Original Article Multilineage hematopoietic progenitor activity generated autonomously in the mouse yolk sac: analysis using angiogenesis-defective embryos CHRISTINE RAMPON and PHILIPPE HUBER* CEA, Laboratoire de Développement et Vieillissement de l’Endothélium, Inserm EMI 02-19, DRDC, Grenoble, France ABSTRACT The capacity of the yolk sac to generate multilineage, adult-type hematopoiesis was investigated in vivo using vascular endothelial-cadherin deficient embryos. In these mutants, the yolk sac is not connected to the vasculature of the embryo and therefore all hematopoietic activity detected therein is intrinsic to the yolk sac and not derived from intraembryonic sources. At embryonic days 9.5 and 10.5, the yolk sac contains blood cells from the first wave of hematopoiesis, i.e. primitive erythrocytes and monocytes, but also multipotent progenitors from definitive hematopoiesis and a few granulocytes. Reverse transcription-polymerase chain reaction analysis revealed expression of specific genes of all lineages except lymphoid cells. Moreover, hematopoi- etic colony assays showed the existence of committed progenitors of the second wave of embryonic hematopoiesis, namely for definitive erythrocytes, megakaryocytes, granulocytes and monocytes. Conversely, the number of lymphocytes after lymphoid culture was insignificant. Our data provide evidence for multilineage hematopoiesis (but not lymphopoiesis) in the yolk sac in the absence of seeding from the embryo. The small number of definitive mature blood cells indicates however that the yolk sac is not an effective environment for the terminal differentiation of committed progenitors from the second wave of hematopoiesis. KEY WORDS: hematopoiesis, yolk sac, progenitor, transgenic mice, vascular endothelial-cadherin Int. J. Dev. Biol. 47: 273-280 (2003) 0214-6282/2003/$25.00 © UBC Press Printed in Spain www.ijdb.ehu.es *Address correspondence to: Dr. Philippe Huber. CEA, Laboratoire de Développement et Vieillissement de l’Endothélium, Inserm EMI 02-19, DRDC, 17, rue des Martyrs, 38054 Grenoble, France. Fax +33-43-878-4964. e-mail: phuber@cea.fr Abbreviations used in this paper: AGM, aorta-gonad-mesonephros; βH1, em- bryonic β-globin; β-maj, adult β-globin; BFU, burst-forming unit; CFU, colony-forming unit; E, embryonic day; FCS, fetal calf serum; GEMM, granulo-erythro-myelo-megakaryocytic; HPRT, Hypoxanthine phosphorybosyltransferase; IL, interleukin; mAb, monoclonal antibody; MGG, May-Grünwald-Giemsa; Mk, megakaryocyte; MPO, myeloperoxydase; PBS, phosphate buffer saline; PECAM, platelet/endothelial cell adhesion molecule; PF4, platelet factor 4; P-Sp, paraaortic splanchnopleura; SCF, stem cell factor; VE-cadherin, vascular endothelial-cadherin; YS, yolk sac. Introduction During mouse development, the first site of active hematopoie- sis is the yolk sac (YS), where red blood cells can be detected as early as embryonic day (E)7.5 (Moore and Metcalf, 1970). This primary hematopoiesis appears in mesoderm-derived blood is- lands in the YS wall and produces primitive nucleated erythro- cytes and macrophages. Once the vitello-embryonic circulation is established at E8.5, these cells are delivered into the circulatory system of the embryo until they are replaced at E10.5 by a second wave of blood cells resulting from adult-type hematopoiesis (for review, see Morales-Alcelay et al., 1998; Cumano and Godin, 2001; Orkin and Zon, 2002). At this time of development, hemato- poiesis is established in the fetal liver and generates multiple blood cell types, i.e., enucleated erythrocytes synthesizing adult globins, monocytes, granulocytes, megakaryocytes and lympho- blasts. The liver is the principal organ supporting blood cell production until hematopoiesis initiates in the bone marrow at the end of gestation. It is well established that the liver is not a site of emergence of hematopoietic progenitors and depends on coloni- zation by circulating cells. However, the origin of adult hematopoi- etic progenitors has been a question of debate for the last thirty years. The kinetic data of Moore and Metcalf (Moore and Metcalf, 1970) led to the hypothesis that committed adult-type progenitors found in the liver originated initially in the YS. Furthermore, the existence of definitive erythroid progenitors in the YS prior to vascular communication supported this view (Wong et al., 1986). Later on, two groups identified an intraembryonic site of emer- gence of definitive hematopoietic progenitors in the paraaortic