Isolation, characterisation and evolution of zebrafish (Danio rerio) bmp9, bmp10 and gdf11

Abstract

Three members of the TGFβ superfamily, bmp9, bmp10 and gdf11 were isolated from zebrafish genomic and cDNA libraries to further elucidate the role of these genes during embryogenesis. Zebrafish bmp9 and bmp10 belong to the BMP9/10 subfamily. The level of sequence similarity between the zebrafish and mammalian BMP9/10 subfamily members prompted an investigation into the evolutionary relationships of the zebrafish genes. The orthology of bmp9 and bmp10 were examined by phylogenetic and syntenic analysis. Phylogenetic analysis grouped bmp9, DSLI, Bmp9 and BMP9 into a single clade and bmp10, Bmp10 and BMP10 into another. Zebrafish bmp9 and human BMP9 were mapped to linkage group 12 and chromosome 17, respectively, and share syntenic relationships with a number of other genes mapped onto these respective chromosomes. Zebrafish bmp10 and human BMP10 were mapped to linkage group 5 and chromosome 2, respectively, and share syntenic relationships with mcm6/MCM6 and pax8/PAX8. The phylogenetic and syntenic analyses support the orthology of bmp9 and bmp10 to human BMP9 and BMP10, respectively. The phylogenetic analysis also suggests that chick DSL1 is not a unique member of the TGFβ superfamily but is the chicken ortholog of BMP9/Bmp9. The expression patterns of bmp9 and bmp10 were analysed by RT-PCR, Northern analysis, and by whole mount in situ hybridisation. No specific expression pattern was detected for either bmp9 or bmp10 by whole mount in situ hybridisation, indicating the low expression levels of these genes. The lower than expected sequence similarity to the mammalian orthologs and the low level of expression suggest a lack of evolutionary pressure between subfamily members. Zebrafish Gdf11 shows a high level of amino acid identity over the length of the entire protein to mouse GDF11. Phylogenetic analysis grouped gdf11, Gdf11 and GDF11 into a single clade. Zebrafish gdf11 and human GDF11 were mapped to linkage group 11 and chromosome 12, respectively, and share syntenic relationships with atp5b/ATP5b, dspg3/DSPG3, hoxcb/HOXC, and plasticin/PRPH on LG11/Hsa12. The phylogenetic and syntenic analyses, therefore, support the orthology of gdf11 to human GDF11. The expression pattern of gdf11 was analysed by whole mount in situ hybridisation. Expression of gdf11 was detected in both the dorsal and vegetal tail bud progenitors and during segmentation stages, expression becomes restricted to the caudal-most chordamesoderm of the tail bud. During the pharyngula stage, dynamic expression of gdf11 was detected in neural structures including the ventral hindbrain, midbrain and the forebrain. Expression of gdf11 was absent in the tail bud of ntlb195 mutant embryos but present in neural structures, indicating that the expression of gdf11 is regulated by different factors in these tissues. The function of gdf11 was analysed by microinjection of synthetic gdf11 mRNA. Overexpression of gdf11, however, resulted in the severe dorsalisation of the embryo, probably due to activation of a Nodal specific pathway.