Interruption of cenph Causes Mitotic Failure and Embryonic Death, and Its Haploinsufficiency Suppresses Cancer in Zebrafish

Xinyi Zhao,Long Zhao,Tian Tian,Yu Zhang,Jingyuan Tong,Xiaofeng Zheng,Anming Meng

doi:10.1074/jbc.m110.136077

Xinyi Zhao, Long Zhao + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.136077

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Abstract

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.

Highlights

Kinetochore is a structure formed at the outer surface of the centromere of a mitotic chromosome
Compared with wild type sibling embryos, we found that stac mutants have more mitotic cells with aberrant spindles and chromosome missegregation with an arrest of the cell cycle in G2/M phase
From the dent on the p53 antisense morpholino (p53-MO) doses (Fig. 5F). These results confirm that 20-somite stage to 36 hpf, we consistently identified a much the p53 apoptosis pathway plays a role in cell death of stac higher proportion of abnormal mitotic cells in stac mutants mutant embryos

Summary

EXPERIMENTAL PROCEDURES

Gene Trapping and Transposon Mutagenesis—The transposon-based gene trap vector T2BGS was modified from T2KSAG [23] by replacing the original splicing acceptor with a splicing acceptor in the first intron of the zebrafish bcl gene, which was a gift from Dr Jian Zhang. For spindle and active caspase 3 immunofluorescence, embryos fixed in 4% paraformaldehyde were dehydrated in graded methanol at Ϫ20 °C for at least 30 min, followed by rehydration three times for 5 min each in PBST (1ϫ PBS, 0.3% Triton X-100). After washing three times with PBST, embryos were incubated in block solution (2% blocking reagent (Roche Applied Science), 10% fetal calf serum, 1% dimethyl sulfoxide in PBST) for 1 h at room temperature. Embryos were incubated for binding of the primary antibody at 4 °C overnight, rinsed four times for 20 min each in PBST (1ϫ PBS, 0.1% Triton X-100), and incubated in block solution (10% fetal calf serum, 1% dimethyl sulfoxide in PBST) for 1 h at room temperature. Fish were sacrificed 6 months after exposure, and serial step 5-␮mthick sagittal sections were made for each fish and examined for the existence of tumors by hematoxylin and eosin staining

RESULTS

Extensive Apoptosis Occurs in stac

Findings

DISCUSSION