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Characterization and genomic structure of Dnah9, and its roles in nodal signaling pathways in the Japanese flounder (Paralichthys olivaceus)

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Abstract

The nodal signaling pathway has been shown to play crucial roles in inducing and patterning the mesoderm and endoderm, as well as in regulating neurogenesis and left–right axis asymmetry. Here, we present the first complete cDNA and genomic sequences as well as the promoter predication of the Dnah9 gene in the Japanese flounder. The 15,558-bp-long cDNA is divided into 96 exons and spread over 138 kb of genomic DNA. Protein sequence comparison showed that it shares higher identity with other vertebrate orthologs, with an ATP binding dynein motor, AAA domain and microtubule binding stalk of dynein motor. Dnah9 exhibited maternal and ubiquitous expression in all cells of the early development stages, but became concentrated in the head at 1 DAH, as identified by qRT-PCR and in situ hybridization methods. Furthermore, after nodal signaling was inhibited, the level of Southpaw did not change significantly at early development stage (50 % epiboly) but increased significantly at late stages (27-somite stages and 1 DAH), as well as the expression of Lefty, an inhibitor of nodal signaling, increased continuously. On the other hand, the expression level of Dnah9 decreased. The transcription factor binding site of FAST-1 (SMAD interacting protein) was identified in the transcription region of Dnah9 by the promoter analysis, which might format the complexes of SMADs, FAST-1 and the transcription region of Dnah9 served as a bridge of Dnah9 and nodal signaling. All evidences indicated that Dnah9 might be downstream of nodal during the early development stages, and an indirect function through SMADs for nodal signaling pathway.

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Acknowledgments

We appreciate the help of Dr. Sarah E. Webb, The Hong Kong University of Science & Technology (HKUST), for helpful comments on this manuscript. This study was financially supported by a Grant from The National Science Foundation of China (No. 31372511) and The National High Technology Research and Development Program of China (No. 2012AA10A401).

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Authors and Affiliations

  1. Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao, 266003, Shandong, China

    Jingjing Niu, Conghui Liu, Fang Yang, Zhenwei Wang, Bo Wang, Quanqi Zhang, Yan He & Jie Qi

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  1. Jingjing Niu
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  2. Conghui Liu
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  3. Fang Yang
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  7. Yan He
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Correspondence to Yan He or Jie Qi.

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Supplementary material 1 (DOC 112 kb)

Supplementary Fig. 1

The DNAH9 protein sequence of the Japanese Flounder is aligned against those of nine other species. Homo sapiens ENST00000262442; Latimeria chalumnae ENSLACT00000019339; Gadus morhua ENSGMOT00000015923; Lepisosteus oculatus ENSLOCT00000015006; Oreochromis niloticus ENSONIT00000024597; Oryzias latipes ENSORLT00000020555; Takifugu rubripes ENSTRUT00000000142; Tetradon nigroviridis ENSTNIT00000008436; and Xiphorus maculates ENSXMAT00000011889. In the case of 100 % conservation in the ten sequences, then the amino acids are shaded in black. Four different symbols represent the four kinds of functional domains, including the P-loop domain, microtubule binding site, protein kinase C phosphorylation site, and the leucine zipper region. (TIFF 11013 kb)

Supplementary material 3 (TIFF 11012 kb)

Supplementary material 4 (TIFF 11012 kb)

Supplementary material 5 (TIFF 11010 kb)

Supplementary Fig. 2

Molecular phylogenetic analysis of DNAH9. The tree was constructed by MEGA (version 5.1) using Poisson Correction distance based upon the neighbor-joining method, with 1000 bootstrap replicates. Twenty other species with typical representation were chosen for comparison, including Tripneustes gratilla, Branchiostoma floridae, Gasterosteus aculeatu, and Xenopus tropicalis, and the GenBank accession numbers are shown after each species. Paralichthys olivaceus was clustered with Xiphophorus maculates. (TIFF 3278 kb)

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Niu, J., Liu, C., Yang, F. et al. Characterization and genomic structure of Dnah9, and its roles in nodal signaling pathways in the Japanese flounder (Paralichthys olivaceus). Fish Physiol Biochem 42, 167–178 (2016). https://doi.org/10.1007/s10695-015-0127-9

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  • DOI: https://doi.org/10.1007/s10695-015-0127-9

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