Functional characterisation of a novel nucleoporin gene NUP98 in zebrafish embryo.

Abstract

INTRODUCTION: The nucleoporin gene nup98 is important for the regulation of cytoplasmic-nuclear trafficking. Frequent disruptions of NUP98 during chromosomal translocation in acute myeloid leukaemia suggest that it may play a role in normal haematopoiesis. nup98-knockout mice has resulted in early embryonic lethality. Therefore, its role in embryonic haematopoiesis remains unclear. In this study, we have cloned a zebrafish nup98 gene and examined its role in embryonic development, with particular reference to haematopoiesis. METHODS: Two expressed sequence tags with translated sequence homologous to human NUP98 were identified. The gene was cloned by PCR from cDNA of zebrafish embryos. Expression of nup98 in zebrafish embryos was investigated spatially by whole-mount in-situ hybridisation and temporally by RT-PCR. The functions of nup98 were examined by morpholino knockdown and the effects on embryonic development evaluated by gene expression studies and confocal microscopy. Cellular functions of zebrafish nup98 were investigated in HeLa cells. RESULTS: Zebrafish nup98 gene shared 65% identity to human NUP98 homolog in protein sequence. The gene was expressed during early embryonic development since 1-cell stage and diffusely in eyes and the developing brain since 18 hpf. About 30% nup98-knockdown embryos developed intracranial haemorrhage at 48 hpf, resulting from disrupted blood vessels. nup98-knockdown upregulated pu.1 and scl as evaluated by quantitative RT-PCR. Moreover, ectopic expression of zebrafish nup98 rescued the defective mRNA export due to NUP98 knockdown in HeLa cells. CONCLUSION: A novel zebrafish nup98 gene was shown to exhibit conserved function in mRNA trafficking. Its role in embryonic development should be further evaluated.