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Supplementary Materials and MethodsDistel et al. 10.1073/pnas.0903060106SI TextMaintenance of Fish. Raising, spawning, and maintaining of ze-brafish were performed as described (1, 2). All experiments wereconducted according to the guidelines reviewed by the EthicsCommittee of the Helmholtz Zentrum Munchen.
Construction of Vectors. pCR2xUAS, pCR3xUAS, pCR4xUAS. Two, 3, and4 repeats of the Gal4-binding site (CGGAGTACTGTCCTC-CGAG) containing 5� 1 PstI and 3� 1 XbaI restriction site weresynthesized by Entelechon GmbH and were provided in thepCR-Script Amp vector (Stratagene).pSK5xUASE1b. pBSUASE1bNotch:intra (3) was EcoRI-digestedand religated. The 5xUASE1bTATA fragment from this vectorwas isolated by a PstI/EcoRI digest and cloned into a PstI/EcoRI-digested pBluescript SK- (pBSK, Stratagene).pSK1xUASE1b. pSK5xUASE1b was digested with NotI/XbaI toreplace the 5 UAS repeats with a single UAS site using theannealed oligos GGCCGCAGATCTCTGCAGCGGAGTACT-GTCCTCCGAGT and CTAGACTCGGAGGACAGTACTC-CGCTGCAGAGATCTGC.pSK1xUASFFL, pSK5xUASFFL. To clone firefly (Photinus Pyralis)luciferase under control of 1x or 5xUASE1b respectively theORF of the firefly luciferase gene was isolated from pPLLUCII(kind gift of J. Graw) by a XhoI/HindIII digest and cloned intoXhoI/HindIII-digested pSK1xUASE1b or pSK5xUASE1b re-spectively.pSK2xUASFFL, pSK3xUASFFL, and pSK4xUASFFL. pCR2xUAS,pCR3xUAS, and pCR4xUAS were digested with XbaI/ApaI. Afragment containing the basal promoter E1b (3) was isolatedfrom pSK5xUASlzkmRPF by a XbaI/ApaI digest and clonedbehind the 2x, 3x, and 4xUAS sites respectively. Subsequently,the 5xUASE1b-fragment of vector pSK5xUASE1bFFL was re-placed by 2x, 3x, and 4xUASE1b-fragments, which were isolatedby a PstI/HindIII digest from the respective 2x, 3x, and4xUASE1b vectors.14xUASE1bFFL. The ORF coding for firefly (Photinus Pyralis)luciferase was isolated from pPLLUCII (kind gift of J. Graw) bya XhoI/SalI digest and cloned downstream of the 14xUASE1bsites of the pBSK- vector used to generate the UG vector (4).SVTkRenilla. The SV40 enhancer/promoter fragment in front ofthe ORF coding for Renilla (Renilla Reniformis) luciferase of thepRL-SV40 vector (Promega) was removed by a BglII (Klenowblunted)/HindIII digest and replaced by the SVTk enhancer/promoter element from the XbaI (Klenow blunted)/HindIII-digested pSVtklacZ vector (kind gift of Christoph Winkler).pCSGalTA2, 3, and 4. Gal4 activator plasmids with minimal VP16transactivation domains were generated by removing the VP16domain from the CMV-GVP vector (4) by a XbaI (Klenowblunted)/EcoRI digest and inserting the TA2, TA3, and TA4transactivation domains from the ptTA2, 3, and 4 vectors(Clontech catalog K6243–1) as a SmaI/PpuMI (Klenow-blunted)fragment.pCSKalTA4. The Gal4 DNA binding domain from yeast wasresynthesized as a EcoRI/SalI fragment to remove potentialmethylation sites, inhibitory secondary mRNA structures and tooptimize the codon usage for expression in zebrafish by Entele-chon GmbH, Regensburg, Germany starting with a GCCGC-CACC Kozak sequence for efficient ribosome binding. Theresulting EcoRI/SalI Kal fragment was inserted into EcoRI/SalI-digested pCSGalTA2, 3, and 4 plasmids to replace the yeast Gal4DNA-binding domain.
pCSGIKalTA4. The rabbit �-globin intron was isolated from thepXIG vector (kind gift of Adam Amsterdam) by a BamHI/SalIdigest and inserted as Klenow-blunted fragment into the EcoRI(Klenow blunted)-digested pCSKalTA4 vector.pCSKalTA4GI. The rabbit �-globin intron from the pXIG vector wassubcloned as a SalI/XbaI fragment into the pBluescript SK-vector (Stratagene). From here the intron was isolated by aBamHI (Klenow blunted)/SalI digest and inserted into theXhoI/SnaBI-digested pCSKalTA4 downstream of KalTA4.pCSKalTA4SVI. The SV40 intron was isolated by a SpeI (Klenowblunted)/NotI digest from pWI x/b (kind gift of Ava Udvadia andElwood Linney) and cloned downstream of KalTA4 into theXhoI (Klenow blunted)/NotI-digested pCSKalTA4 vector.TG5xR (pBSceI-twhhGalTA45xUASmRFP). A NotI-expression cassetteof 5xUAS-EIb driving the expression of the mRFP fluorescentprotein was cloned behind a NotI-digested expression cassette ofthe notochord-specific twhh promoter element (5) driving theexpression of GalTA4 in pBSK-2xSce [see also Tg(twhh:G-UmRFP) (6)].TK1xC (pTol2Sce-twhhKalTA4GI1xUASKcherryGI). The MCS of thepBluescript SK- vector (Stratagene) with 2 flanking I-SceI siteswas inserted into the NdeI (Klenow blunted)-digested pTol2000vector (7) to result in the plasmid pTol2000Sce. A NotI-expression cassette of 1xUAS-EIb driving the expression of themCherry fluorescent protein flanked 5� by the Kozak sequenceGCCGCCACC and 3� by the rabbit �-globin intron was clonedbehind a NotI-digested expression cassette of the notochord-specific twhh promoter element (5) driving the expression ofKAlTA4GI in pBSK-2xSce. This entire cassette could be re-leased by a I-SceI restriction digest and was inserted intopTol2000-Sce.TK5xC (pTol2Sce-twhhKalTA4GI5xUASKcherryGI). A NotI-expressioncassette of 5xUAS-EIb driving the expression of the mCherryfluorescent protein flanked 5� by the Kozak sequence GCCGC-CACC and 3� by the rabbit �-globin intron was cloned behind aNotI-digested expression cassette of the notochord-specific twhhpromoter element (5) driving the expression of KalTA4GI inpBSK-2xSce. This entire cassette was released by a I-SceIrestriction digest and was inserted into pTol2000-Sce.pCSGI. A BamHI/SalI Klenow-blunted fragment containing therabbit �-globin intron was inserted into the SnaBI site of thepCS2� vector (8).pTolmini. This plasmid was generated by inserting minimizedinverted repeats recognized by the Tol2 transposase amplifiedfrom vector pTol2000 (9) using primers:
473: ATTGGTACCCAGAGGTGTAAAGTACTTGAGTA-ATTTTAC and
474: ATACTCGAGCCGGGCCCAAGTGATCTCCAA and475: ATTCTCGAGATTAGATCTAATACTCAAGTACA-
ATTTTAATGGAG and476: ATTCCGCGGCAGAGGTGTAAAAAGTACTCAA-
AAATinto Asp-718/SacII-digested pBluescript SK- (Stratagene). AMCS from the pBluescript SK- vector flanked by 2 I-SceI siteswas inserted in the XhoI (Klenow blunted) site between the Tol2inverted repeats to allow for the I-SceI-mediated insertion offragments of interest.4xKGFP (pTolmini-4xUASKGFPGI). GFP was amplified frompEGFP-N3 (Clontech) using primers
204: TACTCGAGTTACTTGTACAGCTCGTCCAT and620: TATGAATTCGCCGCCACCATGGTGAGCAAGGGC-GAGGAGCTGTTCAC and inserted in the EcoRI/XhoI site in
Distel et al. www.pnas.org/cgi/content/short/0903060106 1 of 7
front of the rabbit �-globin intron of pCSGI. The KGFPGIpAfragment was isolated from this vector by a EcoRI/Asp-718 digestand cloned into EcoRI/Asp-718 digested pSK4xUASFFL toreplace the luciferase. The resulting 4xUASKGFPGIpA-fragment was isolated as a NotI fragment and cloned intoNotI-digested pTolmini.4xKaloop (pTolmini-4xUASKGFP-T2A-KalTA4GI):. A bicistronic T2A-peptide linked expression cassette (10) of GFP-T2A-KalTA4flanked 5� by the Kozak sequence GCCGCCACC in the pCS-GIvector was cloned as a EcoRI/Asp-718 fragment behind 4xUA-SEIb in pBSK. Subsequently, the entire 4xUASEIb-KGFP-T2A-KalTA4-GI was inserted into pTolminiSce as a NotI fragment.
Further details of cloning procedures are available on request.
Luciferase Assays. Zebrafish Pac2 embryonic fibroblasts (11, 12)were transfected using the ‘‘Nanofectin ’’ transfection kit (PAA)according to the manufacturers‘ specifications. Concentrationsfor determining optimized activator activities were 8 ng/�Lactivator plasmids and 80 ng/�L effector plasmids. Concentra-tions used to determine the activity of different numbers of UASrepeats were either both 80 ng/�L or 80 ng/�L for activator and8 ng/�L for effector plasmids. Luciferase assays were performedusing the ‘‘Dual Luciferase Reporter Assay System’’ (Promega).As reporter firefly (Photinus Pyralis) luciferase was cloneddownstream of the respective 1, 2, 3, 4, 5, or 14xUAS:E1bsequences. Renilla (Renilla Reniformis) luciferase under controlof the SVTk promoter was used as control for transfectionefficacy. Luciferase assays were performed in 96-well plates with3 replicates per experiment and analyzed by a ‘‘MicroplateLuminometer Orion’’ using the simplicity 2.0R1 software (bothBerthold Detection Systems). Obtained light intensities fromfirefly luciferase were corrected for transfection efficacies andbackground in the following way:
Vfirefly � MVLuciferase untransfected cells)/(VRenilla �MVRenilla untransfected cells) � Vfirefly E correctedVfirefly E corrected � MVpCS�XxUAS � V firefly B/E correctedVfirefly � measured firefly signalVRenilla � measured Renilla signalMVRenilla untransfected cells � mean value of measured Renillasignal of non-transfected cellsMVLuciferase untransfected cells � mean value of measured fireflysignal of non-transfected cells.MVpCS � XxUAS � Mean of transfection efficacy corrected
signal of cells transfected with pCS without Gal4 activatorand respective 1, 2, 3, 4, 5, or 14xUAS vector
DNA Extraction and Inverse Nested PCR. To determine the integra-tion sites in the transgenic zebrafish KalTA4 enhancer trapstrains genomic DNA (gDNA) was purified from a singletransgenic zebrafish embryo (around 5 dpf) using the gDNAwizard kit (Promega) according to the manufacturer’s instruc-tions. gDNA was digested using the restriction enzymes AluI,HaeI (both Fermentas) and MboI (NEB) respectively and self-ligated after heat-inactivation of the restriction enzymes byadding T4 DNA ligase (Fermentas). After ligation gDNA wasethanol precipitated and resuspended in water. Inverse nestedPCR was performed for 5� and 3� ends of the enhancer trapconstruct using the primer pairs Tol2–5F1/Tol2–5R1 and Tol2–3F1/Tol2–3R1 for the first round of PCR
Tol2–5F1: AGTACTTTTTACTCCTTACATol2–5R1: GTATTGATTTTTAATTGTACTCAAGTol2–3F1: TTTACTCAAGTAAGATTCTAGTol2–3R1: CTCCATTAAAATTGTACTTGAand Tol2–5F2/Tol2–5R2 respectively Tol2–3F2/Tol2–3R2 as
primers for the second round of the nested PCR.Tol2–5F2: CTCCTTACAATTTTATTTACAGTCTol2–5R2: CTCAAGTAAAGTAAAAATCCCCTol2–3F2: ACTTGTACTTTCACTTGAGTATol2–3R2: GCAAGAAAGAAAATAGAGABoth cycles were carried out with Taq Polymerase (Fermen-
tas) and gDNA from the respective line. PCR cycles were both:94 °C 2�, 35 � (94 °C 30,‘‘ 52 °C 45,’’ 72 °C 1.5�), 72 °C 10�, 4 °CHold). PCR products were either subcloned or sent directly forsequencing. Sequences were blasted against the zebrafish ge-nome using the Ensembl database (http://www.ensembl.org/Danio�rerio/Info/Index).
Rhodamine Dextran Retrograde Labeling. Adult zebrafish brainswere dissected in PBS and fixed onto sylgard plates (WorldPrecision Instruments) in DMEM (Invitrogen) using insect pins.After dissection, the tectum was removed to inject dextrantetramethylrhodamine (MW: 3000 kDa, Invitrogen) into thecentral nucleus of the torus semicircularis. After injection brainswere perfused with CO2 for 5 h to allow for retrograde transportof the rhodamine dextran. Afterward, brains were fixed in 4%PFA over night and vibratome sectioned at 100-�m thickness.
1. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonicdevelopment of the zebrafish. Dev Dyn 203:253–310.
2. Westerfield M (1995) in The Zebrafish Book (University of Oregon Press, Eugene, OR).3. Scheer N, Campos-Ortega JA (1999) Use of the Gal4-UAS technique for targeted gene
expression in the zebrafish. Mech Dev 80:153–158.4. Koster RW, Fraser SE (2001) Tracing transgene expression in living zebrafish embryos.
Dev Biol 233:329–346.5. Du SJ, Devoto SH, Westerfield M, Moon RT (1997) Positivie and negative regulation of
muscle cell identity by members of the hedgehog and TGF-ss gene families. J Cell Biol139:145–156.
6. Babaryka A, Kuhn E, Koster RW (2009) In vivo synthesis of meganuclease for generatingtransgenic zebrafish. J Fish Biol 74:452–457.
7. Kawakami K, et al. (2004) A transposon-mediated gene trap approach identifiesdevelopmentally regulated genes in zebrafish. Dev Cell 7:133–144.
8. Rupp RAW, Snyder L, Weintraub H (1994) Xenopus embryos regulate the nuclearlocalization of Xmyod. Genes Dev 8:1311–1323.
9. Urasaki A, Morvan G, Kawakami K (2006) Functional dissection of the Tol2 transposableelement identified the minimal cis-sequenceand a highly repetitive sequence in thesubterminal region essential for transposition. Genetics 174:639–649.
10. Provost E, Rhee J, Leach SD (2007) Viral 2A peptides allow expression of multipleproteins from a single ORF in transgenic zebrafish embryos. Genesis 45:625–629.
11. Amsterdam A, et al. (1999) A large-scale insertional mutagenesis screen in zebrafish.Genes Dev 13:2713–2724.
12. Chen W, Burgess S, Golling G, Amsterdam A, Hopkins N (2002) High-throughputselection of retrovirus producer cell lines leads to markedly improved efficiency ofgerm line-transmissible insertions in zebra fish. J Virol 76:2192–2198.
Distel et al. www.pnas.org/cgi/content/short/0903060106 2 of 7
Fig. S1. Transgenic zebrafish KalTA4 enhancer trap strains. A schematic representation of the trapping construct TK1xC is shown at the top of the figure. (A–X)Confocal microscopy images of Bodipy ceramide-counterstained (green) embryos from isolated KalTA4 enhancer trap lines. Embryos were screened fortissue-specific mCherry fluorescence (red) between 24 and 36 hpf; images were recorded at approximately 50 hpf using a Zeiss LSM510 and a 20� objective. (A–L,Q–T, and U–W) lateral views (W 6dpf heart), (M–P) ventral views and (X) dorsal view. For detailed description of expression patterns see the database at:http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines�distel/.
Distel et al. www.pnas.org/cgi/content/short/0903060106 3 of 7
Fig. S2. Analysis of isolated zebrafish KalTA4 enhancer trap strains. (A) Numbers of independent transgenic founders in the P0, F1, and F2 generation of theKalTA4 enhancer trap screen. (B) Screenshot of searchable database in which images of different expression patterns from many strains are displayed. In addition,sequences of the respective Gal4-activator and UAS effector vectors as well as a protocol for nested inverse PCR to determine the integration sites of the enhancertrap vector in different lines can be found at: http://www.helmholtz-muenchen.de/en/idg/groups/neuroimaging/lines�distel/. (C) Distribution of expressionpatterns in the isolated tissue specific KalTA4 enhancer trap lines of the F2 generation. (D) Distribution of all observed expression patterns throughout theisolated KalTA4 strains of the F2 generation. Note that many lines show mCherry expression in several tissues.
Distel et al. www.pnas.org/cgi/content/short/0903060106 4 of 7
Fig. S3. Comparative mRNA in situ hybridization analysis to determine the onset of expression in rhombomeres 3 and 5 of (A–C) egr2b in WT embryos, (D–F)kalTA4 and (G–I) gfp in offspring of rh3/5:KalTA4 � 4xKGFP cross. (J–L) mRNA in situ hybridization analysis of downregulation of expression of kalTA4 and (M–O)gfp in rhombomeres 3 and 5 of rh3/5:KalTA4 � 4xKGFP embryos. Arrows indicate expression domains of the respective genes.
Distel et al. www.pnas.org/cgi/content/short/0903060106 5 of 7
Fig. S4. Retrograde labeling of secondary octaval nucleus (SON) neurons. Adult brain cryostat sections (100 �m) of rhodamine dextran injected adultrh3/5:KalTA4 � 4xKaloop fish. (A) Rhodamine dextran (red) injection site in the central nucleus of the torus semicirculars (TS). The arrow demarcates the sitewhere the tectum was removed. (B) Rhodamine dextran (red) positive axons innervate the TS coming from caudal projection nuclei and (C, enlarged area of Bmarked with yellow square) some are positive for GFP expression (white arrow). (D) Rhodamine dextran-positive axons are derived from hindbrain neurons, (E)they cross the midline at the level of r3 and r5 and (F) turn dorsally (G–I, enlarged area of F marked with yellow square) revealing their origin from GFP-expressingsomata of rh3/5:KalTA4 � 4xKaloop labeled neurons. Abbr.: llf: lateral longitudinal fascicle, r: rhombomere, TS: torus semicircularis.
Distel et al. www.pnas.org/cgi/content/short/0903060106 6 of 7
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Distel et al. www.pnas.org/cgi/content/short/0903060106 7 of 7
