Cug2 is essential for normal mitotic control and CNS development in zebrafish
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We recently identified a novel oncogene, Cancer-upregulated gene 2 (CUG2), which is essential for kinetochore formation and promotes tumorigenesis in mammalian cells. However, the in vivo function of CUG2 has not been studied in animal models.
To study the function of CUG2 in vivo, we isolated a zebrafish homologue that is expressed specifically in the proliferating cells of the central nervous system (CNS). Morpholino-mediated knockdown of cug2 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 Cug2 is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug2 deficient embryos may provide a model system for human neurodegenerative disorders.
KeywordsZebrafish Embryo Mitotic Arrest Central Nervous System Development Neurodegenerative Phenotype Normal Mitosis
Cancer-upregulated gene 2 (CUG2) is known to be differentially expressed in multiple human cancer tissues including the ovary, liver, lung, intestines and pancreas . Mammalian cells overexpressing CUG2 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, CUG2 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 (CENP-W) [2, 3]. 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 [5, 6]. 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 [2, 3]. 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 [2, 3]. However, the in vivo function of CUG2 has not been studied in animal models.
To elucidate the endogenous function of CUG2 in vivo, we investigated the expression patterns and potential roles of cug2 in zebrafish during early embryogenesis. Our results indicate that Cug2 is essential for normal mitosis and CNS development, and that loss of Cug2 function lead to neurodegenerative phenotypes.
Identification of the zebrafish cug2homologue
Knock-down analysis of cug2in zebrafish embryos
To further determine whether the phenotypes observed in cug2 morphants were caused by apoptotic cell death, cug2 MO-injected embryos were analyzed by acridine orange staining. Acridine orange-positive cells were clearly and broadly detected throughout the bodies of cug2 morphants at 28 hpf, particularly in the CNS including the eye, brain, and spinal cord (Figure 3G, H). These data suggest that the phenotypes induced by morpholino knockdown of cug2 result from induction of apoptosis during early embryogenesis.
cug2deficiency causes neurodegeneration
To further define the neurodegenerative phenotype at the histological level, hematoxylin-eosin (H&E) staining was performed on serial paraffin sections of the brain, retina, and spinal cord at 3 dpf. Like other vertebrates, the zebrafish retina consists of six layers (ganglion cell, inner plexiform, inner nuclear, outer plexiform, outer nuclear, and pigment cell layers) and contains six types of neurons and one type of glial cell . At 3 dpf, cug2 morphants showed severe disruption in the layer formation and pyknotic cells in the retina compared to control embryos (Figure 4G, H). 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 cug2 MO-injected embryos (Figure 4I, J). Interestingly, cug2 deficiency appeared to affect specific subpopulations of the developing neurons; during primary neurogenesis (3 ss) in the neural plate, ngn1 and delta-positive neuronal precursors were slightly increased by cug2 knockdown, while the huC/elavl3-positive differentiating neurons were decreased (Additional File 2: Figure. S2A-C', H). In contrast, during secondary neurogenesis (20 ss), both neuronal precursors and differentiating neurons were affected (Additional File 2: Figure. S2D-F'). These results indicate that cug2 may function in normal differentiation and/or maintenance of neurons rather than early neuronal precursor determination.
cug2morphants exhibit mitotic defects
Next, cug2 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 H3 (pH3), a mitotic marker , revealed that cug2 morphants experienced a significant increase in the number of pH3-positive cells in the spinal cord at 24 hpf (Figure 5E, F, I). The increased number of pH3-positive cells in the cug2 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 cug2 morphants (Figure 5G, H, J).
Combined, these results support the notion that loss of cug2 function causes defective mitosis, leading to mitotic arrest during early neurogenesis. We speculate that zebrafish cug2 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.
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 cug2 expression in developing embryos caused a dramatic increase in the number of mitotically arrested cells exhibiting abnormal spindle formation and chromosome misalignment (Figure 5), as well as extensive apoptotic cell death associated with neurodegenerative phenotypes (Figures 3 & 4).
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 [2, 3, 6]. Our current study further extends this notion and supports an essential role for zebrafish cug2 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 cug2 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 [16, 17, 18]. 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 (seph hi2737Tg , cenpl hi3634Tg , and cenpn hi3505Tg ) results in neurodegenerative phenotypes  similar to those of the cug2 morphants described in our study (Figure 3D).
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 CUG2 in both cancer and neurodegenerative diseases.
In conclusion, this study suggests that Cug2 is required for normal mitosis during early neurogenesis and has functions in neuronal cell maintenance, thus demonstrating that the cug2 deficient embryos may provide a model system for human neurodegenerative disorders.
Fish stocks and maintenance
Adult fish were maintained at 28.5°C on a 14-hr light/10-hr dark cycle. Tg[huC:GFP] was used in knock-down experiments . Embryonic stages were determined by the postfertilization hour and microscopic observation. Some of the embryos were treated with phenylthiocarbamide (1-phenyl-2-thiourea, PTU; Sigma) to suppress melanin synthesis. Animal work was approved by the internal animal ethics committee at Chungnam National University (No. 2010-3-6).
Cloning of the zebrafish cug2homologue
To isolate the zebrafish cug2 gene, a cDNA fragment from a 24 hpf zebrafish cDNA was amplified by RT-PCR based on the NCBI sequence (Genbank: XM_683789). The 519 bp PCR products were cloned into the pGEM-T easy vector (Promega, USA) and then subcloned into the EcoR I site in a pCS2+ expression vector. To construct the cug2-GFP fusion reporter into pCS2+ GFP expression vector, the specific enzyme-linked primers were designed for PCR amplification. PCR products were subcloned into the NcoI site in pCS2+GFP vector.
Whole-mount in situ hybridization, immunostaining and apoptosis detection
To synthesis the RNA probe, a pGEM-T easy vector harboring the cug2 gene was linearized with Sal I and antisense cug2 RNA was transcribed in vitro using T7 RNA polymerase and digoxigenin or fluorescein-labeled UTP. The full-length cDNA of pcna (Genbank: AF140608) was amplified from a 24hpf cDNA and cloned into a pGEM T-easy vector. Antisense digoxigenin-labeled RNA probes for ngn1 , huC/elavl3 , deltaA , and pcna were produced using a digoxigenin-RNA labelling Kit (Roche, Germany) according to the manufacturer's instructions. Whole-mount in situ hybridization was performed as previously described . Whole-mount immunostaining was carried out as described previously  using anti-α-tubulin, anti-acetylated α-tubulin, anti-BrdU (all from Sigma), and anti-phospho-histone H3 Ser10 (Cell Signaling) antibodies. For BrdU incorporation, dechorionated embryos were incubated with 10 mM BrdU in 15% DMSO/egg water (60 μg/ml sea salts (Sigma) in distilled water) for 20 minutes at 4°C and then for exactly 5 minutes at 28.5°C, followed by 4% paraformaldehyde fixation overnight at 4°C and dehydration in methanol at -20°C. To stain nuclei, the embryos were fixed in 4% paraformaldehyde, stained for 10 minutes with Hoechst 33342 (Sigma) and washed in PBS. For detection of apoptotic cells, embryos were placed in 10 μg/ml acridine orange (Sigma) diluted in egg water for 30 minutes and then washed in egg water.
Paraffin sectioning and H&E staining
Embryos were fixed in 4% paraformaldehyde for 1 day at 4°C and then dehydrated with a graded ethanol series up to 100%. Specimens in xylene were embedded in paraffin and cut at a thickness of 6 μm. Histological hematoxylin-eosin (H&E) staining was carried out using standard protocols.
Microinjection of mRNA and morpholino oligonucleotides
Synthetic capped mRNAs for human CUG2, zebrafish cug2, and cug2-GFP were transcribed in vitro by using the SP6 mMESSAGE mMACHINE Kit (Ambion). The synthesized mRNAs were dissolved in 0.2% Phenol Red as a tracking dye, and then microinjected into one to two cell stage embryos with 100 pg per embryo. Morpholinos were resuspended in 1 × Danieau's buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES, pH 7.6) with 0.1% phenol red and microinjected into embryos at the 1-4 cell stage. Concentration of morpholinos injected into embryos as follows: cug2 translation blocker, 500 pg/embryo; cug2 splicing blocker, control MO, and p53 MO, 2 ng/embryo. Injected embryos were incubated until the indicated stage and analyzed by in situ hybridization or immunostaining.
DNA oligonucleotide and MO sequences
The RT-PCR primers used for cloning zebrafish cug2 cDNA from a 24 hpf zebrafish cDNA library were 5'-ataaaacgcctttcacgccgccaa-3' (forward) and 5'-gggctagatactgtccatcatcca-3' (reverse). The PCR primers for constructing cug2-GFP reporter were 5'- cgccatggggatgtcgtcagtaatctct-3' (forward) and 5'- cgccatggactgagtgtgtgtgtgtgca-3' (reverse). Morpholino antisense oligonucleotides for cug2 translation start site; 5'- CTG CTC TCG GTG CTT TCT TCG ACA T-3', the exon 1 splice donor site; 5'-GAA CCT TCT TCA ACT CAC CAT CAA G-3', standard control MO, 5'- CCT CTT ACC TCA GTT ACA ATT TAT A-3', p53 MO, 5'- TTG ATT TTG CCG ACC TCC TCT CCA C were designed to have no predicted internal hairpins, avoiding the presence of four consecutive G nucleotides, and synthesized by Gene-Tools, LLC (Corvallis, OR, USA).
Acknowledgements and Funding
This work was supported by the National R&D Program for Cancer Control, Ministry for Health and Welfare (1020090) and the Basic Science Research Program (NRF-20100005431) of the National Research Foundation funded by the Korean Government (to S.L.).
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