IL134656A - Antibodies which bind a noggin polypeptide and hybridoma capable of producing such antibodies - Google Patents

Abstract

Novel dorsal growth inducing factors, complexes including the factors, and DNA or RNA coding sequences for the factors are described.

Description

10777/00 134656/2 ίΤ7Ν θ τη] ns"1? ϋΊ¾ιπηπ nnvmrm r;m T Dgg^ig D_,nuni?n D^TJU ANTIBODIES WHICH BIND A NOGGIN POLYPEPTIDE AND HYBRIDOMA CAPABLE OF PRODUCING SUCH ANTIBODIES The present application is a divisional patent application from Israel Patent Application No. 106894 (hereafter "the parent application"). The present application relates to antibodies directed against dorsal tissue affecting factor. e parent invention generally relates to growth factors and neurotrophic factors, and more particularly to a soluble growth factor with dorsai growth inducing activity, to complexes including the factor, and to DMA or RNA coding sequences for the factor.

.Growth factors are substances, such as polypeptide hormones, which affect the growth of defined populations of animal ceils in vivo or in vitro, but which are not nutrient substances. Proteins involved in the growth and differentiation of tissues may promote or inhibit - growth, and promote or inhibit differentiation, and thus the general term "growth- factor." includes cytokines and trophic factors. Among . growth, or neurotrophic factors presently known are/those that can be classified into the insulin family [insulin, insu!in-like growth λ factors (e.g., IGF-!, !GF-!I), mammary stimulating factor (MSF), and nerve growth factor (NGF)]; those classified into the epidermal growth factor family [epidermal growth factor (EGF) and transforming growth factors (TGFa, TGF , TGFy)]; those classified into the piateiet-derived growth factor family [platelet-derived growth factor (PDGF), osteosarcoma-derived growth factor (ODGF), and fibroblast growth factor (FGF)]; the neurotrophins [ner/e growth factor (NGF), brain derived neurotrophic factors (BDNF) neurotrophins 3, 4, 5, (NT-3, NT-4, NT-5)]; and others [colony stimulating factor (CSF), T-ceil growth factor, tumor angiogenesis factor (TAF), DNA synthesis promoting factor (DSF), tumor-derived growth factors, fibrobiast-derived growth factor (FDGF)].

Receptors that affect growth (that is, receptors for growth-associated iigands) are proteins found associated with cell surfaces that specifically bind their growth factors as Iigands.

Growth factor receptors are utilized in various clinical and diagnostic applications.

U.S. Patent 4,857,637, issued August 15, 1989, inventors Hammonds et ai., describes a method for immunizing an animal against its growth hormone receptor through use of vaccinating with antibodies in order to stimulate growth of the animals.

U.S. Patent 4,933,294, issued June 12, 1990, inventors Waterfield et al., describes studies of structural alterations of the human EGF receptor and its gene and a relationship in tumorigenesis for assays and therapies involving the human EGF receptor. For example, such assays can involve detection of structurally altered or abnormally expressed growth factor receptor and the mRNA transcripts and genes which encode them. EGF may have a role in cell proliferation and differentiation since it induces early eyelid opening and incisor development in new born mice.

U.S. Patent 5,030,576, issued July 9, 1991 , inventors Dull et al., describes the role of receptors, such as receptors for growth factors, in designing drugs by the pharmaceutical industry, and discloses use of a receptor hybrid for screening drug purposes, such as in studies of EGF binding domains. U.S. Patent 5,087,616, issued February 1 1 , 1992, inventors Myers and Bichon, describes a method for destroying tumor cells with a composition including a drug conjugate. The conjugate has a growth factor as one moiety and a polymeric carrier with a cytotoxic compound as another moiety. Thus, compositions of the patent are described as binding preferentially to tumor cells bearing EGF-binding receptors (when an EGF growth factor, for example, is used as a first moiety).

U.S. Patent 5,098,833, issued March 24, 1992, inventors Lasky, et ai., describes a DNA isolant capable of hybridizing to the epidermal growth factor domain. Expression systems for recombinant production are said to be useful in therapeutic or diagnostic compositions.

A good background review of a neurotrophic factor related to NGF is provided by W092/05254, published April 2, 1992, which also describes state of the art methods of: preparing amino acid sequence variations, site-directed mutagenesis techniques, ligation of coding DNA into a repiicable vector for further cloning or for expression, choice of promoters for expression vectors, suitable host cells for expression, particularly mammalian cells, protein purification upon recovery from culture medium as a secreted protein, derivatization with bifunctional agents to cross-link protein to a support matrix for use with antibodies, entrapment in systems for drug delivery, preparation of therapeutic formulations, and methods of administration. In addition, preparation of polyclonal and monocionai antibodies are described, such as are usefui in diagnostic assays, These various aspects of isolation, preparation, and applications for a novel neurotrophic factor, as illustrated by the W092/05254 publication, are incorporated herein by reference.

Thus, growth factors, their receptors, and DNA or RNA coding sequences, therefore, and fragments thereof are useful in a number of therapeutic, clinical, research, diagnostic, and drug design applications.

Brief Description of the Drawings Figure 1 . Nucleotide sequence (SEQ ID NO: 1 ) for the human noggin gene and deduced amino acid sequence {SEQ ID NO: 2).

Figure 2 (A). Experimental design: competent animal cap(AC) ectoderm was dissected from staged embryos as shown. Stl 0.5 dorsal and ventral AC and ventral marginal zones (V XZ) were also dissected as shown. Explants were washed once in low Ca/Mg Ringers (LCMR) solution and then placed in treatment medium containing factor diluted in LCMR + 0.5%BSA. Explants cultured to late stages {St20 + ) were removed from treatment medium 8-16 hours after the start of treatment and placed in LCMR. When explants reached the desired stage they were either harvested for RNA, or they were fixed for whole mount in situ hybridization or antibody staining.

(B). Neural induction by noggin in the absence of muscle. Lanes 1 -3 show specific fragments protected by N-CAM, β-tubuiin, and XIF- 3 probes respectively in whole St24 embryo RNA. Lanes 4-8 show protection by the mixture of these three probes while lanes 9-13 5 show protection by an actin probe on tRNA(t), St24 embryo RNA (E), and RNA collected from St9 AC treated with 50pM activin (A), 25% of 20 fold concentrated control CHO ceil medium (C) or 25% of 20 fold concentrated noggin conditioned CHO cell medium (N).

Ubiquitously expressed cytoskeletal actin used as a loading control 10 shows that RNA levels in all treatments are comparable (lanes 1 1 - 1 3) , Figure 3. 12% SDS-PAGE run under reducing conditions.

Proteins were visualized by silver staining. Lane 1 shows moiecuiar size standards. Lanes 2-7 show 0, 0.1 , 0.2, 0.5, 1 , and 2 μg of ^ S purified human noggin.

Figure 4 (A). Time course of animal caps treated with purified noggin vs. activin; direct vs indirect neural induction. Animal caps were dissected as shown in Fig. 2A and treated with LCMR + 0.5% BSA (U), a 20% dilution of activin conditioned COS cell medium (A), 0 or ^ μg/ml purified human noggin(N). RNA isolated from treated animal caps (lanes 2-13) along with St22 whole embryo RNA (lanel ) and tRNA (lane 14) was probed for N-CAM, β-tubuiin, muscle and cytoske!etal actins, coilagen type II, and EF-1 a.

(B). Expression of early mesoderm markers in activin but not 5 noggin induced animal caps. Animal caps were dissected from St8 embryos, treated as described in (A), and harvested at St11. Lanes 1 and 2 respectively show goosecoid and Xbra probe protection by Stl 0.5 whole embryo RNA. Lanes 3-6 show protection by a mix of these two probes. Relative RNA levels are demonstrated by separate EF- 1a probe protection.

(C). P!asmid directed gastrula stage noggin expression directly induces neural tissue. One ce!l stage embryos were injected with 20pg of pCSKA/acZ or pCSKAnoggin into the animal pole. Animal caps from injected embryos were dissected at St8-9 and cultured until St20, when they were harvested for analysis by RNase protection.

Figure 5. Responsiveness of dorsal and ventral animal caps to neural induction by noggin. St 105 ventrai and dorsal animal caps were dissected as shown in Fig. 2. Dorsal and ventral animal caps were treated with activin medium (DA,VA) or 1 (ug/ml human noggin (DN, VN) and harvested at St26 for RNase protection analysis using N- CAM, β-tubulin, and act in as probes.

Figure 6. Dose response of ventral marginal zones and animal caps to human noggin protein. St 10.5 VMZs and St9 animal caps were dissected as shown in Fig. 2A., and treated with 0, 1 , 10, 50, 200, and 1000 ug/mi of human noggin (lanes 3-8 and 10-15 respectively). RNA from treated expiants and control whole embryos aged to St26 was then analyzed by RNase protection, using the probes N-CAM, β-tubufin, actin and collagen type II. In this experiment, muscle induction at the dose of 1 ng/ml is stronger than at 10ng/ml, and there is a low level of muscle actin expression in the uninduced VMZs. This could be due to experimental variability since in repeated experiments we saw muscle induction only at the doses of 50ng/ml and above (data not shown).

Figure 7. In situ hybridization and antibody staining. Tailbud embryos stained for NCAM showing side and dorsal views (a,b); NCAM RNA is only detected in the neural tube, and not the somites. For comparison, somites of a taiibud embryo stain for muscie actin, dorsal view (c). Neural specific 6F11 antibody staining at St30 (d-f). Some cement giand pigment remained in these embryos after bleaching as seen in (d), however this pigment is distinct from antibody staining. The inner mass of staining in the noggin treated animal caps is due to the 6F11 antibody detection. Cement giand specific XA G- 1 transcripts detected at St23 (g-i), and anterior brain otxA trasncripts detected at St35 (j-l) in whole embryos at (d.g.j), human noggin treated (1 ς/ηιΙ) animal caps (e.h.k), and untreated animal caps (f,i,i).

Figure 8. Reverse phase HPLC profile of two refolded isoforms of noggin. The refolded noggin solution was applied onto a Brownlee Aquapore AX-300, 0.46 x 22 cmh'PLC column at a flow rate of 1 ml/min. The column was equilibrated with solvent A containing 0.1 % TFA in water. Solvent B was 0.1% TRA in acetonitriie. The column was developed according to the following protocol: a) 2 min isocratica!ly at 95% of solvent A- 5% of solvent 8; 60 min linear gradient to 65% of solvent 8 and 35% of solvent A. Correctly refolded noggin elutes earlier at 44%-46% solvent B.

Figure 9. Reverse-phase HPLC chromatography characterization of recombinant noggin refolded and purified from E. coli. Conditions as in the legend to Fig. 8.

Figure 10. Recombinant noggin produced in E. coli and in insect cells analyzed by 12.5% SDS-PAGE. Lanes H, L: High and low molecular weight markers of the indicated size, respectively. Lanes 1 ,2: Recombinant noggin produced in £ coli and in insect cells respectvely, treated with 2-mercaptoethanol before electrophoresis.

The slower mobility of noggin from insect csiS corr©ponds to the size ncrease that would occur due to i i-linked giycosyiation at the single consensus site. Lanes 2,3: Recombinant noggin produced E. co/i -und n insect cells respectveiy, not tr@ated with 2- 5 mercaptoetheno! fore electrophoresis.

Figure 1 1. Circular dichrois spectra ©f recombinant noggin produced in £ coif {--), ®nd insect sslls {-).

Figure 12, Ventral marginal so © assay showing induction of muscle actin mRNA after exposure to human noggin CO.01 , 0.05, i O 0.2 //g/ml) produced in bacuiovirus, § mock transfected culture of bacuiovirus {0.02, 1 £/g/ml)' or human noggin produced in E.coii {OA , 0.5, 2, or 10 ^g/mi) .

Figure 1 3. Nucleotide sequence for the mouse noggin gene and deduced amino acid sequence.

In one aspect, the parent invention provides an isolated noggin polypeptide having an amino acid sequence as set forth in Figure 1 {Sequence !.D. no.2) or a functionally equivalent amino acid sequence.

Such a polypeptide may be characterized by one or mors of the following, highly conserved amino acid sequences: QMWLWSQTFCPVLY CSEQ 3D NO:3); .

RFWRRYV YGSC {SEQ. ID N0:4) S RSCSVPEGMVC {SEQ ID NO:5); LRWRCQRR (SEQ ID O:6); and, ISEC CSC (SEQ ID N0:7).

Peptides of the paren invention induce dorsal growth in vertebrates and can be prepared in soluble, physiologically active form for a S number of therapeutic, clinical, and diagnostic applications.

In a preferred embodiment, human noggin protein as set forth in Figure 1 (SEQ ID NO: 2) is prepared for use in therapeutic, ciinicai and diagnostic applications. !n another aspect of the rent invention an oligonucleotide, such as cDNA, is provided having substantial similarity to (or being the same as) SEQ ID NO:8 (deduced amino acid sequence, SEQ ID NO: 9), SEQ ID NO: 10 (deduced amino acid sequence, SEQ ID NO: 11), or SEQ ID NO:1. This oligonucleotide can be single or double stranded, be formed of DNA or RNA bases, and can be in the antisense direction with respect to SEQ ID NOS: 8, 10 or 1. SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO:1 each code for a functional polypeptide that we 'have designated "noggin," which is capable of. inducing dorsal development in vertebrates when expressed.

Noggin or fragments thereof (which also may be synthesized by in vitro methods) may be fused (by recombinant expression or in yitro covaient methods) to an Immunogenic polypeptide and this, in turn, may be used to immunize an animal in order to raise antibodies against a noggin epitope. Theses antibodies are the subject of the present invention. Anti-noggin is recoverable from the serum of immunized animals. Alternatively, monoclonal antibodies may be prepared from cells to the immunized animal in conventional fashion. Antibodies identified by routine screening will bind to noggin but will not substantially cross-react ith "wnt" or other growth factors. ' immobilized anti-noggin antibodies are useful particularly in the diagnosis (in vitro or in vivo) or purification of noggin.

Substitutional, deietional, or insertionai mutants of noggin may be prepared by in yitro or recombinant methods and screened for immuno-crossreactivity with noggin and for noggin antagonist or agonist activity.

Noggin aiso may be derivatized in vitro in order to prepare immobilized noggin and iabeiled noggin, particularly for purposes of diagnosis of noggin or its antibodies, or for affinity purification of noggin antibodies.

The parent invention further provides for expression of bioiogicaily active noggin molecules in prokaryotic and eukaryoiic expression systems. 0 The parent invention further provides for the production of noggin in quantities sufficient for therapeutic and diagnostic applications. Likewise, the anti-noggin antibodies of the present invention may be utilized in therapeutic and diagnostic applications. For mos purposes, it is preferable to use noggin genes or gene products from the same 1 5 species for therapeutic or diagnostic purposes, although cross- species utility of noggin may be useful in specific embodiments of the invention. in additional embodiments; the noggin nucleic acids, proteins, and peptides of the parent invention may be used to induce neural tissue formation in mammals.

Description of the Preferred Embodiments We have discovered a structurally unique growth factor thai is readily available in substantially pure, soluble form. We have named the inventive polypeptide "noggin." This newly isolated neurotrophic factor induces dorsai development in vertebrates.

An earlier described family of proteins that aiso induces dorsal development are the "wnt" proteins. These, however, in contrast to noggin remain tenaciously bound to ceil surfaces. Our initial work with noggin has been in Xenopus embryos; however, noggin is highly conserved among vertebrates, as our work with mouse noggin has demonstrated. The prior known FGF growth factor family is also known to be involved in early embryonic induction, but both the FGF proteins and their receptors are distinctly different from noggin. Noggin modifies the actions of FGF (and also activin), for example by potentiating growth, and is thus particularly suggested in therapeutic compositions for use in combination with other growth factors (as therapeutic adjuvants), such as to modify or potentiate their effects.

We have cloned cDNA for noggin. The noggin cDNA contains a singie reading frame encoding a 26 kDa protein with a hydrophobic amino-terminai sequence. Noggin is secreted. Noggin's cDNA encodes the protein as a 26 kDa protein, but we have determined that noggin is secreted in vivo, apparently as a dimeric glycoprotein with a starting apparent molecular weight of about 33 kDa (as the wild- type subunit). When not glycosylated, the monomeric unit has an apparent molecular weight on SDS PAGE of about 25-30 kDa.

We have cloned the gene for human noggin (Figure 1 ; SEQ !D NO: 1). The sequence codes for a protein which has noggin activity (SEQ ID NO: 2). The carboxy terminal region of noggin shows homology to a Kunitz-type protease inhibitor, indicating that noggin protein, or fragments thereof, may exhibit activities of a protease inhibitor.

We have been able to express biologically active noggin in both eukaryotic and prokaryotic host cells. Two expression systems we have successfully used to express biologically active noggin have been mammalian cell lines (COS and mouse 293). A third expression system is injection of synthetic mRNA into Xenopus oocytes. In addition, we have successfully expressed biologically active human noggin in a prokaryotic system, E. coli, and in bacuiovirus.

Expression in these several different systems aiso illustrates the high degree of conservation for noggin. We have found, for example, substantial sequence similarity between frog noggin and mouse noggin with a number of completely conserved stretches.

Thus, the following amino acid sequences represent completely conserved portions as between frog noggin and mouse noggin: QMWLWSQTFCPVLY (SEQ ID NO:3); RFWPRYVKVGSC (SEQ ID NO:4); SKRSCSVPEGMVCK (SEQ ID NO:5); LRWRCQRR (SEQ ID NO:6); and, !SECKCSC (SEQ !D NO:7).

There is about 87% overall conservation between the mouse and frog sequences, and we have also observed a unique cysteine distribution between the two.

Noggin nucleic acids, or oligonucleotides, encode a noggin polypeptide or hybridize to such DNA and remain stably bound to it under stringent conditions and are greater than about 10 bases in length; provided, however, that such hybridizing nucleic acid is novei and unobvious over any prior art nucleic acid including that which encodes or is complementary to nucleic acid encoding other growth factors.

By "stringent conditions" we mean are those which (1 ) employ low ionic strength and high temperature for washing, for example, 0.15 M NaCl/0.015 sodium citrate/0.1% aDodSo4 at 50°C, or (2) use during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum aibumin/0.1 %Ficoil/0.1 % polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42° C.

By "substantial similarity," when we are referring to a nucleotide sequence, is meant cross hybridization of sequences under conditions of moderate stringency using a probe greater than 100 nucleotides long at 30°C in a standard buffer {Wahi et a!., PNAS,76, 3683) and washes at 37°C in 300 mM NaCI, 30 mM sodium citrate, 0.2% SDS at pH 7. Alternatively, one is able to isolate, by polymerase chain reaction, a fragment of DNA coding for noggin or noggin family members when using primers of degenerate sequence that encode those SEQ ID NOS:3-7.

By "substantial similarity" when reference is made to proteins is that noggin from different species, or noggin family members within a species, will preserve the positions of cysteine residues in at least 80% of positions throughout the protein. Like the neurctrophin family, the sequence of the mature form of noggin and noggin related polypeptides will be identical in at least 40% of positions. Substantial similarity at the protein level includes an ability of a subject protein to compete with noggin for binding to receptors and some (but not all) monoclonal antibodies raised against noggin epitopes.

The cloned cDNA for noggin {derived from frog) is designated herein as SEQ ID NO: 8, partial sequence from mouse as SEQ ID NO: 10 or full sequence of mouse noggin as shown in Figure 13. The human sequence is designated herein as SEQ ID NO: 1 . We have used RNA transcripts from the SEQ !D NO: 8 clone to rescue embryos and return them to substantially normal development when the noggin RNA is injected into ventralized embryos. Sn high doses this results in excessive head development and it is for this reason we named the protein "noggin." In northern blot analysis the noggin cDNA hybridizes to two mRNAs that are expressed both maternally and zygotically.

When using nucleotide sequences coding for part or all of noggin in accordance with this invention, the length of the sequence should be at least sufficient in size to be capable of hybridizing with endogenous mRNA for the vertebrate's own noggin. Typically, sufficient sequence size (for example, for use as diagnostic probes) will be about 15 consecutive bases (DNA or RNA). !n some diagnostic and therapeutic applications, one may wish to use nucleotide noggin coding sequences {analogous to all or a portion of SEQ ID MO: 8, SEQ ID NO: 10, the nucleotide sequence of Figure 3 or SEQ ID NO: ) in the anti-sense direction with respect to either SEQ SD NOS: 8, 0, or 1, or the nucleotide sequence of Figure 13, We suggest as a few preferred primers for amplifying noggin from other species {e.g. human): ' Primer 1 SEQ ID NO: 2 C A A/G A C N T T C/T T G C/T C C N G T N ' Primer 2 SEQ ID NO: 3 T T C/T T G G C C N C/A G N T A C/T G T N A A A/G G T N G G 5' Primer 3 SEQ ID NO: 4 C C N G A A/G G G N AT G G T N T G 3' Primer 1 SEQ ID NO: 15 C A C/G T/A A/G C A C/T T T A/G C A C/T T C 3' Primer 2 SEQ ID NO: 16 C A N A C C A T N C C C/T T C N G G 3' Primer 3 SEQ ID NO: 17 C G/T N C G T T/C T G G'A C A N C G/T C C A where N represents a mixture of ail four nucleotides and mixtures of two nucleotides are represented by alternates (e.g. A/G).

Although noggin transcript is not localized in the oocyte and cleavage stage embryo, zygotic transcripts are initially restricted to the presumptive dorsal mesoderm, and reach their highest levels at the gastrula stage in the dorsal lip of the blastopore (Spemann's organizer). In the neurula, noggin is transcribed in the notochord and prechordal mesoderm.

Without being bound by theory, we have formulated hypotheses about the embryologica! effects of noggin based on where it is expressed, and on the effects of RNA injection in embryos. Since noggin is expressed in the Spemann organizer, we believe noggin to be a mediator of the effects of the Spemann organizer, namely neural induction and dorsalization of the mesoderm. We have shown that noggin is able to directly induce neural tissue formation. Since noggin is expressed in the notochord and head mesoderm, we believe noggin to influence either the dorsal-ventral pattern or anterior- posterior pattern of the neural plate. Since noggin is expressed in the branchial arch neural crest, we believe it may therefore influence whether neural crest cells deposit cartilage and also to influence later branchial arch growth and remodelling. Noggin is expressed in the tail fin neural crest, and since neural crest is required for growth of the fin, noggin may act as a growth factor for epidermis or mesenchyme.

Although much of our experimental work has involved rescue of embryonic development, because expression in the notochord persists in the growing taii bud and a discontinuous line of stained cells (indicating expression of noggin initiated at new sites) runs the length of the roof plate of the neural tube (and is also apparent in the head mesoderm), we believe noggin is expressed as an adult cell function also.

A number of applications for noggin are suggested from its properties.

The noggin cDNA should be useful as a diagnostic tool (such as through use of antibodies in assays for proteins in cell lines or use of oligonucleotides as primers in a PCR test to amplify those with sequence similarities to the oligonucleotide primer, and to see how much noggin is present, e.g. primers such as 5' Primers 1 -3 and 3* ■Primers 1 -3).

Because noggin has a pattern of expression that suggests it is used to regulate cartilage production in the embryonic head, clinical uses to regulate cartilage and bone growth are suggested for noggin in therapeutic compositions and particularly in combination with other growth factors due to a property of noggin to potentiate at least some growth factors. Since neural crest ceils are required for the tadpole fin to grow, noggin seems to be a growth factor for the tissue matrix and epidermis and should prove useful, for example, in wound healing compositions.

Noggin, of course, provides the key to isolate its receptor . Since many receptors mutate to cellular oncogenes, the noggin receptor should prove useful as a diagnostic probe for certain tumor types. Thus, when one views noggin as iigand in complexes, then complexes in accordance with the invention include antibody bound to noggin, antibody bound to peptides derived from noggin, noggin bound to its receptor, or peptides derived from noggin bound to its receptor. Mutant forms of noggin, which are either more potent agonists or antagonists, are believed to be ciinicaiiy useful. Such complexes of noggin and its binding protein partners will find uses in a number of applications.

Practice of the parent invention includes US6 of 3Π oligonucleotide construct comprising a sequence coding for noggin and for a promoter sequence operative! linked to noggin in a mammalian, bacterial or a viral expression vector. Expression and cloning vectors contain a nucleotide sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomes, and includes origins of replication or autonomously replicating sequences. The well-known piasmid pBR322 is suitable for most gram negative bacteria, the 2u piasmid origin for yeast and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

Expression and cloning vectors should, contain a selection gene, also termed a selectable marker. Typically, this is a gene that encodes a protein necessary for the survival or growth of a host eel! transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector wiil not obtain an advantage in growth or reproduction over transformed hosts.

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g. ampicii!in, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies.

Examples of suitable selectable markers for mammalian ceils are dihydrofoiate reductase (DHFR) or thymidine kinase. Such markers enable the identification of ceils which were competent to take up the noggin nucleic acid. The mammalian ceil transformants are placed under selection pressure in which oniy the transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively changed. Amplification is the process by which genes in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells.

Increased quantities of noggin can therefore be synthesized from the amplified DNA.

For example, ceils transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium which contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host ceil in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Uriaub and Chasin, Proc. Nat. Acad. Sci., 77, 4218 (1980). The transformed ceils then are exposed to increased levels of Mtx. This leads to the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding noggin. Alternatively, host cells transformed by an expression yector comprising DNA sequences encoding noggin and aminoglycoside 3* phosphoiransferase (APH) protein can be seiected by ceil growth in medium containing an aminoglycoside antibiotic such as kanamycin or neomycin or G418. Because eukarotic cells do not normally express an endogenous APH activity, genes encoding APH protein, commonly referred to as neo resistant genes, may be used as dominant selectable markers in a wide range of eukaryotic host ceils, by which cells transformed by the vector can readily be identified.

Expression vectors, unlike cloning vectors, should contain a promoter which is recognized by the host organism and is operabiy iinked to the noggin nucleic acid. Promoters are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control. They typically fall into two classes, inducible and constitutive. inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters can be operabiy linked to noggin encoding DNA by removing them from their gene of origin by restriction enzyme digestion, followed by insertion 5' to the start codon for noggin.

Nucleic acid is operabiy Iinked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operabiy Iinked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operabiy linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operabiy linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operabiy jinked means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation at convenient restriction sites, !f such sites do not exist then synthetic oligonucleotide adapters or linkers are used in accord with conventional practice.

Transcription of noggin-encoding DNA in mammalian host cells is controlled by promoters obtained from the genomes of viruses such as polyoma, cytomegalovirus, adenovirus, retroviruses, hepatitis-B virus, and most preferably Simian Virus 40 (SV40) , or from heterologous mammalian promoters, e.g. the actin promoter. Of course, promoters from the host ceil or related species also are useful herein.

In particular embodiments Of the aren invention expression of noggin in E. coii is preferably performed using vectors which comprise the following: a [§£ UV5 promoter which may be controlled by the lactose operon repressor; a strong ribosome binding site, for example, the ribosome binding site of bacteriophage T7; a mutation in the replication control region of the plasmid which may increase copy number; and a mutation which limits the expression of the antibiotic resistance protein.

In a preferred embodiment, noggin is expressed in a high copy number kanamycin resistant pBR322-derived plasmid under the control of a lac UV5 promoter, !n an additional preferred embodiment, noggin is expressed in bacuiovirus under the control of the polyhedrin promoter of Autrographa caiifornica Multiple Nuclear Polyhedrosis yirus in insect host cells.

Noggin is believed to find use as an agent for enhancing the survival or inducing the growth of nerve and muscle ceils. It, therefore, is useful in the therapy of congenital conditions or degenerative disorders of the nervous system {"neurodegenerative diseases"), including such diseases as Alzheimer's disease, Parkinson's disease, Huntington's chorea, ALS, peripheral neuropathies, and other conditions characterized by necrosis or loss of neurons, whether central, peripheral, or motorneurons. In addition, it may be useful for treating damaged nerve ceils, e.g., nerves damaged by traumatic conditions such as burns and wounds, diabetes, kidney dysfunction, and the toxic effects of chemotherapeuiics used to treat cancer and AIDS, it also is useful as a component of culture media for use in culturing nerve cells in vitro.

The capacity of noggin to induce neural tissue may be useful in diseases where neural tissue is formed improperly or incompletely during development. Thus, noggin and the noggin gene are also useful in treating congenital malformations such as anencephafy, or the loss of cerebrai hemispheres which results from failure of closure of the anterior neurai tube during development.

Practice of this invention includes preparation and uses of a diagnostic or therapeutic agent comprising a nucleotide sequence of at least about 1 5 DNA or RNA bases analogous to all or a portion of either SEQ ID NO: 8, SEQ ID NO: 10, the nucleotide sequence of Figure 13, or SEQ ID NO: 1 or of the nucleic acid sequences contained in bacteriophages, hnogX-9 or hnog -10. That is, noggin preparations are useful as standards in assays for noggin and in competitive-type receptor binding assays when labelled with radioiodine, enzymes, fiuorophores, spin labels, and the like. Therapeutic formulations of noggin are prepared for storage by mixing noggin having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, in the form of iyophilized cake or aqueous solutions.

Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides: proteins, such as serum albumin, gelatin or immunoglobulins. Other components, can include glycine, biuiamine, asparagine, arginine,' or lysine: monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitoi or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Piuronics or PEG.

Noggin may be USed according tO the parent invention as described supra. The concentration of the active ingredient used in the formulation wiil depend upon the effective dose required and the mode of administration used. The dose used should be sufficient to achieve circulating plasma concentrations of active ingredient that are efficacious. Effective doses may be,, extrapolated from dose-response curves derived from in vitro or animal model test systems.

By referring to noggin, the parent and present invention also contemplates the use of fragments, derivatives, agonists or antagonists of . noggin molecules.

Noggin may be administered in any pharmacologically acceptable carrier. The administration route may be any mode of administration known in the art, including but not limited to intravenously, intrathecally, subcutaneousiy, by injection into involved tissue, intraarterial'!-/, intranasal!*/, orally, or via an implanted device. The parent invention provides for pharmaceutics compositions comprising noggin in a pharmacologically acceptable carrier.

Administration may result in the distribution of noggin throughout the body or in a localized area. For example, in some conditions which involve distant regions of .the nervous, system, intravenous or intrathecal administration of noggin may be desirable. Alternatively, and not by way of limitation, when localized regions of the nervous system are involved, local administration may be desirable, in such situations, an implant containing noggin may be placed In or near the lesioned area.

Suitable implants include, but are not limited to, ge!foarn, wax, or micropariicle-based Implants.

Depending upon the. mode of administration, the active ingredient may be formulated In a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.

Inventive complexes comprise a ligand characterized by one or more of the SEQ ID NOS:3-7. The ligand can be bound to a protein, such as antibody. Such aniibodies can be poiyciona! or monoclonal. Polyclonal antibodies to noggin generally are raised in animals by muitipie subcuianeous (sc) or intraperitoneal (ip) injections of noggin and an adjuvant. It may be useful to conjugate noggin or a fragment containing the target amino acid sequence to a protein which is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum aibumin, bovine thyroglobuiin, or soybean trypsin inhibitor using a bifunctiona! or derivatizing agent, for example, maleimidobenzoy! sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxy-succinimide {through lysine residues), giutaraldehyde, succinic anhydride, SOCI2, or FP N = C = 1 0 NFL Animals can be immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 μα of conjugate {for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermal^ in multiple sites. One 1 5 month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Fruend's complete adjuvant by subcuianeous injection at multiple sites. Seven to 14 days later animals are bled and the serum is assayed for anti-noggin titer. Animals are boosted υηίίί the titer plateaus. Preferably, the . animal Is boosted with the conjugate of the same noggin polypeptide, but conjugated to a different protein and/or through a different cross-linking agent.

Conjugates also can be made in recombinant cei! culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.

Monoclonal antibodies of the present invention are prepared by recovering spleen cells from immunized animals and : immortalizing the cells in conventional fashion, e.g. by fusion with myeioma ceils or by EB virus transformation and screening for clones expressing the desired antibody. !n a preferred embodiment, a rat monoclonal antibody such as RP57-16, prepared after immunization of a rat with recombinant human noggin, reacts specifically with both Xenopus and human noggin, but not with the neurotrophics BDNF, NT-3 and NT-4.

Noggin antibodies are useful in diagnostic assays for noggin or its antibodies. In one embodiment of a receptor binding assay, an antibody composition which binds to ail of a selected plurality of members of the noggin fami!y is immobilized on an insoluble matrix, the test sample is contacted with the immobilized antibody composition in order to adsorb all noggin family members, and then the immobilized famiiy members are contacted with a plurality of antibodies specific for each member, each of the antibodies being individually identifiable as specific for a predetermined famiiy member, as by unique labels such as discrete fluorophores or the like. By determining the presence and/or amount of each unique label, the relative proportion and amount of each famiiy member can be determined.

Noggin antibodies also are useful for the affinity purification of noggin from recombinant ceil culture or natural sources. Noggin antibodies that do not detectably cross-react with other growth factors can be used to purify noggin free from these other family members.

Aspects of the invention will now be illustrated by the following examples.

FXPFR1MFNTAL PRCCEDURFS Production of Xenoous embrvos Xenopus embryos were prepared by the protocol described by Condie and Hariand {Development, 101 , 93-105, 1987). Embryos were staged according to the table of Nieuwkoop and Faber ("Normal Table of Xenopus laevis"(Daubin), Amsterdam: North Holland, 1 967).

Ventra!ized embryos were produced by irradiation with a Stataiinker (Stratagene), and dorsalized embryos were produced by treatment with LiCI as described by us in our paper on certain "wnt" proteins (designated "Xwnt-8"), Smith and Hariand, Ceil, Vol. 87, pp. 753-765 (1 991 ) (incorporated by reference and occasionally referred to hereinafter as "S&H, supra").

EXA P E .1 Isolation and Sequencing of Noggin cDNA The construction of the size-selected plasmid cDNA library from stage 1 1 LiCI-treated embryos was as follows. Sixty micrograms of poly(A) RNA from stage 1 1 LiCi-ireated embryos was size fractionated on a 10% to 30% sucrose gradient in the presence of methyimercuric hydroxide. First strand cDNA was synthesized from 2 μα of the size-fractionated poly(A) RNAs primed with oiigo(dT) oligonucleotide containing the recognition site for Notl. After synthesis of the second strand, cDNAs were treated with EcoRI methylase, iigated with EcoRI linkers, digested with EcoRI and Not!, and finally Iigated to 125 ng of modified pGEM-5Zf(-) (Promega). The pGEM-SZf(-) used here was modified by the addition of an oligonucleotide into the Nsil site to create an EcoRi site. The vector was not treated with alkaline phosphatase, but the excised polyiinker sequence was removed on a sepharose 4BCL column. The !igated products were used to transform XL-! Blue ceils (Stratagene), and plated to give 100,000 colonies per cm piate. P!asmid DNAs were isolated from piate cultures by the alkaiine-iysis/polyethylene glycol precipitation protocol.

Dorsaiizing activity in the library was assayed by injecting RNA transcripts made from pooled piasmid DNA. Single clones were isolated by a process of sib selection, in this procedure the piasmid library was replated on 12 plates with 10-fold fewer colonies per piate. RNA was synthesized from pooled piasmid DNAs isoiated from each piate and tested for dorsaiizing activity by injection into UV-ventraiized embryos. Those pools with dorsaiizing activity were repiated and screened as described above. This process was repeated until single clones were isolated.

In vitro RNA synthesis, injection assay for dorsal axis rescue and sib-se!ections were also done, as described by us in S&H, supra. The nucleotide sequence of both strands of the isoiated noggin cDNA clone was determined by the dideoxy termination method using modified T7 DNA polymerase (US Biochem). Deletions were prepared in sequencing templates by both restriction enzyme and exonuciease HI digestion (Henikoff, eth. Enzymol, 155, 156-165, 1 987).

In vitro tra ns lation One-half g of in vitro synthesized noggin, Xwnt-8, and goosecoid mRNAs were translated in a nuclease treated rabbit reticulocyte !ysate (Promega) with added ass-methionine according to the manufacturer's instructions. The translation products were visualized by SDS-polyacrylamide gei electrophoresis (12% geis) followed by fluorography. Noggin protein had the molecular weight predicted by the open reading frame.

RNA iso!ation and Analysis Total RNA was isolated from embryos and oocytes by a small scale protocol as described by Condie and Harland, supra. Dorsal lips were dissected from 30 unfixed stage 10.5 embryos and pooled for total RNA preparation. Samples containing either the total RNA equivalent of 2.5 embryos or approximately 2 ο of poly A+ RNA were analyzed by northern blotting. Random primed DNA probes were prepared from a 1 ,323 bp fragment of noggin cDNA from the EcoRi site at nucleotide -83 to an EcoRV site that lies In the vector immediately 3' to the end of the cDNA.

RNAse protection assays were done using a protocol as detailed by Melton et ai. (Nuc. Acids Res., 12, 7035-7056, 1984) with minor modifications (C. intner, Sa!k institute, La Joila, California). A noggin cDNA exonuclease HI deletion done, illustrated by SEQ ID NO: 8 but having a deletion from the 3' end to nucleotide 383, was - used as a template for synthesizing RNA probes. The template DNA was linearized by EcoRI restriction enzyme digestion and a 463 base antisense RNA incorporating 3∑p was synthesized with T7 RNA polymerase. A 387 base antisense EFIa RNA probe was used as a control for amount of RNA per sample. Probes were gei purified prior to use.

In situ hybridization After fixation and storage, the embryos were checked to ensure the blastocoei and archenteron were punctured. Care was taken to puncture the residual blastocoei of neurulae and tadpoles as well as the archenteron. Embryos were rewashed at room temperature in 100% ethanol for two hours to remove residual lipid. After hybridization, staining was allowed to develop overnight and the embryos were then fixed in Bouin's. Newly stained embryos have a high background of pink stain but most of this washes out, leaving the specific stain. Following overnight fixation, the embryos were washed well with 70% ethanol, 70% ethanol buffered with PBS and methanol. Embryos were cleared in Murray's mix and photographed with Kodak Ekiar 25 film, using a Zeiss axioplan microscope (2.5 or 5x objective with 3x12B telescope to assist with focusing).

Lineaoe Tracing Lineage tracing with mRNA that encodes nuclear localized B-galactosidase was as we described in S&H, supra. Ventraiized embryos were coinjected at the 32 cell stage with 0.5 ng B-ga!aciosidase and 25 pg nogginAS' mRNAs. Embryos were fixed and stained with X-gai at approximately stage 22.

Results Noggin cDNA Encodes a Novel Polypeptide The 1833 nucleotide sequence of the selected clone is shown by SEQ ID NO: 3 and sometimes also referred to as "clone A3.a The sequence contains a single long open reading frame encoding a 222 amino acid polypeptide with a predicted moiecuiar weight of 26 kDa. At the amino terminus, the hydrophobic stretch of amino acids suggests that the polypeptide enters the secretory pathway. There is a single potential site for N-!inked giycosylation at amino acid 61. Extensive untranslated regions are located both 5' and 3' to the reading frame (593 and 573 bp, respectively). The 3' untranslated region is particularly rich in repeated dA and dT nucleotides, and contains, in addition to a polyadenylation signal sequence located 24 bp upstream from the start of the poly A tail, a second potential polyadenylation sequence 147 bp further upstream.

Sense RNA synthesized from clone A3 with SF6 RNA polymerase was translated in a rabbit reticulocyte lysate system. The 3S-labeled products were fractionated on a 12% SDS-po!yacryiamide gel and visualized by fiuorography. The major protein product had the expected moiecuiar weight of approximately 26 kDa.

Comparison of the amino acid sequence of the predicted polypeptide to the National Center for Biotechnology information BLAST network (non-redundant data base) did not identify any similar sequence. Thus, this clone encodes the new type of protein we have named "noggin" which is secreted, and which has dorsal inducing activity in Xenopus. Νοσσίη mRNA can Rescue a Complete Dorsal-Ventrai Axis Injection of noggin RNA into a single biastomere of a four cell stage UV-ventralized embryo can restore the complete spectrum of dorsal structures. The degree of axis rescue was dependent upon the amount of RNA injected, with embryos receiving !ow doses having only posterior dorsal structures, while embryos receiving higher doses had excess dorsal-anterior tissue. RNA transcripts from two noggin piasmids were tested. The first contained the full cDNA. The second (pNogginAS') had a deletion removing the first 513 nucleotides of the 5' untranslated region up to the EcoRl site. The resulting embryos from injection of RNA transcripts of these two piasmids, as well as Xwnt-8 RNA for comparison, were scored according to the dorsoanterior index (DAI) scale of Rao and Elinson (Dev. Biol., 127, 64-77, 1988). In this scale, a completely ventraiized embryo is scored as zero, a normal embryo is scored as 5, and the most severely dorsoanteriorized embryos, those having radial dorsoanterior structures, were scored as 10. RNA synthesized from pNogginAS' inogginAS' mRNA) repeatedly gave a higher DA! than the equivalent amount of mRNA synthesized from the complete cDNA. The dose-dependency of axis rescue by nogginAS' mRNA was very similar to that of Xwnt-8 mRNA.

UV ireated embryos were also injected with a higher doses (1 ,000 pg) of the noggin mRNAs. Injection of this dose of noggin mRNA into one biastomere at the four call stage resulted in embryos with very severe hyperdorsalization (DAI >7). However, most of these embryos died at the late gastrula/early neurula stage. Apparently excessively strong gastrulation movements resulted in the thinning and rupture of the blastocoel roof. We have also observed this effect with high doses of injected Xwnt-8 mRNA.

The rescue of dorsal development by both nogginA5' and Xwnt- 8 mRNAs followed a consistent pattern in which increasing amounts of the mRNAs lead to progressively more anterior structures being rescued. For example, embryos that received 1 pg of the RNAs had primarily the posterior and trunk dorsal structures rescued, and for the most part lacked head structures. Higher doses (10 or 100 pg) of both of the RNAs resulted in embryos with more anterior development, and many had either nearly normal or hyperdorsalized phenotypes.

Noagin injected Blastomeres Act as a Nieuwkooo Center The effect of varying the site of noggin mRNA injection was investigated. Thirty-two cell stage UV-treated embryos were injected with either 0.5 ng of B-ga!actosidase mRNA alone or 0.5 ng B-gaiactosidase mixed with 25 pg nogginAS' mRNA. Injection of noggin mRNA into blastomeres of the vegetal tier gave the most strongly dorsoanteriorized embryos, in both of the vegetal injected embryos the nuclear X-Gai staining was found almost exclusively in the endoderm (the mRNA encodes a B-galactosidase that translocates to the nucleus, allowing distinction from the diffuse background stain). One of the embryos shown was strongly hyperdorsalized (DAI approximately 7} as a result of the noggin mRNA injection, and had a severely truncated tail and enlarged head structures. Embryos were also rescued by noqgin mRNA injections into the marginal zone.

In these embryos B-galactosidase staining was observed primarily in the axial and head mesoderm. Injection of noggin mRNA into the animal pole had very little effect on axis formation. Likewise, B-galactosidase mRNA aione was without effect.

Noagin mRNA is Expressed Both Maternally and Zvooticallv In northern blot analysis of RNA from Xenopus embryos two noggin mRNA species of approximate sizes 1.8 and 1.4 kb were observed. A relatively low !eve! of noggin mRNA was detected in oocytes. By stage 11 the level of noggin mRNA was significantly higher, reflecting zygotic transcription (as opposed to the maternally deposited transcripts seen in oocytes). Noggin mRNA remained at the elevated level up to the latest stage examined (stage 45).

We expect that the primary dorsalizing RNA in our library to be elevated in LiCI-treated embryos relative to normal or UV-treated embryos. Lithium ion treatment resulted in a large increase in the amount of noggin mRNA expressed, relative to untreated embryos.

UV treatment had the opposite effect. Noggin mRNA expression was essentially undetectable in total RNA samples from these embryos. Thus, the abundance of noggin mRNA in manipulated embryos parallels the rescuing activity.

We analyzed the distribution of noggin in oocytes and cleavage stage embryos. Since the amount of maternally deposited noggin RNA is too low for in situ hybridization to detect above background, we used an RNAse protection assay. Oocytes were dissected into animal and vegetal halves. No enrichment of noggin mRNA was seen in either hemisphere relative to total oocyte RNA. Four-cell stage embryos were dissected into dorsal and ventrai halves, as well as animal and vegetal halves. Noggin transcripts were found to be distributed evenly between dorsal and ventrai hemispheres as well as animal and vegetal hemispheres. The same result was obtained with embryos that wer tilted 90° immediately following fertilization and then marked with a vita! dye on their uppermost side to indicate the future dorsai side. Older (32 cell stage) biastula embryos were also dissected into dorsal-ventral and animal-vegetal halves. No enrichment of noggin mRNA in any of the hemispheres was seen relative to the totai embryo. In addition, treatment did not alter the abundance of maternally deposited noggin RNA. indicating no preferential degradation in ventral tissues. Samples with known amounts of in vitro synthesized noggin mRNA were included in the RNAase protection assay. From these and other data we estimate that there is approximately 0.1 pg of noggin mRNA per biastula stage embryo and 1 pg per gastrula stage embryo.

The localization of noggin transcripts was investigated in early gastrula stage embryos. Dorsai Sips were dissected from stage 1 0.5 embryos. A northern blot of equal amounts of totai RNA from intact embryos, dissected dorsal !ips, and from the remaining embryo after dissection of the dorsai lip was hybridized with a noggin probe and then re-hybridized with an EF!a probe, as a control for amount of RNA loaded per sampie. The autoradiograph of the biot showed that noggin mRNA at this stage is enriched in the dorsal iip. n situ Hybridization : Zygotic Expression of Noooin i n the Spemann Organizer .

The localization of noggin transcripts in developing embryos was examined in greater detaii using whole mount in situ hybridization. Whole fixed embryos were hybridized with digoxigenin containing RNA probes.

Hybridized RNA probe was then visualized with an alkaline phosphatase-conjugated anti-digoxigenin antibody. The specificity of hybridization seen with antisense noggin probes was tested both by hybridizing embryos with sense noggin probes, and by using two non-overiapping antisense probes. Due both to the low level of expression, and to background staining, noggin mRNA could not be detected unequivocally before the late blastula stage. The increased ieve! of noggin mRNA that was detected by northern blot following activation of zygotic transcription was apparent in in situ hybridization at stage 9 as a patch of staining cells on the dorsai side of the embryo. Viewed from the vegetal pole, this patch of ceils was restricted to a sector of about 600. A side view of the same embryo shows that the staining ceils were located within the marginal zone (i.e., between the animal and vegetal poies and within the presumptive dorsai mesoderm forming region). Transcripts are largely restricted to the nucleus at this stage.

A side view of an early gastrula stage embryo 30 (approximate stage 10.5) shows specific hybridization primarily in the involuting mesoderm at the dorsai !ip. A vegetal view of the same embryo (blastopore !ip arrowed) shows that noggin mRNA is most abundant on the dorsai side, but expression extends at the lower level to the ventrai side of the embryo. This method of in situ hybridization does not detect transcripts in the most yolky endodermai region of embryos, therefore we cannot rule out expression in more vegetai regions than those seen in the Figure. Treatments which are known to affect the size of the dorsal lip (LiCI treatment, UV irradiation) had a profound effect on the pattern of noggin in situ hybridization, !n LiCI treated embryos the staining is intense throughout the marginal zone. UV treatment reduced the hybridization signal to low levels. This result is consistent with amounts of noggin mRNA seen by northern blot analysis. The UV treated embryo also is a negative control for specificity of hybridization.

As gastruiation proceeds, noggin mRNA staining follows the involuting dorsal mesoderm, and is highest in the presumptive notochord. By the late neuruia stage (approximately 18) noggin mRNA expressing cells are clearest in the most dorsal mesoderm, primarily in the notochord but aiso extending more anteriorly into the pre-chorda! mesoderm. The anterior tip of the notochord is arrowed. During tailbud stages expression of noggin in the dorsal mesoderm declines, through expression in the notochord persists in the growing taiibud. Expression of noggin initiates at several new sites, which become progressively clearer as the tadpole matures. A discontinuous line of stained ceils runs the length of the roof plate of the neural tube. Staining is aiso apparent in the head mesoderm, primarily in the mandibular and gill arches. We suspect that this expression corresponds to ske!etogenic neural crest ceils.

Furthermore, subsets of these ceils express hcmeobox genes that mark different anterior-posterior levels of the head neural crest, for example En-2 in the mandibular arch is seen by antibody staining. Ceils with stellate morphology stained from noggin mRNA in the tail fin. These stellate ceils are aiso likely to be derived from the neurai crest. None of these patterns were seen with the sense probe, or with a number of other probes.

EXAMPLE 2 Νοσσίπ cDNA Transfected into COS Ceils Produces Active Conditioned Medium For COS cells the noggin cDNA was inserted into a COS cell expression vector. COS cells were transfected, and medium harvested after allowing expression of the introduced noggin genes. This medium has been tested in an animal cap assay for mesoderm inducing or dorsalizing activity. We have tested two transfection protocols, a standard one, where cells recover and then are transferred to serum-free medium, and an alternate where cells are transferred to a defined medium lacking serum but containing transferrin, insuiin, and BSA. Ceils remain healthy in the supplemented medium and a coiransfected β-galactosidase gene gives 100 fold more activity than in the unsupplemented medium. The results of treating ceils with these media is shown below in Table 1 . Animal caps were taken from ventraiized animals, treated and at the end of neurulation they were scored for elongation, usually a sign that n.oiochcrd or neural tissues have been induced. Elongation is indicated in Table 1 by a "+" and even greater elongation a "+÷ " In addition, they are scored for a molecular marker by Northern blotting.

As shown by the data of Table 1 , the noggin cDNA has a large effect on the COS cell conditioned medium. However, noggin is probably interacting with something else in the medium, since COS-ceil conditioned medium alone has some activity, it is possible that noggin is causing the cells to secrete something that they normally would not, but the experiments do indicate that noggin is secreted and is responsible for some of the activity.

TABLE! Elongation N-CAM expression Transferred to serum free medium + transferrin, BSA, and insulin 1 , Vector only + 2. Noggin cDNA -H- Transferred to serum free medium without supplements 1 . Vector oniy 2. Noggin cDMA Noooin mRNA Injected into Oocytes Produces Active Secreted Noggin Protein A second approach to studying whether protein can be secreted in active form is to inject oocytes with mRNA and take materia! secreted by the oocyte. A particular advantage of this method is that the injected mRNA is efficiently translated, and most of the translation of the oocyte can be taken up by the injected mRNA. A new protein, whose synthesis is directed by injected noggin mRNA is secreted into the medium. Noggin cleariy synergizes with activin to produce elongated explants that express elevated levels of muscie actin.

Biochemical Properties of Noggin Injected oocytes are injected with mRNA, and labelled with 35 S methionine. Most of the radioactive protein secreted into the medium is from the injected mRNA. The noggin protein, which is almost isotopical!y pure, can then be analyzed. From this analysis we have determined that noggin is a dimeric glycoprotein. When run under reducing conditions, and treated with N-glycanase to remove sugar residues, noggin migrates only slightly slower than its predicted molecular weight of 26 kDa. The removal of sugar side chains results in a loss of about 4 kDa from a starting apparent molecular weight of 33 kDa. When run under non-reducing conditions it migrates at double this value.

We do not yet know if the dimer of the protein is the active species, or if there is a proteoiyticai!y processed form which is active. In a control experiment with activin mRNA, oocytes produce activin activity, but the bulk of the radioiabe!led protein migrates as the precursor form. Only a smail amount of processed protein (15 kDa) was detected. It is possible that noggin injected oocytes secrete predominantly unprocessed protein and a trace of extremely active processed protein that we have not detected. Despite the caveats, the main point from analysis of injected oocytes and transfected COS cells is that active noggin can be obtained as a freely soluble secreted polypeptide. This sets it apart from the other group of genes with dorsalizing activity, the wnts. Wnt proteins have not been available in soluble form and this has greatly hampered the analysis of their biological activities, and of the receptor that binds to them.

EXAMPLE 3 Cloning of the Mouse Noagin Homoloa It is currently impossible to eliminate zygotic noggin transcription from developing Xenopus embryos. Sn contrast, it should be possible to generate homozygous null mutations in the mouse. We have cloned the mouse noggin cDNA (SEQ ID NO: 10). This is useful to generate mutant mice. In addition to generating the probes and tools to make mutant mice, a comparison of the noggin sequences should be a useful predictor of conserved domains and functions. The C -terminal 80 amino acids are 87% identical between SEQ !D NOS: 8 and 10.

Mouse noggin was isolated from an embryonic cDNA library by probing with a radioiabel!ed frog noggin cDNA under conditions of moderate stringency (as defined earlier). Subsequently a genomic clone was isolated by probing a genomic library with the mouse noggin cDNA 1 5 under conditions of high stringency {as defined, but hybridized at 42° C and washed at 50° C in 1 5 mM NaC), 1 .5 mM sodium citrate). The full nucleotide sequence of mouse noggin cDNA as well as the deduced amino acid sequence are shown in Figure 13. There are only two amino acid differences between mouse noggin and human noggin.

EXAMPLE 4 Cloning of the Human Nocoin Homoioq ' Materials and Methods Probe oreoaration Two oligonucleotides were synthesized based on the mouse noggin sequence (supra). The sequence of the oligonucleotides is noggin 51 : 5'-CAG ATG TGG CTG TGG TCA-3' (SEQ !D NO: 18) corresponding to amino acids QMWLWS (SEQ ID NO: 19) and noggin 3' : 5'-GCAGGAACACTTACACTC-3' (SEQ ID NO: 20) corresponding to amino acids EC CSC (SEQ ID NO: 21 ) of the mouse noggin protein .

The oligonucleotides were used for PGR amplification of a segment of DNA of 260 nucleotides using as a template a mouse cDNA clone prepared as set forth in Example 3. The amplified fragment had a nucleotide sequence that corresponds to nucleotides 2 through 262 of the mouse sequence as set forth in SEQ ID NO: 10. After amplification, the PGR reaction was e!ectrophosed in agarose gels, the DNA band of 260 nts purified by Magic PGR (Promega), and used as template for the probe labeling reaction. The probe was labeled using a standard PGR reaction (Perkin-Eimer) on 20 ng of DNA template and 0.2 m Curie of alpha 32P-dCTP (Du Pont 3000 Ci/mmol) instead of dCTP. Unincorporated label was separated from the probes on a G5Q NICK column (Pharmacia). The excluded voiume of the reaction contained a total of 1.8 x108 cpm.

In addition, one degenerated oligonucleotide, named noggin D. corresponding to conserved mouse and Xenopus noggin sequences, was synthesized as follows: Noggin D: 5'- GARGG!ATGGTITGYAARCC- 3' (SEQ ID NO: 22). Noggin D (SEQ ID NO: 22) was labeled by kinase reaction using T4 polynucleotide kinase and gamma 32P ATP. The labeled oligonucleotide was purified by NAPS (Pharmacia) column and used for library hybridization.

Library screening A human placental genomic library (Clontech Gat#HL1067J, average insert size 15 kb) in vector EMBL-3 was plated according to manufacturer specifications in NM 538 E.coli. Approximately 3 million plaques were transferred to nitrocellulose filters (BA-85 Schieicher and Schuell) in three repiicas (named A, B and C) and screened according to aniatis, et ai.[Sambrook, et a., Molecular cloning a laboratory manual, CSH Lab Press, New York (1989)]. The replica filters A and C were hybridized in a buffer containing 0.5 sodium phosphate, pH 7.2, 7% sodium dodecyi sulphate, 1% crystalline BSA, 1 mM EDTA, 40 m g/mi denatu rated salmon sperm D A and about 1 06 cpm/mi of the PCR probe (supra). After hybridization for 12 h at 65 °C, the filters were washed twice at room temperature in 2x SSC (30 mM sodium citrate, 0.3 M NaC!), 0.1% SDS and then at 65°C in 2xSSC, 0.1% SDS for 20 min and exposed to Kodak X-OMAT AR fiim. The filter repiica B were hybridized with the labeled oligonucleotide noooin D in 6xSSC , 0.1 % SDS at 51 °C for 12 h followed by wash at 2xSCC, 0.1% SDS at room temperature, and in SxSSC, 0.1% SDS at 50°C and exposed to Kodak X- OMAT AR fiim. Positive plaques from all replicas were isolated and purified by re-screening as above. Purified positive plaques were suspended in 500 μΙ SM (100 mM NaCI, 10mM MgS04 x 7H20, 50 mM Tris HC! pH 7.5, 0.01% gelatin). 160 μΙ of phage suspension was mixed with 0.5 mi saturated NM538 culture, incubated for 20 min at 37°C and then inoculated into 250 mi LB containing 10 mM g S04, 0.2% maltose. The cultures were incubated untii ceil lysis (7-8 hr) at 37°C. The phage lysates were used for phage DNA purification by the Qiagen procedure according to the manufacturers recommendations (Qiagen).

Sequencing, Sequencing was performed by using the Applied Biosystems Mode! 373A automatic sequencer and Applied Biosystems Taq DyeDeoxy™ Terminator Cycle Sequencing Kit.

Results Filters hybridized to the PGR mouse noggin probes (SEQ ID NOS: 13 and 20) showed two strong signals corresponding to phage piaques named hnogX-9 and hnog-10. These plaques also hybridized to degenerate oligonucleotide probe ηοσσίηΡ (SEQ ID NO: 22) revealed thai these clones correspond to the human noggin gene. In addition, two other plaques named hnogX-5 and hnog -7 produced slightly weaker signals when hybridized to the PGR probes. These clones correspond to either human noggin or related gene(s). All of the human DNA inserts can be excised from the vectors using known restriction sites as described in the literature regarding each particular library.

A 1.6 kb Sa fragment from clone hnog -9 containing the human noggin gene was subcloned and the nucleotide sequence determined as set forth in Figure 1. The amino acid sequence for human noggin, as deduced from the nucleotide sequence, is aiso set forth in Figure 1. The gene or cDNA may be expressed in various eukaryotic or prokaryotic expression systems to produce biologically active human noggin protein, !t is expected that the human protein will exhibit neurotrophic activity similar to that exhibited by Xenopus noggin protein.

EXAMPLE 5 Tissue Localization of message for human ηοσσίη Materials and Methods Probe preparation Probes were prepared as set forth in Exampie 4, The oligos used are as follows: SEQ ID NO: 23: ' GACJCGAGT.CGA.CAT.CGC.AGA.TGT.GGC.TGT.GGT.CAC SEQ ID NO: 24: ! CCA.AGC.TTC.TAG.AAT.TCG.CAG.GAA.CAC.TTA.CAC.TCG.G (The underlined sequence represent mouse noggin sequence; the . rest of the sequence are tails containing restriction sites for cloning.) A DNA fragment of approximately 300 bp was obtained by PGR amplification of a mouse cDNA clone prepared as described in Example 3.

RNA Preparation and Northern Blots Selected tissues were dissected from Sprague-Dawley rats and immediately frozen in liquid nitrogen. RNAs were isolated by homogenization of tissues in 3 M LiCI, 6 M urea, as described in Bothwell, et at. 1990 (Methods of Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett). RNAs (10 μg) were fractionated by electrophoresis through quadruplicate 1% agarose-formaldehyde gels (Bothwell, et ai., 1990, Methods of Cloning and Analysis of Eukaryotic Genes, Boston, MS, Jones and Bartlett) followed by capillary transfer to nylon membranes (MagnaGraph, Micron Separations inc.) with 10xSSC (pH7). RNAs were UV-cross-linked to the membranes by exposure to ultraviolet light (Strataiinker, Straiagen, inc.) and hybridized at S8°C with radiolabied probes in the presence of 0.5 M N P04 (pH 7), 1 % bovine serum albumin (fraction V, Sigma, Inc.) 7% SOS, 1 mM EDTA [ ahoudi, et ai., Biotechniques 7 :331 -333 (1939)3, 100 μgί(τ\\ sonicated, denatured salmon sperm DNA. Filters were washed at 88°C with 3xSSC: 0.1 % SDS and subjected to autoradiography for 1 day to 2 weeks with one or two intensifying screens (Cronex, DuPont) and X-ray film (AR-5, Kodak) at 70°C. Ethidium bromide staining of the gels demonstrated that equivalent levels of total RNA were being assayed for the different samples ias in Maisonpierre, et al., Science 247:1446-1451 (1990)].

RNA was prepared from the following human cell lines: Neuroblastoma Neuroepithelioma CHP-134 SK-N-MC LA-N-1 CHP-100 LA-N-5 !ARC-EW! IMR-32 SK-N-LO SHSY5Y SK-ES SKNSH DADY SHEP Hematoooetic Small Cell Luna Carcinoma Cervicai Carcinoma K562 Calif 3 HeLa U937 SKLu Ml NCI-H69 TF1 SK ES BAF B9 Sympathoadrenal Precursor Hepatoblastoma stiuNQbtastQma MAH HEPG2 adsen Med U266 Pheochromocvtoma PC12 RESULTS We have amplified a DNA fragment from the mouse noggin plasmid, corresponding to the region conserved between. Xenopus and mouse noggin.

The amplified fragment of approximately 300bp was used as probe to hybridize to northerns, with RNAs prepared from aduit and embryonic tissues, as well as from various ceil lines. Noggin transcript of about 2Kb in size was detected in adult rat brain, and in a cell line, SKMES, a small ceil lung carcinoma.

Expression of noggin transcripts was examined in various tissues from rat and mouse at different stages of development and in adult. In the mouse, noggin transcripts can be detected in embryos or head from E9 to E12, as well as in newborn brain and adult brain. There was no detectable signai in peripheral tissues examined except in skeletal- muscle. Abundant level of expression was also found in hippocampal astrocytes isolated from postnatal mouse.

In the rat, noggin transcripts were detectable in embryos or head from E9 to E18, as well as in brain from P1 , P19 and adult brain. In the cerebellum, expression of noggin appeared to be higher in E18 and P1 ; in the spinal cord, expression of noggin mRNA peaked at P1. Examination of noggin expression in all of the CNS regions, especially the olfactory bulb, midbrain, hindbrain and cerebellum. In the adult, noggin mRNA could be detected in ail CNS regions, especially the olfactory bulb and cerebellum. There also appeared to be low levels in the skin.

EXAMPLE 6 Neural Induction bv Noggin Materials and Methods Preparation of Xenopus noggin CHO ce!l conditioned medium Xenopus noggin CHO conditioned medium was made by selecting for stably transfected CHO cells. Dihydrofolate reductase (DHFR) deficient CHO parental cells { J. Papkoff, Syntex Research) were transfected with a Xenopus noggin expression plasm id containing noggin in tandem with the dihydrofolate reductase gene. Growth in nucleoside free medium was used to select for successfully transfected ceils. Nine colonies of transfectants were picked and grown up individually. The noggin gene in these cells was amplified by slowly increasing the dose of methotrexate, an inhibitor of DHFR. The presence of noggin transcripts was first tested by Northern analysis. Subsequently, two clones, B3 and C3, were shown to secrete noggin protein, since conditioned medium from these lines was capable of dorsalizing ventral marginal zones. Furthermore, by labeling B3 cellular proteins with 35S-methionine, noggin protein could be identified as a band of about 30kD on reducing SDS-PAGE, and a band of 60kD on non-reducing SDS-PAGE indicating it forms the expected dimer. These properties matched those of the noggin protein previously produced in Xenopus oocytes supra, {Smith et al., Nature 361 , 547-49, 1993). B3 conditioned medium was collected in a mixture of 1 part alpha MEM and 9 parts CHO-S-SF il (Gibco-BRL). The cells were allowed to condition the medium for 3 days. Control medium from parental cells (CHO dhfr-) was collected identically. Twenty fold concentrated medium was made using Centriprep 10 concentrators, where the 20 fold change is measured by volume.

Purification of human nonpin from COS ceils Human noggin protein was purified by a cationic exchange column. COS/M5 ceils were transiently transfected with . a human noggin expression plasmid, pCAEI I . Ceils were allowed to condition DME (Specialty Media) for two to three days, after which the medium was removed. Particulates from the medium were removed by a centrifugation step and subsequent passage through a 0.2um cellulose acetate filter. This cieared medium was pumped onto a MonoS (Pharmacia) coiumn which was washed with several volumes 40mM sodium phosphate (pH 7.3), 150mM NaCI, 1 mM EDTA. Proteins were then eluted in a linear gradient with 40mM sodium phosphate (pH 8.5), 1.3M NaCI, 1 mM EDTA. Noggin protein e!utes at 0.8M NaCI and is ≥ 90% pure by SDS-PAGE.

Xenoous otx Isolation To isolate Xenopus Otx clones a tadpole head cDNA library (Hemmati-Brivanlou, et al., Development 106, 61 1 -617, 1989) was screened with a mouse otx cDNA (S-L Ang and Rossant, Toronto) at iow stringency. The clones that were picked fell into two classes. One class, which we have designated otxA, included pXOT21.2, the probe used here. By in situ hybridization, transcripts are first detected prior to gastrulation in the superficial layer on the dorsal side. During neurulation a large anterior domain expressed the gene, and includes both neural and non-neural tissues. After a decline in expression in the tailbud tadpole, the gene is reexpressed specifically in the brain and eyes.

Ventral marginal zone assay Embryo oreoaration Xenopus laevis embryos are fertilized and de-jei!ied as described (Condie and Harland, 1987. Development 101 , 93-105), routinely the evening before dissections. Embryos are cultured overnight at 15°C. The vitelline membrane surrounding each developing embryo is manually removed the following morning at the late biasiula stage. Untii dissection, the embryos are maintained in 1/3x modified ringers in agarose coated dishes.

Ventral marginal zone dissection Embryos are oriented with their yolky vegetai hemisphere up so the dorsai side can be identified. The dorsal side of the eariy gastruia is marked by the presence o a small arc of dense pigment called the "dorsal lip" which marks the start of involution of dorsal mesoderm. The ventral marginal zone (VMZ) is found directly opposite the dorsal lip, and is dissected. Since the vitelline membrane has been removed, the embryo tends to flatten. Using a specially constructed knife made of an eyebrow, mounted onto a glass pipet with wax, two cuts are made through the flattened embryo from the top facing vegetal pole through to the animal pole. The cuts are made such that they isolate approximately 30-60 degrees of the ventral side away from more lateral tissues. A third cut which is perpendicular to the first two cuts completely isolates the ventral marginal zone tissue away from the rest of the embryo. This third cut is at the level of approximately two thirds of the radius of the embryo from the center. Prior to treatment the VMZ is washed 1x in the culture medium.

Assay Approximately between 5 to 10 VMZs are used per assay. The washed VMZs are dropped gently (trying to minimize transfer of liquid) into eppendorf tubes containing the desired treatment protein medium for assay. The VMZs are allowed to develop to the late neurula or early tailbud stage as assessed by control whoie embryo development. At this time RNA is isolated from the VMZs and control whole embryos as described (Condie and Hariand, ibid). The expression of muscle actin in VMZs indicates a derealization event (Lettice and Slack, 1933. Development, 1 17, 263-72) . RNA from each sample is run on a formaldehyde-agarose gel and blotted to gene screen. The blot is then hybridized with a Xenopus muscle actin probe (Dworkin-Rastl et al., 1986. J. Embryol. exp. Morph. 91 , 153- 8) . Quantitation of dorsaiization can be carried out by normalizing muscie actin signal to that of the ubiquitously expressed EF-1 a (Krieg et al., 1989. Devi. Bioi. 133, 93-100) . Quantitation is done using phosphor imaging.

RNase protection assay RNase protection was carried out as described (D.A. Melton et ai., Nucieic Acids Res 12,7035-56, 1984), with the modification that digestion was carried out at room temperature (22° C) using RNase T1 oniy (Ca!biochem 556785) at 10 units/mi. 20-30 animal caps were harvested for each lane, of this 80% was used for neural markers and 10% for muscle actin and collagen type II. For goosecoid and brachvury 20 caps were used. Exposures ranged from 12 hours to 5 days. In al! cases, films were preflashed. !n cases where a marker was not expressed, the result was confirmed with greater sensitivity using phosphor imaging.

Results.

The development of vertebrate embryos requires several inductive interactions. Mesoderm, which eventually forms tissues such as notochord, muscie, heart, mesenchyme and blood, is induced in the equatorial region of the embryo (Nieuwkoop, Wiihelm Roux' Arch. Entw ech. Org, 162, 341 -373, 1969) . This inductive event is well studied, and there are several candidates for the endogenous inducer(s) including members of the fibroblast growth factor(FGF) family and activin (Jesseil and Melton, Cell 68, 257-70 1992; Sive, Genes Dev 7, 1 -12, 1993) and TGFb family (Asashima, et al., Roux's Arch. Dev. Bioi. 198, 330-335, 1990; Asashima, et al., Naturwissenschaften 77, 8, 389-91 , 1990; Green and Smith, Nature 347, 391 -394, 1990; Smith, et al.f Nature 345, 8277, 729-31 , 1990; Thomsen, et a!., Ceil 63, 485-493, 1990; van, et ai., Nature 345, 6277, 732-4, 1990) . The use of dominant negative receptors for both FGF (Amaya, et ai., Cell 66, 257-270, 1991 ) and activin (Hemmati-Brivanlou and Me!ton, Nature 359, 609-614, 1992) in Xenopus embryos strongly suggests that the signaling pathways activated by these molecules are essential for proper mesoderm formation . Molecules such as wnts {Christian, et a!., Development 1 1 1 , 1045-1055, 1991 ; McMahon and Moon, Cell 58, 1075-84, 1989; Smith and Harland, Ceil 67, 753-765, 1991 ; Sokoi, et ai., Cell 67, 741 -752, 1991 ) and noggin (Smith, et a!. , Nature 361 , 547-49, 1993) modify the kinds of mesoderm made without inducing mesoderm directly. in a subsequent induction, the dorsai mesoderm of the Spemann organizer signals nearby lateral mesoderm to take on a more dorsal fate (Dale and Slack, Development 100, 2, 279-35, 1 987; Lettice and Slack, Development, 117, 263-271 , 1993; Spemann and Mangold, Arch, mikrosk. Anat. EntwMech. 100, 599-S38, 1924; Stewart and Gerhart, Development 109, 363-372, 1990) , The only known factor which is expressed in the organizer and can mimic its dorsalizing activity is noggin.

Dorsal mesoderm of the Spemann organizer also signals nearby ectoderm to become neural tissue. Neural induction by dorsai mesoderm has been demonstrated in amphibians (Dixon and Kintner, Development 106, 749-757, 1989; Doniach, et al., Science 257, 5069, 542-5, 1992; Hamburger, The Heritage of Experimental Embryology: Hans Spemann and the Organizer, 1988; Kintner and Melton, Development 99, 311 -25, 1987; Spemann, Arch, mikrosk.

Anat. EntwMech. 100, 599-838, 1938) , birds (Kintner and Dodd, Development 113, 1495-1506, 1991 ; Tsung, et a!., Acta Biol exp Sinica 10, 69-80, 1965) , and recently in mice (Ang and Rossant, Development 118, 139-149, 1993) . Despite decades of effort, little is known about the molecular nature of the factors responsible for this induction. Among known inducers, activin can promote formation of neural tissue, but this is due to a secondary induction by the dorsai mesoderm that activin induces (Green, et al., Development 108, 1 , 173-83, 1990; Green and Smith, Nature 347, 391-394, 1990; Kintner and Dodd, Development 113, 1495-1506, 1991 ) . Thus, activin cannot promote formation of neural tissue when added to gasiruia ectoderm; however, such ectoderm remains competent to be neuralized by dorsal mesoderm untii the end of gastru!ation (Sharpe and Gurdon, Development 109, 765-74, 1990) .

Direct Neural induction bv Nooain Candidates for the endogenous inducer are expected to induce neural tissue in the absence of dorsal mesoderm. Competent animal cap ectoderm from late biastula stage embryos (St9) was used to test noggin's neural inducing capacity. Xenopus noggin protein conditioned medium was collected from stably transfected CHO cells and twenty fold concentrated medium was used to treat St 9 animal caps. Markers used in an RNase protection assay were N- CAM (Jacobson and Rutishauser, Developmental Biology 116, 524- 31 , 1986; Kintner and Melton, Development 99, 311-25, 1987) , a neural ceil adhesion molecule, a neural specific isoform of b-tubuiin (Good, ei ai. Nucleic Acids Res 17, 8000, 1989; Good, et al., Dev Biol 137, 414-8, 1990; Richter, ei ai., Proc Natl Acad Sci USA 85, 8086-90, 1988) that is expressed in the hind brain and spinal cord, and XIF3, a neurally expressed intermediate filament gene (Sharpe, et al., Development 107, 701 -14, 1989) to assay for neural induction. All these markers are restricted to neural tissue, however, only NCAM is expressed throughout the nervous system. We found that Xenopus-noggin conditioned medium induces high levels of N-CAM and XIF3 expression[Fig. 2.; Iane8] in treated animal caps, without inducing muscle actin(!ane 13) (Dworkin-Rastl, et al., J. Embryol. exp, orph. 91 , 153-168, 1986; Mohun, et al., Nature 3 , 716-721 , 1984) . Control CHO cell medium induces neither muscle nor neural tissues (lanes 7,12). St 9 activin treated animai caps express muscle actin(ianei 1 ) and all three neural markers(iane 6), demonstrating activin's ability to generate neural tissue indirectly. It is interesting to note that noggin induces very little, if any b-tubuiin expression, while inducing high levels of N-CAM, but activin induction has nearly the converse effect.

To determine whether noggin protein is sufficient to- induce neural tissue, COS cells were transfected with pCAEH , a human noggin expression piasmid, and the conditioned medium was purified by cation exchange chromatography resulting in noggin preparations that were 90% pure [Fig. 3.]. Such purified human noggin protein is aiso able to induce neural tissue in animai caps [Fig. 4a., see below].

We have shown that noggin does not induce muscle in late blastula stage animal caps, however, it is possible that noggin induces other types of dorsai mesoderm. To address this concern, we asked whether noggin could induce the expression of the ear!y mesoderm markers goosecoid {Blumberg, et ai., Science 253, 194-6, 1991 ; Cho, et al., Cell 67, 11 11-20, 1991 ) , a marker of organizer tissue and subsequently head mesoderm or X-brachyury (Smith, et al., Cell 67, 79-87, 1991 ) , which appears to be expressed in all mesodermal precursors early, and subsequently is expressed in posterior mesoderm and notochord. Animal caps were treated at stage 9 and collected at stage 11 , when expression of goosecoid and brachyury in the normal embryo is high. Neither marker is turned on by purified human noggin (Fig. 4b. lane 5) at a dose with demonstrated neural inducing activity (Fig 6 Iane15); in contrast animal caps treated in the same fashion with activin show both goosecoid and X-bra expression (Fig. 4b. iane 4) as expected for this mesoderm inducing factor (Cho, et ai., Ceil 67, 1111-20, 199 ; Smith, et ai., Cell 67, 79-87, 1991 ) . Untreated animal caps show no expression of these mesodermal markers (lane 3), and RNA levels in the collected animal caps are shown to be comparable using EF-l levels .(Krieg, et ai., Dev Biol 133, 93-100, 1989) .

Since purified human noggin is capable of driving neural induction, no additional factors which may have been present in the crude conditioned medium are required. Furthermore, Xenopus and human noggin, with 80% amino acid identity, can both act to induce neural tissue in Xenopus, suggesting a conserved function for these two proteins. However, for noggin to be a candidate endogenous neural inducer it must be able to induce neural tissue at a stage when neural induction occurs in normai whole embryos. It is unclear when the first instructive signals are sent from dorsal mesoderm to ectoderm in embryos. However, it is known that by early gastruia stages, dorsal ectoderm has already been specified to become neurai tissue (Jones and Woodland, Development 107, 785-91 , 1989) .

The neural inducing signal is therefore likely to start before this stage. The latest stage at which animal caps have been shown to be competent to respond to neural-inducing mesoderm is the early neurula (St13-14) (Sharpe and Gurdon, Development 109, 765-74, 1990) . . Thus, a candidate endogenous neural inducer must be able to induce neural tissue from gastrula stage competent ectoderm.

Neurai induction at the Gastrula Stage In order to assess the competence of ectoderm to respond to noggin we treated animal caps taken from biasiula (St8), late blastu!a (Si9), early gastrula (St10) and ventral animal caps from • mid-gasiruia (St O.5) stage embryos with purified human noggtn[Fig. 2.]. We also treated similarly staged animal caps with activin to demonstrate its mesoderm inducing and secondary neurai inducing activities, and to contrast activin's effects with those of noggin[Fig. 4a. ]. Activin treated animal caps show neural induction only in conjunction with induction of dorsal mesoderm, such as muscle and notochord (lanes 3,6,9). In a number of experiments, we confirmed that activin's ability to induce dorsal mesoderm, and consequently neurai tissue, declines rapidly at the gastrula stage (lane 12) (Green, et ai., Development 108, 173-83, 1990; Kintner and Dodd, Development 113, 1495-1506, 1991 ) . in the experiment shown here a larger than usual dose of activin was given. Under these conditions, only a small amount of neurai tissue is made, perhaps because so much mesoderm is induced that there is not much competent ectoderm, left in the explant to be neuraiized. In contrast noggin can induce neural tissue in animai caps taken from ail of these stages without inducing the notochord and somite marker, collagen type H (Amaya, et a!., Development 1 18, 477-87, 1993; Bieker and Yazdani-Buicky, J Histochem Cytochem 40, 1 1 7-20, 1992) , or muscle actin (lanes 4,7,10,13). This gives additional support to the proposal that noggin is a direct neurai inducer, since it can act in the absence of both early and late mesoderm markers. Furthermore, we have shown that noggin can induce neura! tissue in competent ectoderm at a time when mesoderm inducers are inactive. In some experiments, noggin addition to gastrula (but not biastu!a) animal caps resulted in induction of muscle (data not shown). This occurred at stages when activin could no longer induce muscle. We interpret this as a resuit of a dorsalizing action by noggin on tissues that have received a weak mesoderm-inducing signal, The mesoderm-inducing signal which spreads into the gastrula animal cap is not enough to induce mesoderm, but in the presence of Xwnt-8 or noggin, muscle Is formed. One interesting corollary of the induction of muscle is that the kinds of neurai tissue seen in the expiant are modified. Induction in expiants that contain no muscle usually yields N-CAM expression, but if muscle Is present, expression of both N-CAM and b-tubuiin is seen. This phenomenon is demonstrated in the secondary neurai induction by activin in St. 9 animal caps[Fig. 2.] and in the comparison of neurai tissue induced by noggin in ventrai marginal zones versus animal caps [Fig. 6.]. In the ventrai marginal zones and animal caps in which muscie is present, both N-CAM and b-tubuiin are expressed, whereas induced animal caps without muscle, show only N-CAM expression.

Neural induction after injpr†jon of DNA coding for nogg in To confirm our conclusions using a different experimental approach, we have directed noggin expression to gastruia stage animal caps by injecting the plasmid pCSKA-noggin into the animal pole of a one ceil stage embryo. This plasmid, in which noggin is under the control of the cytoskeletai actin promoter, turns on the expression of noggin mRNA at the onset of gastrulation (Smith, et a!., Nature 361 , 547-49, 1993) . At the blastuia stage, the animal caps are dissected and then matured to tailbud stages for molecular analysis. Animal caps injected with the noggin plasmid show expression of N-CAM in the absence of muscle or notochord markers (Fig. 4c. lane 2). A control plasmid directing the expression of lac 2 showed no neural or mesodermal induction as expected (lane 1 ). This experiment demonstrates that ectopic noggin expression can directly induce neural tissue in gastruia stage ectoderm, a stage when neural induction is taking place in whole embryos.

Differences in competence between dorsal and ventral animal cans.

Animal caps taken from the dorsal side of gastruia stage . embr/os show greater competence to form neural tissue than yentral animal caps (Otte and Moon, Ceil 68, 1021 -29, 1992; Sharpe, ei al., Ceil 50, 749-58, 1987) , when involuted anterior mesoderm is used as the inducer. This type of mesoderm, however, has weaker inducing capacity than the rest of the involuted mesoderm (Sive, et al., Ceil 58, 171-180, 1989) . Furthermore, the ventral side of an embryo can support the formation of a complete secondary axis when the organizer is placed on that side (Gimiich and Cooke, Nature 306, 471-3, 1983; Smith and Slack, J. Embryoi. Exp. Morph. 78, 299-317, 1983; Spemann, Arch, mikjrosk. Anat. EntwMech. 100, 599-838, 1938) , indicating that there is no qualitative difference in competence. Thus, while a weak inducer might unmask slight differences in competence of the ectoderm, it has been suggested that a robust neurai inducer would show little difference in its effects on dorsal and ventral ectoderm (Servetnick and Grainger, Development 112, 177-88, 1991 ) . Therefore we tested noggin's effects on dorsal and ventral ectoderm from the early gastruia. No difference in N-CAM expression is detected (Fig. 5, lanes 4,6), while the ventral animal caps treated with noggin show a greater amount of muscle actin expression (presumably through dorsa!ization of tissues that received a low-grade mesoderm induction). Activin treated dorsal caps show induction of roughly the same level of muscle actin expression (lane 5) as the ventrai noggin treated caps, however, activin treatment did not induce detectable neural specific transcripts (lanes 3,5). This indicates that muscle tissue induced at this stage is not sufficient to secondarily induce neural tissue, and that noggin must be present to induce neural tissue.

We conclude that there is no dcrsai-ventrai difference in noggin mediated neurai induction, suggesting that noggin behaves like the robust neural inducing signal of the Spemann organizer, not like the weaker signal from early anterior mesoderm.

Dose Dependence To determine what levels of noggin protein are required for neurai inducing activity, we carried out a dose response experiment. In addition to determining the doses required for neural induction in animal caps, we have aiso carried out a dose response of the dorsaiization of ventral marginal zones in order to compare the doses required for these two types of inductions. Stage 9 animal caps or St. 10.5 VMZ were treated with purified human noggin, and N-CAM and β-tubuiin were used to assay neural induction, while muscle actin was used as a marker of dorsal mesoderm. This experiment shows that neural induction occurs at a dose of Ι μς/ml, which is a twenty fold higher dose than required for dorsaiization of VMZ [Fig. 5j. There are several observations that may account for the apparently high dose requirement. First, to get a maximal neural response from dorsal mesoderm, the tissues must be left in contact through most of neurulation (Sharpe and Gurdon, Development 109, 765-74, 1990) ; in contrast, the animal caps treated with noggin ciose up rapidly, this inhibits factor access, and consequently they receive only a brief effective dose. Second, it is likely that noggin is not the only neurai inducer active in the embryo; it has been shown in a variety of amphibians that the somites (Hemmati-Brivanlou, et aL Science 250, 800-802, 1990; Jones and Woodland, Development 107, 785-91 , 1989) and the neurai plate have neurai inducing activity (Hamburger, The Heritage of Experimental Embryology: Hans Spemann and the Organizer, 1988; Servetnick and Grainger, Dev Biol 147, 73-82, 1991 ) and noggin transcripts are not detected there. Thus it is plausible that noggin is one of several neural-inducing activities. In this connection it is worth noting that noggin is equally potent in inducing neurai tissue in ventral marginal zones as in dorsaiizing them to generate muscle. Numerous other experiments (see Fig. 5) show that induction of a similar amount of muscle at this stage by activin does not resuit in neurai induction. Fourth, it may be that only a small fraction of the purified protein is active, and that the experiment overestimates the amount of protein needed for neural induction. Finally, it is possible that the accessibility of exogenously added solubie noggin is significantly lower than noggin protein being secreted endogenousiy.

Patterning Embryonic neural tissue develops an anteroposterior (A-P) pattern, with various brain structures, eyes, and the spinal cord, it is thought that A-P neural pattern requires the presence of dorsal mesoderm, whether it be adjacent to the responding ectoderm in a planar configuration (Dixon and Kintner, Development 106, 749- 757, 1989; Doniach, et al., Science 257, 542-5, 1992; Kintner and Melton, Development 99, 311-25, 1987; Ruiz i Altaba, Development 108,595-604, 1990) , or directly beneath it in a vertical interaction {Dixon and Kintner, Development 106, 749-757, 1989) (Hemmati-Brivanlou, ei a!., Science 250, 800-802, 1990; Sharpe and Gurdon, Development 109, 765-74, 1990; Sive, et al., Cell 58, 171 -180, 989) . Both of these types of interactions occur in normal development, and both probabiy contribute to the resulting pattern. To deiermine if noggin induces patterned neural tissue, and if so, what neural regions are represented, we used Xenopus otx as a marker of forebrain and mid brain; En-2 (Hemmati-Brivanlou, et al., Development 1 1 , 715-724, 1991 ) as a marker of the mid brain- hind brain boundary, and Krox-20 (Wilkinson, et al., Nature 337, 461 - 4, 1989) as a marker of the third and fifth rhombomeres of the hind brain in in situ hybridization (Hariand, Methods in Cell Biology, 36, 675-685, 1991 ) . Antibodies directed against XIHbox S (Wright, et ai., Development 109, 225-34, 1990) mark posterior hind brain and spinai cord structures. Prior to the use of these markers, we observed the formation of cement giands in noggin treated animai caps. Since cement glands are induced organs of ectodermal origin found anterior to the neural plate, this resuit suggests that noggin induces anterior structures. In situ hybridization confirms this by showing the presence of a cement gland specific transcript, XAG-1 (Sive, et ai., Ceil 58, 171 -180, 1989) in noggin treated animai caps, but not in control treated animal caps[Fig. 7.]. !n situ hybridization with the region specific neural markers[Fig. 7.] show that noggin induces forebrain type tissue as seen by the expression of otx in noggin treated animal caps. We have not detected En-2, Krox20, or XIHbox, suggesting that these more posterior markers are not induced by noggin.

Expression of neural anlinens We have demonstrated that noggin directly induces the expression of neural specific transcripts. A further demonstration is to use antibodies against neurai specific antigens to show that the noggin induced tissue is phenotypicaily neurai. To this end, we have treated animal cap tissue with noggin and cultured them to a late stage (St 35) for antibody staining. We have used the 8F1 1 anti-N-CA antibody, which stains the entire neural tube of a normai embryo. Noggin treated animai caps express this antigen [Fig. 7.] while control untreated animai caps do not. This indicates that noggin can induce the production of neural specific proteins in treated animal caps. We have failed to detect the expression of numerous other antigens that are characteristic of various subclasses of differentiated neural cells. These included 2G9, which 83 siains most neural tissue, including peripheral neurons, Tor 24.55, which stains sensory neurons, and Tor 23, which stains a variety of neurons including motor neurons.

EXAMPLE 7 Production of recombinant human ηοσσϊη from E. coli and baculovirus Materials and Methods Genetic Engineering and Ceil Cuiture A lactose inducible expression piasmid was constructed by replacing the Swa1/Bsm1 region of pRPN40 (Masiakowski et ai , J. Neurochem. 57, 1003-1012, 1991 ) with the Swa1 /Bsm1 region of the human noggin gene obtained by PCR and spanned by the same restriction sites, resulting in plasmid pRG301 . pRG301 is a high copy number kanamycin resistant plasmid derived from pBR322 with the human noggin gene under the control of the lacUVS promoter. A piasmid containing the high copy number kanamycin resistant gene was deposited with the Agricultural Research Collection (NRRL), Peoria, Illinois, and bears accession number B-1 S600. This piasmid was described in United States Patent Application Serial No. 07/478,338, which is incorporated by reference herein in its entirety. E. coli W31 10laciq cells transformed with pRG301 were grown at 37°C, induced with lactose, harvested by centrifugation, washed once with 100 mM Tris-HCI, 50 mM EDTA pH 8 and stored frozen, essentially as described (Masiakowski et al, ibid. ).

Recovery from inclusion bodies E. coii cell paste (32 g) was suspended in ten volumes (v/w) of 50 mM TrisHCI-pH 8.0-5 mM EDTA, lyse i in a French Press at 8,000 psi and 8°C and centrifuged at 8,000xg for 30 min at 4°C. The pellet containing noggin was suspended in the original volume of 2 M urea-20 mM TrisHCI, pH 8.0 and stirred for 30 min. The suspension was centrifuged at 8,000xg at 4°C for 30 min and the pellet consisting mostly of inclusion bodies (IB) was suspended in 20 volumes (v/w) of 6 M guanidine HCI, 50 mM TrisHCI.1 mM EDTA, 50 mM DTT and stirred for one hour at room temperature. After centrifugation at 8,000xg for 30 min, the supernatant containing 0.45-0.50 g denatured and reduced noggin was diafiitered against 10 volumes of 6 M urea-50 mM sodium acetate pH 4.5-1 mM EDTA-0.1 mM DTT using Omega 10,000 MW cut-off membranes. The diafiltrate containing 0.4- 0.44 g noggin was ioaded at a flow rate of 30 ml/min onto a 2.6 x 10 cm column of S-Sepharose (Pharmacia), equilibrated in 6 M urea-50 mM sodium acetate- 1 mM EDTA-0.1 mM DTT pH 4.5 . The column was first washed with the same buffer and then with a one liter gradient (0-1 MaCI) at a flow rate of 30 ml/min. Fractions containing noggin were identified by gei electrophoresis and pooled. The yield was 0.2-0.25 g noggin.

Refolding.

The denatured and reduced noggin solution was adjusted to .05- .2 mg/mi protein concentration and brought to 1.5-2.5 M guanidineHCI-0.1 M TrisHCI pH 8.0-0.1 mM EDTA-0.2-2 mM reduced glutathione-0.02-0.2 mM oxidized glutathione (preferably at a ratio of 10:1 reduced to oxidized glutathione) at 4°C under slow stirring. After 24-72 hours, two refolded noggin isoforms were identified by RP-HPLC chromatography (Fig. 8). The refolded noggin solution was diafiltered against 20 volumes of 0.05 M sodium acetate pH 4.5, brought to 50 mM potassium phosphate pH 7.2 and stirred slowly at 4°C for 1 hour minimum. Misfolded noggin precipitated and was removed by centrifugation for 30 min at 8,000 xg.

Reverse ohase HPLC chromatography.

Refolded noggin can be purified by chromatography on a 12 mm C8, 1 x 25 cm Dynamax 300 A column equilibrated in solvent A (0.1% TFA in water). After loading, the column was washed with solvent A and was developed at a flow rate of 4 ml/min according to the following protocol: (a) 10 min isocratically at 70% of solvent A, 30 % of solvent 8 (0.1% TFA in acetonitriie); 30 min linear gradient to 60 % solvent B and 40 % solvent A. Correctly refolded noggin e!utes earlier at 44%-46% solvent B. The yield was 0.07-0.1 g noggin.

Production of human ποασίη in Bacuiovirus cell culture.

The SF21 line of Spodoptera frugiperda was routinely maintained as cell monolayers in Grace's Insect Cell medium supplemented with !acialbumin hydroiysate and yeastoiate (Gibco).

This medium completed with 10% v/v heat-inactivated fetai calf serum (Irvine Scientific) is identified as T NFH-10. Cells were also cultured in serum-free medium (SF-900-!l; Gibco) after adaptation. Suspension cultures in either medium were raised in microcarrier culture flasks (Bellco) using a stirring speed of 80 rpm. All cultures were maintained at >96 % viability, as judged by trypan blue exclusion.

Construction of recombinant har.iilnvirus.

Sequences corresponding to human noggin were excised as a 5'-BamH1 — Psi1 -3" fragment from an expression plasmid containing the human noggin gene. This fragment was inserted into BamH1 — Pst1 digested pVL1393 (invitrogen). The resulting plasmid, pTR 1009, has the human noggin sequence immediately downstream of the polyhedrin promoter of Autrographa californica Multiple Nuclear Poiyhedrosis Virus (AcMNPV), and this promoter-heterologous gene fusion is flanked in turn by recombination targets derived from the Ac NPV polyhedrin region. Recombinant plasmid DNA was purified by aikaiine lysis and CsCl centrifugation. SF21 cells were co-t ans fected with plasmid and viral DNA by the following method: Plasmid DNA (3 mg) was mixed with 0.5 mg linearized, deleted vi ral DNA (Bacuio Gold™, Pharminigen), and precipitated with ethanoi. Dried DNA was then resuspended in water (50 ml), mixed with 1 .5mi Grace's medium, and 30 ml Lipofectin™ cationic liposomes (BRL). The DNA-!iposome mixture was vortexed, allowed to stand at room temperature for 15 minutes and added dropwise to a monolayer of SF21 ceils (2x106 cells/60 mm plate). After incubation at 27°C f o r four hours, 2 mi T NFH-10 was added and the culture returned to incubation for 5 days. Tissue culture medium was harvested and used as a source of virus for plaque isolation.

Recombinant virus was isolated by multiple rounds of piaque purification on SF21 ceils. Diluted virus (0.5 mi) was adsorbed t o ceil monolayers (2x106 ceils/60 mm plate) for a period of one hour at 27°C, aspirated, and virus plaques were allowed to develop with an overlay of 0.5% agarose in TMNFH-10 medium for a period of 6 days. Virus piaques were picked after microscopic inspection, and eiuted into 2ml SF900-II medium. Virus stocks were amplified by low multiplicity (0.1 pfu/cell) infection. Virus clones expressing noggin were identified by metabolic labeling of infected cultures with 35S-methionine and 35S-cysteine and analysing total labeled protein by poiyacrylamide gel electrophoresis and autoradiography. A labeled protein of the expected apparent Mr of 20,000-30,000 was detected by this method in candidate clones but not in control cultures.

Expression and purification of baculovirus-derived ηοοσι' η .

SF21 cells were cultured in suspension flasks to a density of approximately 1 .8x1 C6/ml in SF900-II medium. Cultures (500 mi) were collected by centrifugation at 1000xg for 1 0 min and resuspended in 20ml of growth medium containing 5- 1 0 pfu/cell recombinant virus. Virus was allowed to adsorb for 1 hour at room temperature with gentle mixing. Infected cells were then diluted to their original volume with fresh growth medium, and incubated at 27°C for 3 days. Ceils and debris were subsequently clarified from the growth medium by centrifugation at 10OOx g for 20 min.

Cell supernatants were brought to pH 8.0, passed through a 0.45 mm Mi!!ipak 60 filter and applied to a Fast S column that had been equilibrated in 25 m HEPES pH 3.0. The column was washed with the same buffer and developed with a linear NaCI gradient t o remove other medium components. Noggin eiuted from this column at 1 M NaCI.

Results Characterization of human nogoin produced in E. coii and i n ft¾QulQvirvi5 Reverse-phase HPLC chromatography shows that recombinant noggin refolded and purified from E. coli elutes in a singie sharp peak, indicating the presence of one predominant isoform (Fig. 9), Electrophoresis on 15 % polyacryiamide-SDS-reducing gels shows that noggin from either E. coii or insect ceils is better than 95 % pure and migrates in a single band corresponding to a protein of 20-30 kD. Noggin from insect cells shows s!ightly slower mobility, apparently due to additional mass from N-linked glycosyiation at the singie consensus site (Fig. 10). Treatment with Endo F converts the mobility of insect-produced noggin to that of the bacterially produced protein (data not shown).

In the absence of reducing agents, noggin produced either i n E.coii or in bacuiovirus behaves as a disuifide-!inked oiigomeric protein (Fig, 10). However, by gei filtration analysis and mass spectroscopy noggin is primarily a dimeric protein (data not shown).

Circular dichroism studies show that recombinant noggin refolded and purified from E. coii as well as noggin purified fro m insect ce!!s have very similar conformations (Fig. 11 ). Secondary structure determined by this method indicates that noggin consists of 48% alpha-helix, 0 % beta-structure, and 52 % random coil.

Biolooical Activity of human noggin produced in E. coii and i n bacuiovirus Biological activity of human noggin produced in E. coli or in bacuiovirus was determined by assay of muscie actin expression in the ventral marginal zone assay, as described supra. Results shown 89 in Fig. 12 indicate a positive dose response for induction of muscle aciin mRNA in VMZ exposed to either bacteriaily produced human noggin, or baculovirus produced human noggin.

EXAMP S 3 Production and characterization of rat monoclonal antibody R P57-1 6 reactive with human noagin .

Materials and Methods Production of antibody RP57-16 rat monoclonal antibody reactive with recombinant human and Xenopus noggin was produced by the immunization of a female Lewis rat with four 35 μα injections of pu rified recombinant human noggin (produced in E. coii) over a two month period. For the initial immunization, the protein was injected in the rear foot pad in Freund's complete adjuvant. Subsequent injections were given in the same foot pad in Freund's incomplete adjuvant. The rai was euthanized 3 days after the fourth injection.

Lymph node cells from the immunized rat were mixed with SP2/0-E.O. mouse myeloma cells at a ratio of 2:1 . After centrifugation, the cell mixture was resuspended in 0.25 ml of 42% (w/v) PEG 3350 (Baker) in phosphate-buffer-saline with 10% (v/v ) dimethyisuifoxide (Sigma) for a total of 3 minutes in a 37°C water bath. Ceils were plated at a density of 5 X 0 lymphocytes per well in 96-we!l plates (Falcon 3072) in DME /F-12 ( ediatech, inc.) containing 10% FBS (supplemented with streptomycin, penicillin , pyruvate, and giutamine) and H GT (1.6 x 10-3 M thymidine, 4.0 1 0 -4 methotrexate, 1 .3 x 10-3 sodium bicarbonate and 1.0 x 10-2 hypoxanthine). After 10 days in culture, supernatants were harvested and assayed for antibody activity against recombinant human noggin by indirect ELISA. Supernatant from COS-M5 ce l ls iransfected with the piasmid containing the human noggin gene was air dried overnight in Probind 96-well assay plates (Falcon 3915). Non-specific binding was eliminated by 2 hour incubation at ambient temperature with PBS/1 % BSA (Sigma). Plates were washed 2 times with PBS/0.02 % Tween 20. Culture supernants were then added and incubated at ambient temperature for 1 hour. Plates were washed 4 times with PBS/0.02 % Tween 20. Secondary antibody, Goat anti-Rat IgG (H÷L) alkaline phosphatase conjugate(Caltag) diluted 1 :2000 in PBS/1 % BSA was added to each well and the plates incubated at ambient temperature for 1 hour. Plates were again washed 4 times with PBS/0.02 % Tween 20. Antibody binding was visualized by 1 hour incubation at ambient temperature in the dark with pNPP (p-nitrophenyl phosphate, Sigma) 1 mg/ml in diethanolamtne buffer, pH 9,8. The reaction was stopped by the addition of an equal volume o f 100 mM EDTA. Absorbance was read at 405 nm on a Thermomax icroplate Reader {Molecular Devices). A reaction was considered positive if the absorbance was 2 times that of the negative control (diluent alone followed by secondary antibody and substrate). Positive clones were expanded and culture supernatant containing monoclonal antibody was collected for specificity analysis.

RP57-16 was cloned in soft agar. Cloned hybrid cells were expanded in DMEM/F-12 (Mediatech, Inc.) containing 10% FBS (supplemented with strepto mycin , pen icillin , pyruvate, and giutamine). Supernatant containing antibody was aiiquoted and stored at -70°C until use.

Specificity Analysis EUSA 100 ng of purified recombinant human noggin, Xenopus noggin, BDNF, NT-3, and NT4 protein was individually passively adsorbed t o Probind 96-well assay plates by overnight incubation at 4°C in 50 m bicarbonate buffer, pH 9.6. BDNF, NT-3 and NT-4 were used t o assess non-specific binding of rat monoclonal antibody RP57-1 8. Supernatants from COS- 5 cells transfected with either the plasmid containing the human noggin gene or the plasmid containing the fig C - terminal tagged Xenopus noggin gene were air dried to Probind 9 8 - weil piates overnight. Non-specific binding was eliminated by 2 hour incubation at ambient temperature with PBS/1% BSA (Sigma). Plates were washed 2 times with PBS/0.02 % Tween 20. Undiluted RP57-16 was added and incubated at ambient temperature for 1 hour. Plates were washed 4 times with PBS/0.02 % Tween 20. Secondary antibody, Goat anti-Rat igG (H+L) alkaline phosphatase conjugate(Caitag) diluted 1 :2000 in PBS/1 % BSA was added to each well and the plates incubated at ambient temperature for 1 hour. Plates were again washed 4 times with PBS/0.02 % Tween 20. Antibody binding was visualized by 1 hour incubation at ambient temperature in the dark with pNPP (p-nitrophenyl phosphate, Sigma) 1 mg/ml in diethanolamine buffer, pH 9.8, The reaction was stopped by the addition of an equal volume of 100 mM EDTA. Absorbance was read at 405 nm on a Thermomax icropiaie Reader (Moiecular Devices). A reaction was considered positive if the absorbance was 2 times that of the negative control {diiuent aione foilowed by secondary antibody and substrate).

Electrophoresis and Western Blotting Rat monoclonal antibody RP57-16 was also analyzed by Western blotting. 50 ng of recombinant human noggin, non-reduced and reduced, were electrophoresed on 12.5% SDS-polyacryiamide gels and electroblotted on nitrocellulose membranes. Membranes were blocked with PBS/1 % Casein/0.1 % Tween 20, and then incubated for 2 hours with undiluted RP57-16 culture supernatant. Following 4 washes in PBS/0.02% Tween 20, the membranes were incubated with a 1 :5000 dilution of Goat anti-Rat IgG (H+L) horseradish peroxidase conjugate (Pe!freeze) in PBS/1% BSA/0.1 % Tween 20. Membranes were washed 4 times with PBS/0.02% Tween 20. Proteins were visualized with ECL Western Blotting Reagents (Amersham) according to the manufacturer's instructions. Membranes were then exposed to XAR 5 Scientific imaging film (Kodak) for 5 seconds.

RESULTS Rat monoclonal antibody RP57-16 reacts with both recombinant human and Xenopus noggin and with recombinant human noggin produced in E. coli, in insect cells, and in COS-M5 cells. The antibody does not react with the neurotrophins BDNF, NT-3 and NT-4.

Western biotting showed that the antibody detects both reduced and non-reduced protein.

The following were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockviile, Maryland 20852 under the terms of the Budapest Treaty: ATCC Accession Date of No. Deposit phage hncgX-5. 7531 1 9-23-92 phage hnog -7 75309 9-23-92 phage hnogX-S 75310 9-23-92 phage ηηοσλ-1 0 75308 9-23-92 hybrldoma RP57-16 CRL 1 1446 3- 25-93 It is to be understood thai while the invention has been described above in conjunction with prefe rred specific embodiments, the description and examples are intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.

Passages of the description which are out of ambit of the appended claims do not constitute part of the claimed invention.

SEQUENCE -LISTINGS INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 699 base pairs (S) TYPE: nucleic acid (C) STRANDEDNESSs unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA {ix) FEATURE ; (A) NAME/KEY: CDS (3) LOCATION: 1. ,639 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG GAG CGC TGC CCC AGC CTA GGG GTC ACC CTC TAG GCC TG GTG GTG 48 Met Glu Arg C a Pro Ser Leu Gly Val Thx Lsu Tyr Ala Lau Val Val 1 5 10 15 GTC CTG GOG CTG CGG GCG ACA CCG GCC GGC GGC CAG CAC TAT CTC CAC 96 Val Leu Gly Lsu Arg Ala Thr Pro Ala Cly Gly Gin Bis Tyr Leu Hia 25 30 ATC CGC CCG GCA CCC AGC GAC AAC CTG CCC CTG GTG GAC CTC ATC GAA 144 He Arg Pro Ala Pro Ser Asp Asn Leu Pro Lau Val Asp Leu He Glu 40 45 CAC CCA GAC CCT ATC TTT GAC CCC AAG GAA AAG GAT CTG AAC GAG ACG 192 His Pro Asp Pro He Phe Asp Pro Lys Glu Lys Aso Leu Asn Glu Thr 50 55 60 CTG CTG CGC TCG CTG CTC GGG GGC CAC TAC GAC CCA GGC TTC ATG GCC 240 Leu Leu Arg Ser Leu Leu Gly Gly Hi3 Tyr Aso Pro Gly Phe Met Ala 65 70 75 80 ACC TCG CCC CCC GAG GAC CGG CCC GGC GGG GGC GGG GGT GCA GCT GGG 288 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly Ala Ala Gly 85 90 95 GGC GCG GAG GAC CTG GCG GAG CTG GAC CAG CTG CTG CGG CAG CGG CCG 336 Gly Ala Glu Asp Leu Ala Glu Leu Aso Gin Leu Lsu Arg Gin Arg Pro 100 105 110 TCG GGG GCC ATG CCG AGC GAG ATC AAA GGG CTA GAG TTC TCC GAG GGC 384 Ser Gly Ala Met Pro Ser Glu He Lys Gly Leu Glu Phe Ser Glu Gly 115 120 125 TTG GCC CAG GGC AAG AAG CAG CGC CTA AGC AAG AAG CTG CGG AGG AAG 432 Leu Ala Gin Gly Lys Lys Gin Arg Lau Ser Lv3 Lys Leu Arg Arg Lys 130 135 " 140 TTA CAG ATG TGG CTG TGG TCG CAG ACA TTC TGC CCC GTG CTG TAC GCG 4S0 Leu Gin Met Tr~o Lau Trp Ser Gin Thr Phe Cys Pro Val Leu Tyr Ala 145 * 150 155 160 TGG A C GAC CTG GGC AGC CGC TTT TGG CCG CGC TAC GTG AAG GTG GGC 528 Trp Asn Aso Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Glv 165 170 175 AGC TGC TTC AGT AAG CGC TCG TGC TCC GTG CCC GAG GGC ATG GTG TGC 576 Ser Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys ISO 185 190 AAG CCG TCC AAG TCC GTG CAC CTC ACG GTG CTG CGG TGG CGC TGT CAG 624 Lvs Pro Ser Lys Ser Val His Leu Thr Val Lau Arg Trp Arg Cys Gin 195 200 205 CGG CGC GGG GGC CAG CGC TGC GGC TGG ATT CCC ATC CAG TAC CCC ATC '672 Arg Arg Gly Gly Gin Arg Cys Gly Trp He Pro lie Gin Tyr Pro lie ■ 210 215 220 ATT TCC GAG TGC AAG TGC TCG TGC TAG 699 He Ser Glu Cys Lys Cys Ser Cys 225 230 INFORMATION FOR SEQ ID NO: 2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 232 amino acids (B) TYPE: amino acid (D) TOPOLOGY: unknown INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: He Ser Glu Cys Lys Cys Ser Cys 1 S INFORMATION FOR SEQ ID NO: 3: ¾i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1834 base pairs (B) TYPE: nucleic acid {C) STRANDEDNESS : unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: ONA (lit) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 595..1253 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTAATAAATC CTAAGTAGCC AGAGGGACGA GCTACAGACT GGTTGCGGCG CAGGGTTTAT 60 CCAGGGCAGA GAGGAGCAGC AAAAGCACAT TGCGCAGCTC TCACTCCCCC TTTCCTTCTG 120 CTTCACTCTA TAAGGGCTCC TGCAAATGAA AGAGACCTGC GGGGATTTGC GCGGACAGAT 180 GTAAAGGAGA TCCTGCAACT TTCTCTGGTT GCATCCCTGG GAGTCGCTCC GCGCCGCTGG 240 CTGATTGCGA CTGTTGCTTT CCACAGCTCC CTTCTTCCGC AGTTTCTTCT AGGAGCAGAT 300 CGAGTCTCTG GTTACCATGG TGATCGAGCT GAAAGTGAAG AATATTTAAG AGAGGGGAGG 360 CTGGAGCCAG CAGGCAGACA AAGTGGTGCC ACCAAGGACT GTGCGTAAAG GGTGAGCGCA 420 TTGGAGACAG ACAGGGGCTC TGCTGAACTT CCACTTGACT GCGATGAGAG GGGGGAATCC 480 CCAATTCGCT AGGTGCCCCT GAACCCCCCA GAATTCCTCC TCTGATGCAT TATTTATGAT 540 CTCTGGCAAG AAATCGGGAG CACCCAACTC TTATTTTGTG CAGCTGTGTG CAGC ATG 597 Met 1 GAT CAT TCC CAG TGC CTT GTG ACT ATA TAT GCT CTG ATG GTC TTC TTG S 5 Aso His Ser Gin Cys Leu Val Thr He Tvr Ala Lau Met Val Phe Lau 10 15 GGA CTT AGA ATA GAC CAA GGG GGT TGC CAA CAT TAT CTG CAC ATC ¾GA 693 Gly Leu Arc He Asp Gin Gly Gly Cys Gin His Tyr Leu His He Arg 25 30 CCG GCT CCT AGT GAA AAC CTA CCA CTG GTG GAC CTT ATT GAG CAC CCG 741 Pro Ala Pro Ser Glu Asn Leu Pro Leu Val ASD Leu He Glu His Pro 40 45 GAT CCC ATC TAT GAT CCC AAG GAG AAG GAT CTT AAC GAG ACC TTG CTG 789 Asp Pro He Tyr Asp Pro Lys Glu Lys ASD Leu Asn Glu Thr Leu Leu 50 55 * 60 65 AGG ACT TTA ATG GTT GGA CAC TTT GAC CCC AAC TTT ATG GCC ACC ATC 837 Arg Thr Leu Met Val Gly His Phe Asa Pro Asn Phe Met Ala Thr ∑le 70 " 75 80 CTG CCA GAG GAG AGA CTT GGA GTG GAG GAC CTT GGG GAG TTG GAT CTC 885 Leu Pro Glu Glu Arg Leu Gly Val Glu Asp Leu Gly Glu Leu Asp Leu 85 90 .95 CTT CTT AGG CAG AAG CCC TCG GGG GCA ATG CCA GCG GAA ATC AAG GGA 933 Leu Leu Arg Gin Lys Pro Ser Gly Ala Met Pro Ala Glu He Lys Glv 100 105 110 CTG GAG TTT TAC GAG GGG CTT CAG AGC AAA AAG CAC AGA CTG AGC AAG 981 Leu Glu Phe Tyr Glu Gly Leu Gin Ser Lys Lys His Arg Leu Ser Lye 115 120 125 AAA CTC AGG AGA AAG TTG CAG ATG TGG CTC TGG TCC CAG ACC TTC TGT 1029 Lys Leu Arg Arg Lys Leu Gin Met Trp Leu Trp Ser Gin Thr Phe Cys 130 135 140 145 CCT CTC CTT TAC ACA TGG AAT GAC CTA GCG ACC AGG TTT TGG CCT CGC 1077 Pro Val Leu Tyr Thr Trp Asn Aso Leu Gly Thr Arg Phe Trp Pro Arg 150 " 155 160 TAT GTG AAA GTA GGG AGC TGC TAC AGT AAG AGG TCT TGT TCT GTG CCA 1125 Tyr Val Lys Val Gly Ser Cys Tyr Ser Lys Arg Ser Cys Ser Val Pro 165 1 0 175 GAG GGC ATG GTT TGC AAA GCT GCC AAG TCT ATG CAT TTG ACC ATC TTA 1173 Glu Gly Met Val Cys Lys Ala Ala Lys Ser Met His Leu Thr lie Leu 180 185 190 AGG TGG AGA TGT CAA CGC AGG GTT CAG CAG AAG TGT GCG TGG ATA ACC 1221 Arg Trp Arg Cys Gin Arg Arg Val Gin Gin Ly3 Cys Ala Trp lie Thr 195 200 205 ATT CAG TAC CCT GTC ATT TCC GAG TGC AAA TGC TCA TGC TGAGACTCTT 1270 He Gin Tyr Pro Val He Ser Glu Cys Lys Cys Ser Cys 210 215 220 GGACTAATGC AAAAAGACAG TAGCTTCATG GTTCAAGCTT CATGTTATAT GCACTGTAAT 1330 ATGTAGAAAT GTATATGTGT GTATATATGG CATTGGTCTA AATTACTATT AAAAGG CAG 1390 TATTATTCTT TTAAATAACC AGTGTCTACT GTATTTCCAA CACTATTATC CTGGTTGTGT 1450 TTTATTTTAA TAATATTAT ATTATTTTTT TTTTGCCTAA TGTATCTCTA TTTATATCCA 1510 AAAAAAGAGC ACTTCGCTTG GCGAAGCATT TTTTTTTAAA GAAAAAAAAA ACAAATTTAA 1570 TAGTTTAATA ATATAGAAGC ATTTTTTTCC TTTAATGGAA AATGTGCCTT TTTTTGATGG 1630 ACCTCAAAAA AAAAATGAAT AAAAACCAGA GCAAGATATA ATTTTCAGTT TATTGTACAT 16S0 AGAAAGAGAA CACATTTTGA AAGATGAAAA ATTTTACTCT TGTAAATGAG AACTATCTGC 1750 TATTTATTCT TTTATTTTTT TTTTCCTCCC CCTGTAGAGT GCAGAATAAA AGTAAACCAC 1810 TAAAATATTA AAAAAAAAAA AAAA 1334 INFORMATION FOR SSQ ID NO: 9: {i) SEQUENCE CHARACTERISTICS .' (A) LENGTH: 222 amino acids {S) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Aso His Ser Gin Cys Leu Val Thr He Tyr Ala Leu Met Val Phe 1 * 5 10 15 Leu Gly Leu Arg He Asp Gin Gly Gly Cys Gin His Tyr Leu His He 25 30 Arg Pro Ala Pro Ser Glu Asn Leu Pro Leu Val Asp Leu He Glu His 40 45 Pro Aso Pro He Tyr Aso Pro Lys Glu Lys Aso Leu Asn Glu Thr Leu 50 * 55 60 Leu Arg Thr Leu Met Val Gly His Phe ASD Pro Asn Phe Met Ala Thr 65 70 75 80 lie Leu ?ro Glu Glu Arg Leu Gly Val Glu Aso Leu Gly Glu Leu Asp 85 90 * 95 Leu Leu Leu Arg Gin Lys Pro Ser Gly Ala Met Pro Ala Glu lie Lys 100 105 110 Gly Leu Glu Phe Tyr Glu Gly Leu Gin Ser Lys Lys His Arg Leu Ser 115 120 125 Lys Lys Leu Arg Arg Lys Leu Gin Met Trp Leu Trp Ser Gin Thr Phe 130 135 140 Cys Pro Val Leu Tyr Thr In Asn Asp Leu Gly Thr Arg Phe Trp Pro 145 150 " 155 " 160 Arg Tyr Val Lys Val Gly Ser Cys Tyr Ser Lys Arg Ser Cys Ser Val 165 170 175 Pro Glu Gly Met Val Cys Lys Ala Ala Lys Ser Met His Leu Thr lie 180 185 . 190 Leu Arg Tr-o Arg Cys Gin Arg Arg Val Gin Gin Lys Cvs Ala Trp lie 195 200 205 Thr lie Gin Tyr Pro Val lie Ser Glu Cys Lys Cys Ser Cys 210 ■ 215 220 t INFORMATION FOR S2Q ID NO: 10: {i) SEQUENCE CHARACTERISTICS : { } LENGTH: 356 base pairs (B) TYPE: nucleic acid FEATURE: (A) NAME/KEY : modified base (B) LOCATION: IS (D) OTHER INFORMATION: /mod base= OTHER /label= Y /note=> "ϊ = c or I."

Claims (9)

134656/2 -90- CLATMS

1. An antibody which binds a noggin polypeptide which has the amino acid sequence set forth in Figure 1 (SEQ ID NO:2) or a fragment or derivative thereof which exhibits noggin activity by promoting the induction of neural tissue in a Xenopus animal cap assay.

2. An antibody according to claim 1 which specifically binds a noggin polypeptide encoded by the DNA of hnog -9 (ATCC 75310) or hnog -10 (ATCC 75308).

3. An antibody according to claim 1 or 2 which is monoclonal.

4. An antibody according to claim 1 or 2 which is polyclonal.

5. A hybridoma capable of producing monoclonal antibodies according to claim 3.

6. A hybridoma according to claim 5 which is RP57- 16 (ATCC CRL 11446).

7. A monoclonal antibody obtainable from the hybridoma of claim 6.

8. An antibody according to any one of claims 1 to 4 or 7 for use in a method of treatment or diagnosis of the human or animal body.

9. An antibody according to claim 1 which is substantially as hereinbefore described with reference to Example 8. LUZZATTO 4 LUZZ TO : 'V