . in such optimized regimes , the medicaments , including the granulation inhibitor may be applied in any sequence and in any combination . bone loss resulting from injury or disease can occur locally , for example , in the case of a broken bone , or may be more systemic , for example in a person suffering from osteoporosis . depending on the bone , nature of the disease or injury , one skilled in the art would select a particular route and method of administration of the sost antagonist . the sost antagonists of the present invention may also be included in slow release formulations for prolonged treatment following a single dose . in one embodiment , the formulation is prepared in the form of microspheres . the microspheres may be prepared as a homogenous matrix of a sost antagonist with a biodegradable controlled release material , with optional additional medicaments as the treatment requires . the microspheres are preferably prepared in sizes suitable for infiltration and / or injection , and injected systemically , or directly at the site of treatment . the formulations of the invention are also suitable for administration in all body spaces / cavities , including but not limited to pleura , peritoneum , cranium , mediastinum , pericardium , bursae or bursal , epidural , intrathecal , intraocular , etc . some slow release embodiments include polymeric substances that are biodegradable and / or dissolve slowly . such polymeric substances include polyvinylpyrrolidone , low - and medium - molecular - weight hydroxypropyl cellulose and hydroxypropyl methylcellulose , cross - linked sodium carboxymethylcellulose , carboxymethyl starch , potassium methacrylate - divinylbenzene copolymer , polyvinyl alcohols , starches , starch derivatives , microcrystalline cellulose , ethylcellulose , methylcellulose , and cellulose derivatives , β - cyclodextrin , poly ( methyl vinyl ethers / maleic anhydride ), glucans , scierozlucans , mannans , xanthans . alzinic acid and derivatives thereof , dextrin derivatives , glyceryl monostearate , semisynthetic glycerides , glyceryl palmitostearate , glyceryl behenate , polyvinylpyrrolidone , gelatine , agnesium stearate , stearic acid , sodium stearate , talc , sodium benzoate , boric acid , and colloidal silica . slow release agents of the invention may also include adjuvants such as starch , pregelled starch , calcium phosphate mannitol , lactose , saccharose , glucose , sorbitol , microcrystalline cellulose , gelatin , polyvinylpyrrolidone . methylcellulose , starch solution , ethylcellulose , arabic gum , tragacanth gum , magnesium stearate , stearic acid , colloidal silica , glyceryl monostearate , hydrogenated castor oil , waxes , and mono -, bi -, and trisubstituted glycerides . slow release agents may also be prepared as generally described in wo 94 / 06416 . the amount of sost antagonist administered to an individual will depend , in part , on the disease and extent of injury . methods for determining an effective amount of an agent to administer for a diagnostic or a therapeutic procedure are well known in the art and include phase i , phase ii and phase iii clinical trials . generally , an agent antagonist is administered in a dose of about 0 . 01 to 200 mg / kg body weight when administered systemically , and at a concentration of approximately 1 μm , when administered directly to a wound site . the total amount of sost antagonist can be administered to a subject as a single dose , either as a bolus or by infusion over a relatively short period of time , or can be administered using a fractionated treatment protocol , in which the multiple doses are administered over a more prolonged period of time . one skilled in the art would know that the concentration of a particular sost antagonist required to provide an effective amount to a region or regions of injury depends on many factors including the age and general health of the subject as well as the route of administration , the number of treatments to be administered , and the nature of the sost antagonist , including whether the sost antagonist is an antibody , a peptide , or a non - peptide small organic molecule . in view of these factors , the skilled artisan would adjust the particular dose so as to obtain an effective amount for efficaciously promoting bone deposition for therapeutic purposes . all publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference . although the foregoing invention has been described in some detail by way of illustration and example for clarity and understanding , it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims . as can be appreciated from the disclosure provided above , the present invention has a wide variety of applications . accordingly , the following examples are offered for illustration purposes and are not intended to be construed as a limitation on the invention in any way . those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results . isolation of sost cdna . sost was isolated from a mouse day 11 cdna library using touchdown pcr ( 1 cycle , denature 92 ° c . 2 ′; 5 cycles , 92 ° c . 1 ′, anneal 68 ° c . 1 ′, extend 72 ° c . 2 ′; 30 cycles , 92 ° c . 1 ′, anneal 64 ° c . 1 ′, extend 72 ° c . 2 ′; 1 cycle , extension 72 ° c . 10 ′). forward primer , cgtgcctcatctgcctacttgtgca ; reverse primer , gaagtccttgagctccgactggttgtg . 1 ul of dmso was added to the reactions . the full length mouse sost cdna was created using successive pcr reactions based on gi : 13161022 . wise mutant mouse ( 129sv / ev & amp ; c57bl6 ). a neolacz cassette containing stop codons at the 3 ′ end was inserted into a wise gdna sequence isolated bac from a 129svev mouse . the cassette was inserted into the first exon of wise using smai and ecori sites . the modified bac was then used for homologous recombination after electroporation into 129svev mouse es cells . specific integrants were selected from southern analysis using a 3 ′ probe within exon 2 of wise . ecori digests yielded either a 6 . 8 kb fragment associated with homologous recombination , or a 9 kb fragment associated with a random integration event . bioinformatics . phylogenetic tree : sost and wise cystein knot protein sequences were blasted ( ncbi ) and all significant sequences were isolated . the cystein knots from all sequences were manually aligned using the software t - coffee and then analyzed with phylip bootstrap neighbor joining methods . chromosomal location : wise and sost dna sequences where blasted against the mouse ( mus musculus ) ensembl database ( http :// www . ensembl . org / mus — musculus / blastview ). xenopus assays . capped rna synthesis - 5 ug linear dna template ( sost , noggin , beta - catenin , wnt8 , wise ) was added to the following ; 5 × transcription buffer ( p118b promega ), 0 . 1m dtt , 0 . 1m atp , 0 . 1m ctp , 0 . 1m utp , 0 . 1m gtp , 5 mm cap ( nen 514045 ), rnasin , and polymerase of choice . rna used in cap assays : 250 pg noggin ; sost 300 pg , 600 pg , 900 pg ; 5 pg wnt8 . 1 rna for ventral marginal zones : sost 300 pg , wnt8 100 pg , beta - catenin 200 pg . bmp assay . atdc - 5 at low passage were consistently grown at subconfluency in dmem / f12 media supplemented with 10 % heat inactivated fetal calf serum , 100 units / ml penicillin , and 100 mg / ml streptomycin . in 96 well plates ( corning ) inhibitors were diluted . a constant amount of bmp ( r and d systems ) was added to each well and incubated for 1 hour at 37 ° c . atdc - 5 cells were counted and plated at 2 × 10 5 cells / ml . heparin was added to the plate containing bmp4 at a final concentration of 2 μg / ml . l - ascorbic acid was added to the plate containing bmp6 at a final concentration of 50 μg / ml . cells were then incubated for 3 days at 37 ° c . cell layers were washed twice with pbs and lysed in 0 . 15 m nacl , 3 mm nahco 3 , and 0 . 1 % triton x - 100 at ph 9 . 3 . cell layers were incubated at 37 ° c . for 30 mins . 20 p . 1 of each sample was incubated with 1 mg / ml of p - nitrophenyl phosphate ( sigma ) in 1 m diethanolamine ( sigma ) with 0 . 5 mm mgcl 2 at a ph of 9 . 8 and then incubated at 22 ° c . for 8 mins . reaction was stopped by the addition of 0 . 5 n naoh . optical density was measured at 405 nm . immunoprecipitation . a 10 mm dish of 293 cells was transfected with 10 ug of lrp5 , lrp6 , sost or wise , using fugene 6 ( roche ). pcs2 + lrp5 contains the extracellular portion of the human sequence between ecori - xbai . pcs2 + sost contain the whole reading frame and has been modified at the 3 ′ end to have a kozak sequence and flag tag . wise - flag , and lrp6 - igg - fc are described in itasaki et al . ( 2003 ). pcs2 + with an igg - fc insert is the vector control . the supernatants were collected on day1 , day 2 , and day3 . the supernatant was concentrated through an appropriate molecular weight amicon ultra ( millipore ) spin column . a protein concentration was taken on the concentrated supernatants , and 50 ug of each sample was used in each ip . anti - flag m2 affinity gel ( sigma ) was used for the wise and sost ips . tooth radioactive in situs . c57b6 j mice ( jackson laboratories ) were mated . the day of identification of a vaginal plug was considered e0 . 5 , and the day of birth p0 . embryos were harvested at day 16 . 5 , embedded in oct ( vwr ), and quick frozen in isopentane on dry ice . 14 um cryostat sections were collected on probe - on plus slides and stored at − 80 ° c . with dessicant prior to hybridization . sections were equilibrated to room temperature , fixed in 4 % paraformaldehyde in pbs for 20 min , rinsed twice in pbs with 0 . 1m glycine , once in pbs alone , acetylated in 0 . 1m triethanolamine ( ph 8 . 0 ) and 0 . 25 % acetic anhydride for 10 min , rinsed twice more in pbs , and then dehydrated . hybridization was carried out using 35 s - labeled antisense probes . hybridization and post hybridization protocol as described by gall et al . ( 1995 ). 23 slides were dipped in kodak ntb - 2 liquid emulsion diluted 1 : 1 with distilled water , and exposed for 21 - 28 days at − 80 ° c . developed sections were then h and e stained . x - rays & amp ; teeth dissections . the mice were dissected and the jaws were placed in a proteinase k solution ( 2 × ssc , 0 . 2 % sds , 10 mm edta , and 100 ul of 10 mg / ml proteinase k ) overnight at 55 ° c . the next day the jaws are air - dried and a digital faxitron was used for capturing x - ray images of the mouse maxilla . the teeth were removed using tweezers . bone & amp ; retinal immunochemistry . chick hh45 femurs were harvested and fixed in 3 . 5 % pfa . the tissue was then processed for cryosectioning . mouse retinas were harvested from p0 and p2 . 5 month . the retinas were fixed in formalin and processed for paraffin sectioning . immunochemistry was preformed using pbs ( with or without triton ) and 10 % gs . the primary antibodies used were a custom made peptide antibody against chick wise ( 1 : 100 ), chick pax6 ( 1 : 10 ) and mouse 2h3 ( 1 : 10 ) from hybridoma bank . bone density . bone densitometry was measured using a piximus mouse densitometer ( ge medical syetms ). bone minieral and body composition are measured using dual xray absorptiometry ( dexa ). trap and alp . staining was preformed on crysectioned mouse femurs . trap ( tartrate resistant acid phosphatase ) staining was done as per manufactured protocol for sigma acid phosphatase kit ( 181a ). alkaline phospatase ( alp ) staining was carried out for 15 minutes at rt in 100 mm tris - maleate ( ph 9 . 2 ), naphthol as - mx phosphate and fast red tr . bone ish . sum cryosections of mouse femurs were dried for 2 hours to overnight at room temperature . rinse slides in 30 ° c . water to melt gelatin . rinse in pbs , 2 × ssc . hybrize using 1 ug / ml dig - labelled probe in a humidified chamber containing depc water . coverslip slides and incubate overnight at 65 ° c . posthyb wash for 2 × 10 minutes in 50 % formamide , 1 × ssc , 0 . 1 % tween 20 . wash 2 × mabt 10 minutes , incubate 30 minutes with blocking buffer ( 20 % goat serum , 20 % bbr , 60 % mabt ). add anti - dig ap 1 : 2000 and incubate overnight at room temp . wash mabt 5 minutes , ntmt 10 minutes , then reveal in ntmt with nbt / bcip . stop reaction with pbst . 1 . itasaki , n . et al . wise , a context - dependent activator and inhibitor of wnt signalling . development 130 , 4295 - 305 ( 2003 ). 2 . brunkow , m . e . et al . bone dysplasia sclerosteosis results from loss of the sost gene product , a novel cystine knot - containing protein . am j hum genet 68 , 577 - 89 ( 2001 ). 3 . kusu , n . et al . sclerostin is a novel secreted osteoclast - derived bone morphogenetic protein antagonist with unique ligand specificity . j biol chem 278 , 24113 - 7 ( 2003 ). 4 . balemans , w . et al . increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein ( sost ), hum mol genet 10 , 537 - 43 ( 2001 ). 5 . sasai , y . et al . xenopus chordin : a novel dorsalizing factor activated by organizer - specific homeobox genes . cell 79 , 779 - 90 ( 1994 ). 6 . piccolo , s . et al . the head inducer cerberus is a multifunctional antagonist of nodal , bmp and wnt signals . nature 397 , 707 - 710 ( 1999 ). 7 . hsu , d . r ., economides , a . n ., wang , x ., eimon , p . m . & amp ; 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