Generation of Transgenic Zebrafish A plasmid with the enhanced green Yuorescent protein (EGFP) under the control of the zebrafish spon1b promoter (Accession# NM_131517) was constructed. The recombinant spon1b:GFP construct was obtained by a combination of assembly PCR and traditional cloning. By PCR, using the appropriate primers, a 10.3-kb DNA sequence from the 59 promoter region of the spon1b gene, obtained from the genomic CH211-20O6 clone (BACPAC Resources Center, Children’s Hospital Oakland Research Insti- tute, Oakland, CA), and a 0.86-kb fragment containing the 39 flanking region of spon1b, were placed upstream and downstream, respectively, of the EGFP sequence. The resulting 12.2-kb construct was transferred into a modified pNEB193 plasmid containing two meganuclease I-SceI sites. The one-cell stage zebrafish embryos were co-injected with the plasmid DNA containing the construct and the meganuclease I-SceI, as recommended [69]. Injected embryos were screened for the presence of GFP fluorescence between 48 and 72 hpf. Positive embryos were raised to sexual maturity and out-crossed with wild type fish to identify transgenic carriers. The presence of green fluorescence in the F1 offspring identified transgenic founder zebrafish. Out of 69 adult GFP-positive fish, two had germ-line transmission of spon1b:GFP, with similar expression patterns in transgenic F1 progeny. The line that had the stronger fluorescence signal was used for the studies described here. Real-time Quantitative Reverse-Transcriptase PCR (qPCR) Total RNA was extracted and purified from whole embryos at desired stages and adult brains using RNAeasy Qiagen (Qiagen, Chatsworth, CA, USA), according to the manufacturer’s protocol. RNA extraction from brain tissue was conducted using QIAzol Lysis Reagent (Qiagen, Chatsworth, CA, USA) before RNA isolation using RNAeasy Qiagen kit. The quantity and quality of RNA was determined spectrophotometrically, at 260 nm and 260/280 nm. The same amount of RNA from each sample was converted into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instruction. qPCR was performed using a TaqManH Universal PCR Master Mix and ABI Prism 7300 Real Time PCR System (ABI, Foster City, CA, USA). The TaqManH primers and probes {59 FAM, 39 TAMRA} for spon1a and spon1b were designed based on previously reported sequences of zebrafish genes and obtained from ABI: spon1a Forward, 59- GTGACCAAGAGGAGGATTATGCT-39, Reverse, 59-TGTC- ATGAATTGCGTGTCTTCCT-39; {ATCGATTATCTGGAA- ATTT}, spon1b Forward, 59-GAAGGAGAGCCAGAAACTTA- CCAA -39, Reverse, 59-CAATGAGGGTAAAACCACGAAA- GT-39; {CACCTACAGAGTGAGTTTG}, and b-actin Forward, 59-GCTGTTTTCCCCTCCATTGTTG -39, Reverse, 59-TTT- CTGTCCCATGCCAACCA-39; {CCCAGACATCAGGGAG- TG}. Gene expression was normalized using b-actin expression level for each individual fish sample. A second housekeeping gene, EGF-1, was also used in some of the assays to control for lack of non-specific changes in mRNA abundance Forward, 59- TCCTTGCGCTCAATCTTCCAT -39, Reverse, 59-GCACGG- TGACAACATGCT-39; {ACCAGCCCATGTTTGAG}. Rela- tive mRNA expression level was calculated using the standard comparative delta-Ct method. Within each experiment, embry- onic or adult brain tissue samples were collected and processed in parallel, and the expression was measured within the same microplate, each sample in triplicate. F-Spondin Expression in Zebrafish Tissue Preparation for RNA and Protein Staining To obtain tissue samples, adult zebrafish were anesthetized using tricaine (MS-222, Sigma). When they stopped breathing, they were decapitated and the heads fixed in 4% paraformalde- hyde in 0.1 M phosphate buffer (PFA) for at least 2 hours. Then, the brains were dissected out and re-fixed in 4% PFA overnight. The following day, the brains were cryopreserved in a solution of 30% sucrose until they sank. Then, they were fast frozen in isopentane, embedded in OCT (Tissue-Tek, Torrance, CA, USA), and serially cryosectioned in the coronal plane every 20 mm. Sections were stored at 280uC until processed for in situ hybridization or immunohistochemistry. Embryos were collected without removing the chorion, at different developmental stages, and fast frozen in liquid nitrogen for qPCR analysis. In situ Hybridization The plasmid containing the sequence for spon1b was kindly provided by Drs. Halpern and Gamse. After digestion with EcoRI, in vitro transcription was conducted using T7 RNA polymerase and digoxigenin labeling mix to generate antisense RNA probes. For in situ hybridization, the sections were brought to room temperate (RT) for 45 minutes. Then they were rehydrated in PBS 2 x 10 minutes. After triethanolamine/acetic anhydride treatment for 10 minutes, the sections were permeabilized in 1% Triton X-100/ PBS for 30 minutes and washed with PBS. Then, the slides were pre-hybridized for 2 h at RT in a solution containing 10% of 50%- Dextran sulfate (American Bioanalytical), 20% 20X SSC buffer (Ambion), 50% formamide (American Bioanalytical), 2% 50X Denhardt’s (Sigma), 5% fish sperm DNA (Invitrogen) and 15% DEPC water, followed by overnight hybridization with DIG- labeled RNA probes (0.2 ng/ul) at 65uC. The next day, the sections were incubated in 0.2X SSC at 65uC for 2 hours, followed by 2 more washes of 0.2XSSC at RT for 10 min each. Sections were blocked in 2% normal sheep serum in TBST for 1 hour at RT, and then incubated with anti-DIG alkaline phosphatase conjugate (1:7000, Roche) overnight at 4uC. The third day, the sections were washed with TBST 3610 minutes, and incubated in color reaction buffer (0.1 M Tris, 0.1 M NaCl, 0.05 MgCl2, 0.05% Tween-20) for 10 minutes. Then, the signal was visualized with BM Purple substrate solution (Roche) according to the manufacturer’s protocol, and the reaction stopped with PBS. Whole-mount in situ hybridization was performed essentially as previously described (Musson et al., 2009). Briefly, specimens were rehydrated, digested with Proteinase K, pre-hybridized for 2 hours at 70uC in solution containing 50% formamide, 56 SSC buffer, 1% sodium dodecyl sulfate, 50 mg/ml yeast RNA and heparin, followed by overnight hybridization with DIG-labeled RNA probes (0.2 ng/ml). Samples were then incubated with anti-DIG alkaline phosphatase conjugate overnight at 4uC. Signal was visualized with centrifuged BM Purple substrate solution (Roche Diagnostics), according to the manufacturer’s protocols. Immunohistochemistry Immunohistochemical procedures were performed following standard protocols [70]. In brief, sections were brought to RT and rehydrated in 0.1M KPBS. Cryosections were blocked in 5% Normal Donkey Serum in KPBS for 60 min (only for double fluorescent immunostaining), and incubated in primary antibodies diluted in 0.4% Triton-X100/KPBS for 48 h at 4uC. Then, sections were washed and incubated in secondary antibodies diluted in 0.4% Triton-X100/KPBS for 1 hour at RT. For double immunostaining experiments, sections were washed before repeating the same procedure with the following antibody. Slides were coverslipped using Immuno-mount (Vector). Modifications PLoS ONE | www.plosone.org 19 June 2012 | Volume 7 | Issue 6 | e37593