US20070117098A1

US20070117098A1 - 'signalin' family of TGFbeta signal transduction proteins and uses related thereto

Info

Publication number: US20070117098A1
Application number: US11/251,061
Authority: US
Inventors: Jonathan Graff; Tod Woolf; Ping Jin; Douglas Melton
Original assignee: Harvard College; Curis Inc
Current assignee: Harvard College; Curis Inc
Priority date: 1995-12-20
Filing date: 2005-10-14
Publication date: 2007-05-24
Also published as: CA2239126A1; IL124860A0; KR20000064501A; US7034114B2; NZ326596A; DE868513T1; AU1520497A; AU726918B2; US20020146773A1; WO1997022697A1; EP0868513A1; US6428977B1; JP2000506373A

Abstract

The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here signalin-related genes, which are involved in a signal transduction induced by members of the TGFβ superfamily. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 10/095,492, filed Mar. 12, 2002, which is a continuation of U.S. application Ser. No. 08/580,031, filed Dec. 20, 1995 (U.S. Pat. No. 6,428,977). The foregoing applications are hereby incorporated by reference in their entirety.

FUNDING

Work described herein was supported by funding from the National Institutes of Health. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68:185-199; Jessell, T. M. et al., (1992) Cell 68:257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
Several classes of secreted polypeptides are known to mediate the cell-cell signaling that determines tissue fate during development. An important group of these signaling proteins are the TGFβ superfamily of molecules, which have wide range of functions in many different species. Members of the family are initially synthesized as larger precursor molecules with an amino-terminal signal sequence and a pro-domain of varying size (Kingsley, D. M. (1994) Genes Dev. 8:133-146). The precursor is then cleaved to release a mature carboxy-terminal segment of 110-140 amino acids. The active signaling moiety is comprised of hetero- or homodimers of the carboxy-terminal segment (Massague, J. (1990) Annu. Rev. Cell Biol. 6:597-641). The active form of the molecule then interacts with its receptor, which for this family of molecules is composed of two distantly related transmembrane serine/threonine kinases called type I and type II receptors (Massague, J. et al. (1992) Cell 69:1067-1070; Miyazono, K. A. et al. EMBO J. 10:1091-1101). TGFβ binds directly to the type II receptor, which then recruits the type I receptor and modifies it by phosphorylation. The type I receptor then transduces the signal to downstream components, which are as yet unidentified (Wrana et al, (1994) Nature 370:341-347).
Several members of the TGFβ superfamily have been identified which play salient roles during vertebrate development. Dorsalin is expressed preferentially in the dorsal side of the developing chick neural tube (Basler et al. (1993) Cell 73:687-702). It promotes the outgrowth of neural crest cells and inhibits the formation of motor neuron cells in vitro, suggesting that it plays an important role in neural patterning along the dorsoventral axis. Certain of the bone morphogenetic proteins (BMPs) can induce the formation of ectopic bone and cartilage when implanted under the skin or into muscles (Wozney, J. M. et al. (1988) Science 242:1528-1534). In mice, mutations in BMP5 have been found to result in effects on many different skeletal elements, including reduced external ear size and decreased repair of bone fractures in adults (Kingsley (1994) Genes Dev. 8:133-146). Besides these effects on bone tissue, BMPs play other roles during normal development. For example, they are expressed in non skeletal tissues (Lyons et al. (1990) Development 109:833-844), and injections of BMP4 into developing Xenopus embryos promote the formation of ventral/posterior mesoderm (Dale et al (1992) Development 115:573-585). Furthermore, mice with mutations in BMP5 have an increased frequency of different soft tissue abnormalities in addition to the skeletal abnormalities described above (Green, M. C. (1958) J. Exp. Zool. 137:75-88).
Members of the activin subfamily have been found to be important in mesoderm induction during Xenopus development (Green and Smith (1990) Nature 47:391-394; Thomsen et al. (1990) Cell 63:485-493) and inhibins were initially described as gonadal inhibitors of follicle-stimulating hormone from pituitary cells. In addition, antagonists of this signaling pathway can be used to convert embryonic tissue into ectoderm, the default pathway of development in the absence of TGFβ-mediated signals. BMP-4 and activin have been found to be potent inhibitors of neuralization (Wilson, P. A. and Hemmati-Brivanlou, A (1995) Nature 376:331-333).
Further evidence for the importance of a TGFβ family member in early vertebrate development comes from a retroviral insertion in the mouse nodal gene. This insertion leads to a failure to form the primitive streak in early embryogenesis, a lack of axial mesoderm tissue, and an overproduction of ectoderm and extraembryonic ectoderm (Conlon et al. (1991) Development 111:969-981; Iannaccone et al (1992) Dev. Dynamics 194:198-208). The predicted nodal gene product is consistent with previous studies showing that nodal is related to activins and BMPs (Zhou et al. (1993) Nature 361:543-547). A role for TGFβ family members in the development of sex organs has also been described; Mullerian inhibitory substance functions during vertebrate male sexual development to cause regression of the embryonic duct system that develops into oviducts and uterus (Lee and Donahoe (1993) Endocrinol. Rev. 14:152-164).
Members of this family of signaling molecules also continue to function post-development. TGFβ has antiproliferative effects on many cell types including epithelial cells, endothelial cells, smooth muscle cells, fetal hepatocytes, and myeloid, erythroid, and lymphoid cells. Animals which cannot produce TGFβ1 (homozygous for null mutations in the TGFβ1 gene) have been found to survive until birth with no apparent morphological abnormalities (Shull et al. (1992) Nature 359:693-699; Kulkami et al. (1993) Proc. Natl. Acac. Sci. 90:770-774). The animals do die around weaning age, however, owing to massive immune infiltration in may different organs. These data are consistent with the inhibitory effects of TGFβ on lymphocyte growth (Tada et al. (1991) J. Immunol 146:1077-1082). In another system, the expression of a TGFβ transgene in the mammary tissue of mice has been shown to inhibit the development and secretory function of mammary tissue during sexual maturation and pregnancy (Jhappan, C. et al. (1993) EMBO J. 12:1835-1845; Pierce, D. F. et al. (1993) Genes Dev. 7:2308-2317). In addition to these inhibitory effects, TGFβ can also promote the growth of other cell types as evidenced by its role in neovascularization and the proliferation of connective tissue cells. Because of these activities, it plays a key role in wound healing (Kovacs, E. J. (1991) Immunol Today 12:17-23)

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a novel family of genes, and gene products, expressed in vertebrate organisms, which genes referred to hereinafter as the “signalin” gene family, the products of which are referred to as signalin proteins. The products of the signalin gene have apparent broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, and can be used or manipulated to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.
In general, the invention features isolated vertebrate signalin polypeptides, preferably substantially pure preparations of one or more of the subject signalin polypeptides. The invention also provides recombinantly produced signalin polypeptides. In preferred embodiments the polypeptide has a biological activity including: an ability to modulate proliferation, survival and/or differentiation of mesodermally-derived tissue, such as tissue derived from dorsal mesoderm; the ability to modulate proliferation, survival and/or differentiation of ectodermally-derived tissue, such as tissue derived from the neural tube, neural crest, or head mesenchyme; the ability to modulate proliferation, survival and/or differentiation of endodermally-derived tissue, such as tissue derived from the primitive gut. Moreover, in preferred embodiments, the subject signalin proteins have the ability to modulate intracellular signal transduction pathways mediated by receptors for members of the Transforming Growth Factor β superfamily of molecules.
In one embodiment, the polypeptide is identical with or homologous to a signalin protein. Exemplary signalin proteins are represented by SEQ ID No. 14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. Related members of the vertebrate signalin family are also contemplated, for instance, a signalin polypeptide preferably has an amino acid sequence at least 60% homologous to a polypeptide represented by any of SEQ ID Nos:14-26, though polypeptides with higher sequence homologies of, for example, 70, 80%, 90% or are also contemplated. The signalin polypeptide can comprise a full length protein, such as represented in the sequence listings, or it can comprise a fragment corresponding to particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or 200 amino acids in length. In preferred embodiments, the polypeptide, or fragment thereof, specifically modulates, by acting as either an agonist or antagonist, the signal transduction activity of a receptor for a transforming growth factor β.
In certain preferred embodiments, the invention features a purified or recombinant signalin polypeptide having a molecular weight in the range of 45 kd to 70 kd. For instance, preferred signalin polypeptide chains of the α and β subfamilies, described infra, have molecular weights in the range of 45 kd to about 55 kd, even more preferably, in the range of 50-55 kd. In another illustrative example, preferred signalin polypeptide chains of the γ subfamily have molecular weights in the range of 60 kd to about 70 kd, even more preferably in the range of 63-68 kd. It will be understood that certain post-translational modifications, e.g., phosphorylation and the like, can increase the apparent molecular weight of the signalin protein relative to the unmodified polypeptide chain.
In another embodiment, the signalin polypeptide comprises a signalin motif represented in the general formula shown in SEQ ID No:28. In a preferred embodiment the signalin motif corresponds to a signalin motif represented in one of SEQ ID Nos:14-26. In another embodiment, the signalin polypeptide of the invention comprises a ν domain represented in the general formula SEQ ID No:27. In a preferred embodiment the ν region corresponds to a ν domain represented in one of SEQ ID Nos:14-26. In another preferred embodiment, the signalin polypeptide of the invention comprises a χ domain represented in the general formula SEQ ID No:29. In a further preferred embodiment the χ region corresponds to a χ domain represented in one of SEQ ID Nos:14-26. In another preferred embodiment, the signalin polypeptide can modulate, either stimulate or antagonize, intracellular pathways mediated by a receptor for a TGFβ. In still another embodiment, the polypeptide comprises an amino acid sequence represented in the general formula: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV. In still a further embodiment, the signalin polypeptide of the present invention comprises an amino acid sequence represented by the general formula: LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV. In an additional embodiment, the signalin polypeptide of the present invention comprises an amino acid sequence represented by the general formula: LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
In another preferred embodiment, the invention features a purified or recombinant polypeptide fragment of a signalin protein, which polypeptide has the ability to modulate, e.g., mimic or antagonize, a the activity of a wild-type signalin protein. Preferably, the polypeptide fragment comprises a signalin motif.
Moreover, as described below, the preferred signalin polypeptide can be either an agonist (e.g. mimics), or alternatively, an antagonist of a biological activity of a naturally occurring form of the protein, e.g., the polypeptide is able to modulate differentiation and/or growth and/or survival of a cell responsive to authentic signalin proteins. Homologs of the subject signalin proteins include versions of the protein which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which inactivate an enzymatic activity associated with the protein.
The subject proteins can also be provided as chimeric molecules, such as in the form of fusion proteins. For instance, the signalin protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the signalin polypeptide, e.g. the second polypeptide portion is glutathione-S-transferase, e.g. the second polypeptide portion is an enzymatic activity such as alkaline phosphatase, e.g. the second polypeptide portion is an epitope tag.
In a preferred embodiment the signalin polypeptide of the present invention modulates signal transduction from a TGFβ receptor. For example, the signalin polypeptide may modulate the transduction of a TGFβ receptor for a member of the dpp family, e.g., dpp, BMP2, or BMP4. In another preferred embodiment, the signalin polypeptide modulates the signaling of a TGFβ other than a dpp family member. For instance, the signalin polypeptide may be involved in signalling from one or more of BMP5, BMP6 BMP7, BMP8, 60A, GDF5, GDF6, GDF7, GDF1, Vg1, dorsalin, BMP3, GDF10, nodal, inhibins, activins, TGFβ1, TGFβ2, TGFβ3, MIS, GDF9 or GDNE.
In yet another embodiment, the invention features a nucleic acid encoding a signalin polypeptide, or polypeptide homologous thereto, which polypeptide has the ability to modulate, e.g., either mimic or antagonize, at least a portion of the activity of a wild-type signalin polypeptide. Exemplary signalin polypeptides are represented by SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. In another embodiment the nucleic acid of the present invention hybridizes under stringent conditions with one or more of the nucleic acid sequences in SEQ ID No:1-13. In preferred embodiments, the nucleic acid encodes a polypeptide which specifically modulates, by acting as either an agonist or antagonist, the signal transduction activity of a receptor for a transforming growth factor β.
In another embodiment, the nucleic acid encodes an amino acid sequence which comprises a signalin motif represented in the general formula shown in SEQ ID No:28. In preferred embodiment the signalin motif corresponds to a signalin motif represented in one of SEQ ID Nos:14-26. In another embodiment, the nucleic acid of the invention encodes an amino acid sequence which comprises a ν domain represented in the general formula SEQ ID No:27. In a preferred embodiment the encoded ν region corresponds to a ν domain represented in one of SEQ ID Nos:14-26. In another embodiment, the nucleic acid encodes a signalin polypeptide of the invention which comprises a χ domain represented in the general formula SEQ ID No:29. In a preferred embodiment the encoded χ region corresponds to a χ domain represented in one of SEQ ID Nos:14-26. In still a another embodiment, the nucleic acid sequence encodes a polypeptide which comprises an amino acid sequence represented in the general formula: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV. In another embodiment, the nucleic acid of the present invention encodes a polypeptide which comprises an amino acid sequence represented by the general formula, LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV. In an still another embodiment, the nucleic acid encodes a polypeptide which comprises an amino acid sequence represented by the general formula, LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
Another aspect of the present invention provides an isolated nucleic acid having a nucleotide sequence which encodes a signalin polypeptide. In preferred embodiments, the encoded polypeptide specifically mimics or antagonizes inductive events mediated by wildtype signalin proteins. The coding sequence of the nucleic acid can comprise a sequence which is identical to a coding sequence represented in one of SEQ ID Nos:1-13, or it can merely be homologous to one or more of those sequences.
Furthermore, in certain preferred embodiments, the subject signalin nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, which regulatory sequence is operably linked to the signalin gene sequence. Such regulatory sequences can be used in to render the signalin gene sequence suitable for use as an expression vector. This invention also contemplates the cells transfected with said expression vector whether prokaryotic or eukaryotic and a method for producing signalin proteins by employing said expression vectors.
In yet another embodiment, the nucleic acid hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of either sense or antisense sequence of one or more of SEQ ID Nos:1-13; though preferably to at least 25 consecutive nucleotides; and more preferably to at least 40, 50 or 75 consecutive nucleotides of either sense or antisense sequence of one or more of SEQ ID Nos:1-13.
Yet another aspect of the present invention concerns an immunogen comprising a signalin polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a signalin polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g. a unique determinant, from a protein represented by one of SEQ ID Nos. 14-26.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the signalin immunogen.
The invention also features transgenic non-human animals, e.g. mice, rats, rabbits, chickens, frogs or pigs, having a transgene, e.g., animals which include (and preferably express) a heterologous form of a signalin gene described herein, or which misexpress an endogenous signalin gene, e.g., an animal in which expression of one or more of the subject signalin proteins is disrupted. Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed signalin alleles or for use in drug screening.
The invention also provides a probe/primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide comprises a region of nucleotide sequence which hybridizes under stringent conditions to at least 12 consecutive nucleotides of sense or antisense sequence of SEQ ID No:1-13, or naturally occurring mutants thereof. Nucleic acid probes which are specific for each of the classes of vertebrate signalin proteins are contemplated by the present invention, e.g. probes which can discern between nucleic acid encoding an α, β, or γ signalin. In preferred embodiments, the probe/primer further includes a label group attached thereto and able to be detected. The label group can be selected, e.g., from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors. Probes of the invention can be used as a part of a diagnostic test kit for identifying dysfunctions associated with mis-expression of a signalin protein, such as for detecting in a sample of cells isolated from a patient, a level of a nucleic acid encoding a subject signalin protein; e.g. measuring a signalin mRNA level in a cell, or determining whether a genomic signalin gene has been mutated or deleted. These so called “probes/primers” of the invention can also be used as a part of “antisense” therapy which refers to administration or in situ generation of oligonucleotide probes or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject signalin proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation. Preferably, the oligonucleotide is at least 12 nucleotides in length, though primers of 25, 40, 50, or 75 nucleotides in length are also contemplated.
In yet another aspect, the invention provides an assay for screening test compounds for inhibitors, or alternatively, potentiators, of an interaction between a signalin protein and a signalin binding protein or nucleic acid sequence. An exemplary method includes the steps of (i) combining a signalin polypeptide or fragment thereof, a signalin binding element, and a test compound, e.g., under conditions wherein, but for the test compound, the signalin protein and binding element are able to interact; and (ii) detecting the formation of a complex which includes the signalin protein and the binding element either by directly quantitating the complex or by measuring inductive effects of the signalin protein. A statistically significant change, such as a decrease, in the formation of the complex in the presence of a test compound (relative to what is seen in the absence of the test compound) is indicative of a modulation, e.g., inhibition, of the interaction between the signalin protein and its binding element.
Yet another aspect of the present invention concerns a method for modulating one or more of growth, differentiation, or survival of a mammalian cell responsive to signalin induction. In general, whether carries out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a signalin polypeptide so as to alter, relative to the cell in the absence of signalin treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell. Accordingly, the method can be carried out with polypeptides mimics the effects of a naturally-occurring signalin protein on the cell, as well as with polypeptides which antagonize the effects of a naturally-occurring signalin protein on said cell. In preferred embodiments, the signalin polypeptide provided in the subject method are derived from vertebrate sources, e.g., are vertebrate signalin polypeptides. For instance, preferred polypeptides includes an amino acid sequence identical or homologous to an amino acid sequence (e.g., including bioactive fragments) designated in one of SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, or SEQ ID No:12, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26. Furthermore, the present invention contemplates the use of other metazoan (e.g., invertebrate) homologs of the signalin polypeptides or bioactive fragments thereof equivalent to the subject vertebrate fragments.
In one embodiment, the subject method includes the treatment of testicular cells, so as modulate spermatogenesis. In another embodiment, the subject method is used to modulate osteogenesis, comprising the treatment of osteogenic cells with a signalin polypeptide. Likewise, where the treated cell is a chondrogenic cell, the present method is used to modulate chondrogenesis. In still another embodiment, signalin polypeptides can be used to modulate the differentiation of neural cells, e.g., the method can be used to cause differentiation of a neuronal cell, to maintain a neuronal cell in a differentiated state, and/or to enhance the survival of a neuronal cell, e.g., to prevent apoptosis or other forms of cell death. For instance, the present method can be used to affect the differentiation of such neuronal cells as motor neurons, cholinergic neurons, dopanergic neurons, serotenergic neurons, and peptidergic neurons.
The present method is applicable, for example, to cell culture technique, such as in the culturing of neural and other cells whose survival or differentiative state is dependent on signalin function. Moreover, signalin agonists and antagonists can be used for therapeutic intervention, such as to enhance survival and maintenance of neurons and other neural cells in both the central nervous system and the peripheral nervous system, as well as to influence other vertebrate organogenic pathways, such as other ectodermal patterning, as well as certain mesodermal and endodermal differentiation processes. In an exemplary embodiment, the method is practiced for modulating, in an animal, cell growth, cell differentiation or cell survival, and comprises administering a therapeutically effective amount of a signalin polypeptide to alter, relative the absence of signalin treatment, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of one or more cell-types in the animal.
Another aspect of the present invention provides a method of determining if a subject, e.g. a human patient, is at risk for a disorder characterized by unwanted cell proliferation or aberrant control of differentiation. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a signalin protein, e.g. represented in one of SEQ ID Nos:14-26, or a homolog thereof; or (ii) the mis-expression of a signalin gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a signalin gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of a signalin gene, e.g. a nucleic acid represented in one of SEQ ID Nos: 1-13, or naturally occurring mutants thereof, or 5′ or 3′ flanking sequences naturally associated with the signalin gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the signalin gene and, optionally, of the flanking nucleic acid sequences. For instance, the probe/primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a signalin protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the signalin protein.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the model system used to test the biological activities of the signalin proteins described in the present invention.
FIG. 2 shows the morphology of animal cap explants from control, signalin1 injected, or signalin2 injected embryos.
FIG. 3 illustrate the histologic analysis of animal cap explants from control, signalin1 injected, or signalin2 injected embryos.
FIG. 4 is an autoradiogram which shows the expression of various marker RNAs in the injected embryos as detected by polymerase chain reaction. Brachyury is a general mesodermal marker; Goosecoid is a marker of dorsal mesoderm; Xwnt-8 is a marker of ventral-lateral mesoderm; globin is a marker of ventral mesoderm; actin is a marker of dorsal mesoderm; NCAM is a marker of neural tissue; and EF-1α is ubiquitously expressed and serves as a control for the amount of RNA included in each reaction. The lane marked “E” contains total RNA harvested from whole embryos and is a positive control. The lane marked “−RT” is identical to the positive control lane, except that reverse transcriptase was not included and serves as a negative control. The lanes designated “S1” and “S2” correspond to samples from embryos injected with xe-signalin1 and xe-signalin 2, respectively.
FIG. 5 is a matrix illustrating a possible grouping of the signalin family into at least three different sub-families. Blacked-out boxes represent >10 mismatches over the signalin motif.
FIG. 6 depicts an alignment of a portion of the signalins identified in both humans and Xenopus.

DETAILED DESCRIPTION OF THE INVENTION

Of particular importance in the development and maintenance of tissue in vertebrate animals is a type of extracellular communication called induction, which occurs between neighboring cell layers and tissues (Saxen et al. (1989) Int J Dev Biol 33: 21-48; and Gurdon et al. (1987) Development 99:285-306). In inductive interactions, chemical signals secreted by one cell population influence the developmental fate of a second cell population. Typically, cells responding to the inductive signals are diverted from one cell fate to another, neither of which is the same as the fate of the signaling cells. Inductive signals are transmitted by key regulatory proteins that function during development to determine tissue patterning. For example, signals mediated by the TGFβ superfamily have been shown to play a variety of roles, including participating in vertebrate tissue induction.
The present invention concerns the discovery of a family of vertebrate genes, referred to herein as “signalins”, which function in intracellular signal transduction pathways initiated by members of the TGβ-superfamily, and have a role in determining tissue fate and maintenance. For instance, the results provided below indicate that proteins encoded by the vertebrate signalin genes may participate in the control of development and maintenance of a variety of embryonic and adult tissues. For example, during embryonic induction, certain of the signalins are implicated in the differentiation and patterning of both dorsal and ventral mesoderm.

The family of vertebrate signalin genes or gene products provided by the present invention apparently consists of at least seven different members which can be grouped into at least three different subclasses within the signalin family. The vertebrate signalins are related, apparently both in sequence and function, to the drosophila and C. elegans MAD genes (Sekelsky et al. (1995) Genetics 139:1347). The cDNAs corresponding to vertebrate signalin gene transcripts were initially cloned from Xenopus and are, arbitrarily, designed as Xe-signalin 1-4. As described in the appended examples, degenerate primers from the cloning of the Xenopus signalins were also used to clone human homologs of this gene family. As a result, cDNA's for at least seven different human signalin transcripts have been identified, and are designated herein, again arbitrarily, as Hu-signalin 1-7. Provided in Table 1 below is a guide to the designated SEQ ID numbers for the nucleotide and amino acid sequences for each signalin clone.

TABLE 1


Guide to signalin sequences in Sequence Listing

	Nucleotide	Amino Acid

Xe- signalin 1	SEQ ID No. 1	SEQ ID No. 14

Xe- signalin 2	SEQ ID No. 2	SEQ ID No. 15

Xe- signalin 3	SEQ ID No. 3	SEQ ID No. 16

Xe- signalin 4	SEQ ID No. 4	SEQ ID No. 17

Hu- signalin 1	SEQ ID No. 5	SEQ ID No. 18

Hu- signalin 2	SEQ ID No. 6	SEQ ID No. 19

Hu- signalin 3	SEQ ID No. 7	SEQ ID No. 20

Hu- signalin 4	SEQ ID No. 8	SEQ ID No. 21

Hu- signalin 5	SEQ ID No. 9	SEQ ID No. 22

Hu- signalin 6	SEQ ID No. 10	SEQ ID No. 23

Hu- signalin 7	SEQ ID No. 11	SEQ ID No. 24

From the apparent molecular weights, the family of vertebrate signalin proteins apparently ranges in size from about 45 kd to about 70 kd for the unmodified polypeptide chain. For instance, Xe- signalin 1 and 3 have apparent molecular weights of about 52.2 kd, Xe-signalin 2 has an apparent molecular weight of about 52.4 kd, and Xe-signalin 4 has an apparent molecular weight of about 64.9 kd.
Analysis of the vertebrate signalin sequences revealed no obvious similarities with any previously identified domains or motifs. However, the fact that each full-length clone lacks a signal sequence, along with the observation that signalin proteins can be detected in both the nucleus and the cytoplasm, indicates that the vertebrate signalin genes encode intracellular proteins.
The above notwithstanding, careful inspection of the clones suggests at least two novel domains, one or both of which may be characteristic of the vertebrate signalin family. The first apparently conserved structural element of the signalin family occurs in the N-terminal portion of the molecule, and is designated herein as the “ν domain”. With reference to xe-signalin-1, the ν domain corresponds to amino acid residues Leu37-Val130. By alignment of the vertebrate signalin clones, the element is represented by the consensus sequence: LVKKLK-X(1)-CVTI-X(2)-RXLDGRLQVXXRKGXPHVIYXRWXWPDL-X(3)-VCXNPYHYXRV (SEQ ID No. 27), wherein X(1) represents from about 17-25 residues, X(2) represents from about 1-35 residues, and X(3) represents about 20-25 residues, and each of the other X's represent any single amino acid, though more preferably represent an amino acid residue in the corresponding vertebrate signalin sequences of the appended sequence listing.
Within the ν domain, there is a motif which is highly conserved not only amongst the vertebrate signaling, but also amongst the related drosophila and C. elegans MAD polypeptides. In particular, this motif (referred to herein as a “signalin-motif”) includes the consensus sequence LDGRLQVXXRKGXPHVIYXRWXWPDL (SEQ ID No. 28). Again, each occurrence of X independently represent any single amino acid, though more preferably represent an amino acid residue in the corresponding vertebrate signalin sequences of the appended sequence listing.
Another apparent motif occurs in the C-terminal portion of the signalin family. Referred to herein as the “χ motif”, it corresponds to amino acid residues Leu405-Leu450 of xe-signalin-1. Again, by alignment of the vertebrate clones presently sequenced, the χ motif can be represented by the consensus sequence LXXXCXXRXSFVKGWGXXXXRQXXXX-TPCWIEXHLXXXLQXLDXVL (SEQ ID No. 29), wherein each occurrence of X independently represent any single amino acid, though more preferably represent an amino acid residue in the corresponding vertebrate signalin sequences of the appended sequence listing.
Not wishing to be bound by any particular theory, analysis of one of the apparently conserved motifs (the signalin motif) suggests that the signalin protein family can be grouped into at least three different sub-families. As FIGS. 5 and 6 illustrate, xe-signalins 1 and 3 and hu- signalins 1, 3 and 7 apparently form one sub-family of signalins (the “α-subfamily” or “α-signalins”). Likewise, xe-signalin 4 and hu-signalins 4 and 2 form a second apparent sub-family (the β-subfamily” or “β-signalins”), and xe-signalin 2 and hu-signalins 5 and 6 form a third sub-family (the “γ-subfamily” or “γ-signalins”). Comparison of the amino acid sequence around the signalin motif amongst members of the α-subfamily demonstrates a consensus sequence for a signalin motif represented by

Claims

1-71. (canceled)

72. A method for modulating one or more of growth, differentiation, or survival of a mammalian cell responsive to signalin-mediated induction, comprising treating the cell with an effective amount of an agent which modulates the signal transduction activity of a signalin polypeptide thereby altering, relative to the cell in the absence of the agent, at least one of (i) rate of growth, (ii) differentiation, or (iii) survival of the cell.

73. The method of claim 72, wherein said agent mimics the effects of a naturally-occurring signalin protein on said cell.

74. The method of claim 72, wherein said agent antagonizes the effects of a naturally-occurring signalin protein on said cell.

75. The method of claim 72, wherein the cell is a testicular cell and the agent modulates spermatogenesis.

76. The method of claim 72, wherein the cell is an osteogenic cell, and the agent modulates osteogenesis.

77. The method of claim 72, wherein the cell is a chondrogenic cell, and the agent modulates chondrogenesis.

78. The method of claim 72, wherein the agent modulates the differentiation of neuronal cells.

79-88. (canceled)

89. A diagnostic assay for identifying a cell or cells at risk for a disorder characterized by unwanted cell proliferation or differentiation, comprising detecting, in a cell sample, the presence or absence of a genetic lesion characterized by at least one of (i) aberrant modification or mutation of a gene encoding a signalin protein, and (ii) mis-expression of said gene; wherein a wild-type form of said gene encodes a signalin protein characterized by an ability to modulate the signal transduction activity of a TGFβ receptor.

90. The assay of claim 89, wherein detecting said lesion includes:

i. providing a diagonistic probe comprising a nucleic acid including a region of nucleotide sequence which hybridizes to a sense or antisense sequence of said gene, or naturally occurring mutants thereof, or 5′ or 3′ flanking sequences aurally associated with said gene;

ii. combining said probe with nucleic acid of said cell sample; and

ii. detecting, by hybridization of said probe to said cellular nucleic acid, the existence of at least one of a deletion of one or more nucleotides from said gene, an addition of one or more nucleotides to said gene, a substitution of one or more nucleotides of said gene, a gross chromosomal rearrangement of all or a potion of said gene, a gross alteration in the level of an mRNA transcript of said gene, or a non-wild type splicing pattern of an mRNA transcript of said gene.

91. The assay of claim 90, wherein hybridization of said probe further comprises subjecting the probe and cellular nucleic acid to a polymerase chain reaction (PCR) and detecting abnormalities in an amplified product.

92. The assay of claim 90, wherein hybridization of said probe further comprises subjecting the probe and cellular nucleic acid to a ligation chain notion (LCR) and detecting abnormalities in an amplified product.

93. The assay of claim 90, wherein said probe hybridizes under stringent conditions to a nucleic acid designated by one or more of SEQ ID Nos. 1-13.

94. A method for screening for compounds that modulate an activity of a signalin polypeptide, comprising

providing a signalin polypeptide and a molecule;

contacting said signalin polypeptide and said molecule with a compound; and

assaying binding of said signalin polypeptide to said molecule in the presence versus the absence of said compound, thereby identifying a compound that modulates the activity of a signalin polypeptide.

95. The method of claim 94, wherein the activity of a signalin polypeptide comprises modulating signaling via the TGFβ signaling pathway.

96. The method of claim 95, wherein the compound is an antagonist that inhibits signaling via the TGFβ signaling pathway.

97. The method of claim 95, wherein the compound is an agonist that promotes signaling via the TGFβ signaling pathway.

98. The method of claim 94, wherein the molecule is a protein.

99. The method of claim 94, wherein the molecule is a nucleic acid.

100. The method of claim 94, wherein the compound promotes binding of the signalin polypeptide to the molecule.

101. The method of claim 94, wherein the compound inhibits binding of the signalin polypeptide to the molecule.

102. A method for screening for compounds that modulate an activity of a signalin polypeptide, comprising

providing a cell that expresses a signalin polypeptide, which cell is responsive to TGFβ signaling;

contacting said cell with a compound; and

assaying signalin inductive responses in the cell in the presence versus the absence of said compound, thereby identifying a compound that modulates the activity of a signalin polypeptide.

103. The method of claim 102, wherein assaying signalin inductive responses in the cell comprises assaying cell proliferation or cell differentiation.

104. The method of claim 102, wherein assaying signalin inductive responses comprises assaying signaling via the TGFβ signaling pathway.

105. The method of claim 102, wherein assaying signalin inductive responses comprises measuring expression of genes which are regulated by the TGFβ signaling pathway.

106. The method of claim 102, wherein the activity of a signalin polypeptide comprises modulating signaling via the TGFβ signaling pathway.

107. The method of claim 106, wherein the compound is an antagonist that inhibits signaling via the TGFβ signaling pathway.

108. The method of claim 106, wherein the compound is an agonist that promotes signaling via the TGFβ signaling pathway.

109. The method of claim 102, wherein the cell expresses a recombinant signalin polypeptide.