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 experi￾ments, 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 single￾cell 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 study￾ing 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 homolo￾gous 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