MXPA00000242A - Murine and human cerberus-like proteins and compositions comprising them - Google Patents

Abstract

Purified mammalian cerberus proteins and process for producing them are disclosed. DNA molecules encoding the mammalian cerberus proteins are also disclosed. The proteins may be used for inducing formation, growth, differentiation, proliferation and/or maintenance of neurons and/or related neural cells and tissues such as Schwann cells, glial cells and astrocytes, and for other tissue repair, including cardiac and endoderm.

Description

PROTEINS SIMILAR TO CERBERUS BE MAMÍFERO, AND COMPOSITIONS The present invention relates to new members of the family of cerberus proteins, to the DNA that encodes them, and to processes to obtain them. These proteins can be used to induce the expression of factors in and / or differentiation of tissues and organs and, particularly, to induce the formation, growth, differentiation, proliferation and / or maintenance of nervous, endodermal and cardiac tissue. Therefore, these proteins can be useful in the treatment of wounds, tumors and in the improvement and / or inhibition of the formation, growth, differentiation, proliferation and / or maintenance of cells of other tissues and organs, for example, epidermal, pancreatic, liver, spleen, lung, kidney, brain and / or other tissue. These proteins can also be used to increase the activity of other factors of tissue regeneration and differentiation, such as BMPs. The protein has been termed by the inves as similar to mammalian cerberus.

Amiecedemites of the I_? Ve ?? ció_n The search for the molecule or molecules responsible for the formation, proliferation. Differentiation and maintenance of tissues and organs, such as neurons and related tissues and nerve cells, has been intense in virtue of the tremendous need for useful factors for the treatment of conditions that involve degradation or damage to these tissues. A Xenopus protein previously identified in embryos, cerberus. It seems to be involved in the induction of the head. Bouwmeester et al., Nature, 382: 595-601 (1996).

Objectives Inves have discovered novel mammalian members of the cerberus family of proteins and have surprisingly discovered that members of the family of cerberus proteins are also capable of inducing, enhancing and / or inhibiting formation, growth. proliferation, differentiation, maintenance of neurons and / or cells and related nervous tissue, such as Schwann cells, glial cells and astrocytes. Accordingly, the present invention provides methods for inducing the formation of neurons and / or cells and related nervous tissue, such as Schwann cells, glial cells and astrocytes. the administration to progenitor cells comprising a composition of at least one protein that is a member of the family of cerberus proteins. The present invention relates to a family of proteins designated as cerberus, which appears to be a pioneer protein, with a residue pattern of 9 cisterns, which is present in the embryo. In cerberus mRNA, Xenopus, is expressed at low levels in the egg without fertilization, and zygotic transcripts begin to accumulate in early gastrula. The expression continues during gastrulation and early neuralation, declining quickly during the neuralación. Importantly, the expression of the cerberus begins approximately one hour after that of the cordina, suggesting that the cerberus could act current below the cordina signal. The cerberus domain of organizer includes the leading edge of the most anterior organizing cells and extends ithe lateral mesoderm. The leading edge causes the liver, pancreas, and bowel in its middle part, and the more lateral region motivates the mesoderm of the heart in later stages of its development. In preferred embodiments, the composition may comprise a protein having the amino acid sequence of SEQ ID NO: 2, starting at amino acid 1, 18 to 24, 41. 85 to 91 or 162, and ending at amino acid 241 or 272; in SEQ ID NO: 8, beginning with amino acid 1, 18 to 25, 41, 85 to 91 or 162, and ending at amino acid 241 or 267. In one embodiment, the method comprises administering the composition to a patient in vivo. Alternatively, the method may comprise administering the composition to cells in vitro and recovering neurons and / or cells and related nervous tissue, such as Schwann cells, glial cells and astrocytes, which may be subsequently administered to a patient. The composition may further comprise a suitable carrier for administration. The present invention also provides novel DNA sequences that encode new members of the family of cerberus proteins. In particular embodiments, the present invention provides novel DNA sequences encoding cerberit proteins of mammal, such as murine and human cerberus. In the List of Sequences XXgm. £ ^^ ¿? ¿^^ X¿X¡¿ £. they provide the nucleotide sequences and the corresponding amino acid sequences encoded by these DNA sequences. In particular, the present invention comprises an isolated DNA sequence encoding a mammalian cerberits protein comprising a DNA sequence selected from the group consisting of: nucleotides starting at # 58, 109, 178, 313, 316, 319, 322, 325, 328 or 541 and ending at # 780 or 873 of SEQ ID NO: 1; nucleotides starting at nucleotide # 1, 52, 55, 58, 61, 64, 67, 70, 73 121, 256, 259, 262, 265, 268, 271 or 484 and ending at nucleotide # 723 or 801 of SEQ ID NO: 7; or nucleotides encoding amino acids starting at # 1, 18, 41, 85 to 91 or 162 and ending at # 241 or 272 of SEQ ID NO: 2; or amino acids starting at # 1, 18 to 25, 41, 85 to 91 or 162 and ending at # 241 or 267 of SEQ ID NO: 8, as well as fragments and variants of the above sequences and which are easily obtainable from the previous ones, and which maintain cerberus activity. The present invention further comprises sequences that hybridize to these sequences under conditions of astringent hybridization and that encode a protein exhibiting cerberus activity. It is expected that the mammalian cerberus protein, as expressed by mammalian cells such as CHO cells, exists as a heterogeneous population of active cerberus species with variable N termini. Based in part on the von Heginje signal peptide prediction algorithm, the first 17 to 24 amino acids appear to be involved in signaling for the secretion of the mature peptide. It is expected that the active species can optionally include the signal peptide and will include amino acid sequences starting with the amino acid residues # 1, 18, 19, 20, 21, 22, 23, 24 or 25 of SEQ ID NO: 2 or of the SEQ ID NO: 8. Thus, DNA sequences encoding active mammalian cerberus proteins are expected to include those comprising nucleotides # 109, 112, 115, 118, 121, 124, 127 or 130 to # 780 u 873 of SEQ ID NO: 1, or comprising nucleotides # 1, 52, 55, 58, 61, 64, 67, 70 or 73 to # 723 or 801 of SEQ ID NO-7. Accordingly, the active species of the cerberus-like protein are expected to include those comprising amino acids # 1, 18, 19, 20, 21, 22, 23, 24 or 25 to # 241 or 272 of SEQ ID NO: 2; or amino acids # 1, 18, 19, 20, 21, 22, 23, 24 or 25 to # 241 or 267 of SEQ ID NO: 8.

As described hereinafter, it is further expected that the cerberus and cerberus-like proteins may be proteolytically processed by cells to further form active species. For example, putative cleavage proteolytic processing sites, which are typically characterized by the formula RXK / RR, are found in the 5 amino acids 37 to 40 and 82 to 85 of SEQ ID NO: 2 or SEQ ID NO. : 8. In this way, it is expected that DNA sequences encoding mammalian cerberus proteins include those comprising nucleotides # 178 or 313 to # 780 or 873 of SEQ ID NO: 1; and # 121 or 256 to # 723 or 801 of SEQ ID NO: 7. Consequently, it is expected that additional active species of cerberus-like protein include those that comprise the amino acids that begin at # 41 or 86 and end at # 241 or 272 of SEQ ID NO: 2, or that include amino acids that begin at # 41 or 86 and end at # 241 or 267 of SEQ ID NO: 8 The above sequences of SEQ ID NO: 1 and 2 are used to isolate and sequence the amino acid and DNA sequences similar to Cerberus. In another embodiment, the present invention comprises a method for altering the Regulation of genes in a patient in need thereof, which comprises administering to said patient an effective amount of the above compositions. The alteration of the regulation of nerve genes can be carried out by stimulating or inhibiting the binding by cerberus proteins of receptor proteins, including bone morphogenetic proteins [BMPs]. Thus, the family of cerberus and cerberus-like proteins may be able to induce the formation of nervous, bone, cartilage or other tissue. The family of cerberus and cerberus-like proteins may also be able to inhibit, increase or otherwise affect the activity of their receptor molecules to which they bind, including molecules of the BMP family. In other embodiments, the present invention comprises vectors comprising the Previous DNA molecules in operative association with an expression control sequence for them, as well as host cells transformed with these vectors. In still other embodiments, the present invention comprises methods for producing purified mammalian cerberus proteins, new mammalian cerberus proteins, and compositions containing the mammalian cerberus proteins. These methods can understand the steps of: cultivating a transformed host cell with a sequence of DNA encoding a mammalian cerberus protein as described above; and recovering and purifying said mammalian cerberus protein from the culture medium. The present invention further comprises the mammalian cerberus polypeptide. purified, which is produced by the above methods, as well as purified mammalian cerberus polypeptides, comprising an amino acid sequence encoded by the above DNA sequences. The proteins of the present invention may comprise the amino acid sequence beginning at amino acid # 1, 18, 19, 20, 21. 22, 23, 24, 25, 86, 87, 88, 89, 90, 91 or 162 and ends in amino acid # 241 or 272 of SEQ ID NO: 2; # 1, 18, 19, 20, 21, 22, 23, 24, 25, 86, 87, 88, 89, 90, 91 or 162 and ending in # 241 or 267 of SEQ ID NO: 8; or a mammalian cerberus protein having a molecular weight of about 20-30 kd, said protein comprising an amino acid sequence highly homologous to the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 8, and possessing the capacity of regulating the transcription of one or more genes. A cerberus-like protein species is a mature peptide that is contemplated to comprise the amino acid sequence from amino acid 18 to amino acid 272 of SEQ ID NO: 2; or from amino acid 18 to amino acid 267 of SEQ ID NO: 8, each being expected to have a molecular weight of approximately 28.6 kd. Other protein species similar to active cerberus is a divided peptide that is contemplated to comprise the amino acid sequence from amino acid 86 to amino acid 91 and terminates at amino acid 272 of SEQ ID NO: 2, particularly from amino acid 18 or 90 to amino acid. amino acid 272 of SEQ ID NO: 2, or from amino acid 86 to 91 terminating at amino acid 267 of SEQ ID NO: 8. particularly from amino acid 18 or 90 to 267 of SEQ ID NO: 8. Expected that the mature polypeptide for each of these proteins has a molecular weight of about 20.7 kd.

Description of Sequences SEQ ID NO: 1 is a nucleotide sequence of mammalian cerberus DNA, particularly murine cerberus DNA. SEQ ID NO: 2 is an amino acid sequence of the mammalian cerbeus protein encoded by SEQ ID NO: 1.

SEQ ID NO: 3 to 5 are consensus nucleotide sequences of probes for cerberus and cerberus-like proteins. SEQ ID NO: 6 is a genomic DNA sequence that encodes a protein similar to human Cerberus. The symbol "N" indicates that the nucleotide residue can be any one of A, C, T or G. SEQ ID NO: 7 is the cDNA sequence that codes for protein similar to human cerberus. SEQ ID NO: 8 is the amino acid sequence of human cerberus-like protein encoded by SEQ ID NO: 7.

Description of the ATCC Deposits A strain of E. coli DH5a transformed with pGIMCerb, which comprises the mammalian cerberus coding sequence described in SEQ ID NO: 1, has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, and has been assigned the accession number ATCC 98347.

Beíallada Besciipcióin de la Invención! As used herein, the terms certerus "or" cerberus-like "are both used to indicate the family of proteins that comprise the cerberus and cerberus-like proteins." Cerberus or cerberus-like protein "refers to cerberus or similar proteins. mammalian cerberus, such as murine or human cerberus proteins, and other proteins that share sequence homology for the highly conserved cysteine pattern of the C-terminated portion of mammalian cerberus proteins.A specific member of the cerberus protein family is the murine cerberus-like protein, which possesses the amino acid sequence specified in SEQ ID NO: 2, as well as homologs of this protein found in other species, and other proteins that are closely related in a structural and / or functional manner with murine cerberus, it is also expected that proteins related to cerberus exist in other species ecies, including family members in Xenopus and Drosophila, C. elegans, zebrafish, as well as in mammals, for example, rats, mice and humans. "Cerberus or cerberus-like proteins" also include variants of cerberus proteins, such as allelic variants or variants induced by mutagenesis or deletions, and fragments of cerberus proteins whose variants and fragments possess cerberus activity. "Cerberus and similar to Cerberus" is also used to indicate the family of proteins that share structural and / or functional similarity, including those proteins described hereinafter. As used herein the term "cerberus or cerberus-like activity" refers to one or more of the activities that are exhibited by the mammalian cerberus-like proteins of the present invention. In particular, the "activity of cerberus or similar to cerberus" includes the ability to induce, improve and / or inhibit formation, growth. proliferation, differentiation, maintenance of neurons and / or cells and related nervous tissue such as brain cells, Schwann cells, glial cells and astrocytes. The activity "cerberus or similar to cerberus" also includes the ability to induce molecular markers of neuroendocrine or ectodermal tissue, such as OTX2, N-CAM, MASH. chromagranin and AP2, as well as the ability to induce the formation of neurons and / or cells and related nervous tissue, such as brain cells, Schwann cells, glial cells and astrocytes. The "activity of cerberus or similar to cerberus" may also include the ability to regulate the interaction of ligands and their protein receptors. For example, "cerberus or cerberus-like activity" may include the ability to bind to one or more members of the bone morphogenetic protein family [BMP] and / or the protein families w nt, and thus inhibit , increase or otherwise affect the activity of such molecules. The "activity of cerberus or cerberus-like" may also include the ability to regulate the formation, differentiation, proliferation and / or maintenance of other cells and / or tissue, eg, connective tissue, organs and wound healing. In particular, the "activity of cerberus or cerberus-like" may include the ability to improve and / or inhibit the formation, growth, proliferation, differentiation and / or maintenance of cells and cardiac tissue, of the spleen, of the liver, of the pancreas, of the stomach, the kidney, the lung, and the brain, as well as osteoblasts and bone, chondrocytes and cartilage, tendon, epidermis and muscle. The "activity of cerberus or similar to cerberus" also includes the activities of the sfs_ jM_¡_ < aia TÉjS ^^ AfctoiaiM ^ protein cerberus and similar to cerberus in the tests described in the examples and specified here. Cerberus cDNA and cerberus-like cDNA should be useful as a diagnostic tool (such as through the use of antibodies in assays for proteins in cell lines or the use of oligonucleotides as primaries in a PCR assay to amplify those with sequence similarities to the oligonucleotide primer, and to determine how much of the cerberus is present). The cerberus can act on its target cells through its own receptor. The cerberus, therefore, may be useful for the isolation of the receiver. In addition, the cerberus or its receptor would be useful as a diagnostic probe for certain types of tumors. In this way, cerberus, its receptor or antibodies to any may be potent agonists or antagonists that may be clinically useful. In addition, complexes of cerberus and its receptor, cerberus and antibodies to it, or the cerberus receptor and antibodies to it, may each be useful in a large number of in vitro, ex vivo or clinical uses. The present invention also includes protein variants and functional fragments of the amino acid sequence of the mammalian cerberus protein shown in SEQ ID NO: 2, which maintain cerberus activity. The present invention also includes antibodies to a purified mammalian cerberus protein, such as the above. The composition of the present invention comprises a therapeutically effective amount of at least one of the above mammalian cerberus proteins. It is expected that such protein variants and functional fragments of cerberus or cerberus-like proteins will include amino acid sequences that share important homology with the amino acid sequence of SEQ ID NO: 2, more preferably, at least 80% or 90% identity. of amino acids. Variants and functional fragments that retain activity similar to cerberus are expected to include those that retain the cysteine standard found in SEQ ID NO: 2. For example, a truncated polypeptide comprising amino acids # 162 to # 241 of SEQ ID NO: 2, or from amino acid # 162 to amino acid # 241 of SEQ ID NO: 8, each will retain the complete 9-cysteine pattern found in the carboxy-termination portion of the cerberus sequences or similar to cerberits of SEQ ID NO: 2 and SEQ ID NO: 8, respectively.

In still another embodiment, the present invention comprises a method for altering the regulation of genes in a patient in need, which comprises administering to said patient an effective amount of the aforementioned compositions. For example, altering the regulation of neuronal genes can be carried out by stimulating or inhibiting the binding of receptor proteins, for example, the binding between the mammalian cerberus protein and its receptor protein, such as a protein w nt, or a BMP protein. In this way, cerberus proteins may be able to regulate the binding interaction of ligands to their respective receptor proteins, as well as the interaction of transcriptional factors in cells. The present invention also encompasses hybrid or fusion vectors comprising the coding DNA sequences of the present invention and other cerberus coding sequences, linked to a specific tissue or regulatory sequence susceptible to induction, such as a promoter or operator. . In a preferred embodiment of the invention. the coding sequence for the mammalian cerberus-like protein is operably linked to one or more promoters, enhancers and / or regulatory elements from genes that are selectively expressed in neurons and / or cells and related neural tissues, such as Schwann cells, glial cells and astrocytes. For example, the promoter of the GFAP gene, which is known to be expressed in astrocytes and neuronal cells; and the promoter of the OTX2 gene, which is known to be expressed in the forebrain, are appropriate for the specific tissue production of the cerberus. Additionally, the DNA sequence encoding mammalian cerberus can be operatively linked to one or more regulatory sequences from GFAP or OTX2 proteins, as well as other proteins that are selectively produced in neurons and / or cells and related neural tissue such as Schwann cells, glial cells and astrocytes. In other preferred embodiments of the invention, the coding sequence for mammalian cerberus-like protein is operably linked to the isolated promoter from other genes, organs or cells of interest. Vectors that employ other selective tissue regulatory elements and regulatory elements susceptible to induction, for the selective expression or susceptible to induction of proteins, may also be useful. proteins similar to mammalian cerberus of the present invention.

Another aspect of the invention provides pharmaceutical compositions containing a therapeutically effective amount of mammalian cerberus-like protein, in a pharmaceutically acceptable vehicle or carrier. These compositions of the invention can be used in the formation of neurons and / or cells and related neural tissue, such as Schwam cells, glial cells and astrocytes, as well as tissue and cells of the liver, pancreas, lung, heart, kidney, spleen, stomach and heart, as well as tissue and connective cells, including osteocytes, chondrocytes, myocytes, tendon cells, epidemial cells and adipocytes. These compositions may also be used for the purpose of improving and / or inhibiting the formation, growth, proliferation, differentiation and / or maintenance of bone, osteoblasts, caitilage, chondrocytes, beta cells and other types of cells typically found in the islets. of Langerhans or other pancreatic cells, as well as other tissues of organs such as tissue of the epidermis, spleen, brain, lung and kidney. Compositions comprising protein similar to mammalian cerberus can be used to treat precursor or progenitor cells, such as endoderm cells. which are capable of differentiating into cells comprising differentiated tissue or organs, such as cardiac or neural cells, for the purpose of improving the formation, differentiation, proliferation and / or maintenance of such cells, tissues or organs. Methods for the formation and maintenance of pancreatic cells are described, for example, in WO93 / 00441, the description of which is incorporated herein by reference. In addition, the compositions can be used to regulate embryonic development, for example, by affecting the development of embryonic cells and tissue within the endoderm phenotype. The compositions of the invention may comprise, in addition to a protein similar to mammalian cerberus, other therapeutically useful agents, including growth factors such as the epidermal growth factor (EGF), fibroblast growth factor (FGF), Transforming growth (TGF-a and TGF-ß), Wnts, urchins, including sonic, indian and desert urchins, activins, inhibins, bone morphogenetic proteins (BMPs), and insulin-like growth factor (IGF). The compositions may also include an appropriate matrix, for example, to support the composition and provide a surface for inward growth of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes, or for other growth .....__ jti ^ 2a ^? _____ * a_-. of tissue or cells. The matrix can provide a slow release of the protein similar to mammalian cerberus and / or the appropriate environment for the presentation thereof. Compositions containing protein similar to mammalian cerberus can be employed in methods for the treatment of a number of tissue defects and for the healing and maintenance of various types of tissues and wounds. Tissues and wounds that can be treated include repair or induction of neurons and / or related neural tissues and cells, such as Schwann cells, glial cells and astrocytes. It also includes the treatment of cardiac tissue, liver, pancreas, spleen, lung, kidney, brain and stomach, and may also include cartilage tissue, epidermis, muscle, including cardiac muscle, other connective tissues, such as bone, tendon and ligament and other tissues and other injuries. These methods, in accordance with the present invention, involve the administration to a patient that requires such tissue formation, wound healing or tissue repair, an effective amount of mammalian cerberus protein. Compositions containing the mammalian cerberus-like protein can also be used to treat or prevent degenerative nerve conditions such as Parkinson's Disease, Alzheimer's Disease and Lou Gehrig's Disease, as well as other degenerative nerve diseases and other conditions involving defects in nervous tissue. The compositions may also be useful for treating other conditions such as osteoporosis, rheumatoid arthritis, osteoarthritis and other connective tissue abnormalities, or other organs or tissues, such as muscle tissue, of the pancreas, liver, spleen, lung, heart, brain and kidney, and other tissues and organs. These methods may also involve the administration of a protein of the present invention in conjunction with the administration of at least one other protein, for example growth factors that include EGF, FGF, TGF-α, TGF-β, BMP. Wnts, urchins, including sonic, Indian and desert urchins, activin, inhibin and IGF. In a particular embodiment of the present invention, the mammalian cerberus-like gene or protein can be used to increase the activities of BMPs or other members of the TGF-β superfamily. Still a further aspect of the invention are DNA sequences that encode the expression of mammalian cerberus-like protein. Such sequences include the nucleotide sequence in a 5 'to 3' direction illustrated in SEQ ID NO: 1 or SEQ ID NO: 7, DNA sequences which, by the degeneracy of the genetic code, are identical to the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 7, and coding for the protein of SEQ ID NO: 2 or SEQ ID NO: 8. DNA sequences are also included in the present invention that hybridize under astringent conditions with the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 7 and coding for a protein having activity similar to cerberus. Preferred DNA sequences include those that hybridize under stringent conditions [see, T. Maniatis et al, Molecular Cloning (A Laboratorv Manual). Cold Spring Harbor Laboratory (1982), pages 387 to 389]. It is generally preferred that such DNA sequences encode for a polypeptide that is at least about 80% homologous, and more preferably at least about 90% homologous, to the mature mammalian cerberus-like amino acid sequence, which is shown in SEQ. ID NO: 2 or in SEQ ID NO: 8. In addition, allelic or other variations of the sequences of SEQ ID NO: 1 or SEQ ID NO: 2 are also included in the present invention, whether the nucleotide changes result in changes in the peptide sequence or not, but where the peptide sequence still has activity similar to cerberus. The present invention also includes functional fragments of the DNA sequence of mammalian cerberus-like proteins shown in SEQ ID NO: 1 or SEQ ID NO: 7, which code for a polypeptide that retains cerberus-like protein activity. . The determination of whether a particular variant or fragment of the mammalian cerberus-like protein of the present invention, such as that shown in SEQ ID NO: 2 or SEQ ID NO: 8, maintains activity similar to cerberus, is routinely performed using the assays described in the examples and the present description. The DNA sequences of the present invention are useful, for example, as probes for the detection of mRNA that codes for other cerberus-like proteins in a given population of cells. DNA sequences may also be useful for the preparation of vectors for gene therapy applications as will be described below.

A further aspect of the invention includes vectors comprising a DNA sequence as described above, in operative association with an expression control sequence for it. These vectors can be used in a new process for the production of a recombinant protein similar to mammalian cerberus, of the invention and ; ^ S__É_¡_ __ * ____ in which a cell line transformed with a DNA sequence encoding a mammalian cerberus-like protein in operative association with an expression control sequence for it, is cultured in a culture medium appropriate and mammalian cerberus-like protein is recovered and purified from there. This process can employ a large number of cells known both prokaryotic and eukaryotic as host cells for the expression of the polypeptide. Vectors can also be used in gene therapy applications. In such use, the vectors may be transfected into cells of an ex vivo patient, and the cells may be re-introduced into a patient, or the vectors may be introduced into a patient in vivo through target transfection. Alternatively, the expression of homologous cerberus gene can be upregulated by known recombinant techniques to insert high expression regulatory elements into the genome in the vicinity of the coding sequence of the cers described herein. In a preferred embodiment of the invention, vectors are prepared using one or more non-native regulatory elements, such as promoters and / or enhancers operatively associated with the coding sequence for mammalian cerberus-like, in order to achieve expression of similar to mammalian cerberus in desired cell tissue and / or at a desired time in development. For example, a vector can be constructed using the promoter element from genes, which is known to be constitutively expressed in neuronal development. By operatively associating the promoter from appropriate genes with the coding sequence for the cerberus-like, and by transforming appropriate cells, such as neuronal progenitor cells, one can express similar to mammalian cerberus in these cells, thereby promoting the effects desired training, growth. proliferation, differentiation and / or maintenance of cells such as neurons and / or cells and related neural tissues, such as Schwann cells, glial cells and astrocytes, either in vitro or in vivo cultures. Still a further aspect of the present invention are proteins or polypeptides similar to mammalian cerberus. Such polypeptides are characterized by having an amino acid sequence that includes the sequence illustrated in SEQ ID NO: 2 or SEQ ID NO: 8, variants of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8 , including TiliHllÉtílí- - '«MB & allelic variants that occur naturally, and other variants in which the protein retains activity similar to cerberus, for example, the ability to improve and / or inhibit the formation, growth, proliferation, differentiation and / or maintenance of neurons and / or related cells and neural tissues, such as Schwann cells, glial cells and astrocytes, and may also affect the formation, growth, proliferation, differentiation and / or maintenance of tissue of the pancreas, liver, stomach, cardiac or other tissue such as that of bone, osteocytes, chondrocytes and / or cartilage tissue, or tissue from other organs, such as the spleen, lung, brain and kidney tissue, characteristics of the cerberus-like protein. Preferred polypeptides include a polypeptide that is at least about 80%, and more preferably at least about 90% homologous to the mature mammalian cerberus-like amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8. In addition, allelic variants or other variants of the sequences of SEQ ID NO: 2 or SEQ ID NO: 8 are also included in the present invention., whether such changes in amino acids are induced by mutagenesis, chemical alteration or by alteration of the DNA sequence used to produce the polypeptide, wherein the peptide sequence still possesses activity similar to cerberus. The present invention also includes fragments of the mammalian cerberus-like amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8, which retains the activity of the cerberus-like protein. One skilled in the art can easily produce such variants and protein fragments similar to mammalian cerberus using techniques known in the art, and can easily test them for their activity, as described herein. The purified proteins of the present invention can also be used to generate antibodies, either monoclonal or polyclonal, for proteins similar to mammalian cerberus and / or other related proteins, using methods that are known in the art of antibody production. In this way, the present invention also includes antibodies to cerberus proteins and / or other proteins similar to mammalian cerberus. The antibodies may be useful for the purification of mammalian cerberus-like proteins, or for the inhibition or prevention of the effects of cerberus proteins in vitro or in vivo. Mammalian cerberus-like proteins may be useful for inducing the growth and / or differentiation of embryonic cells and / or progenitor cells. Thus, 5AAAAAA) ia, ^ - ** 3i *? ± «iA *. * Proteins or compositions of the present invention may also be useful for the treatment of cell populations, such as populations of embryo cells or progenitor cells , to improve, enrich or to inhibit the growth and / or differentiation of cells. For example, mammalian cerberus-like proteins may be useful for treating cell populations to improve and / or inhibit the formation, proliferation, differentiation and / or maintenance of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes and / or other cells and tissues. The treated cell populations can be useful for, among other things, gene therapy applications, as described below. Thus, the proteins of the present invention can be useful in the healing of wounds, tissue and in processes of repair and regeneration of organs, as well as in the differentiation of tissue, for example, in the development of the embryo. In particular, it has been observed by the inventors that the mammalian cerberus-like protein can be useful for the induction, formation, growth, proliferation, differentiation and / or maintenance and repair of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes. Cerberus-like proteins are normally present as secreted proteins and have been shown to have effects on the growth and differentiation of neuronal cells and other types of nervous cells and tissues. In this way, these proteins and compositions containing them may be useful in the treatment of nervous and brain disorders, such as Parkinson's disease, Alzheimer's disease, and in the improvement and / or inhibition of formation, growth, proliferation. , differentiation and / or maintenance of cells, for example in the formation of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes. Mammalian cerberus-like proteins provided herein include factors encoded by sequences similar to those of SEQ ID NO: 1 or SEQ ID NO: 7. but within which modifications or deletions have been provided in a natural form (e.g. , allelic variants in the nucleotide sequence that can result in amino acid changes in the polypeptide) or deliberately designed. For example, synthetic polypeptides can totally or partially duplicate continuous sequences of the amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 8. These sequences, by virtue of sharing primary, secondary or tertiary conformational and conformational characteristics with polypeptides similar to mammalian cerberus of SEQ ID NO: 2 or SEQ ID NO: 8 may possess biological properties in common with them. Thus, these modifications and deletions of the cerberus-like native mammal can be employed as biologically active substitutes for polypeptides similar to mammalian cerberus that occur naturally, in therapeutic processes. It can be readily determined if a given variant or fragment of mammalian cerberus-like protein maintains the biological activity of the cerberus by subjecting both the cerberus-like and the variants or fragments of the mammalian cerberus-like to the assays described herein. Other specific mutations of mammalian cerberus-like protein sequences described herein involve modifications of glycosylation sites. These modifications may involve glycosylation sites with O-bond or N-bond. For example, the absence of glycosylation or only partial glycosylation results from substitution or deletion of amino acids at glycosylation recognition sites attached to asparagine. The glycosylation recognition sites attached to asparagine comprise tripeptide sequences that are specifically recognized by appropriate cell glycosylation enzymes. These tripeptide sequences are either asparagine-X-threonine or asparagine-X-serine, where X is usually any amino acid. A variety of amino acid substitutions or deletions in one or both of the first or third amino acid positions of a glycosylation recognition site (and / or amino acid deletion in the second position) results in null glycosylation in the modified tripeptide sequence. Such variants of the mammalian cerberus-like one are comprised within the present invention. Additionally, bacterial expression of mammalian cerberus-like proteins will result in the production of a non-glycosylated protein, even if the glycosylation sites are left unmodified. Said versions of the mammalian cerberus-like, produced in bacterial form are within the scope of the present invention. The present invention also encompasses the new DNA sequences, free of association with DNA sequences encoding other proteinaceous materials, and encoding the expression of mammalian cerberus-like proteins. These DNA sequences include those described in SEQ ID NO: 1 or SEQ ID NO: 7 in a 5 'to 3' direction and those sequences that hybridize them under stringent hybridization conditions [eg, 0.1X SSC, 0.1 % SDS at 65 ° C; see T. Maniatis et al., Molecular Cloning (A Laboratorv Manual, Cold Spring Harbor Laboratory (1982), pages 387 to 389] and code for a protein that possesses activity similar to cerberus.Astringent hybridization conditions also refer to conditions of initial low stringency hybridization, followed by high stringency wash conditions.These DNA sequences also include those comprising variants and fragments of the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 7 that hybridize them under conditions of astringent hybridization and encode a protein having cerberus-like activity Similarly, DNA sequences encoding mammalian cerberus-like proteins encoded by the sequences of SEQ ID NO: 1 or SEQ ID NO : 7, or mammalian cerberus-like proteins comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8, but differing in coding sequence Because of the degeneracy of the genetic code or allelic variants (base changes that occur naturally in the population of species that may or may not result in an amino acid change), they also encode the new factors described here. Variations in the DNA sequences of SEQ ID NO: 1 or SEQ ID NO: 7 that are caused by point mutations or by induced modifications (including insertion, deletion and substitution) to improve the activity, half-life or production of the Coded polypeptides are also included in the invention. Another aspect of the present invention provides a novel method for the production of proteins similar to mammalian cerberus. The method of the present invention involves the cultivation of a suitable cell line, which has been transformed with a DNA sequence coding for a mammalian cerberus-like protein of the invention, under the control of known regulatory sequences. The transformed host cells are cultured and the mammalian cerberus-like protein is recovered and purified from the culture medium. The purified proteins are substantially free of other proteins with which they are co-produced, as well as other contaminants. Appropriate cells or cell lines can be mammalian cells such as Chinese hamster ovary (CHO) cells. The selection of the appropriate mammalian host cells and the methods for transformation, culture, amplification, screening, production and purification of the product are known in the art. See, for example, Gething and Sambrook, Nature, 293: 620-625 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol. 5 (7): 1750-1759 (1985) or Howley et al, U.S. Patent: No. 4,419,446. Another suitable mammalian cell line, which is described in the accompanying examples is the COS-1 monkey cell line. The mammalian cell line CV-1 is also suitable. Also bacteria cells can be suitable hosts. For example, the various strains of E. coli (eg, HB101, MC 1061) are well known as host cells in the field of biotechnology. Several strains of B. subtilis, Pseudomonas, other bacilli and the like can also be employed in this method. For the expression of the protein in bacterial cells, DNA encoding the cerberus-like polypeptide is generally not necessary. Many strains of cells known to those skilled in the art may also be available as host cells for the expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be used as host cells in the method of the present invention. See, for example, Miller et al, Genetic Engineering, 8, 277-298 (Plenum Press 1986) and references cited here. Another aspect of the present invention provides vectors for use in the method of expressing these novel polypeptides similar to mammalian cerberus. Preferably the vectors contain the new complete DNA sequences that were described here and which code for the novel factors of the invention. Additionally, the vectors contain appropriate expression control sequences that allow the expression of cerberus-like protein sequences. Alternatively, vectors incorporating modified sequences as described above are also embodiments of the invention. Additionally, the sequence of SEQ ID NO: 1 or SEQ ID NO: 7 or other sequences encoding mammalian cerberus-like proteins could be engineered to express a protein similar to mature mammalian cerberus by deletion of similar propeptide sequences to mammalian cerberus and replacing them with sequences encoding the complete propeptides of other proteins similar to cerberus or other suitable propeptides. Thus, the present invention includes chimeric DNA molecules that _? - -f * and * t- encode a propeptide from a member of the cerberus-like family in a correct reading frame to a DNA sequence encoding a mammalian cerberus-like polypeptide. The vectors can be used in the method of transforming cell lines and contain selected regulatory sequences in operative association with the DNA coding sequences of the invention that are capable of directing the replication and expression of them in the selected host cells. Regulatory sequences for such vectors are known to those skilled in the art and can be selected depending on the host cells. Said selection is routine and does not form part of the present invention. In order to produce proteins similar to rat cerberus, human or other mammal, the encoding DNA is transfected into an appropriate expression vector and introduced into mammalian cells or other preferred eukaryotic or prokaryotic hosts by genetic engineering techniques conventional It is contemplated that the preferred expression system for mammalian cerberus-like, recombinant and biologically active mammalian cells are stably transformed. One skilled in the art can construct mammalian expression vectors using the sequences of SEQ ID NO: 1, SEQ ID NO: 7 or other DNA sequences encoding cerberus-like proteins or other modified sequences and known vectors, such as pCD (Okayama et al., Mol Cell Biol., 2: 161-170 (1982), pJL3, pJL4 [Gough et al., EMBO J. 4: 645-653 (1985) and pMT2 CXM.) The mammalian expression vector. pMT2 CXM is a derivative of p91023 (b) (Wong et al, Science 228: 810-815, 1985) differing from the latter in that it contains the ampicillin resistance gene instead of the tetracycline resistance gene and also contains an Xhol site for the insertion of cDNA clones The functional elements of pMT2 CXM have been described (Kaufman, RJ, 1985, Proc.Nat.Acid.Sci.USA 82: 689-693) and include adenovirus VA genes, the SV40 origin of replication including the 72 bp enhancer, the adenovirus major late promoter including the site of 5 'splice and most of the tripartite adenovirus leader sequence present in adenovirus late mRNAs, a splice acceptance site 37 a DHFR insert, the early SV40 polyadenylation site (SV40) and pBR322 sequences required for spreading in E. Coli.

Plasmid pMT2 CXM is obtained by digestion with EcoRI of pMT2-VWF, which has been deposited with the American Type Culture Collection (ATCC), Rockville, MD (USA) under accession number ATCC 67122. Digestion with EcoRI cuts the cDNA insert present in pMT2-VWF, producing pMT2 in linear form, which can be ligated and used to transform E. Coli HB101 or DH-5 for ampicillin resistance. The plasmid DNA pMT2 can be prepared by conventional methods. The pMT2 CXM is then constructed using exterior / interior circuit mutagenesis [Morinaga, et al, Biotechnology 84: 636 (1984)]. This removes the bases 1075 to 1145 relative to the Hin III site near the sequences of SV40 origin of replication and sequences of the enhancer pMT2. It also inserts the following sequence: 5 'PO-CATGGGCAGCTCGAG-3' at nucleotide 1145. This sequence contains the recognition site for the restriction endonuclease Xho I. A derivative of pMT2CXM, designated pMT23, contains recognition sites for the endonucleases of Restriction PstI, Eco Rl, Salí and Xhol, The plasmid DNA pMT2 CXM and pMT23 can be prepared by conventional methods. PEMC2ßl derived from pMT21 may also be suitable in the practice of the invention. PMT21 is derived from pMT2 which is derived from pMT2-VWF. As described above, digestion with EcoRI cuts the cDNA insert present in pMT-VWF, producing pMT2 in linear form, which can be ligated and used to transform E. coli HB101 or DH-5 for ampicillin resistance. The plasmid DNA pMT2 can be prepared by conventional methods. PMT21 is derived from pMT2 through the following two modifications.

First, 76 bp of the 5 'untranslated region of the DHFR cDNA including an expansion of 19 G residues from the G / C tail for the cloning of cDNA is eliminated. In this process, an Xhol site is inserted to obtain the following sequence immediately above DHFR: 5'-CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3 'PstI Eco Rl Xhol Second, a single Clal site is introduced by digestion with EcoRV and Xbaí, treatment with Klenow fragment of DNA polymerase I, and binding to a Clal linker (CATCGATG).

This removes a 250 bp segment from the region (VAI) of the associated RNA of the adenovirus but does not interfere with the expression or function of the VAI RNA gene. PMT21 is digested with EcoRI and Xhol, and used to derive the vector pEMC2Bl. A portion of the EMCV leader is obtained from pMT2-ECATl [S.K. Jung et al., J. Virol 63: 1651-1660 (1989)] by digestion with EcoRI and PstI, resulting in a 2752 bp fragment. This fragment is digested with Taql producing an EcoRI-Taql fragment of 508 bp that is purified by electro forestry in low melting agarose gel. A 68 bp adapter and its complement chain are synthesized with a 5 'Taql overhang and a 3' Xhol overhang having the following sequence: '-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTC CTTT Taql GAAAAACACGATTGC-3 'Xhol This sequence matches the EMC virus leader sequence from nucleotide 763 to 827.

It also changes the ATG at position 10 within the EMC virus leader to an ATT and is followed by an Xhol site. A three-way linkage of the EcoRJ-XhoI fragment of pMT21, the EcoRi-TaqI fragment of the EMC virus, and the TaqI-XhoI adapter of the 68 bp oligonucleotide adapter resulting in the vector pEMC2ßl. This vector contains the SV40 origin of replication and enhancer, the adenovirus major late promoter, a cDNA copy of most of the adenovirus tripartite leader sequence, a small hybrid intern sequence, a polyadenylation signal SV40 and the adenovirus VA I gene, DHFR and ß-lactamase markers and a sequence of EMC, in appropriate ratios to direct the high level expression of the desired cDNA in mammalian cells. The construction of the vectors may involve the modification of mammalian cerberus DNA sequences. For example, mammalian cerberus cDNA can be modified by eliminating the non-coding nucleotides at the 5 'and 3' endings of the coding region. The deleted non-coding nucleotides may or may not be replaced by other known sequences and are known to be beneficial for expression. These vectors are transformed into host cells suitable for the expression of mammalian cerberus proteins. Additionally, the sequence of SEQ ID NO: 1 and other sequences encoding mammalian cerberus proteins can be manipulated to express a mature mammalian cerberus protein by deleting the mammalian cerberus coding propeptide sequences and replacing them with coding sequences. the complete propeptides of other proteins. One skilled in the art can manipulate the sequences of SEQ ID NO 1 or SEQ ID NO: 7 by eliminating or replacing the mammalian regulatory sequences flanking the coding sequence with bacterial sequences to create bacterial vectors for intracellular or extracellular expression by cells from bacteria For example, the coding sequences could be further manipulated (ie, linked to other known or modified linkers by deleting non-coding sequences therefrom or by altering nucleotides by other known techniques). The modified mammalian cerberus coding sequence could then be inserted into a known bacterial vector using methods such as that described in T. Taniguchi et al Proc Nati. Acad. Sci. USA. 77-5230-5233 (1980). This exemplary bacterial vector could then be transformed into bacterial host cells and in this way a protein would be expressed. For a strategy to produce extracellular expression of mammalian cerberus proteins in bacteria cells, see for example, the European patent application EPA 177,343. Similar manipulations can be performed for the construction of an insect vector (see, for example, the procedures described in published European patent application 155,476) for expression in insect cells A yeast vector could also be constructed using regulatory sequences from yeast for intracclulai and extracellular expression of the factors of the present invention by yeast cells [See, for example, the methods described in PCT application WO86 / 00639 and European Patent Application EP 123,289] .A method for the production of High levels of a mammalian cerberus protein of the invention in mammalian cells can involve the construction of cells containing multiple copies of a heterologous mammalian cerberus gene.The heterologous gene is ligated to a label susceptible to amplification, eg, the the dihydrofolate reductase (DHFR) for which cells it contains n copies of increased genes can be selected for propagation at increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol., 159: 601-629 (1982). This approach can be employed with a number of different cell types. For example, a plasmid containing a DNA sequence for a mammalian cerberus protein in operative association with sequences of other plasmids making it possible to express it and the DHFR expression plasmid pAdA26SV (A) 3 [Kaufman and Sharp, J. Mol. Biol., 2: 1304 (1982) can be co-introduced into CHO cells deficient in DHFR, DUKX-BII, by various methods including co-precipitation with calcium phosphate and transfection, electrophoresis or protoplast fusion. Transformants of DHFR expression are selected to grow the alpha medium with dialyzed fetal serum, and subsequently selected by amplification for growth at increasing concentrations of MTX (eg, sequential steps at 0.02, 0.2, 1.0 and 5uM MTX) as it is described in Kaufman et al., Mol Cell Biol., 5: 1750 (1983). The transformants are cloned, and the expression of the mammalian cerberus, biologically active, is monitored by assay in one of the tests described in the examples and the description. The expression of mammalian cerberus protein should be increased with increasing levels of MTX resistance. The mammalian cerberus polypeptides are characterized using standard techniques known in the art such as pulse labeling with [35S] methionine or cysteine and polyacrylamide gel electrophoresis. Similar procedures can be followed to produce other related cerberus proteins. A mammalian cerberus protein of the present invention, which demonstrates cerberus activity, has application in the induction, formation, growth, proliferation, differentiation and / or maintenance and healing of cells and tissues such as neurons and / or cells and neural tissues. related, such as Schwann cells, glial cells and astrocytes, and other tissues, in humans and other animals. Said preparation employing mammalian cerberus protein may have prophylactic use in the treatment of Parkinson's disease, Alzheimer's disease, as well as in the prevention of neural tumors and other neural tissue disorders. The formation of new neurons and / or related neural cells and tissues such as Schwann cells, glial cells and astrocytes, and other neural phenotype cells, induced by a cerberus protein contributes to the repair of congenital tissue conditions or defects, induced by trauma or oncology. The mammalian cei'berus protein can also be used in the treatment of neural disease. and in other tissue and organ repair processes. Such agents can provide an environment that attracts suitable progenitor cells, stimulate the growth and proliferation of neuron-forming cells or induce the differentiation of progenitors of neuron-forming cells, and may also sustain the regeneration of other tissues and organs. The mammalian cerberus polypeptides of the invention may also be useful in the treatment of organ disorders. The proteins of the invention may also be useful in the healing of wounds and in the repair of related tissue. Types of injuries include, but are not limited to, burns, cuts and ulcers. (See, for example, PCT Publication WO84 / 01106 for a description of wound healing and related tissue repair). It is also contemplated that proteins of the invention may increase the survival of neuronal, astrocytic and / or glial cells and, therefore, be useful in the transplantation and treatment of conditions that present a decrease in neuronal survival and repair. The proteins of the present invention may be further useful for the treatment of conditions related to other types of tissues, such as the epidermis, muscle, connective tissue, such as bone, cartilage, tendon and ligament, and other organs such as the pancreas, liver, spleen, lung, cardiac, brain and kidney. The proteins of the present invention may also have value as a dietary supplement, or as additives for cell culture medium. For this use, the proteins can be used intact, or they can be pre-digested to provide a more easily absorbed supplement. The proteins of the invention may also have useful characteristic properties of the Cei'berus family of proteins. Such properties include angiogenic properties. chemoattractants and / or chemoattractants, and effects on cells that include differentiation responses, cell proliferative responses and responses that involve cell adhesion, migration and extracellular matrices. These properties make the proteins of the invention potential agents for the healing of wounds, reduction of fibrosis and reduction of tissue formation of scar tissue. The proteins of the invention may also be useful for the induction of the formation of cells capable of secreting valuable hormones, including endocrine hormones and expermas. A further aspect of the invention is a therapeutic method and a composition for the treatment of disorders of neurons and / or related neural tissues and cells such as Schwann cells, glial cells and astrocytes, and other conditions related to tissue, disorders or neural diseases. and of neurons. The invention further comprises therapeutic methods and compositions for wound healing and tissue repair. Such compositions comprise a therapeutically effective amount of at least one mammalian cerberus protein of the present invention in admixture with a pharmaceutically acceptable carrier, carrier or matrix. It is further contemplated that the compositions of the invention may increase the neuronal survival of glial cells and astrocytes and. therefore, be useful in the transplant and treatment of conditions that present a decrease in neuronal survival. It is expected that cerberus and cerberus-like proteins may exist in nature as homodimers or heterodimers. To promote the formation of protein-like dimers with increased stability, one can genetically engineer the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 7 to provide one or more additional cysteine residues to increase potential formation of dimer. The resulting DNA sequence would be able to produce an "added cysteine variant" of cerberus-like protein. Alternatively, one could produce "cysteine aggregate variants" of cerberus-like proteins by altering the sequence of the protein at the amino acid level, for example, by altering the amino acid sequences of one or more amino acid residues for Cis. The production of "added cysteine variants" of proteins is described in U.S. Patent 5,166,322, the disclosure of which is incorporated herein by reference. It is expected that the proteins of the invention may act in concert with or perhaps synergistically, with other proteins and related growth factors. Such combinations may comprise separate molecules of the cerberus or cerberus-like proteins and other proteins or heteromolecules composed of different fractions. For example, a method and composition of the invention may comprise a disulfide-linked dimer comprising a subunit of cerberus protein and a subunit derived from the "BMP" proteins. Thus, the present invention includes a purified cerberus-like polypeptide, which is a heterodimer wherein a subunit comprises an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 8, and a subunit comprises a sequence of amino acids for a bone morphogenetic protein selected from the group consisting of BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP- 11, BMP-12 or BMP-13, described in PCT application WO 95/16035, or BMP-15 described in PCT application WO 96/36710 ° BMP-16, described in the co-pending patent application with number series 08 / 715,202, filed September 18, 1996. A further embodiment may comprise a heterodimer of cerberus fractions, for example, of Xenopus cei'berus and a mammalian homolog of Xenopus cerberus or another cerberus-like protein. Additional methods and therapeutic compositions of the invention, therefore, comprise a therapeutic amount of at least one mammalian cerberus protein of the invention with a therapeutic amount of at least one other protein, such as a member of the TGF-β superfamily of proteins, which includes bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and other proteins. The composition may further include other growth factors and agents such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-a). and TGF-β), Wnts, urchins, including sonic, Indian and desert urchins, activins, inhibins. ) fibroblast growth factor k (kFGF), parathyroid hormone (PTH), leukemia inhibitory factor (LIF / HILDA / DIA), insulin-like growth factors (IGF-I and IGF-II). Portions of these agents can also be used in compositions of the present invention. It is within the skill of the art to prepare and formulate such physiologically acceptable protein compositions, with due regard to pH, isotonicity, stability and the like. Therapeutic compositions are also currently valuable for veterinary applications due to the lack of species specificity in the Cei'berus proteins. Particularly, domestic animals and thoroughbred horses in addition to humans, are desirable patients for such treatments with the cerberus proteins of the present invention. The therapeutic method includes the administration of the composition topically, sitymically or locally as an injection or implantation. When administering the therapeutic composition for use in this invention is, of course, in a physiologically acceptable and pyrogen-free form. In addition, the composition may desirably be in an encapsulated or injectable viscous form for delivery to the site of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes or other damaged tissue. Topical administration may be adequate to heal wounds and repair tissues. Therapeutically useful agents other than cerberus proteins that may also optionally be included in the composition described above, they may alternatively or additionally, be administered simultaneously or sequentially with the cerberus composition in the methods of the invention. For implantation, the composition preferably includes a matrix capable of liberal-mammalian cerberus proteins to the site of neurons and / or related neural cells and tissues such as Schwann cells, glial cells and astrocytes or other damaged tissues, providing a structure for the development of tissue and optionally capable of being reabsorbed within the body. The matrix can provide slow release of mammalian cerberus protein and / or other proteins, as well as an appropriate presentation and a suitable environment for cell infiltration. Such matrices can be formed from materials currently in use for other medical implant applications. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the mammalian cerberus compositions will be defined by the appropriate formulation. The dosage regimen will be determined by the attending physician, considering several factors that modify the action of the mammalian cerberus protein, for example, amount of tissue to be formed, the site of the tissue damage, the condition of the damaged tissue, the size of the lesion, type of damaged tissue, the age, sex and weight of the patient, the severity of the infection, time of administration and other clinical factors. The dose can «s. Vai-iar with the type of matrix used in the reconstitution and the types of mammalian cerberus proteins found in the composition. The addition of other known growth factors, such as IGF I (insulin-like growth factor) to the final composition can also affect the dose. Progress can be monitored through periodic evaluations of tissue growth and / or repair. The advance can be monitored, for example, by x-rays, histomorphometric determinations and tetracycline labeling. The following examples illustrate the practice of the invention in the recovery and characterization of mammalian cerberus protein and using DNA to recover other cerberus proteins, obtaining human proteins and expressing the proteins via recombinant techniques. As will be recognized, numerous variants of the materials and methods described can be carried out and are within the scope of the present invention.

Example 1: OMM Cluster of? _n MMIG___O Homologue for Xenopus cerberus The cysteine-rich, carboxy-terminated domain of xenopus cerberus was used to screen mammalian and ESTS libraries reported in GENBANK. One EST, AA 120122. derived from the embryonic region library of day 7.5 of Beddington was identified by sequence homology for xenopus cerberus. Although the partial clone had almost no sequence conservation in the N-terminus half of the molecule, the C-terminus domain and specifically the nine-cysteine pattern showed sequence conservation. The full-length cDNA encoding the murine cerberus-like protein was isolated from a cDNA library of murine embryonic cells and the gene isolated from a murine genomic library. CDNA similar to murine cerberus (SEQ ID NO: 1) encodes a protein of 272 amino acids with MW predicted of 30.5 Kd (SEQ ID NO: 2). The murine cerberus-like gene, Mcer-1, has a single 2Kb intron at position 564 of SEQ ID NO: 1 of cDNA. The predicted protein contains a hydrophobic signal sequence at its amino terminus, indicating that the molecule is secreted. Analysis by Sigcleave indicates that the first 17 residues are separated from the mature molecule during secretion (Sigcleave record = 7.6). The protein similar to cerberus de minino, mature, is predicted to i ^^ - r'¡ ??? ie? & * < - * «~ ~ ~ ~, - ~: is a protein of 255 MW residues of 28.6 Kd that includes residues 18-272 of SEQ ID NO: 2. Cerberus-like cDNA was expressed in COS cells and labeled with 3S-Met / 3S-Cys. The resulting protein was secreted into the culture medium and resulted in a stained band of MW 38-44 Kd when fractionated in 16% polyacrylamide reduction gels. Stained bands indicate that the protein is glycosylated consistent with the two putative N-linked glycosylation sites in the sequence. The expression of the protein in reticulocyte systems shows a 33Kd band consistent with predicted protein in the absence of glycosylation. When fractionated in non-reducing polyacrylamide gels, approximately half of the COS expressed runs of material with a molecular weight of 78-84 Kd indicating that the protein is capable of forming homodimers attached to cysteine. The protein sequence contains a hydrophobic signal sequence at its amino terminus and a cysteine-rich domain near its carboxy terminus. The cysteine-rich domains of the Xenopus cerberus and murine cerberus-like proteins are 58% identical and the total amino acids are 31% identical. The pattern of nine cysteines in the carboxy terminus is conserved between the Xenopus cerberus and the protein similar to mammalian cerberus.

Example 2; Similar to mammalian cerberus encodes mima Secreted Protein To test if the full-length cDNA encoded a secreted protein, the human 293T cell line was transiently transfected with the DNA sequence of SEQ ID NO: 1, which codes for a similar protein to mammalian cerberus, was cloned into a eukaryotic expression vector and labeled with S-methionine. A broad band was secreted into the culture medium, as well as a minor form. Similar to the expression of COS cells, the fractionation of the material expressed in the 293T cells evidenced the formation of dimers linked to cysteine. The cerberus-like protein is presumably glycosylated since the translated protein in the reticulocyte system (in the absence of membranes) resulted in a molecular weight band of 33 kD corresponding to the predicted molecular weight from the amino acid sequence. The cerberus-like protein has two putative N-linked glycosylation sites. Accordingly, the DNA of SEQ ID NO: 1 defines a secreted protein with high amino acid identity with Xenopus cerberus in the cysteine-rich domain.

Example 3. Mammalian Cerberus-like Protein Expression In pre-striped mouse embryos (5.5 days post-coitum), transcripts resembling mammalian cerberus were detected on one side of the primordial endoderm, including the far tip of the embryo. In early primordial stria, expression was found in a patch of primordial endoderm cells on one side of the embryonic region and did not extend further to the tip of the embryo. This patch corresponded to the anterior side of the embryo, because in sections it was found in the endoderm opposite to the formation of the primordial stria, which could be recognized as a thickening of the posterior apiblast. In the mean stria, the positive area similar to cerberus remained in the anterior primordial endoderm. In the final stage of the stria, when the node has reached the far tip of the embryo, a second population of expression similar to cerberus was observed in the region surrounding the node. These cells presumably correspond to definitive endoderm cells, which are known to emerge from the node. The endodermal nature of this population of cells was confirmed by histological analysis. In the stage of neural plate, it was found similar to cerberus implicit in the anterior neural plate, in a pattern comparable to that of the expression domain of Otx2 in endoderm. Ang et al., Cell. 78: 561-574 (1994). At this stage, the positive cell population a cerberus-like presumably consisted of both primitive and definitive endoderm, and did not include the node itself. Importantly, cells expressing the secreted factor similar to cerberus are in direct contact with Coi-affection, which subsequently originate the front and middle region of the CNS brain. In the early stage of headfold, the signal similar to cerberus in the endoderm begins to weaken. In late-head folding, expression similar to cerberus is confined to the middle and contiguous endoderm. The expression is found in all the cells from the middle part of the rostral end of the embryo to the vicinity of the node and includes the endoderm of the anterior intestine and the mesoderm from the precordal to the notochordal plates. The expression of cerberus-like mRNA in the anterior endoderm remains until the beginning of somitogenesis and subsequently becomes undetectable. By way of hA¡sfc < g «4 ... i¿j! ^^ _. ^ __ í_¿ = -i _ ^ _.! ^ _ i. &. .., ^ __ ^ _ 8_a __? __ JM wrfa-- concomitantly, a late phase of expression begins in the mesoderm of the somites. In this way, the similar to cei'berus defines an anterior domain of the endoderm in early mouse embryos. The expression was found in the anterior endoderm in direct contact with the future neural plate, but was never detected in cells of the posterior endoderm in contact with the primordial stria, providing additional evidence of support of the role of cerberus-like in the induction of anterior neuroectoderm.

Example 4: Tests to Beteraminate the Fund of Cerberus or Similar to Cerberus in the Mouse. To determine the function of cerberus or cerberus-like genes in the mouse embryo, the transgenic knockout and mis-expression mice can be made by manipulating murine embryo (ES) progenitor cells and injection into blastula using standard procedures. The murine knockout can be achieved by replacing a central portion of the cerberus or cerberitic-like genes with a selectable marker (eg, neo), transfecting the construct into ES cells and selecting for double crossing. For erroneous expression, cerberus-like Cei'berus genes may be manipulated to be expressed from generic (eg, actin) or tissue-specific promoters (eg, IDX) and reintroduced into murusio ES cells. Transgenic animals can be generated from said cells designed using classical methods. To determine the function of cerberus or cerberus-like proteins in the adult mouse, the protein can be injected directly into tissues or released by viral vectors. For example, cerberus or cei'berus-like genes can be transiently overexpressed in the adult mouse using adenovirus vectors and the function or activity of the proteins investigated by physiological, histochemical and biochemical analysis of the animals.

Example 5: Isolation of Genes Cerberus and Similar to Cerberus de Hiamano Hybridization of the gene similar to murine cerberus to human DNA sequences identified a single Xbal-generated band of approximately 15 Kb demonstrating its capacity as a probe to identify the gene similar to Human cerberus within a human genomic library. Alignment of the Xenopus cerberus and murine cerberus genes has identified three regions of sufficient nucleic acid homology within the cysteine-rich domain to serve as probes for the human cerberus and cei'berus-like genes. The consensus sequences for the three probes are: (1) TGCCCTTCAMYCAGAMYATTGYMCATGAARACTGT [SEQ ID NO: 3] (2) CAGAACAAYCTKTGCTTTGGTAAATGCA [SEQ ID NO: 4] and (3) TGYTCCCAYTGCTYGCCSWCCAAATT [SEQ ID NO: 5]. The first probe encompasses the 2 Kb intron in the mouse gene and is therefore less likely to work well as a probe. Used individually or together, the last two oligos containing degeneration of six times and ten times, respectively, would serve as probes for cerberus and similar genes to human cerberus in genomic or cDNA libraries. The nucleotide and amino acid sequences obtained from human cerberus according to the methods described are shown in SEQ ID NO: 7 and SEQ ID NO: 8. respectively.

Example < ? Identification and Isolation of Cerberus Proieins and Similar to Additional Cerberus. The cerberus and cerberus-like proteins are members of a family that can be recognized by its unique cysteine pattern. This family includes cerberus protein and similar to cerberus. Dan's protein and Norrie's protein. The Dan protein is a tumor suppressor candidate and defects in the Norrie protein result in congenital defects that include blindness and deafness. The members of the Cerberus family therefore seem to play an important role in cell differentiation and proliferation and, in this way, it is important to find other members of this family of proteins. The members of the family can not necessarily be recognized by amino acid homology, which demonstrates a significant amount of diversity, but can be recognized by a unique pattern of nine cysteines. The consensus cysteine pattern in each of these proteins is C (X 13.15) C (X9) CxGxC (X14.23) CXXC (X] 3) C (Xis-iß) CXC, where Xy indicates the number of residues »Rluil? M? ÍÍ? Í ^? ¡?, A ^ K? * g-'Mto "• * ^ -» - «" «-« - «b non-cysteine amino acid found between each cysteine in the conserved cysteine pattern of the family of cerberus and cerberus-like proteins. By investigating mammalian ESTs for this motive, new members of the Cerberus family have been identified including ESTs N35377 from human and EST AA289245 from murine. Using standard procedures, full-length genes can be isolated from genomic or cDNA libraries. It is expected that these genes code for signaling proteins that function to model the embryo, control cell differentiation or cell proliferation and thus be candidate proteins in medical therapy. Within the cysteine-rich domains of the Xenopus cerberus and murine cerberus-like proteins there are two discrete subregions that are highly conserved cysteine motifs and may be particularly useful for the identification and isolation of cerberus and cerberus-like proteins of other species, as well as related members of families of cerberus and cerberus-like proteins. It is expected that the use of these highly conserved motifs to screen mammalian libraries and ESTSs reported in GENBANK will identify additional proteins from the families of cerberus and cerberus-like proteins from humans and other species, as well as to identify additional family members. . The first region is the motif C-X-G-X-C. which corresponds to Cys-Phe-Gly-Lys-Cys found at amino acid residues 186 to 190 of SEQ ID NO: 1. Thus, the oligonucleotides degenerate to the TGC TTT sequence GGC AAA TGC at nucleotide positions 613 to 627 of SEQ ID NO: 1, and contiguous regions. they may be useful to identify and isolate other genes that share the C-X-G-X-C motif of cerberus and cerberus-like proteins. A second highly conserved region within the cysteine-rich domains of the Xenopus cerberus and murine cerberus-like proteins is the CXXC motif, which corresponds to Cys-Ser-His-Cys found at amino acid residues 206 to 209 of the SEQ ID NO: 1. In this way degenerate the oligonucleotides to the TGC TTC CAC TGC sequence at nucleotide positions 673 to 684 of SEQ ID NO: 1, and contiguous regions, it may be useful to identify and isolate other genes they share the CXXC motif of cerberus and cerberus-like proteins. Using the above motifs, families of cerberus and cerberus-like proteins can be identified from other species, and additional members of the family from humans, mice or frogs can be found. The use of two previous motives in concert can also provide specific identification and isolation of additional members of the Cerberus family and similar to Cerberus.

Example 7. Assays for Activity Microinjection and Cerberus Similar to injection mRNA encoding Xenopus cerberus in Xenopus blastomeres at different stages (4 cell, 8-cell and 32 cells) has profound effects on the expression pattern of 10 specific genes and on the morphology resulting from the development of the embryo. The incorrect expression of cerberus inhibits the development of the precordal plate. notochord and mesoderm of the ventral trunk and their respective molecular markers, goosecoid, collagen II and x-globin. The microinjection of cerberus induces the anterior neuroectodermal structures such as the brain, olfactory placodes and cement gland 15 (cement gland). The expression of the gene induced by cerberus includes N-CAM (brain), Otx2 (forebrain), CG-13 (cement gland) and Nkx-2.5 (primordium of the heart). Induction of neural tissue by cerberus was specific to the anterior region of the brain as indicated upregulation Otx2 marker but no subsequent markers including In-2 (midbrain-hindbrain junction), Krox-20 (hindbrain) and XlHbox-6 (spinal cord). The injection of cerberus into specific blastomere cells of 32 cells resulted in the induction of ectopic heads and double heart and liver. Similarly, similar to what happens with cerberus, the microinjection of cerberus-like mRNA inside animal protective cover explants induces CNS in xenopus embryos. However, microinjection similar to cerberus mammalian mRNA in Xenopus embryos did not induce the formation of structures ectopically head, for example, containing forebrain, ciclópeos eyes, placodes olfactory and cement glands, suggesting overlapping but not identical functional effects. In this way, the cerberus-like factor is a neutralization factor, which leads to the formation of forebrain in xenopus trials.

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Additional Test Example heard Embryonic Stem Cell In order to test the effects of cei'berus like proteins of the present invention, it is possible to test the effects of growth and differentiation in vitro in a number of cell lines available embryonic stem. One such cell line is ES-E14TG2, which is available at the American Type Culture Collection in Rockville, Md. In order to conduct the tests, the cells can be propagated in the presence of 100 units of LIF to keep them in a state without differentiation. The assays are arranged by first eliminating the LIF and adding the cells in suspension, in what is known as embryoid bodies. After 3 days the embryo bodies are plated on gelatin-coated dishes (12-well dishes for PCR analysis, 24-well plates for immunocytochemistry) and treated with the proteins to be tested. The cells are supplemented with nutrients and treated with the protein factor every 2-3 days. The cells can be adapted so that the assays can be conducted in medium supplemented with 15% Fetal Bovine Serum (FBS) or defined medium of CDM containing much lower amounts of FBS. At the end of the treatment period (varying from 7-21 days) RNA was harvested from the cells and analyzed by quantitative multiplex PCR for the following markers: Branchyury, a marker of the mesoderm, AP-2, a marker of the ectoderm, and HNF -3a, a marker of the endoderm. Through immunocytochemistry it is also possible to detect the differentiation of neuronal cells (glia, astrocytes and neurons), muscle cells (cardiomyocytes, skeletal and smooth muscle), and various markers of other phenotype such as core protein proteoglycan (cartilage), and cytokeratins (epidermis). Since these cells have a tendency to differentiate autonomously when the LIF is removed, the results are always quantified by comparison with a control without treatment.

Example 9: Expression of Human Cerberus Protein The human Cerberus cDNA sequence (SEQ ID NO: 7) has been expressed in COS cells using mammalian expression vectors as described in the application. The observed protein was secreted, with a molecular weight of about 35-45 kD, consistent with a glycosylated protein of 250 (amino acids # 18 to # 267 of SEQ ID NO: 8) amino acids in length. The above description details preferred embodiments of the present invention. Numerous modifications and variations in the practice of it are expected to occur to those skilled in the art when considering this description. It is believed that those modifications and variants are within the appended claims. All publications and patents referred to herein are incorporated herein by reference, as if they had been fully set forth in this disclosure. "í &?. - ^^ ---; ^ A-fe ^^ i? ^ s .. '. r ^^^, ^? -? m, w? i < ^' ^ ifi.ii: , - LISTING ÍM SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Follettie, Maximillian DeRobertis, Edward M. (ii) TITLE OF THE INVENTION: Protein similar to mammalian Cerberus and compositions (iii) NUMBER OF SEQUENCES: 8 (ív) ADDRESS FOR CORRESPONDENCE: (A) SENDER: GENETICS INSTITUTE, INC. (B) STREET: 87 CAMBRIDGEPARK DRIVE (C) CITY: CAMBRIDGE (D) STATE: Massachusetts (E) COUNTRY: USA (F) POSTAL CODE: 02140 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT REQUEST DATA: (A) NUMBER OF APPLICATION: TBD (B) DATE OF PRESENTATION: with this (C) CLASSIFICATION: (viii) INFORMATION OF THE POWDER / AGENT: (A) NAME: Lazar, Steven R. (B) REGISTRATION NUMBER: 32,618 (C) NUMBER OF REREFENCE / FILE: Gl 5290APCT (íx) INFORMATION FOR TELECOMMUNICATIONS: (A) TELEPHONE: (617) 498-8224 (B) TELEFAX: (617) 876-5851 (2) INFORMATION FOR SEQ ID NO.:l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1003 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CD S (B) LOCATION: 58..873 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GAATTCGGCC AAAGAGGCCT ATGTGAATCT AACCTCAGTC TCTGGGAATC AGGAAGC 57 ATG CAT CTC CTC TTA GTT CAG CTG CTT GTT CTC TTG CCT CTG GGG AAG 105 Met His Leu Leu Leu Val Gln Leu Leu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 GCA GAC CTA TGT GTG GAT GGC TGC CAG AGT CAG GGC TCT TTA TCC TTT 153 Wing Asp Leu Cys Val Asp Gly Cys Gln Ser Gln Gly Ser Leu Ser Phe 20 25 30 CCT CTC CTA GAA AGG GGT CGC AGA GAT CTC CAC GTG GCC AAC CAC GAG 201 Pro Leu Leu Glu Arg Gly Arg Arg Asp Leu His Val Wing Asn His Glu 35 40 45 GAG GCA GAA GAC AAG CCG GAT CTG TTT GTG GCC GTG CCA CAC CTC ATG 249 Glu Wing Glu Asp Lys Pro Asp Leu Phe Val Wing Val Pro His Leu Met 50 55 60 GGC ACC AGC CTG GCT GGG GAA GGC CAG AGG CAG AGA GGG AAG ATG CTG 297 Gly Thr Ser Leu Wing Gly Glu Gly Gln Arg Gln Arg Gly Lys Met Leu 65 70 75 80 TCC AGG CTT GGA AGA TTC TGG AAG AAA CCT GAG ACC GAA TTT TAC CCC 345 Ser Arg Leu Gly Arg Phe Trp Lys Pro Glu Thr Glu Phe Tyr Pro 85 90 95 CCA AGG GAT GTG GAA AGC GAT CAT GTC TCA TCG GGG ATG CAG GCC GTG Í93 Pro Arg Asp Val Glu Ser Asp His Val Ser Ser Gly Met Gln Ala Val 100 105 110 ACT CAG CCA GCA GAT GGG AGG AAA GTG GAG AGA TCA CCT CTA CAG GAG 441 Thr Gln Pro Wing Asp Gly Arg Lys Val Glu Arg Ser Pro Leu Gln Glu 115 120 125 GAA GCC AAG AGG TTC TGG CAT CGG TTC ATG TTC AGA AAG GGC CCG GCG Glu Ala Lys Arg Phe Trp His Arg Phe Met Phe Arg Lys Gly Pro Wing 130 135 140 TTC CAG GGA GTC ATC CTG CCC ATC AAA AGC CAC GAA GTA CAC TGG GAG 537 Phe Gln Gly Val lie Leu Pro lie Lys Ser His Glu Val His Trp Glu 145 150 155 160 ACC TGC AGG ACT GTG CCC TTC AAC CAG ACC ATT GCC CAT GAA GAC TGT 585 Thr Cys Arg Thr Val Pro Phe Asn Gln Thr lie Wing His Glu Asp Cys 165 170 175 CAA AAA GTC GTT GTC CAG AAC AAC CTT TGT TTT GGC AAA TGC AGT TCC 633 Gln Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Ser Ser 180 185 190 ATT CGT TTT CCC GGA GAA GGG GCA GAT GCC CAC AGC TTC TGC TCC CAC 681 lie Arg Phe Pro Gly Glu Gly Wing ASp Wing His Ser Phe Cys Ser His 195 200 205 TGC TCG CCC ACC AAA TTC ACC ACC GTG CAC TTG AGG CTG AAC TGC ACC 729 Cys Ser Pro Thr Lys Phe Thr Thr Val His Leu Arg Leu Asn Cys Thr 210 215 220 AGC CCA ACC CCC GTG GTC AAG ATG GTG ATG CAA GTA GAA GAG TGT CAG 777 Ser Pro Thr Pro Val Val Lys Met Val Met Gln Val Glu Glu Cys Gln 225 230 235 240 TGC ATG GTG AAG ACG GAA CGT GGA GAG GAG CGC CTC CTA CTG GCT GGT 825 Cys Met Val Lys Thr Glu Arg Gly Glu Glu Arg Leu Leu Leu Wing Gly 245 250 255 TCC CAG GGT TCC TTC ATC CCT GGA CTT CCA GCT TCA AAA ACA AAC CCA 873 Ser Gln Gly Ser Phe lie Pro Gly Leu Pro Wing Ser Lys Thr Asn Pro 260 265 270 TGATTACCTC AACAGAAAGC AAAACCTCAA CAGAATAAGT GAGGGTTATT CAATCTGGAA 933 ATGTTATGTG A GTTATATAA AGATCAGTGG AAAAAAAAAA AAAAAAAAAAA AAAAAAAAAAA 993 AAGCGGCCCCC 1003 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 272 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (Xl) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: Met His Leu Leu Leu Val Gln Leu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 Wing Asp Leu Cys Val Asp Gly Cys Gln Ser Gln Gly Ser Leu Ser Phe 20 25 30 Pro Leu Leu Glu Arg Gly Arg Arg Asp Leu His Val Wing Asn His Glu 35 40 45 Glu Wing Glu Asp Lys Pro Asp Leu Phe Val Wing Val Pro His Leu Met 50 55 60 Gly Thr Ser Leu Wing Gly Glu Gly Gln Arg Gln Arg Gly Lys Met Leu 65 70 75 80 Ser Arg Leu Gly Arg Phe Trp Lys Lys Pro Glu Thr Glu Phe Tyr Pro 85 90 95 Pro Arg Asp Val Glu Be Asp His Val Be Ser Gly Met Gln Ala Val 100 105 110 Thr Gln Pro Wing Asp Gly Arg Lys Val Glu Arg Ser Pro Leu Gln Glu 115 120 125 Glu Ala Lys Arg Phe Trp His Arg Phe Met Phe Arg Lys Gly Pro Wing 130 135 140 Phe Gln Gly Val lie Leu Pro lie Lys Ser His Glu Val His Trp Glu 145 150 155 160 Thr Cys Arg Thr Val Pro Phe Asn Gln Thr lie Wing His Glu Asp Cys 165 170 175 Gln Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Ser Ser 180 185 190 lie Arg Phe Pro Gly Glu Gly Wing Asp Wing His Ser Phe Cys Ser His 195 200 205 Cys Ser Pro Thr Lys Phe Thr Thr Val His Leu Arg Leu Asn Cys Thr 210 215 220 Ser Pro Thr Pro Val Val Lys Met Val Met Gln Val Glu Glu Cys Gln 225 230 235 240 Cys Met Val Lys Thr Glu Arg Gly Glu Glu Arg Leu Leu Leu Wing Gly 245 250 255 Ser Gln Gly Ser Phe lie Pro Gly Leu Pro Wing Ser Lys Thr Asn Pro 260 265 270 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (n) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 TGCCCTTCAM YCAGAMYATT GYMCATGAAR ACTGT 35 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs ^^ iht ^^^? aí ^^? a ^^^^ (B) TYPE: nucleic acid (C) STRING: simple (D) TOPOLOGY: linear (11) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEO ID NO: 4: CAGAACAAYC TKTGCTTTGG TAAATGCA 28 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (li) ) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: TGYTCCCAYT GCTYGCCSWC CAAATT 26 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3595 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (n) ) TYPE OF MOLECULE: DNA (genomic) (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 117..623 (ix) CHARACTERISTICS: (A) NAME / KEY: intron (B) LOCATION: 624 ..2402 (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 2403..2699 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: TCCGGCCAGG CAGGTATCTA TATATCCGAT TTCCTTTTTC CCAAGTCCTG CAGAAGAATG 60 AGCCTCTCCT TTGGGCCTCA TCATTTTACC AAAAAGAAGC TTGGGCCCCT GACAGCATGC 120 ATCTCCTCTT ATTTCAGCTG CTGGTACTCC TGCCTCTAGG AAAGACCACA CGGCACCAGG 180 ATGGCCGCCA GAATCAGAGT TCTCTTTCCC CCGTACTCCT GCCAAGGAAT CAAAGAGAGC 240 TTCCCACAGG CAACCATGAG GAAGCTGAGG AGAAGCCAGA TCTGTTTGTC GCAGTGCCAC 300 ACCTTGTAGC CACCAGCCCT GCAGGGGAAG GCCAGAGGCA GAGAGAGAAG ATGCTGTCCA 360 GATTTGGCAG GTTCTGGAAG AAGCCTGAGA GAGAAATGCA TCCATCCAGG GACTCAGATA 420 GTGAGCCCTT CCCACCTGGG ACCCAGTCCC TCATCCAGCC GATAGATGGA ATGAAAATGG 480 AGAAATCTCC TCTTCGGGAA GAAGCCAAGA AATTCTGGCA CCACTTCATG TTCAGAAAAA 540 CTCCGGCTTC TCAGGGGGTC ATCTTGCCCA TCAAAAGCCA TGAAGTACAT TGGGAGACCT 600 GCAGGACAGT GCCCTTCAGC CAGGTATGTG TTCTGGGGGG AGAGCAGGTA AGAGTTTGCA 660 GGTGGTAGTG GACAGCTGGG ATGGATGGAG AGTAGGGGAA AAGGCTGTCA GGAGCCTGAC 720 TCTAGCTTAA CTACAGATTT GGTCCTTGGG CATTCATCAT AGGATTTGGC AAAGATTAAG 730 TTTCCTTCTG GCCTTTCCAT TTTTTCTTGG CATTGTGGAA ATGCTGCAAG AATGATATGA 340 TGATACTGTC AATATCAGTA ATCATTCATT CACACTGAAG ACACAGAGCT CTGTTTTATT 900 TATTTATTTT TGCATTGGAG GTGATCTACT CAGAGATATA AGTCAGACTG TACCCTCAGT 960 TAGGAAACTG AGAATTTAGA GAATCACCAG AACTCCTCTG TAGCTATCTT TCTGCACTCT 1020 ATTAATATGG GATGAGCAGG TCAACTCCCA TTTGTTGATA AAGTGGGGTG CATTGGACTC 1080 CTTCCCAAAT ACTCTCATAT CCATTTACGA TGGTCTTAAT CCCCATAGTC CATACTTAAT 1140 TACTTTATAG GTTTATGAGG GACTTCTTTA ATAGCTTGCT AAAGCTTATC CCACAACCTC 1200 AAAGTACGTT GAGGTTCTCA GGCAAAAGTT GTCATATCAT TTCTAGTATT ATGATAGCAA 1260 AAAAGTGATT TTCTTTCACT TATTTTCTCA TATGAGCTTT TTAAAAAATC AATCTTGATG 1320 TGAGATCATA TCTCCTCCCC TTAGAAGTAC CTTTCTCCTG ATTCATGTTG TGTTGGCTGA 1330 TTTGTAGTTA TTATGATCAA TTCCATGCTA TTAAGACAAA GGGACATCCT ACTGTCTACT 1440ATATCTACAT TCCAAATGTT AAATTAAAAT TGAGAACTTG CATTAGGTCC 1500 TTAAACATGA AGATATTGAA CCAAAAACAT GCAGGGTAGA GTAAAATTTT ATAGTCGAGT 1560 AATGCTACCC AATTAAGCAA GCAATAGAAT AGGGCAATTG ACTGTTCAAG GCAGTTAAGT 1620 ATTCTGCCTG AAAAGGCAAG GATATGTAGC AATGGCAAGT CAATTATCAA ATAATAATGA 1680 CTACTCTGTT GGCCATGTGC AATTAGAAAA TTACCCCTAA GAATCAGGCA ATCAAATTTC 1740 TTTTGAAATT CTTCTTTTGA ATTCTATTGC TAATTAAATT AAAACTAAGA TGTTTGACTC 1800 TTACATATTT TGAAAGGCAT ATAAAGCTAG GTGCTTGGAG TTATGAGAGG TAAAGGTGAT 1360 GTAATATNCA ATGATTTGCA GGCATATGCA TTGTAACTCT GCTTGCATAC AACTTCATAG_1_2C • > ? ¿A? 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ACTTGAATGT ACTACAGGTC TTGCAGAATA GGATAGAATT AAACCTAGAA TGTTCTGATC 530 TATTCTACGA TCAATGTAAC AAATATGTAT TGGGAGCCTA CTATGCACAA AGCCCTGTGA 2040 GGAATAAAAA AGTAAGGCAC ATTACTTATG TAAGATAATT ACCATTAGAA TTTTTCAATC 2100 GCTCACATCC AATTAGACAA AATTGCTTAA GGTTTTGCAC GAATAATGTA GAGTTAAATA 2 60 TTTTTTTATGT TAACTTAGGG ATTCCCTAAA GGCTGTTTAA TAATTTACTC AATAAAGAAA 2220 ATTTAATTGA GGTGGTTCTG TGCCCTTATA GATACCATCA CTTGCATATT GCAAATTGTA 2280 TCCAAAATTG GAAAGCTTTG AAATTTTTAA ATTATCCTCA GATTTACAGT CCATAGCTTC 2340 TGCATTATGT GTGTTAAAGA AATAATTCAA AATAACGTAA TGGAAATGTG TTTGCTTTTT 2400 AGACTATAAC CCACGAAGGC TGTGAAAAAG TAGTTGTTCA GAACAACCTT TGCTTTGGGA 2460 AATGCGGGTC TGTTCATTTT CCTGGAGCCG CGCAGCACTC CCATACCTCC TGCTCTCACT 2520 GTTTGCCTGC CAAGTTCACC ACGATGCACT TGCCACTGAA CTGCACTGAA CTTTCCTCCG 2530 TGATCAAGGT GGTGATGCTG GTGGAGGAGT GCCAGTGCAA GGTGAAGACG GAGCATGAAG 26.0 ATGGACACAT CCTACATGCT GGCTCCCAGG ATTCCTTTAT CCCAGGAGTT TCAGCTTGAA 2 OC GAGCTATCCC ACTATTACCT TTGAAAAGCA AAACCACAAC AGCAAAGATG CTGATTATTC 2 / 6Í AGTCTGAAAA TGTTAAGTGG GTACATAACA TTTTCAGGGA AAGGTGACTT GAAACGTAGT 2820 TTTAAATTAG AACGATAGAG GAAATGATAT TAGTCTAGTT ATTGGTACAC GTTTGAGACC 2830 TTGTCTCAGC TCTGCCACTA ACTAGCCGTA GAATGTTAAG TTGTAAAACC TTTCTCCATC 29.0 TAAAGATTTT CATCTATAAA TGACGGACCC GACCTAGATG ATTGCTAAAA TCCTTTCCAC 3000 TACTAATATT CCGTGATGCA TTTTCTCCAA GTTTGGGTAA AAGCCCTCCA TCTAAAGAAG 3060 GAAAAGAAAT AAGCGAGACC ATAAAAATGG GCTTCTTTAA TGTGTGTCAA ATCACCAGCA 3120 AGGAAAGAAG CAAGATAGAG AGGGAGGAAG GAAGGAAGGA AGGAAGGAAA GAAGGAAGGA 3180 AGGAAGGAAG GAAGGAAGGA AGGAAGGAAG GAAGGAAGGA AGGAAGGCAG GCAGGCAGGC 32 ^ 0 AGGCAGGCAG GCAGGCACGC AGGCAGGCAG GGAGGCAGGC TACGTGAAAT ATTTGTAGGA 3300 AAGATTCTCA TACTTATAGT TACTTTTGCA ACCCAAACAG TGTTTTACTT GACTTCTATC 3360 TGATGATTAA GTCTTTCCAC AGATGTAAGG AGTAACTTGC TTGGTTGCCT CCTTTTTATA 3420 AATACTCCTC ATATAAAGTA CTTAATGTCA GGTCTCTGAC TTTGAAGAAG GAACAGTGAT 2430 GTTAATTTTA GTAGTTTATA TAGGGAAGAG GAACAATCAC TGGTAGCCAA ACAAGTACCT 3540 ATATTATGAG GAAGGAAAAA TACATGACTA CTACCAGGTT TAGAGATCCG AATTC 3595 (2) INFORMATION FOR SEQ ID N0: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 804 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1.804 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: ATG CAT CTC CTC TTA TTT CAG CTG CTG GTA CTC CTG CCT CTA GGA AAG 48 Met His Leu Leu Leu Leu Phe Gln Leu Leu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 ACC ACA CGG CAC CAG GAT GGC CGC CAG AAT CAG AGT TCT CTT TCC CCC 96 Thr Thr Arg His Gln Asp Gly Arg Gln Asn Gln Ser Leu Ser Pro 20 25 30 GTA CTC CTG CCA AGG AAT CAA AGA GAG CTT CCC ACA GGC AAC CAT GAG 144 Val Leu Leu Pro Arg Asn Gln Arg Glu Leu Pro Thr Gly Asn His Glu 35 40 45 GAA GCT GAG GAG AAG CCA GAT CTG TTT GTC GCA GTG CCA CTC CTT GTA 192 Glu Wing Glu Glu Lys Pro Asp Leu Phe Val Wing Val Pro His Leu Val 50 55 60 GCC ACC AGC CCT GCA GGG GAA GGC CAG AGG CAG AGA GAG AAG ATG CTG 240 Wing Thr Ser Pro Wing Gly Glu Gly Gln Arg Gln Arg Glu Ly s Met Leu 65 70 75 80 TCC AGA TTT GGC AGG TTC TGG AAG AAG CCT GAG AGA GAA ATG CAT CCA 288 Ser Arg Phe Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro 85 90 95 TCC AGG GAC TCA GAT AGT GAG CCC TTC CCA CCT GGG ACC CAG TCC CTC 336 Ser Arg Asp Ser Asp Ser Glu Pro Pro Phe Pro Gly Thr Gln Ser Leu 100 105 110 ATC CAG CCG ATA GAT GGA ATG AAA ATG GAG AAA TCT CCT CTG CGG GAA 384 lie Gln Pro lie Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg Glu 115 120 125 GAA GCC AAG AAA TTC TGG CAC CAC TTC ATG TTC AGA AAA ACT CCG GCT 432 Glu Ala Lys Lys Phe Trp His His Phe Met Phe Arg Lys Thr Pro Wing 130 135 140 TCT CAG GGG GTC ATC TTG CCC ATC AAA AGC CAT GAA GTA CAT TGG GAG 480 Ser Gln Gly Val lie Leu Pro lie Lys Ser His Glu Val His Trp Glu 145 150 155 160 ACC TGC AGG ACA GTG CCC TTC AGC CAG ACT ATA ACC CAC GAA GGC TGT 528 ^^^ _ s_al-faith »Aa, _lalBat > -. - ^ ^ ^ ^, Thr Cys Arg Thr Val Pro Phe Ser Gln Thr lie Thr His Glu Gly Cys 165 170 175 GAA AAA GTA GTT GTT CAG AAC AAC CTT TGC TTT GGG AAA TGC GGG TCT 576 Glu Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser 180 185 190 GTT CAT TTT CCT GGA GCC GCG CAG CAC TCC CAT ACC TCC TGC TCT CAC 624 Val His Phe Pro Gly Ala Wing Gln His Ser His Thr Ser Cys Ser His 195 200 205 TGT TTG CCT GCC AAG TTC ACC ACG ATG CAC TTG CCA CTG AAC TGC ACT 672 Cys Leu Pro Ala Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr 210 215 220 GAA CTT TCC TCC GTG ATC AAG GTG GTG ATG CTG GTG GAG GAG TGC CAG 720 Glu Leu Ser Ser Val He Lys Val Val Met Leu Val Glu Glu Cys Gln 225 230 235 240 TGC AAG GTG AAG ACG GAG CAT GAA GAT GGA CAC ATC CTA CAT GCT GGC 768 Cys Lys Val Lys Thr Glu His Glu Asp Gly His He Leu His Wing Gly 245 250 255 TCC CAG GAT TCC TTT ATC CCA GGA GTT TCA GCT TGA S04 Ser Gln Asp Ser Phe He Pro Gly Val Ser Wing 260 265 (2) INFORMATIONFOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 268 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Met His Leu Leu Leu Phe Gln Leu Leu Val Leu Leu Pro Leu Gly Lys 1 5 10 15 Thr Thr Arg His Gln Asp Gly Arg Gln Asn Gln Ser Ser Leu Ser Pro 20 25 30 Val Leu Leu Pro Arg Asn Gln Arg Glu Leu Pro Thr Gly Asn His Glu 35 40 45 Glu Wing Glu Glu Lys Pro Asp Leu Phe Val Wing Val Pro His Leu Val 50 55 60 Wing Thr Ser Pro Wing Gly Glu Gly Gln Arg Gln Arg Glu Lys Met Leu 65 70 75 80 Be Arg Phe Gly Arg Phe Trp Lys Lys Pro Glu Arg Glu Met His Pro 85 90 95 Ser Arg Asp Ser Asp Ser Glu Pro Phe Pro Pro Gly Thr Gln Ser Leu 100 105 110 He Gln Pro He Asp Gly Met Lys Met Glu Lys Ser Pro Leu Arg Glu 115 120 125 Glu Ala Lys Lys Phe Trp His His Phe Met Phe Arg Lys Thr Pro Ala 130 135 140 Ser Gln Gly Val He Leu Pro He Lys Ser His Glu Val His Trp Glu 145 150 155 160 Thr Cys Arg Thr Val Pro Phe Ser Gln Thr He Thr His Glu Gly Cys 165 170 175 Glu Lys Val Val Val Gln Asn Asn Leu Cys Phe Gly Lys Cys Gly Ser 180 185 190 Val His Phe Pro Gly Ala Wing Gln His Ser His Thr Ser Cys Ser His 195 200 205 Cys Leu Pro Wing Lys Phe Thr Thr Met His Leu Pro Leu Asn Cys Thr 210 215 220 Glu Leu Ser Ser Val He Lys Val Val Met Leu Val Glu Glu Cys Gln 225 230 235 240 Cys Lys Val Lys Thr Glu His Glu Asp Gly His He Leu His Wing Gly 245 250 255 Ser Gln Asp Ser Phe He Pro Gly Val Ser Wing 260 265 . * xmimJm ttÉ¡iSB £ r * r. _ > > ^? -_MÉtti__MBtíta > «ALj» a-

Claims (25)

1. Novelty of the Invention 1. An isolated DNA sequence comprising a DNA sequence selected from the group consisting of: (a) nucleotides starting from nucleotides starting at # 1, 52, 55, 58, 61, 64, 67. 70. 73 , 121, 256, 259, 262, 265, 268, 271 or 484 and ending at # 723 or 801 of SEQ ID NO: 7; and (b) sequences that hybridize to (a) under stringent hybridization conditions and that code for a protein exhibiting cerberus activity.
2. 2. An isolated DNA sequence comprising a DNA sequence selected from the group consisting of: (a) nucleotides encoding amino acids starting with amino acids starting at # 1, 18 to 25, 41, 85 to 91 or 162 and ending in # 241 or 267 of SEQ ID NO: 8; and (b) sequences that hybridize to (a) under stringent hybridization conditions and that code for a protein exhibiting cerberus activity.
3. 3. A vector comprising a DNA molecule of claim 1 in operative association with an expression control sequence for this.
4. 4. A vector comprising a DNA molecule of claim 2 in operative association with an expression control sequence for this.
5. 5. A host cell transformed with the vector of claim 3.
6. 6. A host cell transformed with the vector of claim 4.
7. 7. An isolated DNA molecule comprising the DNA sequence selected from the group consisting of: (a) nucleotides # 268 to # 801 of SEQ ID NO: 2; and (b) allelic sequences that occur naturally and equivalent sequences of (a) degenerative codon.
8. 8. A vector comprising a DNA molecule of claim 7 in operative association with an expression control sequence for this.
9. 9. A host cell transformed with the vector of claim 8. 1 (0).
10. An isolated DNA molecule that encodes a mammalian cerberits protein, said DNA molecule comprising nucleotides # 268 to # 801 of SEQ ID NO: 7.
11. 11. An isolated DNA molecule according to claim 10. further comprising a nucleotide sequence encoding a 5 'propeptide suitable for and bound in frame to the DNA coding sequence. .
12. 12. A vector comprising a DNA molecule of claim 11 in operative association with an expression control sequence for this.
13. 13. A host cell transformed with the vector of claim 12.
14. 14. A method for producing purified mammalian cerberus protein, said method comprising the steps of: (a) culturing a transformed host cell with a DNA sequence in accordance with claim 1, comprising a sequence of nucleotides that encode a mammalian cerberus protein; and (b) recovering and purifying said mammalian cerberus protein from the culture medium.
15. 15. A method for producing purified mammalian cerberus protein, said method comprising the steps of: (a) culturing a transformed host cell with a DNA sequence according to claim 2, comprising a sequence of nucleotides that encode a protein cei 'mammalian berus; and (b) recovering and purifying said mammalian cerberus protein from the culture medium.
16. 16. A method for producing purified mammalian cei'berus protein, said method comprising the steps of: (a) culturing a transformed host cell with a DNA sequence according to claim 10, comprising a nucleotide sequence coding for a mammalian cerberus protein; and (b) recovering and purifying said mammalian cerberus protein from the culture medium.
17. 17. A purified mammalian cerberus polypeptide comprising an amino acid sequence according to SEQ ID NO: 8.
18. 18. A purified mammalian cerberus protein produced by the steps of: (a) culturing a transformed cell with a DNA comprising the nucleotide sequence of nucleotide # 268 to # 801 as shown in SEQ ID NO: 7; and (b) recovering and purifying from the culture medium a protein comprising the amino acid sequence of amino acid # 90 to amino acid # 267 as shown in SEQ ID NO: 8.
19. 19. A composition comprising a therapeutic amount of at least one mammalian cerberus polypeptide according to claim 18. 2®.
20. A method for altering the regulation of neuronal genes in a patient in need thereof, comprising administering to said patient, an effective amount of the composition of claim 19.
21. 21. A purified mammalian cerberus protein comprising the sequence of amino acids of amino acid # 1 to # 267 of SEQ ID NO: 8.
22. 22. Antibodies to a purified mammalian cerberus protein according to claim 21.
23. 23. A purified mammalian cerberus protein comprising the sequence of amino acids of amino acid # 90 to # 267 of SEQ ID NO: 2.
24. 24. Antibodies to a purified mammalian cerberits protein according to claim 23.
25. 25. A purified mammalian cerberus protein produced by the steps of (a) culturing a transformed cell with a DNA comprising the nucleotide sequence of nucleotide # 52 to # 801 as shown in SEQ ID NO: 7; and (b) recovering and purifying from the culture medium a protein comprising the amino acid sequence of amino acid # 18 to amino acid # 267 as shown in SEQ ID NO: 8. esnmen de la Bescripción Protected mammalian cerberus proteins and processes to produce them are described. DNA molecules encoding mammalian cerberus proteins are also described. The proteins can be used to induce the formation, growth, proliferation, differentiation and / or maintenance of neurons and / or related neural cells and tissues, such as Schwann cells, glial cells and astrocytes, and for the repair of other tissues, including cardiac and endoderm. M-l M 7¿? ^ r ^ ¿^ ^ ^ ^ ÍH *. "? 7ít l ~ .. < , ~