expansion of the notochord. These morphological changes are associated with a marked expansion in the ntl mRNA expression domain and shh mRNA expression domain. Overexpression of IGF-2a also caused a widening in the space between the myoD and myogenin in the anterior region of an embryo. Many of these embryos had duplicated or partial duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. Brown et al. (2009) reported that knockdown of either igf-2a of igf-2b led to defects in dorsal midline development, although knockdown of igf-2b also induced ectopic fusion of the nephron primordia [12]. Collectively, these studies suggest that that IGF signaling plays a critical role in midline formation and notochord development. 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. Given the central role of IGF signaling in growth regulation, it will be important to investigate whether this high rate of gene retention in genes belonging to the IGF signaling pathway in the ray-fin fish plays any role in the continuous growth pattern of the teleosts. Materials and Methods Chemicals and reagents All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. Restriction endonucleases were purchased from New England BioLabs (Beverly, MA). Superscript II reverse transcriptase (RT) and Oligonucleotide primers were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). RNase-free DNase was purchased from Promega (Madison, WI). The anti-Akt and anti- Phospho-Akt antibodies (Ser473) were purchased from Cell Signaling (Danvers, MA). Experimental animals Zebrafish (Danio rerio) were maintained at 28 C on a 14 h:10 h (light:dark) cycle, and fed twice daily. Embryos were generated by natural crosses. Fertilized eggs were raised in embryo medium at 28.5 C and staged according to the standard criteria of Kimmel et al. [62]. For in situ hybridization analysis, embryo medium was supplemented with 0.003% (w/v) 2-phenylthiourea to inhibit embryo pigment formation. All experiments were conducted following guidelines approved by the University of Michigan Committee on the Use and Care of Animals. Molecular cloning Searching the zebrafish EST and genome databases (Ensembl Zebrafish: http://www. ensembl.org/Danio_rerio/blastview) and the NCBI database (http://www.ncbi.nlm.nih.gov/) identified two distinct IGF-2 mRNAs (GenBank Accession No. XM_001338006, 3404 bp; NM_131433, 2065 bp, respectively) and one IGF-1 mRNA (GenBank Accession No. NM_131825, 1521 bp). We further searched these databases by BLAST searches using the IGF-1 sequence as a query. Two IGF-1-like EST sequences (GenBank Accession No. XM_684982, 636 bp; CO353655, 600 bp, respectively) were obtained. Based on these sequences, the following gene-specific primers were designed to clone and confirm the predicted IGF cDNA sequences. igf-1a: 59 RACE primer 59- TGTCTGCATAAGGCTTTTGAA- TAATCC-39, 59 nest RACE primer 59-CTGTCGGTTTGCTG- AAAT-39; 39 RACE primer: 59- ATAGCCACTCTTCCTGTAAG- G-39 and 39 nest RACE primer 59- TAGAGGACAGCGGGAG- GAATGAACTGA-39. Multiple IGF-1 and -2 Genes igf- 1b: 59 RACE primer 59-AAGCCYCTGTCTCCRCACA- CAAACTSCAG-39, 59 nest RACE primer 59- TCTGGCCCCT- TCAGTGTTGTCTGGCAG-39; 39 RACE primer: 59- CTGSA- GTTTGTGTGYGGAGACAGRGGCTT-39 and 39 nest RACE primer 59- CGACAACTGAGACGCCAACA-39. igf-2a: 59 RACE primer 59- CATACATCGCGTGTCTTA- CATACACGT-39, 59 nest RACE primer 59- CTCCKTARCCT- CTGAGCAGC -39; 39 RACE primer: 59- CCATCCTTCTTCC- CACAG-39 and 39 nest RACE primer 59- TGAAAAGACAG- GATCGTTTGCACAGAG-39. igf-2b: 59 RACE primer 59- AGCAAAGGCTTTAACTT- CATTCTCCA-39, 59 nest RACE primer 59- CTCCKTARCCT- CTGAGCAGC -39; 39 RACE primer: 59- CAACCTGCCATC- GTCCCA-39 and 39 nest RACE primer 59- TGATGGCCAGC- CATCCCAAATGGCA-39. Total RNA was isolated from 10 hpf zebrafish embryos and adult liver using the TRIzol reagent (Invitrogen) and used as template. The 59 and 39 ends of the IGF mRNA were amplified using a SMART RACE cDNA amplifica- tion kit (Clontech, USA) following the manufacturer’s protocol. PCR products were gel purified and ligated into the T/A cloning vector pGEM-T Easy (Promega, USA) and transformed into E. coli DH5a competent cells. Positive clones were examined by PCR and direct sequencing. Phylogenetic analysis and physical mapping The genomic structure and chromosomal location of each zebrafish igf gene were determined by searching the zebrafish genome (http://www.ensembl.org/Danio_rerio/index.html). Ami- no acid sequences of IGFs were aligned by CLUSTAL X. Phylogenetic analysis was done using B and A domain sequences by Neighbor-joining method in the MEGA3 program. Gap sites in the alignment were not used in the phylogenetic reconstruction. The reliability of the estimated tree was evaluated by the bootstrap method with 1000 pseudo-replications. Synteny analysis was carried out based on Danio rerio Zv6 and Homo sapiens Build 36.3 (http:// www.ensembl.org/Homo_sapiens/index.html) and data from zeb- rafish and human synteny map. Genomic structure was determined by searching the zebrafish genome (http://www.ensembl.org/ Danio_rerio/index.html). Plasmid construction The open reading frame of each zebrafish IGF gene was amplified by PCR using Pfu DNA polymerase (Stratagene, La jolla, CA, USA). The following primers were used: igf-1a 59- GTGGATCCAC- CATGTCTAGCGGTCATTTCTTCCAG-39 and 59-GCTCTA- GATCACATGCGATAGTTTCTGCCCCCTGTGT-39, igf-1b 59- GTGGATCCACCATGGTGCCCAGCTGGCGGAGTGT-39 and 59- GCTCTAGATCACCGGATGTGAGAGATGAATGTTGGC- GT-39, igf-2a 59- GTGGATCCACCATGGAGGACCAACTAAAA- CATCAT-39and 59- GCTCTAGATCACTTGTGGCTAACGTA- GTTTTCTGTGG-39, igf-2b 59- GTGGATCCACCATGGATGA- TTACCATGTATTCTGTGC-39and 59- GCTCTAGATCATTT- TCGGGATGTGCTGATCTGGACGT-39. The resulting cDNA was cloned into the pGEM-T Easy vector (Promega, USA). To gene- rate the IGF peptide expression constucts, the above PCR product was digested with BamHI and XbaI and subcloned into BamHI and XbaI sites of pCS2+. Reverse transcription (RT)-PCR and whole mount in situ hybridization Total RNA was isolated from embryo or adult tissues using the TRIzol reagent (Invitrogen). One mg of RNA was reverse- transcribed to single strand cDNA using SuperScript II reverse PLoS ONE | www.plosone.org 10 September 2009 | Volume 4 | Issue 9 | e7026