BRIEF COMMUNICATIONS Generating parabiotic cells of the aorta floor through a specific transformation that we have called the endothelial hematopoietic transition. These first zebrafish embryos for cell His s then enter the bloodstream to see a transient embryon migration and homing where they expand and differentiate before colonizing the thymus and kidney, the definitive sites of hematopoiesis in fish.5.8. studies For the study of hematopoietic stem and progenitor cells(HSPCs), their behavior and the influence of successive microenvironments Doris Lou Demy,,4, Zachary Ranta,4,4, on their lineage commitment, parabiosis has been a powerful tool in mouse 9,0 and quail-chick chimeras. In parabiosis experi Jean-Michel Giorgi, Magali Gonzalez ments, two genetically marked organisms are surgically conjoined a Philippe Herbomel1, 2 Karima KissaI-3 to share a common blood circulation, making it possible to analyze the interactions of the circulating cells from one partner with the g anabiosis, the surgical generation of conjoined organisms sharing potential homing sites in the other partner; this strategy provides a common bloodstream, has been a powerful tool for studying the means to discriminate whether a mutation that perturbs these hematopoietic cell migration and interaction with stromal interactions acts on the HSPCs or on the homing site. However, niches in rodent and avian systems. we describe a technique to parabiosis has not yet been applied to zebrafish. Here we describe a a generate parabiotic zebrafish embryos based on blastula fusion. methodology based on the fusion of zebrafish blastulae that leads to cell migration and homing to niches and peripheral tissues in This methodology can be applied to live imaging studies at single zebrafish parabiotes of different genetic backgrounds. cell resolution for the study of, for example, the effects of diffusible signals on cell lineage specification or the cell-autonomous versus E In zebrafish, as in mammals, hematopoiesis occurs in two waves. non-cell autonomous effects of specific genes. We illustrate the their interactions with stromal niches 2 tissues to become tissue-resident leukocytes.2. The 'definitive wave involves long-term hematopoietic stem cells(HSCs)that will zebrafish blastulae and allowing them to develop as partially fused e generate all blood cell lineages. In mammals, HSCs originate from embryos that share a common blood circulation an intraemb ronic region called the aorta-gonad-mesonephros and enter the bloodstream to colonize the fetal liver. where they expand and differentiate and subsequently seed the definitive hematopoietic organs, the thymus and bone marrow In zebrafish, previous studies*-6 have established that the thin space separating the dorsal aorta and axial vein in the trunk region is homolo- gous to the mammalian aorta-gonad-mesonephros. Recently we described how zebrafish HSCs derive directly from endothelial Figure 1 Zebrafish parabiosis by blastula fusion: experimental procedure. (a-g) Images of zebrafish blastulae(a-c; e-g, top) and parabiotes(d: e-g, bottom) through a dissecting microscope under transmitted light (a-c, e-g)or red fluorescence merged with transmitted Light(d).The boxed region in a is shown enlarged in b and c. Dechorionated embryos HoOdoo were placed in methylcellulose(a), scratched with a glass needle(b) 97hpf. 118 h-pf. and allowed to fuse(c). Rhodamine dextran-injected embryos were pink under transmitted light(a-c; e-g, top) and fluorescent( d). A, animal pole; M, margin; I, intermediate region(between A and M). Distinct fusion patterns resulted from different blastula fusion orientations: A-M(e), M-I(f)and I-I(g)fusions are shown. Scale bars, 250 um See also Supplementary Video 1 Recherche Scientifique, Unite de Recherche Associee 2578, Paris, France. Unite Mixte de Recherche 5235, Dynamique des Interactions Membranaires Normales et Pathologiques. Universite Montpellier 2, Montpellier, France. These authors contributed equally to this work. Correspondence should be addressed to KK(karima kissa @univ-montp2 fr) RECETVED 5 MARCH 2012: ACCEPTED 11 JANUARY 2013: PUBLISHED ONLINE 3 FEBRUARY 2013: DOL: 10.1038/NMETH 2362 NATURE METHODS I ADVANCE ONLINE PUBLICATION| 1
© 2013 Nature America, Inc. All rights reserved. brief communications nature methods | ADVANCE ONLINE PUBLICATION | cells of the aorta floor through a specific transformation that we have called the endothelial hematopoietic transition7. These first HSCs then enter the bloodstream to seed a transient embryonic site of hematopoiesis, the caudal hematopoietic tissue (CHT)4,8, where they expand and differentiate before colonizing the thymus and kidney, the definitive sites of hematopoiesis in fish4,5,8. For the study of hematopoietic stem and progenitor cells (HSPCs), their behavior and the influence of successive microenvironments on their lineage commitment, parabiosis has been a powerful tool in mouse9,10 and quail-chick chimeras11. In parabiosis experiments, two genetically marked organisms are surgically conjoined to share a common blood circulation, making it possible to analyze the interactions of the circulating cells from one partner with the potential homing sites in the other partner; this strategy provides the means to discriminate whether a mutation that perturbs these interactions acts on the HSPCs or on the homing site. However, parabiosis has not yet been applied to zebrafish. Here we describe a methodology based on the fusion of zebrafish blastulae that leads to parabiosis between embryos of two genetic backgrounds of interest. This methodology can be applied to live imaging studies at singlecell resolution for the study of, for example, the effects of diffusible signals on cell lineage specification or the cell-autonomous versus non–cell autonomous effects of specific genes. We illustrate the value of zebrafish parabiosis by following HSPCs in vivo and studying their interactions with stromal niches. The technique consists of fusing two differentially marked zebrafish blastulae and allowing them to develop as partially fused embryos that share a common blood circulation. Generating parabiotic zebrafish embryos for cell migration and homing studies Doris Lou Demy1,2,4, Zachary Ranta1,2,4, Jean-Michel Giorgi3, Magali Gonzalez3, Philippe Herbomel1,2 & Karima Kissa1–3 Parabiosis, the surgical generation of conjoined organisms sharing a common bloodstream, has been a powerful tool for studying hematopoietic cell migration and interaction with stromal niches in rodent and avian systems. We describe a technique to generate parabiotic zebrafish embryos based on blastula fusion. This procedure permits the in vivo visualization of hematopoietic cell migration and homing to niches and peripheral tissues in zebrafish parabiotes of different genetic backgrounds. In zebrafish, as in mammals, hematopoiesis occurs in two waves. The ‘primitive’ wave produces erythrocytes and myeloid cells; the latter differentiate in the yolk sac and then invade the embryonic tissues to become tissue-resident leukocytes1,2. The ‘definitive’ wave involves long-term hematopoietic stem cells (HSCs) that will generate all blood cell lineages. In mammals, HSCs originate from an intraembryonic region called the aorta-gonad-mesonephros and enter the bloodstream to colonize the fetal liver, where they expand and differentiate and subsequently seed the definitive hematopoietic organs, the thymus and bone marrow3. In zebrafish, previous studies4–6 have established that the thin space separating the dorsal aorta and axial vein in the trunk region is homologous to the mammalian aorta-gonad-mesonephros4. Recently we described how zebrafish HSCs derive directly from endothelial 1Institut Pasteur, Unité Macrophages et Développement de l’Immunité, Département de Biologie du Développement et Cellules Souches, Paris, France. 2Centre National de la Recherche Scientifique, Unité de Recherche Associée 2578, Paris, France. 3Unité Mixte de Recherche 5235, Dynamique des Interactions Membranaires Normales et Pathologiques, Université Montpellier 2, Montpellier, France. 4These authors contributed equally to this work. Correspondence should be addressed to K.K. (karima.kissa@univ-montp2.fr). Received 5 March 2012; accepted 11 January 2013; published online 3 February 2013; doi:10.1038/nmeth.2362 a 3.5 h.p.f. 3.5 h.p.f. 3.5 h.p.f. 24 h.p.f. 4 h.p.f. 4.5 h.p.f. 118 h.p.f. I-I 120 h.p.f. A-M M-I 3.5 h.p.f. 97 h.p.f. b c d e f g Figure 1 | Zebrafish parabiosis by blastula fusion: experimental procedure. (a–g) Images of zebrafish blastulae (a–c; e–g, top) and parabiotes (d; e–g, bottom) through a dissecting microscope under transmitted light (a–c, e–g) or red fluorescence merged with transmitted light (d). The boxed region in a is shown enlarged in b and c. Dechorionated embryos were placed in methylcellulose (a), scratched with a glass needle (b) and allowed to fuse (c). Rhodamine dextran–injected embryos were pink under transmitted light (a–c; e–g, top) and fluorescent (d). A, animal pole; M, margin; I, intermediate region (between A and M). Distinct fusion patterns resulted from different blastula fusion orientations: A-M (e), M-I (f) and I-I (g) fusions are shown. Scale bars, 250 µm. See also Supplementary Video 1
BRIEF COMMUNICATIONS Figure 2 Live imaging of hematopoietic cell pui: gfp/galaTa:dsred egfp/wild type behavior and homing in parabiotic zebrafish embryos and larvae (a-h)Fluorescence images of pul:gfp//gatala: dsred parabiotes with separate trunks and tails. GFP+ and DsRed+ myeloid cells from either embryo are seen in the yolk sac(a, inset, arrowheads )and tail (f, arrowheads)of the conjoined embryo; DsRed+ erythrocytes circulate in the yolk sac(a, b). 48 h-pf.d 48 h.p.f. heart(b, arrowhead)and tail of the pul: gfp(c, g)or atala: dsred(d, h) parabiotes. In contrast, nonmigratory GFP+ muscle cells(e) and DsRed* epidermal mucous cells(d, blue arrowheads) are present in only the respective bomb∥cd4gp transgenic embryo. This was observed in 24 of 24 embryos, pooled from three experiments (i-k)Fluorescence images of karl gfp/wild-type parabiotes. Fluorescent HSPCs from the kdr: p aorta are seen in the caudal hematopoietic tissue(i,j, arrowheads), thymus(k, arrowhead) and kidney(k, inset, dotted region) of the kdrtgfp//wild type(wild-type tail) 3 nontransgenic wild-type parabiote. This was 18 h.p.f. 72 h.p. g two experiments.(I)Fluorescence images of cd41: gfp//mib parabiotes. cd41:gfp GFP+ pro-thymocytes are seen in the thym rudiments of both the cd41: gfp(pink box and set)and the nontransgenic, noncirculating mind bomb mutant(blue box and inset). This was observed in 3 of 3 embryo pairs, in which the mind bomb mutant partner lacked circulation. Zebrafish parabiotes were imaged with a stereomicroscope(a-j, )or confocal microscope(k), under transmitted light(l) fluorescence(c-f l, insets)or both(a, b, g-k). Scale bars: 250 um(a, b, k, ) 100 um(c-j: k, inset; L, pink inset), 50 um(a, inset; L, blue inset) n, notochord; t, thymus; y, yolk sac:*, urogenital opening: WT, wild type. See also Supplementary Videos 2 and 3 backgrounds at the 128-cell blastula stage(Online Methods). of the embryos overnight( Supplementary Fig. 1). Therefore, in Using a Pasteur pipette, we collect both embryos and transfer them all subsequent experiments, blastulae were fused between the gether into a methylcellulose drop covered with high-calcium 256-to 512-cell stage and just before 30% epiboly Ringer's solution containing antibiotics. With a fine paintbrush, we We quantified the effect of blastula orientation on fusion s then gently move the methylcellulose around both embryos, bring- success and the resulting parabiosis patterns(Fig. le-g and (Fig. la and Online Methods; detailed protocol in Supplementary mostly gave rise to viable but malformed embryo pairs(10 of 17 Note). Using a pulled glass micropipette, we detach a few cells from Supplementary Fig. 2a, d, e). Blastulae fused by their blastoderm both blastulae at their contact point(Fig. Ib and Supplementary margins or by margin to intermediate region mostly separated Video 1). The methylcellulose is then moved again around the during the experiment (27 of 38 pairs). Among the 1l successful blastulae so as to press the wounds against each other and pro- fusions, 4 were malformed or died and 8 survived. Of these eight mote fusion(Fig. 1c). The next day, we replace the high-calcium to survive to parabiosis, five gave rise to parabiotes with separate nger's solution containing dissolved methylcellulose with heads(Fig. If and Supplementary Fig 2c-e). In contrast, fusions mbryo water containing antibiotics(Online Methods). Between between the animal pole of one blastula and the intermediate or 17%and 56% of the blastula pairs, depending on the develop- marginal region of the other led to a high rate of viable, well- mental stage(Supplementary Fig. 1), developed into viable and formed parabiotes by 2 d p f (22 of 51); of these, 19 were fused partially fused embryos that shared a common blood circulation by their heads( Fig. le and Supplementary Figs. 2b, d, e and 3) Cparabiotes)and that otherwise displayed normal overall mor- Blastulae fused by their intermediate regions also yielded a high phology and spontaneous movement(Fig. 1d-g) proportion of viable parabiotes by 2 d p f. (43 of 95), most of We found that the developmental stage at which blastula fusion which(31 of 43)had separate heads(Fig. 1g and Supplementary is performed is critical for successful parabiosis. Blastula fusion Fig 2d, e). Thus, by selecting the proper orientation upon blastula must be performed between the 256-cell and the 30% epiboly fusion, one can favor the generation of parabiotic pairs fused in stages to result in viable parabiotic embryos. Before the 256-cell patterns appropriate to the biological question to be addressed stage, fusion usually led to a single embryo of both genetic back- To assess the frequency of shared circulation of the fused grounds surrounding the two yolk sacs(data not shown). Blastula embryos, we fused transgenic gatala: dsred blastulae, in which fusions performed at the 256-to 512-cell stages led to 40% viable erythroid cells and early primitive myeloid cells are labeled, with parabiotic embryos by 1 d post-fertilization(d p f ),49% when nontransgenic partners. We analyzed 24 parabiotes displaying performed at 1,000-cell/high stages and 56% when done at sphere/ different fusion patterns(fused heads or fused tails)at 2 d p f, dome stage. The success rate then decreased abruptly to 17% by and all of them displayed shared blood circulation as evidenced by 2 I ADVANCE ONLINE PUBLICATION I NATURE METHODS
© 2013 Nature America, Inc. All rights reserved. | ADVANCE ONLINE PUBLICATION | nature methods brief communications First, we remove the chorions from embryos of two different backgrounds at the 128-cell blastula stage (Online Methods). Using a Pasteur pipette, we collect both embryos and transfer them together into a methylcellulose drop covered with high-calcium Ringer’s solution containing antibiotics. With a fine paintbrush, we then gently move the methylcellulose around both embryos, bringing them closer to each other in the proper orientation for fusion (Fig. 1a and Online Methods; detailed protocol in Supplementary Note). Using a pulled glass micropipette, we detach a few cells from both blastulae at their contact point (Fig. 1b and Supplementary Video 1). The methylcellulose is then moved again around the blastulae so as to press the ‘wounds’ against each other and promote fusion (Fig. 1c). The next day, we replace the high-calcium Ringer’s solution containing dissolved methylcellulose with embryo water containing antibiotics (Online Methods). Between 17% and 56% of the blastula pairs, depending on the developmental stage (Supplementary Fig. 1), developed into viable and partially fused embryos that shared a common blood circulation (‘parabiotes’) and that otherwise displayed normal overall morphology and spontaneous movement (Fig. 1d–g). We found that the developmental stage at which blastula fusion is performed is critical for successful parabiosis. Blastula fusion must be performed between the 256-cell and the 30% epiboly stages to result in viable parabiotic embryos. Before the 256-cell stage, fusion usually led to a single embryo of both genetic backgrounds surrounding the two yolk sacs (data not shown). Blastula fusions performed at the 256- to 512-cell stages led to 40% viable parabiotic embryos by 1 d post-fertilization (d.p.f.), 49% when performed at 1,000-cell/high stages and 56% when done at sphere/ dome stage. The success rate then decreased abruptly to 17% by the 30% epiboly stage, mainly because of separation and/or death of the embryos overnight (Supplementary Fig. 1). Therefore, in all subsequent experiments, blastulae were fused between the 256- to 512-cell stage and just before 30% epiboly. We quantified the effect of blastula orientation on fusion success and the resulting parabiosis patterns (Fig. 1e–g and Supplementary Fig. 2). Blastulae fused by their animal poles mostly gave rise to viable but malformed embryo pairs (10 of 17; Supplementary Fig. 2a,d,e). Blastulae fused by their blastoderm margins or by margin to intermediate region mostly separated during the experiment (27 of 38 pairs). Among the 11 successful fusions, 4 were malformed or died and 8 survived. Of these eight to survive to parabiosis, five gave rise to parabiotes with separate heads (Fig. 1f and Supplementary Fig. 2c–e). In contrast, fusions between the animal pole of one blastula and the intermediate or marginal region of the other led to a high rate of viable, wellformed parabiotes by 2 d.p.f. (22 of 51); of these, 19 were fused by their heads (Fig. 1e and Supplementary Figs. 2b,d,e and 3). Blastulae fused by their intermediate regions also yielded a high proportion of viable parabiotes by 2 d.p.f. (43 of 95), most of which (31 of 43) had separate heads (Fig. 1g and Supplementary Fig. 2d,e). Thus, by selecting the proper orientation upon blastula fusion, one can favor the generation of parabiotic pairs fused in patterns appropriate to the biological question to be addressed. To assess the frequency of shared circulation of the fused embryos, we fused transgenic gata1a:dsred blastulae, in which erythroid cells and early primitive myeloid cells are labeled, with nontransgenic partners. We analyzed 24 parabiotes displaying different fusion patterns (fused heads or fused tails) at 2 d.p.f., and all of them displayed shared blood circulation as evidenced by pu1:gfp//gata1a:dsred kdrl:gfp//wild type (wild-type tail) a Eye 25 h.p.f. Eye y 48 h.p.f. 48 h.p.f. 48 h.p.f. 72 h.p.f. * * * * * * * * y d f h c e g 48 h.p.f. y gata1a:dsred gata1a:dsred pu1:gfp pu1:gfp pu1:gfp tail gata1a:dsred tail b c d e f g h i j kdrl:gfp//wild type kdrl:gfp Eye Eye y n y 4.75 d.p.f. Ear WT 72 h.p.f. k mind bomb//cd41:gfp mind bomb y 72 h.p.f. t t Eye y y t t Ear Ear Eye Eye cd41:gfp l Figure 2 | Live imaging of hematopoietic cell behavior and homing in parabiotic zebrafish embryos and larvae. (a–h) Fluorescence images of pu1:gfp//gata1a:dsred parabiotes with separate trunks and tails. GFP+ and DsRed+ myeloid cells from either embryo are seen in the yolk sac (a, inset, arrowheads) and tail (f, arrowheads) of the conjoined embryo; DsRed+ erythrocytes circulate in the yolk sac (a,b), heart (b, arrowhead) and tail of the pu1:gfp (c,g) or gata1a:dsred (d,h) parabiotes. In contrast, nonmigratory GFP+ muscle cells (e) and DsRed+ epidermal mucous cells (d, blue arrowheads) are present in only the respective transgenic embryo. This was observed in 24 of 24 embryos, pooled from three experiments. (i–k) Fluorescence images of kdrl:gfp//wild-type parabiotes. Fluorescent HSPCs from the kdrl: gfp aorta are seen in the caudal hematopoietic tissue (i,j, arrowheads), thymus (k, arrowhead) and kidney (k, inset, dotted region) of the nontransgenic wild-type parabiote. This was observed in 7 of 7 parabiote pairs pooled from two experiments. (l) Fluorescence images of cd41:gfp//mib parabiotes. cd41:gfp GFP+ pro-thymocytes are seen in the thymic rudiments of both the cd41:gfp (pink box and inset) and the nontransgenic, noncirculating mind bomb mutant (blue box and inset). This was observed in 3 of 3 embryo pairs, in which the mind bomb mutant partner lacked circulation. Zebrafish parabiotes were imaged with a stereomicroscope (a–j,l) or confocal microscope (k), under transmitted light (l), fluorescence (c–f; l, insets) or both (a,b,g–k). Scale bars: 250 µm (a,b,k,l), 100 µm (c–j; k, inset; l, pink inset), 50 µm (a, inset; l, blue inset). n, notochord; t, thymus; y, yolk sac; *, urogenital opening; WT, wild type. See also Supplementary Videos 2 and 3
BRIEF COMMUNICATIONS DsRed* erythroid cells circulating in both embryos. This indicates colonizing the CHT and thymus of mib mutants( data not shown) that their vascular systems had anastomosed in the fused region. whereas in parabiotes in which the mib embryo had no blood circu By 4 d p f, 22 of the 24 parabiotes were still alive, and in 18 of lation, we observed only thymus colonization(n=3, Fig. 21). These them DsRed+ cells were still circulating in both embryos results show that Notch signaling deficiency does not affect the We applied this technique to follow the behavior of primitive ability of the Cht and thymus to attract and host wild-type HSPCs hematopoietic cells in parabiotes. We fused two embryos harbor- They also confirm and extend previous results showing that blood different reporter transgenes: Pul: gfp2, which labels primitive circulation is required for CHT but not thymus colonization,6. myeloid cells(macrophages and granulocytes), and gatala: dsred 3 We here demonstrate the usefulness of the zebrafish blastula Fig. 2a-h; note: pul is also known as spilb) At 18.5 h post fertiliza- fusion technique for studying hematopoietic cells and their inter tion(h p f )in parabiotic embryos displaying separate trunks and actions with stromal niches. Beyond hematopoietic cells, this tails, GFP primitive myeloid cells and DsRed* primitive erythroid technique can be used for studying other migratory cells such as and myeloid cells were detected in only the embryo of origin, at neural crest cells or circulating signals and their interaction with their normal location for this developmental stage(Supplementary their target tissue or processes such as innervation or vascular bud Fig 3). By 23 h p f, primitive myeloid cells born in the yolk sac of formation. It can be a powerful alternative to cell transplantation either embryo had started to invade the other embryo(Fig 2a and experiments for investigating cell-autonomous versus non-cell Supplementary Video 2). By 25 h.p. f, blood circulation started, autonomous gene function notably as both situations to be tested and the DsRed+ erythroid cells born in the trunk of the gatala: (mutant-cell migration into wild-type tissue and vice versa)occur dsred embryo were then seen circulating within both embryos, within every parabiotic pair. Finally, reverse genetic tools such as the shared circulation e parabiotes(Fig 2a-h, antisense morpholinos and mRNA injection can be applied to either 9 Supplementary Fig 3 and Supplementary Video 2). By 48 h.p.f., partner before fusion, thereby further extending the range of poten- a GFP+ myeloid cells and DsRed* myeloid and erythroid cells were tially valuable applications of this simple and powerful technique g found in the CHT of both parabiotes(100%, n=35; Fig 2c-h) Next we investigated the migration and homing of defini- METHODS tive HSPCs in parabiotes In the kdr: gfp transgenic line, GFP is Methods and any associated references are available in the online expressed in all vascular endothelial cells and their HSPC progeny version of the pape upon fusion of a kdr: gfp with a nontransgenic wild-type embryo, Note: Supplementary information is available in the online version of the paper. g colonization of the wild-type ChT by GFP+ HSPCs was detected 7 of 7 parabiotic pairs with separate trunks and tails examined, ACKNOWLEDGMENTS 是mxd31A3dpP6p山 expanded over the油可的乙la时t的 starting at 48h. p f. (Fig. 2i), and this HSPCs were found to colo- nize the thymic rudiment of the wild-type parabiote(Fig. 2k and La Recherche sur le Cancer. Z R was supported by a short-term fellowship from 9 a pattern remarkably similar to what has been previously observed the mia itere re e enseignement Sup. ieua et d e a rea chehe and thAe oin dation s the pronephric kidney of the wild-type parabiote(Fig. 2k). Thus, from la Region Languedoc-Roussillon o HSPCs born from the aorta of the karl: fp partner were able to AUTHOR CONTRIBUTIONS each and settle in the successive hematopoietic niches of both K.K. designed the experiments. D.L. D, Z.R. J -M G, M.G. and K.K. performed the parabiotes with the same timeline as in normal development. experiments. K.K. wrote the manuscript with input from D L D. and PH Another useful aspect of this approach is the opportunity to track COMPETING FINANCIAL INTERESTS circulating or migrating cells marked by widely expressed reporter The authors declare no competing financial interests transgenes In pul: gfpllgatala: dsred parabiotes-that is, parabiotes witheachembryoharboringaseparatetransgene-gfp+myeloidPublishedonlineathtpi://www.nature.com/doifinder/10.1038/nmeth.2362. cells could be clearly observed in the atala: dsred tail(Fig. 2f) com/reprints/index. html. without the strong ectopic GFP expression observed in the muscles of the pul gfp embryo(Fig. 2e); conversely, observation of Ds Red+ 1. HerbomeL P, Thisse, B.& Thisse, C. Dew. Biol. 238, 274-288(2001) myeloid and erythroid cells was easier in the pul;gfP partner tail 2. Le Guyader, D. et al. Blood 111, 132-141(2008) 3. Godin, I.& Cumano, A. Nat. Rev. Immunol. 2, 593-604(2002) (Fig. 2c), where DsRed was not expressed in epidermal mucous 4. Murayama, E. et aL Immunity 25, 963-975(2006) cells(Fig 2d). In kdr: gfp//wild-type parabiotes, tracking GFP+ 5. Jin, H, Xu, J& Wen, Z Blood 109, 5208-5214(2007) 6. Gering, M.& Patient, R Dev. Cell 8, 389-400(2005 HSPCs in the CHT, thymus and kidney was also easier in the 7. Kissa, K& Herbomel, P Nature 464, 112-115(2010) nontransgenic parabiote(Fig 2i-k and Supplementary Video 3) 8. Kissa, K et al. Blood 111, 1147-1156(2008) owing to the lack of vascular GFP expression. 9. Goldman, D.C. et al Blood 114, 4393-4401(2009) Last, we applied the blastula fusion technique to study the role of 10. Wright, D.E. Wagers. A.d., Gulati, A P, Johnson, F.L.& Weissman, L.L. in which Notch signaling is din ing the mind bomb(mib)mutant, 11. Dieterlen-Lievre, F. Martin, C. Beaupain, D. Foti biol(Praho)25 genes affecting hematopoiesis upted and definitive HSPCs do not 93-295(1979) form. We fused mib mutant blastulae with cd41: gfp transgenic 12. Hsu, K et al Blood 104, 1291-1297(2004) blastulae, in which HSPCs weakly express GFP6(note: cd41 is 13. Traver, D. et al Nat. Immunol. 4, 1238-1246(2003) Miller, C T, Schilling, T F, Lee, K, Parker, J.& KimmeL, C.B. also known as itga2b) In parabiotes with separate trunks and tails Development127.3815-3828(200 that shared a common bloodstream, we observed GFPlow HSPCs 15. Carmany-Rampey, A.& Moens, C.B. Methods 39, 228-238(2006). NATURE METHODS I ADVANCE ONLINE PUBLICATION3
© 2013 Nature America, Inc. All rights reserved. nature methods | ADVANCE ONLINE PUBLICATION | brief communications DsRed+ erythroid cells circulating in both embryos. This indicates that their vascular systems had anastomosed in the fused region. By 4 d.p.f., 22 of the 24 parabiotes were still alive, and in 18 of them DsRed+ cells were still circulating in both embryos. We applied this technique to follow the behavior of primitive hematopoietic cells in parabiotes. We fused two embryos harboring different reporter transgenes: pu1:gfp12, which labels primitive myeloid cells (macrophages and granulocytes), and gata1a:dsred13 (Fig. 2a–h; note: pu1 is also known as spi1b). At 18.5 h post fertilization (h.p.f.) in parabiotic embryos displaying separate trunks and tails, GFP+ primitive myeloid cells and DsRed+ primitive erythroid and myeloid cells were detected in only the embryo of origin, at their normal location for this developmental stage (Supplementary Fig. 3). By 23 h.p.f., primitive myeloid cells born in the yolk sac of either embryo had started to invade the other embryo (Fig. 2a and Supplementary Video 2). By 25 h.p.f., blood circulation started, and the DsRed+ erythroid cells born in the trunk of the gata1a: dsred embryo were then seen circulating within both embryos, demonstrating the shared circulation of the parabiotes (Fig. 2a–h, Supplementary Fig. 3 and Supplementary Video 2). By 48 h.p.f., GFP+ myeloid cells and DsRed+ myeloid and erythroid cells were found in the CHT of both parabiotes (100%, n = 35; Fig. 2c–h). Next we investigated the migration and homing of definitive HSPCs in parabiotes. In the kdrl:gfp transgenic line, GFP is expressed in all vascular endothelial cells and their HSPC progeny5. Upon fusion of a kdrl:gfp with a nontransgenic wild-type embryo, colonization of the wild-type CHT by GFP+ HSPCs was detected in 7 of 7 parabiotic pairs with separate trunks and tails examined, starting at 48 h.p.f. (Fig. 2i), and this population expanded over the next days (Fig. 2j). At 3 d.p.f., GFP+ HSPCs were found to colonize the thymic rudiment of the wild-type parabiote (Fig. 2k and Supplementary Video 3) by migrating through the mesenchyme in a pattern remarkably similar to what has been previously observed in unmanipulated larvae6. By 4.75 d.p.f., HSPCs were detected in the pronephric kidney of the wild-type parabiote (Fig. 2k). Thus, HSPCs born from the aorta of the kdrl:gfp partner were able to reach and settle in the successive hematopoietic niches of both parabiotes with the same timeline as in normal development. Another useful aspect of this approach is the opportunity to track circulating or migrating cells marked by widely expressed reporter transgenes. In pu1:gfp//gata1a:dsred parabiotes—that is, parabiotes with each embryo harboring a separate transgene—GFP+ myeloid cells could be clearly observed in the gata1a:dsred tail (Fig. 2f) without the strong ectopic GFP expression observed in the muscles of the pu1:gfp embryo (Fig. 2e); conversely, observation of DsRed+ myeloid and erythroid cells was easier in the pu1:gfp partner tail (Fig. 2c), where DsRed was not expressed in epidermal mucous cells (Fig. 2d). In kdrl:gfp//wild-type parabiotes, tracking GFP+ HSPCs in the CHT, thymus and kidney was also easier in the nontransgenic parabiote (Fig. 2i–k and Supplementary Video 3) owing to the lack of vascular GFP expression. Last, we applied the blastula fusion technique to study the role of genes affecting hematopoiesis using the mind bomb (mib) mutant, in which Notch signaling is disrupted and definitive HSPCs do not form4. We fused mib mutant blastulae with cd41:gfp transgenic blastulae, in which HSPCs weakly express GFP6 (note: cd41 is also known as itga2b). In parabiotes with separate trunks and tails that shared a common bloodstream, we observed GFPlow HSPCs colonizing the CHT and thymus of mib mutants (data not shown), whereas in parabiotes in which the mib embryo had no blood circulation, we observed only thymus colonization (n = 3, Fig. 2l). These results show that Notch signaling deficiency does not affect the ability of the CHT and thymus to attract and host wild-type HSPCs. They also confirm and extend previous results showing that blood circulation is required for CHT but not thymus colonization2,6. We here demonstrate the usefulness of the zebrafish blastula fusion technique for studying hematopoietic cells and their interactions with stromal niches. Beyond hematopoietic cells, this technique can be used for studying other migratory cells such as neural crest cells or circulating signals and their interaction with their target tissue or processes such as innervation or vascular bud formation. It can be a powerful alternative to cell transplantation experiments14 for investigating cell-autonomous versus non–cell autonomous gene function15, notably as both situations to be tested (mutant-cell migration into wild-type tissue and vice versa) occur within every parabiotic pair. Finally, reverse genetic tools such as antisense morpholinos and mRNA injection can be applied to either partner before fusion, thereby further extending the range of potentially valuable applications of this simple and powerful technique. Methods Methods and any associated references are available in the online version of the paper. Note: Supplementary information is available in the online version of the paper. Acknowledgments We thank B. Robert, Y. Lallemand, D. Montarras and T. Schilling for their critical reading of the manuscript. This work was partially supported by the Caisse Autonome Nationale de Sécurité Sociale dans les Mines and the Association pour la Recherche sur le Cancer. Z.R. was supported by a short-term fellowship from the Weissman International Internship Program; D.L.D. by PhD fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche and the Fondation pour la Recherche Médicale; and J.-M.G. and M.G. by a Chercheur d’Avenir grant from la Région Languedoc-Roussillon. AUTHOR CONTRIBUTIONS K.K. designed the experiments. D.L.D., Z.R., J.-M.G., M.G. and K.K. performed the experiments. K.K. wrote the manuscript with input from D.L.D. and P.H. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/doifinder/10.1038/nmeth.2362. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Herbomel, P., Thisse, B. & Thisse, C. Dev. Biol. 238, 274–288 (2001). 2. Le Guyader, D. et al. Blood 111, 132–141 (2008). 3. Godin, I. & Cumano, A. Nat. Rev. Immunol. 2, 593–604 (2002). 4. Murayama, E. et al. Immunity 25, 963–975 (2006). 5. Jin, H., Xu, J. & Wen, Z. Blood 109, 5208–5214 (2007). 6. Gering, M. & Patient, R. Dev. Cell 8, 389–400 (2005). 7. Kissa, K. & Herbomel, P. Nature 464, 112–115 (2010). 8. Kissa, K. et al. Blood 111, 1147–1156 (2008). 9. Goldman, D.C. et al. Blood 114, 4393–4401 (2009). 10. Wright, D.E., Wagers, A.J., Gulati, A.P., Johnson, F.L. & Weissman, I.L. Science 294, 1933–1936 (2001). 11. Dieterlen-Lièvre, F., Martin, C. & Beaupain, D. Folia biol. (Praha) 25, 293–295 (1979). 12. Hsu, K. et al. Blood 104, 1291–1297 (2004). 13. Traver, D. et al. Nat. Immunol. 4, 1238–1246 (2003). 14. Miller, C.T., Schilling, T.F., Lee, K., Parker, J. & Kimmel, C.B. 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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 rhodaminedextran (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 temperature. Lower, 2–4%, methylcellulose concentrations were also used successfully; a lower concentration allows an easier reorientation of embryos, whereas a higher concentration requires better dexterity but also ensures a higher pressure between both blastulae, 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 penicillinstreptomycin, 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 performed 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–chargecoupled-device video camera (HVD-20; Hitachi), recorded on miniDV 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 glassbottom 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)