Zebrafish IGF Genes: Gene Duplication, Conservation and Divergence, and Novel Roles in Midline and Notochord Development Shuming Zou1,2, Hiroyasu Kamei1, Zubin Modi1, Cunming Duan1,3* 1 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Key Laboratory of Aquatic Genetic Resources, Shanghai Ocean University, Shanghai, China, 3 Laboratory of Molecular Medicine, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China Abstract Insulin-like growth factors (IGFs) are key regulators of development, growth, and longevity. In most vertebrate species including humans, there is one IGF-1 gene and one IGF-2 gene. Here we report the identification and functional characterization of 4 distinct IGF genes (termed as igf-1a, -1b, -2a, and -2b) in zebrafish. These genes encode 4 structurally distinct and functional IGF peptides. IGF-1a and IGF-2a mRNAs were detected in multiple tissues in adult fish. IGF-1b mRNA was detected only in the gonad and IGF-2b mRNA only in the liver. Functional analysis showed that all 4 IGFs caused similar developmental defects but with different potencies. Many of these embryos had fully or partially duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. IGF- 2a, the most potent IGF, was analyzed in depth. IGF-2a expression caused defects in the midline formation and expansion of the notochord but it did not alter the anterior neural patterning. These results not only provide new insights into the functional conservation and divergence of the multiple igf genes but also reveal a novel role of IGF signaling in midline formation and notochord development in a vertebrate model. Citation: Zou S, Kamei H, Modi Z, Duan C (2009) Zebrafish IGF Genes: Gene Duplication, Conservation and Divergence, and Novel Roles in Midline and Notochord Development. PLoS ONE 4(9): e7026. doi:10.1371/journal.pone.0007026 Editor: Michael Hendricks, Harvard University, United States of America Received July 8, 2009; Accepted August 18, 2009; Published September 17, 2009 Copyright: 2009 Zou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NSF Grant IBN 0110864 to CD. SZ is supported in part by a scholarship from China Scholarship Council and HK is supported in part by a postdoctoral fellowship from Japan Society of Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cduan@umich.edu Introduction Insulin-like growth factors (IGFs), including IGF-1 and IGF-2, are evolutionarily conserved peptides that act through a conserved signaling pathway to regulate growth, development, metabolism, and longevity in a wide variety of animals [1]. IGF-1 and -2 are structurally related to insulin and relaxin in vertebrates, and insulin-like peptides (ILP) in invertebrates [2,3,4,5]. IGFs and insulin share 50% sequence identity in amino acid sequence in their B and A domains. Unlike insulin, however, the C-domains of IGFs are not removed by proteolytic processing. In addition, IGF- 1 and IGF-2 contain an E- domain in their carboxy-terminus. The E-domain is removed as a post-translational modification to produce mature IGFs [6]. Activation of the IGF signaling pathway occurs when IGF ligands bind their cognate receptor tyrosine kinases. This leads to activation of a number of downstream signaling cascades, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-Akt pathways [7,8,9]. In mammals and birds, there is a single IGF-1 gene and a single IGF-2 gene, in addition to a single insulin gene. Recent studies have indicated the zebrafish genome contains more than two IGF genes. Chen et al. (2001) reported the presence of a gene encoding an IGF-like peptide [10]. Maures at al. (2002) cloned cDNAs encoding the complete coding sequences for the same IGF-1-like peptide and an IGF-2 peptide [5]. A more recent study by Sang et al. (2008) reported the presence of two distinct IGF-2 genes (igf-2a and -2b) and that these genes exhibited distinct expression patterns [11]. They further showed that knockdown of igf-2a and -2b by antisense morpholinos indicated that they each play a distinct role in early development [12]. Wang et al. (13) reported the cloning of another IGF peptide that they termed as IGF-3, from tilapia and zebrafish [13]. Despite these new findings, there has been no report on the molecular characterization of any of these zebrafish IGF genes or their full-length transcripts. For instance, little is known about the alternative splicing for any of the previously identified zebrafish IGF genes, although alternative splicing has been shown to be an important way of generating multiple forms of IGF transcripts and prepropeptides in mammals and humans. Moreover, zebrafish, like many teleost fish, are believed to have experienced an additional genome wide duplication event [14,15]. As a result, they often have two co-orthologs in contrast to a single copy gene in humans and other mammals. Indeed, there are two distinct insulin genes, two IGF-1R genes, and two insulin receptor genes in zebrafish [5,16,17,18]. To date, there is no report on the possible presence of the fourth IGF gene in zebrafish. In this study, we have cloned and identified 4 distinct genes encoding 4 IGF peptides (IGF-1a, -1b, -2a, and -2b) from zebrafish. The structures of these 4 zebrafish IGF genes and their transcripts have been determined. Our PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e7026