ONLINE METHODS then transferred. The methylcellulose around them was then Zebrafish stocks and embryo rearing Wild-type AB; Pul: gfp l2, moved with a fine paintbrush to bring them close to each other gatala: dsred, karl gfp and cd41: gfp transgenic; and mind and in proper orientation for fusion. a pulled glass micropipette bomba mutant embryos were raised and staged according to was then used to detach a few cells from both blastulae at their Westerfield(http://zfin.org/zf_info/zfbook/zfbk.html).EmbryocontactpointasdemonstratedinSupplementaryVideo1.The water was Volvic water with 280 ug/l methylene blue and 0.003% methylcellulose was then moved again around the blastulae so phenylthiourea(PTU)to inhibit melanin synthesis as to press the wounds against each other. Blastula fusions were performed in this way on successive blastula pairs starting at the Rhodamine-dextran cell labeling. I nl of a 10 mg/ml rhodamine- 512-cell stage up until the 30% epiboly stage. Fusions were per dextran(MW=10,000 Da; Invitrogen) solution was injected to formed at room temperature during a 2. 5-h time window(the the one-to four-cell stag time it takes for embryos at room temperature to go from the 512-cell stage to 30% epiboly), which allowed the generation of Blastula fusion procedure. 4% methylcellulose was prepared by 30-40 blastula fusions per embryo clutch per experimenter. After dissolving 2 g of methylcellulose powder in 50 ml embryo water. blastula fusion, the dishes were left on the bench for 20-30 min Complete dissolution required several hours under agitation as to avoid any shaking that could separate the two blastulae. The well as vigorous vortexing. It was then stored at room tempera- dishes were then fully filled with HCR containing antibiotics and ture. Lower, 2-4%, methylcellulose concentrations were also transferred to the incubator at 28C. The next morning, the HCR used successfully; a lower concentration allows an easier reorien- solution with dissolved methylcellulose was replaced by embryo tation of embryos, whereas a higher concentration requires better water containing PTU and antibiotics as above. g dexterity but also ensures a higher pressure between both blas- Figure 1 and Supplementary Video 1 were generated using a a tula, resulting in a higher success rate of fusion High-calcium MZ16 stereomicroscope(Leica)equipped with a three-charge- g Ringer's solution(200 ml) was prepared by adding 4 ml of 5 M coupled-device video camera(HVD-20; Hitachi), recorded on min- gNaclsolution(116mm),200ulof3MKclsolution(2.9mm),idVtapesandcapturedwithBtvPro(http://www.bensoftware.com/) 400 ul of 5 M CaCl2 solution(10 mM)and 1 ml of 1 M HEPEs and iMovie softwares; Figure 2a-j, l was generated using a Macro Fluo solution(5 mM)to 195 ml embryo water and was stored at 4C (Leica)equipped with a Roper camera and Meta view software for a few weeks Embryos were manually dechorionated at the 256-cell stage Time-lapse confocal fluorescence imaging of live zebrafish 8 with fine(Dumont #5)forceps l6 in two separate scratchless glass embryos and larvae Embryos were anesthetized with tricaine16 s were deposited on a 30-mm plastic Petri dish and c. -cellulose immobilized in 1% low-melting point agarose on 35-mm glass- high-calcium Ringer's containing antibiotics(50 U/ml penicillin- medium and imaged on an SPE confocal inverted microscope g streptomycin, 50 U/ml ampicillin, 0.5 ug/ml kanamycin and (Leica)(Fig. 2k and Supplementary Videos 2 and 3)as described 0.5 ug/ml gentamicin). Two embryos, one of each of the two previously genetic backgrounds to be fused, were collected with a g a Pasteur pipette. The tip of the pipette was used to make a small 16. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of NATURE METHODS doi:10.1038/nmeh2362© 2013 Nature America, Inc. All rights reserved. nature methods doi:10.1038/nmeth.2362 ONLINE METHODS Zebrafish stocks and embryo rearing. Wild-type AB; pu1:gfp12, gata1a:dsred13, kdrl:gfp7 and cd41:gfp8 transgenic; and mind bomb2 mutant embryos were raised and staged according to Westerfield16 (http://zfin.org/zf_info/zfbook/zfbk.html). Embryo water was Volvic water with 280 µg/l methylene blue and 0.003% phenylthiourea (PTU) to inhibit melanin synthesis16. Rhodamine-dextran cell labeling. 1 nl of a 10 mg/ml rhodamine￾dextran (MW = 10,000 Da; Invitrogen) solution was injected to embryos at the one- to four-cell stage. Blastula fusion procedure. 4% methylcellulose was prepared by dissolving 2 g of methylcellulose powder in 50 ml embryo water. Complete dissolution required several hours under agitation as well as vigorous vortexing. It was then stored at room tempera￾ture. Lower, 2–4%, methylcellulose concentrations were also used successfully; a lower concentration allows an easier reorien￾tation of embryos, whereas a higher concentration requires better dexterity but also ensures a higher pressure between both blas￾tulae, resulting in a higher success rate of fusion. High-calcium Ringer’s solution (200 ml) was prepared by adding 4 ml of 5 M NaCl solution (116 mM), 200 µl of 3 M KCl solution (2.9 mM), 400 µl of 5 M CaCl2 solution (10 mM) and 1 ml of 1 M HEPES solution (5 mM) to 195 ml embryo water and was stored at 4 °C for a few weeks. Embryos were manually dechorionated at the 256-cell stage with fine (Dumont #5) forceps16 in two separate scratchless glass Petri dishes. Seven to nine small drops of 4% methylcellulose were deposited on a 30-mm plastic Petri dish and covered with high-calcium Ringer’s containing antibiotics (50 U/ml penicillin￾streptomycin, 50 U/ml ampicillin, 0.5 µg/ml kanamycin and 0.5 µg/ml gentamicin). Two embryos, one of each of the two genetic backgrounds to be fused, were collected with a glass Pasteur pipette. The tip of the pipette was used to make a small well in the methylcellulose drop, to which the embryo pair was then transferred. The methylcellulose around them was then moved with a fine paintbrush to bring them close to each other and in proper orientation for fusion. A pulled glass micropipette was then used to detach a few cells from both blastulae at their contact point, as demonstrated in Supplementary Video 1. The methylcellulose was then moved again around the blastulae so as to press the wounds against each other. Blastula fusions were performed in this way on successive blastula pairs starting at the 512-cell stage up until the 30% epiboly stage. Fusions were per￾formed at room temperature during a 2.5-h time window (the time it takes for embryos at room temperature to go from the 512-cell stage to 30% epiboly), which allowed the generation of 30–40 blastula fusions per embryo clutch per experimenter. After blastula fusion, the dishes were left on the bench for 20–30 min to avoid any shaking that could separate the two blastulae. The dishes were then fully filled with HCR containing antibiotics and transferred to the incubator at 28 °C. The next morning, the HCR solution with dissolved methylcellulose was replaced by embryo water containing PTU and antibiotics as above. Figure 1 and Supplementary Video 1 were generated using a MZ16 stereomicroscope (Leica) equipped with a three–charge￾coupled-device video camera (HVD-20; Hitachi), recorded on min￾iDV tapes and captured with BTV Pro (http://www.bensoftware.com/) and iMovie softwares; Figure 2a–j,l was generated using a MacroFluo (Leica) equipped with a Roper camera and MetaView software. Time-lapse confocal fluorescence imaging of live zebrafish embryos and larvae. Embryos were anesthetized with tricaine16, immobilized in 1% low–melting point agarose on 35-mm glass￾bottom dishes (Iwaki), covered with tricaine containing embryo medium8 and imaged on an SPE confocal inverted microscope (Leica) (Fig. 2k and Supplementary Videos 2 and 3) as described previously7. 16. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio) 4th edn. (University of Oregon Press, 2000)