BACKGROUND
cancer-upregulated gene  <dig>  is known to be differentially expressed in multiple human cancer tissues including the ovary, liver, lung, intestines and pancreas  <cit> .
mammalian cells overexpressing cug <dig> showed hallmarks of neoplasmic transformation in vitro, such as increased cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in nude mice, similar to the effects of the h-ras oncogene  <cit> .
recently, cug <dig> was shown to interact with cenp-t and cenp-a, essential components of the nucleosome complex located at the centromere, and was hence named centromere protein w   <cit> .
the centromere is involved in sister chromatid cohesion and the attachment of spindle microtubules, and is thus responsible for accurate chromosome segregation during mitotic and meiotic cell division  <cit> .
cenp-a, a histone h3-like core protein, is required for the recruitment of many constitutive centromere components as well as transient kinetochore components  <cit> .
we and others have reported that cug2/cenp-w forms a dna-binding complex together with the cenp-t and cenp-a as part of the centromere chromatin structure  <cit> .
sirna-mediated knockdown of cug2/cenp-w in hela cells caused defective mitosis characterized by multipolar spindle formation as well as chromosomal misalignment and hypercondensation, resulting in mitotic arrest  <cit> .
however, the in vivo function of cug <dig> has not been studied in animal models.
to elucidate the endogenous function of cug <dig> in vivo, we investigated the expression patterns and potential roles of cug <dig> in zebrafish during early embryogenesis.
our results indicate that cug <dig> is essential for normal mitosis and cns development, and that loss of cug <dig> function lead to neurodegenerative phenotypes.
RESULTS
identification of the zebrafish cug <dig> homologue
a zebrafish cug <dig> homologue was isolated from a  <dig> hpf embryonic cdna library.
the zebrafish cug <dig>  is composed of  <dig> exons encoding  <dig> amino acids.
the nuclear localization signal in the n-terminus is highly conserved among cug <dig> homologues.
clustal × analysis indicates that the zebrafish cug <dig> amino acid sequence shows 58% and 62% similarity to human  and mouse , respectively .
to more precisely confirm the evolutionary conservation of cug <dig>  we investigated the distribution of genes located adjacent to the cug <dig> locus on chromosomes in zebrafish and human using the online program synteny database  <cit> .
two genes, trmt <dig> and rspo <dig>  were located close to each other on human chromosome  <dig> near cug <dig>  and their zebrafish orthologues were located in the vicinity of cug <dig> on chromosome  <dig> , indicating that zebrafish cug <dig> was an orthologue of human cug <dig>  the predicted fish cug <dig> sequence in the ncbi database  suggests an isoform with alternative splicing of exon  <dig>  and no additional isoforms were detected by rt-pcr.
in addition, no evidence of a second orthologue was found.
to determine the temporal and spatial expression patterns of the cug <dig> gene during zebrafish development, stage- and tissue-specific rt-pcr and in situ hybridization were performed, revealing that zebrafish cug <dig> transcripts have both maternal and zygotic expression patterns .
cug <dig> mrna was detectable at variable levels throughout the early embryonic stages  and in selected tissues in the adult .
whole-mount in situ hybridization showed that cug <dig> transcripts were ubiquitously expressed throughout the embryonic body from cleavage to the early somite stage .
at 24- <dig> hpf, cug <dig> expression was specifically detected in the lateral line primordium, gut, and cns including the telencephalon, midbrain, midbrain-hindbrain boundary, hindbrain, and spinal cord .
at  <dig> hpf, the cug2-expressing region overlapped with a proliferating cell marker, pcna-expressing region, such as those in the ciliary marginal zone in the eyes, tectum, midbrain-hindbrain boundary, neural crest cells, pectoral fin buds, and gut .
however, the expression domains of cug <dig> were not overlapped with that of huc, a differentiating neuronal marker .
these restricted and yet overlapping expression patterns indicate that cug <dig> is expressed mainly in the proliferating cell population of the cns during early embryonic development in zebrafish.
knock-down analysis of cug <dig> in zebrafish embryos
to investigate the endogenous roles of cug <dig>  knock-down analysis was performed using an antisense oligonucleotide morpholino .
the cug <dig> mo was designed to target the splicing donor site of exon  <dig>  resulting in an aberrant transcript with a premature stop codon at residue  <dig> .
rt-pcr confirmed that injection of the cug <dig> mo into zebrafish embryos successfully blocked the splicing of cug <dig> transcripts .
also, we designed other mo to target the translation start site  and then confirmed that the mo specifically inhibits the translation of cug2-gfp containing its targeting region .
after  <dig> dpf, cug <dig> mo-injected embryos consistently displayed a range of characteristic phenotypes including flat head, small eyes, pinched midbrain-hindbrain boundary, thin yolk extension, and curved body .
this phenotype was rescued by co-injection of wild-type cug <dig> mrna, but not by injection of p <dig> mo , confirming that the observed phenotype is specific to the effect of cug <dig> knockdown and not due to p53-dependent cell death, a typical off-target effect of mo  <cit> .
to further determine whether the phenotypes observed in cug <dig> morphants were caused by apoptotic cell death, cug <dig> mo-injected embryos were analyzed by acridine orange staining.
acridine orange-positive cells were clearly and broadly detected throughout the bodies of cug <dig> morphants at  <dig> hpf, particularly in the cns including the eye, brain, and spinal cord .
these data suggest that the phenotypes induced by morpholino knockdown of cug <dig> result from induction of apoptosis during early embryogenesis.
cug <dig> deficiency causes neurodegeneration
the phenotypes of cug <dig> mo-injected embryos are remarkably similar to previously reported neural degenerative mutants in zebrafish, such as psm, terf <dig>  and tub  <cit> .
furthermore, the cug <dig> morphants exhibited extensive apoptotic cell death in the cns .
therefore, we utilized the tg transgenic line that expresses neuron-specific gfp under the control of the huc promoter  <cit>  to determine whether cug <dig> knock-down causes neuronal degeneration.
microinjection of cug <dig> mo into tg transgenic embryos caused a reduction in the expression of gfp-positive neurons in the cns, particularly in the nucleus of the medial longitudinal fasciculus  and neurons of the hindbrain rhombomere at  <dig> hpf .
moreover, the number of huc-positive differentiating neurons was dramatically decreased in cug <dig> morphants .
next, embryos were immunostained with anti-acetylated α-tubulin to investigate whether cug <dig> morphants display architectural defects of the axonal scaffold in the brain.
as expected, the axonal scaffolds in the anterior commissure , olfactory nerve , and nmlf were significantly deteriorated, as evidenced by significantly weaker acetylated α-tubulin staining compared to control.
particularly, organization of the commissural axons in the rhombomere segments was severely disturbed in cug <dig> mo-injected embryos .
in addition, reduced arborization was evident in neurons such as the spinal cord rohon-beard  sensory neurons .
to further define the neurodegenerative phenotype at the histological level, hematoxylin-eosin  staining was performed on serial paraffin sections of the brain, retina, and spinal cord at  <dig> dpf.
like other vertebrates, the zebrafish retina consists of six layers  and contains six types of neurons and one type of glial cell  <cit> .
at  <dig> dpf, cug <dig> morphants showed severe disruption in the layer formation and pyknotic cells in the retina compared to control embryos .
moreover, in the spinal cord, both the number of cells in the neural tube and the size of the neural tube were dramatically decreased in cug <dig> mo-injected embryos .
interestingly, cug <dig> deficiency appeared to affect specific subpopulations of the developing neurons; during primary neurogenesis  in the neural plate, ngn <dig> and delta-positive neuronal precursors were slightly increased by cug <dig> knockdown, while the huc/elavl3-positive differentiating neurons were decreased .
in contrast, during secondary neurogenesis , both neuronal precursors and differentiating neurons were affected .
these results indicate that cug <dig> may function in normal differentiation and/or maintenance of neurons rather than early neuronal precursor determination.
cug <dig> morphants exhibit mitotic defects
cug <dig> was previously identified as a component of the constitutive centromere-associated network  and abnormal mitotic events such as multipolar spindle formation and chromosome misalignment were observed in cug2-depleted mammalian cells  <cit> .
we first examined whether cug <dig> localizes to cell's nucleus in zebrafish embryos, using gfp-tagged cug <dig> protein.
the behavior of cug2-gfp is reminiscent of the behavior of the chromosomes during various stages of cell cycle in living zebrafish embryo , thus suggesting that cug <dig> is involved in mitosis in zebrafish.
to investigate whether cug2-deficient embryos exhibit any defects in mitosis, embryos were double-stained with anti-α-tubulin antibody and hoechst  <dig> dye at the neural plate stage to visualize the mitotic spindles and metaphase chromosomes, respectively.
in cug <dig> mo-injected embryos, α-tubulin-positive spindles were short and disorganized compared to the control embryos .
in addition, cug2-deficient embryos exhibited varying degrees of disorganized metaphasic chromosome alignment .
next, cug <dig> morphants were examined to determine whether there were any changes in the number of metaphase cells.
immunostaining of the embryos with an antibody against phosphorylated histone h <dig> , a mitotic marker  <cit> , revealed that cug <dig> morphants experienced a significant increase in the number of ph3-positive cells in the spinal cord at  <dig> hpf .
the increased number of ph3-positive cells in the cug <dig> morphants likely represents an increase in mitotic arrest, rather than enhanced cell proliferation, since there was no significant difference in brdu incorporation between the control and cug <dig> morphants .
combined, these results support the notion that loss of cug <dig> function causes defective mitosis, leading to mitotic arrest during early neurogenesis.
we speculate that zebrafish cug <dig> is required for the normal function of the mitotic spindle and chromosome arrangement at the metaphase plate, consistent with the results from mammalian cell lines.
discussion
here, we report that a zebrafish orthologue of a recently identified human centromeric protein cug2/cenp-w is crucially important for normal mitosis and neurogenesis during early cns development.
knockdown of cug <dig> expression in developing embryos caused a dramatic increase in the number of mitotically arrested cells exhibiting abnormal spindle formation and chromosome misalignment , as well as extensive apoptotic cell death associated with neurodegenerative phenotypes .
we and others have previously shown that cug2/cenp-w is a component of ccan and participates in the formation of the dna-binding complex together with cenp-a, cenp-c, and cenp-t at the kinetochore in mammalian cells  <cit> .
our current study further extends this notion and supports an essential role for zebrafish cug <dig> in kinetochore assembly, defects of which may elicit the checkpoint control mechanism and result in mitotic arrest.
the genomic instability caused by the loss of cug <dig> affects cell viability, as evidenced by extensive apoptosis, leading to neurodegeneration early in cns development in zebrafish.
a number of studies in mice have shown that null mutations of the genes encoding most of the centromere proteins cause defective or arrested mitosis, and result in a degenerative phenotype and embryonic lethality  <cit> .
in addition, genes encoding other zebrafish centromeric proteins, such as cenpa/seph, cenpl and cenpn, are mainly expressed in the proliferating regions during embryogenesis.
insertional mutation of these genes  results in neurodegenerative phenotypes  <cit>  similar to those of the cug <dig> morphants described in our study .
errors in chromosomal segregation due to compromised mitotic checkpoint control leads to aneuploidy, as often observed in transformed cell lines and human tumors.
it has been postulated that common molecular pathways may be involved in both oncogenesis and neurodegeneration, and that genetic alterations of these pathways can lead to either carcinogenesis or neurodegeneration depending on the cellular context  <cit> .
considering the fundamental importance of genome stability in development, differentiation, growth and homeostasis of an organism, the data presented here support the critical role of cug <dig> in both cancer and neurodegenerative diseases.
CONCLUSIONS
in conclusion, this study suggests that cug <dig> is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug <dig> deficient embryos may provide a model system for human neurodegenerative disorders.

 cancer-upregulated gene is known to be differentially expressed in multiple human cancer tissues including the ovary, liver, lung, intestines and pancreas. mammalian cells overexpressing cug showed hallmarks of neoplasmic transformation in vitro, such as increased cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in nude mice, similar to the effects of the h-ras oncogene. recently, cug was shown to interact with cenp-t and cenp-a, essential components of the nucleosome complex located at the centromere, and was hence named centromere protein w. the centromere is involved in sister chromatid cohesion and the attachment of spindle microtubules, and is thus responsible for accurate chromosome segregation during mitotic and meiotic cell division. cenp-a, a histone h3-like core protein, is required for the recruitment of many constitutive centromere components as well as transient kinetochore components. we and others have reported that cug2/cenp-w forms a dna-binding complex together with the cenp-t and cenp-a as part of the centromere chromatin structure. sirna-mediated knockdown of cug2/cenp-w in hela cells caused defective mitosis characterized by multipolar spindle formation as well as chromosomal misalignment and hypercondensation, resulting in mitotic arrest. however, the in vivo function of cug has not been studied in animal models. to elucidate the endogenous function of cug in vivo, we investigated the expression patterns and potential roles of cug in zebrafish during early embryogenesis. our results indicate that cug is essential for normal mitosis and cns development, and that loss of cug function lead to neurodegenerative phenotypes.  identification of the zebrafish cug homologue a zebrafish cug homologue was isolated from a hpf embryonic cdna library. the zebrafish cug is composed of exons encoding amino acids. the nuclear localization signal in the n-terminus is highly conserved among cug homologues. clustal × analysis indicates that the zebrafish cug amino acid sequence shows 58% and 62% similarity to human and mouse, respectively. to more precisely confirm the evolutionary conservation of cug we investigated the distribution of genes located adjacent to the cug locus on chromosomes in zebrafish and human using the online program synteny database. two genes, trmt and rspo were located close to each other on human chromosome near cug and their zebrafish orthologues were located in the vicinity of cug on chromosome, indicating that zebrafish cug was an orthologue of human cug the predicted fish cug sequence in the ncbi database suggests an isoform with alternative splicing of exon and no additional isoforms were detected by rt-pcr. in addition, no evidence of a second orthologue was found. to determine the temporal and spatial expression patterns of the cug gene during zebrafish development, stage- and tissue-specific rt-pcr and in situ hybridization were performed, revealing that zebrafish cug transcripts have both maternal and zygotic expression patterns. cug mrna was detectable at variable levels throughout the early embryonic stages and in selected tissues in the adult. whole-mount in situ hybridization showed that cug transcripts were ubiquitously expressed throughout the embryonic body from cleavage to the early somite stage. at 24- hpf, cug expression was specifically detected in the lateral line primordium, gut, and cns including the telencephalon, midbrain, midbrain-hindbrain boundary, hindbrain, and spinal cord. at hpf, the cug2-expressing region overlapped with a proliferating cell marker, pcna-expressing region, such as those in the ciliary marginal zone in the eyes, tectum, midbrain-hindbrain boundary, neural crest cells, pectoral fin buds, and gut. however, the expression domains of cug were not overlapped with that of huc, a differentiating neuronal marker. these restricted and yet overlapping expression patterns indicate that cug is expressed mainly in the proliferating cell population of the cns during early embryonic development in zebrafish. knock-down analysis of cug in zebrafish embryos to investigate the endogenous roles of cug knock-down analysis was performed using an antisense oligonucleotide morpholino. the cug mo was designed to target the splicing donor site of exon resulting in an aberrant transcript with a premature stop codon at residue. rt-pcr confirmed that injection of the cug mo into zebrafish embryos successfully blocked the splicing of cug transcripts. also, we designed other mo to target the translation start site and then confirmed that the mo specifically inhibits the translation of cug2-gfp containing its targeting region. after dpf, cug mo-injected embryos consistently displayed a range of characteristic phenotypes including flat head, small eyes, pinched midbrain-hindbrain boundary, thin yolk extension, and curved body. this phenotype was rescued by co-injection of wild-type cug mrna, but not by injection of p mo, confirming that the observed phenotype is specific to the effect of cug knockdown and not due to p53-dependent cell death, a typical off-target effect of mo. to further determine whether the phenotypes observed in cug morphants were caused by apoptotic cell death, cug mo-injected embryos were analyzed by acridine orange staining. acridine orange-positive cells were clearly and broadly detected throughout the bodies of cug morphants at hpf, particularly in the cns including the eye, brain, and spinal cord. these data suggest that the phenotypes induced by morpholino knockdown of cug result from induction of apoptosis during early embryogenesis. cug deficiency causes neurodegeneration the phenotypes of cug mo-injected embryos are remarkably similar to previously reported neural degenerative mutants in zebrafish, such as psm, terf and tub. furthermore, the cug morphants exhibited extensive apoptotic cell death in the cns. therefore, we utilized the tg transgenic line that expresses neuron-specific gfp under the control of the huc promoter to determine whether cug knock-down causes neuronal degeneration. microinjection of cug mo into tg transgenic embryos caused a reduction in the expression of gfp-positive neurons in the cns, particularly in the nucleus of the medial longitudinal fasciculus and neurons of the hindbrain rhombomere at hpf. moreover, the number of huc-positive differentiating neurons was dramatically decreased in cug morphants. next, embryos were immunostained with anti-acetylated α-tubulin to investigate whether cug morphants display architectural defects of the axonal scaffold in the brain. as expected, the axonal scaffolds in the anterior commissure, olfactory nerve, and nmlf were significantly deteriorated, as evidenced by significantly weaker acetylated α-tubulin staining compared to control. particularly, organization of the commissural axons in the rhombomere segments was severely disturbed in cug mo-injected embryos. in addition, reduced arborization was evident in neurons such as the spinal cord rohon-beard sensory neurons. to further define the neurodegenerative phenotype at the histological level, hematoxylin-eosin staining was performed on serial paraffin sections of the brain, retina, and spinal cord at dpf. like other vertebrates, the zebrafish retina consists of six layers and contains six types of neurons and one type of glial cell. at dpf, cug morphants showed severe disruption in the layer formation and pyknotic cells in the retina compared to control embryos. moreover, in the spinal cord, both the number of cells in the neural tube and the size of the neural tube were dramatically decreased in cug mo-injected embryos. interestingly, cug deficiency appeared to affect specific subpopulations of the developing neurons; during primary neurogenesis in the neural plate, ngn and delta-positive neuronal precursors were slightly increased by cug knockdown, while the huc/elavl3-positive differentiating neurons were decreased. in contrast, during secondary neurogenesis, both neuronal precursors and differentiating neurons were affected. these results indicate that cug may function in normal differentiation and/or maintenance of neurons rather than early neuronal precursor determination. cug morphants exhibit mitotic defects cug was previously identified as a component of the constitutive centromere-associated network and abnormal mitotic events such as multipolar spindle formation and chromosome misalignment were observed in cug2-depleted mammalian cells. we first examined whether cug localizes to cell's nucleus in zebrafish embryos, using gfp-tagged cug protein. the behavior of cug2-gfp is reminiscent of the behavior of the chromosomes during various stages of cell cycle in living zebrafish embryo, thus suggesting that cug is involved in mitosis in zebrafish. to investigate whether cug2-deficient embryos exhibit any defects in mitosis, embryos were double-stained with anti-α-tubulin antibody and hoechst dye at the neural plate stage to visualize the mitotic spindles and metaphase chromosomes, respectively. in cug mo-injected embryos, α-tubulin-positive spindles were short and disorganized compared to the control embryos. in addition, cug2-deficient embryos exhibited varying degrees of disorganized metaphasic chromosome alignment. next, cug morphants were examined to determine whether there were any changes in the number of metaphase cells. immunostaining of the embryos with an antibody against phosphorylated histone h, a mitotic marker, revealed that cug morphants experienced a significant increase in the number of ph3-positive cells in the spinal cord at hpf. the increased number of ph3-positive cells in the cug morphants likely represents an increase in mitotic arrest, rather than enhanced cell proliferation, since there was no significant difference in brdu incorporation between the control and cug morphants. combined, these results support the notion that loss of cug function causes defective mitosis, leading to mitotic arrest during early neurogenesis. we speculate that zebrafish cug is required for the normal function of the mitotic spindle and chromosome arrangement at the metaphase plate, consistent with the results from mammalian cell lines. discussion here, we report that a zebrafish orthologue of a recently identified human centromeric protein cug2/cenp-w is crucially important for normal mitosis and neurogenesis during early cns development. knockdown of cug expression in developing embryos caused a dramatic increase in the number of mitotically arrested cells exhibiting abnormal spindle formation and chromosome misalignment, as well as extensive apoptotic cell death associated with neurodegenerative phenotypes. we and others have previously shown that cug2/cenp-w is a component of ccan and participates in the formation of the dna-binding complex together with cenp-a, cenp-c, and cenp-t at the kinetochore in mammalian cells. our current study further extends this notion and supports an essential role for zebrafish cug in kinetochore assembly, defects of which may elicit the checkpoint control mechanism and result in mitotic arrest. the genomic instability caused by the loss of cug affects cell viability, as evidenced by extensive apoptosis, leading to neurodegeneration early in cns development in zebrafish. a number of studies in mice have shown that null mutations of the genes encoding most of the centromere proteins cause defective or arrested mitosis, and result in a degenerative phenotype and embryonic lethality. in addition, genes encoding other zebrafish centromeric proteins, such as cenpa/seph, cenpl and cenpn, are mainly expressed in the proliferating regions during embryogenesis. insertional mutation of these genes results in neurodegenerative phenotypes similar to those of the cug morphants described in our study. errors in chromosomal segregation due to compromised mitotic checkpoint control leads to aneuploidy, as often observed in transformed cell lines and human tumors. it has been postulated that common molecular pathways may be involved in both oncogenesis and neurodegeneration, and that genetic alterations of these pathways can lead to either carcinogenesis or neurodegeneration depending on the cellular context. considering the fundamental importance of genome stability in development, differentiation, growth and homeostasis of an organism, the data presented here support the critical role of cug in both cancer and neurodegenerative diseases.  in conclusion, this study suggests that cug is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug deficient embryos may provide a model system for human neurodegenerative disorders. 
in addition, cug2-deficient embryos contained mitotically arrested cells displaying abnormal spindle formation and chromosome misalignment in the neural plate. therefore, our findings suggest that cug is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug deficient embryos may provide a model system for human neurodegenerative disorders. morpholino-mediated knockdown of cug resulted in apoptosis throughout the cns and the development of neurodegenerative phenotypes. however, the in vivo function of cug has not been studied in animal models. we recently identified a novel oncogene, cancer-upregulated gene, which is essential for kinetochore formation and promotes tumorigenesis in mammalian cells.
 we recently identified a novel oncogene, cancer-upregulated gene, which is essential for kinetochore formation and promotes tumorigenesis in mammalian cells. however, the in vivo function of cug has not been studied in animal models.  to study the function of cug in vivo, we isolated a zebrafish homologue that is expressed specifically in the proliferating cells of the central nervous system. morpholino-mediated knockdown of cug resulted in apoptosis throughout the cns and the development of neurodegenerative phenotypes. in addition, cug2-deficient embryos contained mitotically arrested cells displaying abnormal spindle formation and chromosome misalignment in the neural plate.  therefore, our findings suggest that cug is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug deficient embryos may provide a model system for human neurodegenerative disorders. 