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Link:  Pharm/Biotech Resources


Title:  Neurogenesis inducing genes

United States Patent:  6,921,648

Issued:  July 26, 2005

Inventors:  Mikoshiba; Katsuhiko (Wako, JP); Aruga; Jun (Tsukuba, JP); Nagai; Takeharu (Tokyo, JP); Nakata; Katsunori (Tsuchiura, JP)

Assignee:  The Institute of Physical and Chemical Research (JP)

Appl. No.:  244367

Filed:  September 16, 2002

Abstract

The present invention relates to neurogenesis inducing genes. In particular, the present invention provides neurogenesis inducing genes coding for Zic proteins, vectors containing such genes, host cells containing such vectors, proteins produced by such host cells, antibodies raised to such proteins, and therapeutic agents or agents for gene therapy for nervous diseases.

SUMMARY OF THE INVENTION

The present invention provides neurogenesis inducing proteins, genes coding for the proteins; recombinant vectors comprising the genes, transformants comprising the vectors, an antibodies against the proteins; and therapeutic agents for nervous diseases.

In one embodiment, the present invention relates to a recombinant protein having a protein comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the present invention provides a protein which consists of the amino acid sequence shown in SEQ ID NO: 2 having a deletion, substitution, or addition of at least one amino acid and which has neurogenesis inducing activity. In another embodiment, the present invention relates to a neurogenesis inducing gene encoding for a protein comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:2 or a neurogenesis inducing gene encoding for a protein comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:2 having a deletion, substitution, or addition of at least one amino acid and which has neurogenesis inducing activity. The present invention also relates to a gene which hybridizes with the neurogenesis inducing gene encoding for a protein comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:2.

In another embodiment, the present invention relates to a gene comprising a DNA sequence having at least a portion of the nucleotide sequence set forth in SEQ ID NO: 1. The present invention also relates to a DNA which hybridizes with a DNA having at least a portion of the nucleotide sequence set forth in SEQ ID NO:1, and which codes for a protein having neurogenesis inducing activity.

In another embodiment, the present invention relates to an isolated nucleic acid encoding a protein having at least a portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42 and in SEQ ID NO: 44. In one embodiment, the nucleic acid comprises a nucleotide sequence having at least a portion of the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 41, and SEQ ID NO: 43. In preferred embodiments, the present invention provides a nucleic acid encoding a neurogenesis inducing gene (e.g., early neurogenesis inducing gene). In particular, the present invention provides a nucleic acid encoding a gene selected from the group consisting of Zic1, Zic 2, and Zic3 genes. In one embodiment, the gene is a Xenopus gene.

The present invention further relates to a nucleic acid capable of hybridizing to a nucleic acid comprising a nucleotide sequence having at least a portion of the sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 41, and SEQ ID NO: 43. In a preferred embodiment, the nucleic acid capable of hybridizing under stringent conditions.

In yet another embodiment, the present invention also relates to recombinant vectors comprising at least a portion of genes described above. The present invention further relates to transformants (e.g., a host cell) comprising the recombinant vectors of the present invention.

The present invention also relates to compositions comprising a protein having at least a portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO: 44. In one embodiment, the protein is selected from the group consisting of Zic1, Zic2, and Zic3 proteins. In another embodiment, the protein is a Xenopus protein. In yet another embodiment, the protein is a recombinant protein.

The present invention also provides antibodies against the above described proteins. In particular, the present invention relates to a purified antibody specific to a protein having at least a portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO: 44. The present invention further provides therapeutic agents for nervous diseases associated with the above proteins (e.g., in the presence or absence of the proteins). In preferred embodiments, the present invention relates to therapeutic agents or agents for gene therapy of nervous diseases including, but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Parkinson's disease and cerebral ischemia, although it is not intended that the present invention be limited to therapeutic agents related to these diseases.

Furthermore, the present invention relates to methods for producing a neurogenesis inducing protein, comprising, for example, the steps of culturing a transformant comprising a recombinant vector discussed above and recovering the neurogenesis inducing protein from the resultant culture. In one embodiment, the methods of the present invention comprises the steps of: a) providing a composition comprising a recombinant vector, wherein the recombinant vector comprises a nucleic acid having at least a portion of the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 41 and SEQ ID NO: 43, and a host cell; b) transforming the recombinant vector into the host cell to produce a transformant; and c) culturing the transformant to produce a neurogenesis inducing protein. In one embodiment, the methods of the present invention further comprises the step of d) isolating the neurogenesis inducing protein. In preferred embodiments, the neurogenesis inducing protein is an early neurogenesis inducing protein.

Further, the present invention relates to methods for gene therapy, comprising the steps of: a) providing i) a subject, and ii) a composition comprising a nucleic acid having at least a portion of the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 41 and SEQ ID NO: 43; and b) delivery the composition to the subject. In preferred embodiments, the subject suffers from a disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Parkinson's disease and cerebral ischemia.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to genes capable of inducing neurogenesis (e.g., early neurogenesis). In particular, the present invention relates to Zic1, Zic2, and Zic 3 genes, vectors containing such genes, host cells containing such vectors, proteins produced by such host cells, antibodies raised to such proteins, and therapeutic agents or agents for gene therapy of nervous diseases.

The Zic family was originally identified as a group of genes encoding zinc finger proteins expressed in adult mouse cerebella (Aruga et al., J. Neurochem 63: 1880-1890 [1994], supra). In mice, at least four kinds of Zic genes have been identified (Aruga et al., J. Neurochem 63: 1880-1890 [1994], supra; Aruga et al., J. Biol. Chem. 271: 1043-1047 [1996], supra; Aruga et al., Gene 172: 291-294 [1996], supra). These zinc finger proteins share a highly conserved domain consisting of five tandemly repeated C2H2-type zinc finger motifs. The motifs are highly conserved among various species (Benedyk et al., supra; Cimbora and Sakonju, "Drosophila midgut morphogenesis requires the function of the segmentation gene odd-paired," Dev. Biol. 169: 580-595 [1995]; Yokota et al., "Predominant expression of human Zic in cerebellar granule cell lineage and medulloblastoma," Cancer Res. 56: 377-383 [1996]; Gebbia et al., "X-linked situs abnormalities result from mutations in ZIC3," Nature Genet. 17: 305-308 [1997]; Nakata et al., supra), including their Drosophila homologue odd-paired, which plays important roles in parasegmental subdivision and visceral mesoderm development of the Drosophilia embryo.

The mouse Zic1, Zic 2, and Zic3 genes are expressed in a similar but distinct manner during gestational development (Nagai et al., "The expression of the mouse Zic1, Zic2 and Zic3 gene suggests an essential role for Zic genes in body pattern formation," Dev. Biol. 182: 299-313 [1997]). The expression of these genes was detectable at the primitive streak stage and later in neural tissue, somites and limb buds. Although they are expressed in overlapping sites, their respective expression patterns are not identical. These findings suggest that each Zic gene has specific roles in vertebrate development. This has been confirmed in functional studies. For example, the disruption of mouse Zic1 gene results in malformation of the central nervous system, particularly, the cerebellum (Aruga et al., "Mouse Zic1 is involved in cerebellar development," J. Neurosci. 18: 284-293 [1998]), whereas a mutation in human Zic3 results in disturbance of the left to right body axis (Gebbia et al., supra). The present invention contemplates that Zic genes constitute a multigene family in other vertebrates, and that their respective roles are not identical, although an understanding of the mechanism is not necessary in order to practice the present invention, nor is it intended that the present invention be so limited.

In some embodiments, the present invention relates to the role of Xenopus Zic genes in early vertebrate development. Xenopus Zic3 gene has been cloned (Nakata et al., supra). Expression of Xenopus Zic3 is detected in the prospective neural plate region at gastrulation. The onset of its expression is earlier than those of most proneural genes and follows chordin expression. Zic3 expression is induced by blockade of the BMP4 signal. Overexpression of Zic3 results in hyperplastic neural and neural crest-derived tissue. In an animal cap explant, overexpression of Zic3 induces expression of proneural genes such as Neurogenin (Xngnr-1) and neural crest genes. These findings show that Xenopus Zic3 can determine ectodermal cell fate and promote the earliest step of neural and neural crest development.

Experiments conducted during the development of the present invention further identified and characterized Xenopus Zic-related genes, Zic1 and Zic2, and examined their expression patterns and functions. At least three Zic genes exist in Xenopus laevis, and their respective homologues in mice had been reported. The homologues of Zic1, Zic2 and Zic3 have also been confirmed in some other vertebrates (See e.g., Yokota et al., supra; and Gebbia et al., supra), suggesting that Zic1, Zic2 and Zic3 is important in the vertebrate development.

The present invention has determined the expression patterns of the three Zic genes in Xenopus embryos (Table 1) (See also, Nakata et al., supra). Table 1 shows several similarities and differences among the three Zic genes. First of all, Zic2 was maternally expressed. The definitive roles of Zic2 during this period remains unclear at this time. Although an understanding of the mechanisms is not necessary for the practice of the present invention and the present invention is not limited to any particular mechanism, post-transcriptional regulatory mechanisms may work. Some other genes involved in early neural development have also been detected as maternal messages (e.g., Otx2, Neurogenin; See also, Pannese et al., "The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions," Development 121: 707-720 [1995]). The role of Zic1 and Zic2 before zygotic transcription is clarified further below.

The three Xenopus Zic genes have similar expression patterns in neural tissue. All three genes are expressed in the prospective neural plate region at the time of neural induction. Expression in the medial part of the neural plate was diminished while that in the neural plate border region increased. Thereafter, expression increased on the anterior and dorsal side with regard to the anterior-posterior and dorsal-ventral axes, respectively. These patterns of expression show that Zic genes play a role in neural induction and the patterning of neural tissues in the early phase of neural development.

TABLE 1
Comparative summary of the expression of the three Zic genes:
Stage Tissues Zic1 Zic2 Zic3
 
  9 (blastula) Ectoderm +++ +++ -;
  10.5 (gastrula) Prospective neural plate ++ ++ +++
  Prospective epidermis -; + -;
14-16 (neurula) Neural fold +++ +++ +++
  20 (early tailbud) Dorsal brain ++ ++ ++
  Dorsal spinal cord ++ ++ +
  Eye vesicle -; +++ -;
  Somite +++ ++ -;
  30 (tailbud) Telencephaon ++ + +++
  Diencephalon ++ + +++
  Mesencephalon ++ + ++
  Rhombencephalon ++ + ++
  Dorsal spinal cord ++ ++ +
  Eye (ciliary marginal zone) -; +++ -;
  Somite +++ ++ -;
  Lateral mesoderm (tail) -; -; ++
  Cement gland -; + -;
  Posterior ventral epidermis -; + -;

The differences in spatial Zic gene expression in neural tissues were principally different levels of expression along the anterior-posterior axis and expression in the eye (see Table 1). In addition to the expression in neural tissues, that in somites and their derivatives showed variability. In particular, Xenopus Zic1 is strongly expressed in the somites. It was found that mouse Zic1 is similarly expressed in the somites and plays a critical role in the development of somite derivatives (Nagai et al., supra), suggesting that the Xenopus Zic1 may also play a role in somite development.

The expression in somites is well correlated between Xenopus and mouse Zic genes in that the expression is high for Zic1, moderate for Zic2 and very low for Zic3 both in Xenopus and mouse. As a consequence, the expression patterns of Xenopus Zic1, Zic2, and Zic3 correspond to those of mouse Zic1, Zic2, and Zic3, respectively. Thus, the present invention contemplates that the roles of Zic1, Zic2, and Zic3 are generally well conserved between Xenopus and mouse.

Xenopus Zic1 and Zic2 are novel neuralizing factors. Experiments conducted during the development of the present invention showed that Xenopus Zic1 and Zic2 were capable of inducing neural and neural crest tissues. Zic1 or Zic2 overexpression in embryos resulted in the enlargement of neural plates and neural plate border regions in neurula and the appearance of ectopic pigment cells which were derived from the neural crest. Furthermore, overexpression leads to the induction of neural and neural crest markers in the animal cap explants. Thus, the present invention provides compositions that serve as regulators of neural and neural crest development.

The present invention demonstrates that Xenopus Zic3 is a primary regulator of neural and neural crest development (See also, Nakata et al., supra). The present invention also shows that Zic1 or Zic2 overexpression yielded essentially the same results as observed with Zic3 overexpression. Ectopic pigment cells in embryos overexpressing Zic2 were equivalent to those found in Zic3-overexpressing embryos. However, the induced ectopic pigment cells were less dense in the Zic1-overexpressed embryos than in the Zic2 or Zic3-overexpressed embryos (FIG. 11). In addition, a larger amount of RNA was required to induce neural and neural crest markers in animal cap explants to the same extent (FIG. 14). Although an understanding of the mechanisms is not necessary for the practice of the present invention and the present invention is not limited to any particular mechanism, these results indicate that the potency of neural and neural crest induction by Zic1 is less than that of Zic2 and Zic3. This finding suggests that Zic1 may play a supportive role in the Zic2 and Zic3-mediated neural and neural crust induction. This situation is analogous to the relationship between mouse En1 and En2, in which the En1 and En2 proteins play the same roles in midbrain and hindbrain development (Hanks et al., "Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2," Science 269, 679-682 [995]).

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to Zic related genes. In particular, three Zic related genes, Zic1, Zic2, and Zic3 are provided, identified, and compared with respect to their expression patterns and effects of overexpression. The expression patterns of Xenopus Zic1, Zic2, and Zic3 were found to be distinct, although all three genes expressed in the prospective neural plate region in gastrula. Zic1 and Zic2 were functionally related to Zic3 in that these genes induce neural and neural crest tissues when overexpressed.

Xenopus Zic1 and Zic2 were also found to be very similar to mouse Zic1 and Zic2 in the protein coding region including the zinc finger domain. In early gastrula, Zic1 expression was restricted to the prospective neural plate region whereas Zic2 was expressed widely in the ectoderm. Enhanced neural and neural crest-derived tissue formation were observed in Zic1 or Zic2 overexpressed embryos, as well as in the neural and neural crest marker induction in Zic1 or Zic2 overexpressed animal cap explants. These findings suggest that Zic1 and Zic2 have essentially the same properties as Zic3, and that the Xenopus Zic family act cooperatively in the initial phase of neural and neural crest development.

The Detailed Description of the Invention is divided into seven parts: I) Cloning of Neurogenesis Genes; II) Preparation of Recombinant Vectors and Transformants; III) Analysis of Gene Expression; IV) Protein Production; V) Antibodies Against Zic Proteins; VI) Therapeutic Agents and Agents for Gene Therapy for Nervous Diseases; and VII) Isolation and Characterization of Zic1 and Zic2.

I. Cloning of Neurogenesis Genes

mRNA can be prepared by conventional methods. For example, tissue or cells are treated with a guanidine reagent, phenol reagent or the like to obtain the total RNA. Subsequently, poly(A+)RNA (mRNA) is obtained therefrom by the affinity column method using oligo dT-cellulose or poly U-Sepharose carried on Sepharose 2B or by the batch method. Further, the resultant poly(A+)RNA may be further fractionated by sucrose gradient centrifugation or the like.

A single-stranded cDNA is synthesized using the thus obtained mRNA as a template, an oligo(dT) primer and a reverse transcriptase. Then, a double-stranded cDNA is synthesized from the resultant single-stranded cDNA. The resultant double-stranded cDNA is integrated into an appropriate cloning vector to prepare a recombinant vector. A cDNA library can be obtained by transforming Escherichia coli or the like with the resultant recombinant vector and selecting the transformant using tetracycline or ampicillin resistance as an indicator.

The transformation of E. coli can be performed by the method of Hanahan (Hanahan, J. Mol. Biol. 166: 557-580 [1983]) or the like. Briefly, a method in which a recombinant vector is added to competent cells prepared under the co-existence of calcium chloride, magnesium chloride or rubidium chloride may be used. When a plasmid is used as a vector, the plasmid should contain a drug resistance gene such as tetracycline or ampicillin resistance. Alternatively, a cloning vector other than plasmids (e.g. a phage or the like) may be used.

As a screening method to select clones containing the DNA of interest from the resultant transformants, a method may be given, for example, in which a sense primer and an anti-sense primer corresponding to the amino acid sequence of the zinc finger motif of the mouse Zic gene family are synthesized and a polymerase chain reaction (PCR) is performed using these primers.

As a template DNA to be used in the above PCR, a cDNA which is synthesized from the above-described mRNA by reverse transcription may be given. As primers, 5′-GAGAACCTCAAGATCCACAA-3′ (SEQ ID NO: 5) synthesized based on Glu Asn Leu Lys Ile His Lys (SEQ ID NO: 3) may be used for the same strand; and 5′-TT(C/T)CCATG(A/G)ACCTTCATGTG-3′ (SEQ ID NO: 6) synthesized based on His Met Lys Val His Glu Glu (SEQ ID NO: 4) may be used for the anti-sense strand, for example. However, the present invention is not limited to the use of these primers.

The amplified DNA fragment obtained by the above procedures is labelled with 32P, 35S, biotin or the like, to obtain a probe. This probe is hybridized to a nitrocellulose filter on which the DNA of the transformant is denatured and fixed. Then, screening can be performed by searching for positive clones.

For the resultant clone, the nucleotide sequence is determined. This sequencing is performed by conventional methods such as the chemical modification method of Maxam-Gilbert or the dideoxynucleotide chain termination method using M13 phage. In preferred embodiments, the sequencing is carried out with an automated DNA sequencer (e.g., PerkinElmer Model 373A DNA Sequencer).

SEQ ID NO: 1 illustrates by example of a nucleotide sequence for the Zic3 gene of the present invention, and SEQ ID NO: 2 illustrates by example an amino acid sequence for the associated protein. The present invention contemplates variation of this amino acid sequence. Proteins find use with the present invention as long as the protein has neurogenesis inducing activity, and in particular early neurogenesis inducing activity. Thus, the amino acid sequence may have one or more mutations, such as deletions, substitutions, or additions, of at least one amino acid.

For example, at least 1 amino acid, preferably 1 to about 10 amino acids, more preferably 1 to 5 amino acids may be deleted in the amino acid sequence shown in SEQ ID NO:2; or at least 1 amino acid, preferably 1 to about 10 amino acids, more preferably 1 to 5 amino acids may be added to the amino acid sequence shown in SEQ ID NO:2; or at least 1 amino acid, preferably 1 to about 10 amino acids, more preferably 1 to 5 amino acids may be substituted with other amino acid(s) in the amino acid sequence shown in SEQ ID NO:2.

Accordingly, a gene coding for a polypeptide having the amino acid sequence into which the above-mentioned mutation has been introduced is included in the gene of the invention as long as it has neurogenesis inducing activity (e.g., early neurogenesis inducing activity). Also, a DNA which can hybridize with the gene described above under stringent conditions is provided by the present invention. For example, in some embodiments of the present invention, stringent conditions refers to those conditions in which sodium concentration is 600-900 mM and temperature is 60-68 C., preferably 65 C.

The introduction of a mutation into a gene may be performed by conventional methods such as the method of Kunkel, the gaped duplex method, or variations thereof using a mutation introducing kit (e.g., Mutant-K [Takara] or Mutant-G [Takara]) utilizing site-specific mutagenesis or using a LA PCR in vitro Mutagenesis Series Kit manufactured by Takara, or the like.

Once the nucleotide sequence of the gene of the invention has been determined definitely, the gene of the invention may be obtained by chemical synthesis, by PCR using the cDNA or genomic DNA of the gene of the invention as a template, or by hybridization of a DNA fragment having the above nucleotide sequence as a probe.

II. Preparation of a Recombinant Vector and a Transformant

A. Preparation of a Recombinant Vector

The recombinant vector of the invention may be obtained by ligating (i.e., inserting) the gene of the invention to an appropriate vector. The vector into which the gene of the invention is to be inserted is not particularly limited as long as it is replicable in a desired host. For example, plasmid DNA, phage DNA or the like may be used.

Specific examples of plasmid DNA include E. coli-derived plasmids (e.g., pBR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.) and yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.). Specific examples of phage DNA include A phage and the like. Further, an animal virus vector such as retrovirus or vaccinia virus; or an insect virus vector such as baculovirus may also be used.

For insertion of the gene of the invention into a vector, a method may be employed in which the purified DNA is digested with an appropriate restriction enzyme and then inserted into the restriction site or the multi-cloning site of an appropriate vector DNA for ligation to the vector.

The gene of the invention should be operably linked to the vector. For this purpose, the vector of the invention may contain, if desired, cis elements such as an enhancer, splicing signal, poly(A) addition signal, selection marker, ribosome binding sequence (SD sequence) or the like in addition to a promoter and the gene of the invention. As the selection marker, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene, or the like may be used.

B. Preparation of a Transformant

In some embodiments of the present invention, the transformant is obtained by introducing the recombinant vector of the invention into a host so that the gene of interest is expressed. The host is not particularly limited as long as it can express the DNA of the invention. Specific examples of the host include Escherichia bacteria such as E. coli; Bacillus bacteria such as Bacillus subtilis; Pseudomonas bacteria such as Pseudomonas putida; Rhizobium bacteria such as Rhizobium meliloti; yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe; animal cells such as COS cells, CHO cells; or insect cells such as Sf9 and Sf21 cells.

When a bacterium such as E. coli is used as the host, the recombinant vector of the invention is capable of autonomous replication in the host and, at the same time, it is constituted preferably by a promoter, a ribosome binding sequence, the gene of the invention and a transcription termination sequence. The vector may also contain a gene to control the promoter.

Examples of E. coli that find use with the present invention include, but are not limited to, K12 or DH1 strains. Examples of Bacillus subtilis that find use with the present invention include, but are not limited to, MI 114 or 207-21 strains.

As the promoter, any promoter may be used as long as it can direct the expression of the gene of interest in a host such as E. coli. For example, an E. coli- or phage-derived promoter such as trp promoter, lac promoter, PL promoter or PR promoter may be used. An artificially designed and altered promoter such as tac promoter may also be used.

As a method for introducing the recombinant vector into a bacterium, any method of DNA introduction into bacteria may be used. For example, a method using calcium ions (Cohen et al., Proc. Natl. Acad. Sci., USA, 69: 2110-2114 [1972]) electroporation, or the like may be used.

In some embodiments of the present invention, when a yeast is used as the host, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris or the like is used. In this case, the promoter to be used is not particularly limited. Any promoter may be used as long as it can direct the expression of the gene of interest in yeast. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MF α1 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter or the like may be used.

As a method for introducing the recombinant vector into the yeast, any method of DNA introduction into yeasts may be used. For example, electroporation (Becker et al., Methods Enzymol., 194: 182-187 [1990]), the spheroplast method (Hinnen et al., Proc. Natl. Acad. Sci., USA, 75: 1929-1933 [1978]), the lithium acetate method (Itoh, Bacteriol., 153: 163168 [19893]) or the like may be used.

When an animal cell is used as the host, simian COS-7 or Vero cells, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FL cells or the like may be used, although a variety of other cells find use with the present invention. As a promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter or the like may be used. The early gene promoter of human cytomegalovirus may also be used.

As a method for introducing the recombinant vector into the animal cell, electroporation, the calcium phosphate method, lipofection or the like may be used.

When an insect cell is used as the host, Sf9 cells, Sf21 cells or the like may be used. As a method for introducing the recombinant vector into the insect cell, the calcium phosphate method, lipofection, electroporation or the like may be used.

The recombinant vector of the invention incorporated in E. coli DH5 (designation: Escherichia coli pXenopus Zic3) was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City, Ibaraki Pref., Japan) as FERM BP-6519 under the Budapest Treaty on Mar. 26, 1998.

III. Analysis of Gene Expression

Since the gene of the invention has neurogenesis inducing activity, the expression of this gene can be examined by using embryos of specific developmental stages.

The time of expression of the Zic3 gene of the invention in the embryo can be confirmed by analyzing, for example, expression of the mRNA or the protein in embryos of individual developmental stages. For example, as a method for confirming expression of Zic3 mRNA, RT-PCR, or northern analysis may be used; as a method for confirming expression of ZIC3 protein, western analysis using an antibody against this protein may be used.

Further, the distribution of Zic3 expression in the embryo can be confirmed by analyzing the mRNA by in situ hybridization or the like, or by analyzing the protein by immunohistochemical techniques using an antibody. In situ hybridization can be performed, for example, as described previously (Chitnis et al., Nature 375: 761-766 [1995]) by staining the embryo with digoxigenin or a radioisotope labelled RNA probe.

IV. Protein Production

In some embodiments of the present invention, for example, the protein of the invention is a protein having the amino acid sequence encoded by the Zic3 gene of the invention, or a protein which has the above amino acid sequence having the mutation of at least at 1 amino acid and which has neurogenesis inducing activity. This protein is also called "ZIC3 protein".

ZIC3 protein of the present invention can be obtained by culturing the transformant described above and recovering the protein from the resultant culture. The term "culture" includes any of the following materials: culture supernatant, cultured cells, cultured microorganisms, or crushed cells or microorganisms. The cultivation of the transformant of the invention in a medium is carried out by conventional methods used for culturing a host.

As a medium to culture the transformant obtained from a microorganism host such as E. coli or yeast, either a natural or a synthetic medium may be used as long as it contains carbon sources, nitrogen sources and inorganic salts assimilable by the microorganism and is capable of effective cultivation of the transformant. As carbon sources, carbohydrates such as glucose, fructose, sucrose, starch; organic acids such as acetic acid, propionic acid; and alcohols such as ethanol and propanol may be used. As nitrogen sources, ammonia; ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate; other nitrogen-containing compounds; Peptone; meat extract; corn steep liquor and the like may be used. As inorganic substances, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, iron(II) sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like may be used.

In preferred embodiments, the cultivation is carried out under aerobic conditions (such as shaking culture or aeration agitation culture) at 37 C. for 6 to 24 hrs. During the cultivation, the pH is maintained at 7.0 to 7.5. The pH adjustment is carried out using an inorganic or organic salt, an alkali solution or the like. During the cultivation, an antibiotic such as ampicillin or tetracycline may be added to the medium if necessary.

When a microorganism transformed with an expression vector using an inducible promoter is cultured, an inducer may be added to the medium if necessary. For example, when a microorganism transformed with an expression vector using Lac promoter is cultured, isopropyl-O-D-thiogalactopyranoside (IPTG) or the like may be added. When a microorganism transformed with an expression vector using trp promoter is cultured, indoleacetic acid (IAA) or the like may be added.

As a medium to culture a transformant obtained from an animal cell as a host, commonly used RPMI 1640 medium or DMEM medium, or one of these media supplemented with fetal bovine serum, etc. may be used. Usually, the cultivation is carried out in the presence 5% CO2 at 37 C. for 1 to 30 days. During the cultivation, an antibiotic such as kanamycin or penicillin may be added to the medium if necessary.

After the cultivation, ZIC3 protein of the invention is extracted by disrupting the microorganisms or cells if the protein is produced in the microorganisms or cells. If ZIC3 protein of the invention is produced outside of the microorganisms or cells, the culture fluid is used directly or is subjected to centrifugation to remove the microorganisms or cells. Thereafter, the resultant supernatant is subjected to conventional biochemical techniques used for isolating/purifying a protein. These techniques include ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography. These techniques may be used independently or in an appropriate combination to thereby isolate and purify ZIC3 protein of the invention from the above culture.

V. Antibody Against Zic Proteins

In the present invention, antibody against ZIC proteins of the invention can also be prepared. The term "antibody" means an antibody molecule as a whole which can bind to the peptide of the invention that is an antigen, or a fragment thereof (e.g., Fab or F(ab′)2 fragment). The antibody may be either polyclonal or monoclonal.

The antibody of the invention may be prepared by various methods. Such methods of antibody preparation are well known in the art (See e.g., Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press [1989]).

A. Preparation of a Polyclonal Antibody Against the Protein of the Invention

The following description is provided for the ZIC3 protein and is applicable to other ZIC proteins. In some embodiments of the present invention, ZIC3 protein of the invention is genetically engineered as described above or a fragment thereof is administered as an antigen to a mammal such as rat, mouse or rabbit. The dosage of the antigen administered per animal is 0.1 to 10 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. As an adjuvant, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminium hydroxide adjuvant or the like may be used. Immunization is performed mainly by intravenous, subcutaneous or intraperitoneal injection. The interval of immunization is not particularly limited. In preferred embodiments, immunization is carried out one to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably at intervals of 2 to 5 weeks. Subsequently, 6 to 60 days after the final immunization, antibody titer is determined by, preferably, enzyme immunoassay (EIA), radioimmunoassay (RIA) or the like. Blood is collected from the animal, on the day when the maximum antibody titer is shown, to thereby obtain antiserum. When purification of an antibody from the antiserum is necessary, the antibody is purified by appropriately selecting a conventional method such as ammonium sulfate salting out, ion exchange chromatography, gel filtration, affinity chromatography, or using these methods in combination.

B. Monoclonal Antibodies

(i) Recovery of Antibody-Producing Cells

In some embodiments of the present invention, ZIC3 protein of the invention genetically engineered as described above or a fragment thereof is administered as an antigen to a mammal such as rat, mouse or rabbit. The dosage of the antigen administered per animal is, for example, 0.1 to 10 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. As an adjuvant, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminium hydroxide adjuvant or the like may be used. Immunization is performed mainly by intravenous, subcutaneous or intraperitoneal injection. The interval of immunization is not particularly limited. In preferred embodiments, immunization is carried out one to 10 times, preferably 2 to 5 times, at intervals of several days to several weeks, preferably at intervals of 2 to 5 weeks. Subsequently, 1 to 10 days, preferably 3 days after the final immunization, antibody-producing cells are collected. As antibody-producing cells, spleen cells, lymph node cells, peripheral blood cells, etc. may be enumerated. Among them, spleen cells and local lymph node cells are preferable.

(ii) Cell Fusion

In order to obtain hybridomas, cell fusion between antibody-producing cells and myeloma cells is performed. As the myeloma cells to be fused to the antibody-producing cells, a commonly available cell strain of an animal such as mouse may be used. Preferably, a cell strain to be used for this purpose is one which has drug selectivity, cannot survive in HAT selective medium (i.e., containing hypoxanthine, aminopterin and thymidine) when unfused, and can survive there only when fused to antibody-producing cells. As myeloma cells, mouse myeloma cell strains including, but not limited to, P3X63Ag.8.U1(P3U1), Sp2/0, NS-1 may be used.

Subsequently, the myeloma cells and the antibody-producing cells described above are subjected to cell fusion. Briefly, 1109 cells/ml of the antibody-producing cells and 1108 cells/ml of the myeloma cells are mixed together in equal volumes in an animal cell culture medium such as serum-free DMEM or RPMI-1640, and reacted in the presence of a cell fusion promoting agent. In some embodiments, as the cell fusion promoting agent, polyethylene glycol with an average molecular weight of 1,500 Da may be used. Alternatively, the antibody-producing cells and the myeloma cells may be fused in a commercial cell fusion apparatus utilizing electric stimulation (e.g., electroporation).

(iii) Selection and Cloning of a Hybridoma

A hybridoma of interest is selected from the cells after the cell fusion. As a method for this selection, the resultant cell suspension is appropriately diluted with fetal bovine serum-containing RPMI-1640 medium or the like and then plated on microtiter plates at a density of about 2105 cells/well. A selective medium is added to each well. Then, the cells are cultured while appropriately exchanging the selective medium. As a result, about 14 days after the start of cultivation in the selective medium, the growing cells are obtained as hybridomas.

Subsequently, screening is performed as to whether the antibody of interest is present in the culture supernatant of the grown hybridomas. The screening of hybridomas may be performed by any of conventional methods. For example, a part of the culture supernatant of a well in which a hybridoma is grown is collected and subjected to enzyme immunoassay or radioimmunoassay.

Cloning of the fused cell is performed by the limiting dilution method or the like. Finally, the hybridoma of interest which is a monoclonal antibody-producing cell is established.

(iv) Recovery of the Monoclonal Antibody

In some embodiments of the present invention, a method for recovering the monoclonal antibody from the thus established hybridoma such as conventional cell culture methods or the abdominal dropsy formation method may be employed.

In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as 10% fetal bovine serum-containing RPMI-1640 medium, MEM medium or a serum-free medium under conventional culture conditions (e.g., at 37 C. under 5% CO2) for 2 to 10 days. Then, the monoclonal antibody is recovered from the culture supernatant.

In the abdominal dropsy formation method, about 1107 cells of the hybridoma is administered into the abdominal cavity of an animal syngeneic to the mammal from which the myeloma cells were derived, to thereby propagate the hybridoma greatly. One to two weeks thereafter, the abdominal dropsy or serum is collected.

In the above-mentioned method of recovery of the antibody, if purification of the antibody is necessary, the antibody can be purified by appropriately selecting a conventional method such as ammonium sulfate salting out, ion exchange chromatography, gel filtration, affinity chromatography, or using these methods in combination.

Once the polyclonal or monoclonal antibody is thus obtained, in some embodiments of the present invention, the antibody is bound to a solid carrier as a ligand to thereby prepare an affinity chromatography column. With this column, the peptides of the present invention can be purified from the above mentioned source or other sources. These antibodies further find use in Western blotting to detect the proteins of the present invention.

VI. Therapeutic Agents and Agents for Gene Therapy for Nervous Diseases

Since the proteins and the genes of the present invention have neurogenesis inducing activity, they are useful as a therapeutic agents and as agents for gene therapy, respectively, for nervous diseases. In some embodiments of the present invention, the therapeutic agents or the agents for gene therapy of the present invention is administered to a subject orally or parenterally and systemically or locally.

When the protein or the gene of the present invention is used as a therapeutic agent or an agent for gene therapy for nervous diseases, the disease to be treated is not particularly limited. For example, the proteins or the genes may be used, alone or in combination, for diseases including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Parkinson's disease, cerebral ischemia or the like for the specific purpose of treatment or prevention. These diseases may be in the form of a single disease or may be complicated by one of these diseases or by some disease other than those mentioned above. Any of such forms may be treated with the proteins or the genes of the invention.

In preferred embodiments of the present invention, when the therapeutic agent of the invention is administered orally, the agent may be formulated into a tablet, capsule, granule, powder, pill, troche, internal liquid agent, suspension, emulsion, syrup or the like. Alternatively, the therapeutic agent may be prepared into a dry product which is re-dissolved just before use. In preferred embodiments, when the therapeutic agent of the invention is administered parenterally, the agent may be formulated into a intravenous injection (including drops), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, or the like. Injections are supplied in the form of unit dosage ampules or multidosage containers. These formulations may be prepared by conventional methods using appropriate excipients, fillers, binders, wetting agents, disintegrating agents, lubricating agents, surfactants, dispersants, buffers, preservatives, dissolution aids, antiseptics, flavoring/perfuming agents, analgesics, stabilizers, isotonicity inducing agents, etc. conventionally used in pharmaceutical preparations.

Each of the above-described formulations may contain pharmaceutically acceptable carriers or additives. Specific examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, sodium carboxymethyl amylose, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol and lactose. One or a plurality of these additives are selected or combined appropriately depending of the form of the preparation.

The dosage levels of the therapeutic agent of the invention will vary depending on the age of the subject, the route of administration and the number of times of administration and may be varied in a wide range. When an effective amount of the protein of the invention is administered in combination with an appropriate diluent and a pharmaceutically acceptable carrier, the effective amount of the protein can be in the range from 0.01 to 1000 mg/kg per administration, although other amounts are contemplated, where appropriate. One skilled in the art is capable of determining the therapeutically effective amount appropriate any given circumstances. In some embodiments, the therapeutic agent is administered once a day or in several dosages per day for at least one day.

In some embodiments of the present invention, when the gene of the invention is used as an agent for gene therapy for nervous diseases, the gene of the invention may be directly administered by injection. Alternatively, a vector incorporating the gene of the invention may be administered. Specific examples of a suitable vector for this purpose include an adenovirus vector, adeno-associated virus vector, herpes virus vector, vaccinia virus vector and retrovirus vector. The gene of the invention can be administered efficiently by using such a virus vector. Alternatively, the gene of the invention may be enclosed in phospholipid vesicles such as liposomes, and the resultant liposomes may be administered to the subject. Briefly, since liposomes are biodegradable material-containing closed vesicles, the gene of the invention is retained in the internal aqueous layer and the lipid bilayer of liposomes by mixing the gene with the liposomes (i.e., a liposome-gene complex). Subsequently, when this complex is cultured with cells, the gene in the complex is taken into the cells (i.e., lipofection). Then, the resultant cells may be administered by the methods described below.

In some embodiments of the present invention, as a method for administering the agent for gene therapy of the invention, local administration to tissues of the central nervous system (brain, spiral cord) may be performed in addition to conventional systemic administration such as intravenous or intra-arterial administration. Further, an administration method combined with catheter techniques and surgical operations may also be employed.

The dosage levels of the agent for gene therapy of the invention vary depending on the age, sex and conditions of the subject, the route of administration, the number of times of administration, and the type of the formulation, among other considerations. One skilled in the art is capable of determining the therapeutically effective amount appropriate any given circumstances. Usually, it is appropriate to administer the gene of the invention in an amount of 0.1-100 mg/adult body/day, although other concentrations are contemplated, where appropriate.

According to the present invention, there are provided neurogenesis inducing proteins; a neurogenesis inducing genes (e.g., Zic1, Zic2, and/or Zic3) coding for the proteins; recombinant vectors comprising the genes; transformants comprising the vectors; antibodies against the above proteins; and therapeutic agents for nervous diseases. The Zic genes of the invention find use as a diagnostic agents for nervous diseases, as therapeutic agents for Alzheimer's disease and the like, and as probes to detect nervous diseases, among other applications.

VII. Isolation and Characterization of Zic1 and Zic2

A. Isolation of Xenopus Zic 1 and Zic2

To isolate additional Zic related genes in Xenopus, the Xenopus neurula cDNA library was further screened with the cDNA fragments generated by PCR. Two novel Xenopus Zic-related genes were identified (FIG. 8A, B). A comparison of their predicted amino acid sequences to those of Xenopus Zic3, mouse Zic1, Zic2, Zic3, Zic4 and Drosophila Opa (Nakata et al., supra; Aruga et al., J. Neurochem. 63: 1880-1890 [1994]; Aruga et al., J. Biol. Chem. 271: 1043-1047 [1996]; Aruga et al., Gene 172: 291-294 [1996], supra; Benedyk et al., supra) revealed that one was the most similar to mouse Zic1, and the other was similar to Zic2. Therefore these genes were designated Xenopus Zic1 (SEQ ID NO: 41) and Zic2 (SEQ ID NO: 43). Although significant homology was found in the entire protein coding region, the most extensive homology was found in the zinc finger domains (98% between Xenopus Zic1 [SEQ ID NO: 42] and mouse Zic1, 97% between Xenopus Zic2 [SEQ ID NO: 44] and mouse Zic2) (FIG. 1C). In addition, these novel genes showed significant similarity to Drosophila pair-rule gene, odd-paired (opa).

The zinc finger region, particularly from the 3rd to the 5th zinc finger motif is highly similar to those of the Gli-Ci zinc finger proteins (Ruiz i Altaba, "Catching a Gli-mpse of Hedgehog," Cell 90: 193-196 [1997]), which mediate the hedgehog signal. A crystallographic analysis of Gli protein indicated that the same region actually interacts with the DNA (Pavletich and Pabo, Pavletich, "Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers," Science 261, 1701-1707 [1993]). Taken together with previous data indicating that mouse Zic1 can bind to the Gli-binding sequence (Aruga et al., J. Neurochem. 63: 1880-1890 [1994]), the current evidence suggests that the two protein families, Gli-Ci and Zic-Opa, can bind to highly similar target sequences, although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism.

In addition to the zinc finger domain, it is noted that there were short domains which are conserved in the Xenopus Zic, mouse Zic and Drosophila opa genes. In particular, an amino acid sequence motif, FNSTRDFRXR (SEQ ID NO: 49), which was found in the N-terminal region, was highly conserved (FIG. 8D).

B. Temporal Expression Profiles of Zic1 and Zic2 During Xenopus Development

RT-PCR analyses was performed to compare the temporal expression patterns of the Xenopus Zic genes (FIG. 9). Zic2 was maternally expressed, in contrast to Zic3, which was detected from late blastula but not at earlier stages (Nakata et. al., 1997). Zic1 mRNA was detected from the blastula stage (stage 8) and was peaked in the gastrula. The expression profile is similar to that of Zic3. Zic2 was continuously expressed from the egg to the tailbud stage (stage 30) with an increase in expression at the early gastrula stage (stage 10).

C. Spatial Expression Patterns of Zic1 and Zic2 During Xenopus Embryogenesis

To determine the spatial expression patterns of Xenopus Zic1 and Zic2, whole mount in situ hybridization was performed. At the blastula stage (stage 9), Zic1 and Zic2 were expressed throughout the ectoderm (FIG. 10A, M). At the gastrula stage (stage 10.5), Zic1 expression became restricted to the prospective neural plate, as observed for the Zic3 expression (FIG. 10B, C; arrowhead, Nakata et. al., supra), whereas Zic2 was expressed throughout the ectoderm in the gastrula (FIG. 10N, O).

During the late gastrula to neurula stages, both Zic1 and Zic2 expression gradually diminished in the midline region of the neural plate and increased in the anterior neural folds (FIG. 10D-F, P-R). However, Zic2 continued to be expressed in the posterior medial part of the neural plate (FIG. 10Q, arrowhead). At the neurula stage, staining was seen as four longitudinal lines in the trunk, similar to that of Zic3 (FIG. 10F, R, white and black arrowheads, Nakata, et. al., supra).

In the early tailbud stages (stages 22-23; FIG. 10G, H, S, T), Zic1 and Zic2 were expressed in the dorsal forebrain, midbrain, and hindbrain (FIG. 10H, T). Subsequently, expression was seen in the telencephalon and diencephalon/mesencephalon boundary (stage 30; FIG. 10K, W). In the spinal cord, expression was restricted to the dorsal most region including the roof plate (FIG. 10J, L, V, X). These expression patterns in the central nervous system are essentially the same as Zic3 except for a slight difference in the expression level along the anterior to posterior axis (Nakata et. al., supra).

However, the expression patterns of the three Zic genes varied in somites and eye vesicles. Both Zic1 and Zic2 were expressed in the somites. The level of Zic1 expression was higher than that of Zic2, in comparison to the expression in neural tubes (FIG. 10G, S), and Zic2 expression extended more ventrally than Zic1. (FIG. 10L, X). In contrast, Zic3 expression in the somites was negligible (Nakata et. al., supra). As to the expressions in eye vesicles, Zic2 was expressed whereas Zic1 and Zic3 were not (FIG. 10H, T, Nakata et. al., supra). Zic2 expression in the eyes was restricted to the Ciliary marginal zone of neural retina, with no expression in the lens (FIG. 10J, V).

These expression patterns, when compared to that of Zic3, show that each of the three Xenopus Zic genes is involved in several developmental processes including those of the nervous system and somites.

D. Zic1 and Zic2 Induce Neural and Neural Crest Tissues

The effects of the Zic1 or Zic2 overexpression on the embryos was examined. First, MT-Zic1 (Zic1 tagged with myc epitopes ) or MT-Zic2 mRNA were injected into both blastomeres of 2-cell stage embryos (FIG. 11A-F). Ectopic pigment cells appeared in these embryos (FIG. 11C-F). The appearance of ectopic pigment cells was also found in the MT-Zic3 or Zic3 mRNA injected embryos (FIG. 11B, Nakata et. al., supra). However, the pigment cells induced by MT-Zic1 overexpression were apparently less dense and less frequent than those induced by MT-Zic3 or MT-Zic2 overexpression (FIG. 11) [Ectopic pigment cells were found in 0/10 of MT-Zic1 (100 pg), 16/16 of MT-Zic1 (500 pg), 16/20 of MT-Zic2 (100 pg), 19/19 of MT-Zic2 (500 pg) and 13/13 of MT-Zic3 (100 pg) injected embryos]. Each of the Zic proteins was expressed at equivalent levels in each Zic mRNA injected embryo, suggesting that each Zic protein may have different pigment cell-inducing activities (FIG. 11I).

Next, MT-Zic1 (250 pg) or MT-Zic2 (125 pg) mRNA were injected into a blastomere of a 2-cell stage embryo and sectioned at stages 35-36. Thickening of the neural tubes and the ectopic presumptive mesenchymal tissue with ectopic pigment cells in the injected side of these embryos was observed (FIG. 11G, H). The ectopic pigment cells were also found in animal cap explants overexpressing Mt-Zic1 (8/12), or Mt-Zic2 (11/11) (FIG. 12). To clarify whether these pigment cells were melanocytes derived from neural crest, the animal cap explants derived from the embryos obtained by the mating between albino female and wild type males were used. In this case, the shapes of the pigmented cells appearing in the explants could clearly be observed. As expected, the pigment cells had elaborate processes which were typically found in the melanocytes (FIG. 12). In addition, a neural crest marker twist (Xtwi, Hopwood et. al., "A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest," Cell 59, 893-903 [1989]) was expressed in the mesenchymal tissue beneath the ectopic pigment cells in embryos in which Zic1 or Zic2 were overexpressed as observed with Zic3 overexpression (Nakata et. al., supra). These findings suggest that pigment cells expressed in Zic1 or Zic2 injected embryos were derived from neural crest.

To examine whether Zic1 and Zic2 overexpression results in alterations in cell fate, the expression of a neural marker (NCAM) (Kintner and Melton, "Expression of Xenopus NCAM RNA in ectoderm is an early response to neural induction," Development 99: 311-325 [1987]), a neural crest marker (Xslu) (Mayor et. al., "Induction of the prospective neural crest of Xenopus," Development 121: 767-777 [1995]), and an epidermal antigen (EpA) (Jones and Woodland, "Development of the ectoderm in Xenopus: tissue specification and the role of cell association and division," Cell 44: 345-355 [1986]) were examined at an early neurula stage (stage 14) (FIG. 13). The NCAM-expressing neural plate region increased in the Zic1 or Zic2 mRNA injected side [8/8 of MT-Zic1 (250 pg), 10/14 of MT-Zic2 (125 pg) injected embryos] (FIG. 13A, D). Xslu expression was also increased in the Zic1 or Zic2 mRNA injected side [6/9 of MT-Zic1 (250 pg), 18/18 of MT-Zic2 (125 pg) injected embryos] (FIG. 13B, E). In contrast, the expression of EpA was significantly reduced on the Zic1 or Zic2 mRNA injected side [18/22 of MT-Zic1 (250 pg), 20/24 of MT-Zic2 (125 pg) injected embryos] (FIG. 13C, F). These observations suggest that misexpressed Zic1 or Zic2 altered epidermal cell fate to neural and neural crest cell fate.

Next, the expression of several marker genes in the animal cap explants from Zic1 or Zic2 overexpressing blastula were examined (stage 9) (FIG. 14). Zic1 and Zic2 overexpression induced NCAM, a neuronal differentiation marker (N-rubulin; Chitnis et. al., "Primary neurogenesis in Xenopus embryos regulated by a homologue of the drosophila neurogenic gene Delta," Nature 375, 761-766 [1995]) and Xtwi expression in the explants, as expected based on the above results. In addition, a mid-hindbrain junction marker (En2; Hemmati-Brivanlou et. al., "Cephalic expression and molecular characterization of Xenopus En-2," Development 3: 715-724 [1991]), but not a spinal cord marker [HoxB9 (which is the same as Xlhbox6); Wright et. al., "The Xenopus XlHbox6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons," Development 109: 225-234 [1990]) was induced by Zic1 or Zic2 overexpression. These findings indicate that neural tissue generated by the Zic1 or Zic2 overexpression has characteristics of anterior neural tissue similar to those observed with Zic3 overexpression. These inductions appeared to occur without mesoderm induction since no mesodermal marker (M. actin; muscle actin) was induced in this case.
 

Claim 1 of 8 Claims

1. An isolated nucleotide sequence encoding a protein having the amino acid sequence set forth in SEQ ID NO: 44, wherein the protein is a neurogenesis inducing protein.

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