REVIEWS Signalling pathways that control vertebrate haematopoietic stem cell specification Wilson K. Clements1 and David Traver2 Abstract | Haematopoietic stem cells (HSCs) are tissue-specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Clinically, HSCs form the foundation of transplantation-based therapies for leukaemias and congenital blood disorders. Researchers have long been interested in understanding the normal signalling mechanisms that specify HSCs in the embryo, in part because recapitulating these requirements in vitro might provide a means to generate immune-compatible HSCs for transplantation. Recent embryological work has demonstrated the existence of previously unknown signalling requirements. Moreover, it is now clear that gene expression in the nearby somite is integrally involved in regulating the transition of the embryonic endothelium to a haemogenic fate. Here, we review current knowledge of the intraembryonic signals required for the specification of HSCs in vertebrates. Induced pluripotent Haematopoietic stem cells (HSCs) are self-renewing Establishment of adult haematopoiesis stem cell blood and immune cell precursors capable of produc- In all vertebrates, the establishment of self-renewing (iPS cell). An adult cell ing daughter cells that proliferate and mature to pro- HSCs with the full set of lineage potentials is preceded reprogrammed by one of vide all adult blood effector cells, including erythroid, during development by earlier primitive and definitive several protocols to 1 (FIG. 1a) ‘pluripotency’, a state myeloid and lymphoid cells . Clinically, these cells ‘waves’ of haematopoiesis , which take place in var- competent to form all are the relevant component of bone marrow trans- ious anatomical locations (FIG. 1b). Primitive myeloid and embryonic tissues. plants, which are used to treat patients with leukaemia erythroid waves (BOX 1) transiently generate limited sets of and congenital blood disorders, but the availability of effector cells. In Xenopus laevis, the spatial separation of immune-compatible donors remains a problem2. The cells fated to generate primitive and definitive haemato­ advent of induced pluripotent stem cell (iPS cell) technol- poietic tissues is well defined, with primitive precursors ogy has raised the possibility of making HSCs from a occupying the ventral blood islands (VBIs) and definitive patient’s own non-haematopoietic tissues3,4, but it is not blood arising from the dorsal lateral plate mesoderm (DLP 1Department of Hematology, possible so far to convert pluripotent cells to HSCs that mesoderm)7–10; separation occurs as early as the 32‑cell Division of Experimental are capable of long-term self-renewal and generation stage7. Interestingly, these populations retain significant Hematology, St Jude of normal distributions of the complete set of mature plasticity with respect to their potential to adopt primi- Children’s Research Hospital, 5,6 262 Danny Thomas Pl., blood cell lineages . This suggests that key specification tive or definitive haematopoietic fates, as transplantation Memphis, Tennessee 38105, requirements are unknown. Attempts to generate HSCs of VBI cells to the DLP mesoderm and vice versa until USA. in vitro could be informed by understanding the nor- mid-neurula stages results in cells taking on the identity 2Department of Cellular and mal in vivo mechanisms that generate these cells during specified by the site of engraftment11. These results indi- Molecular Medicine and Section of Cell and embryonic development. A clear understanding of the cate that the signalling environment has a major role in Developmental Biology, developmental specification of HSCs might moreover assignment of primitive versus definitive haematopoietic University of California at provide insight into the causes of congenital diseases, fates until relatively late in development. San Diego, 9500 Gilman Dr., such as aplastic anaemias and congenital neutropenia. Definitive haematopoiesis can be split into two waves. La Jolla, California Recent work in multiple species has uncovered several An earlier wave proceeds through a multipotent progeni- 92093‑0380, USA. Correspondence to D.T. previously unknown signalling inputs required for HSC tor — known as the erythromyeloid progenitor (EMP) e‑mail: [email protected] specification; here, we review these advances and place — with lineage potential limited to erythrocytes, mega- doi:10.1038/nri3443 them in a developmental context. karyocytes and myeloid cells12–19. This wave is followed 336 | MAY 2013 | VOLUME 13 www.nature.com/reviews/immunol © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS a Mouse (embryonic day) E7.0 E8.0 E9.0 E10.0 E11.0 E12.0 E13.0 E14.0 Primitive macrophages Primitive haematopoiesis Primitive erythrocytes EMPs Definitive haematopoiesis HSCs 18 h 22 h 26 h 30 h 34 h 38 h 42 h 46 h Zebrafish (hours post fertilization) b Primitive Definitive c Mouse (E10.5) Ventral blood islands haematopoiesis haematopoiesis A ventroposterior region of the Aorta–gonads–mesonephros Placenta Sclerotome embryo in Xenopus laevis and Blood Dorsal islands other species that houses aorta Mesonephros primitive haematopoiesis, in particular erythropoiesis. Mouse Yolk Gonad Dorsal lateral plate sac Dorsal mesoderm aorta Yolk sac Hind gut (DLP mesoderm). Trunk E7.5 E10.5 mesoderm in Xenopus laevis Zebrafish (32 hours post fertilization) that is lateral to the more Anterior lateral plate Caudal d mesoderm axial notochord and somitic Yolk haematopoietic Notochord tissue mesoderm. The DLP Hypochord mesoderm contains tissue Sclerotome fated to form haemogenic Haemogenic endothelium (and thus Zebrafish Dorsal endothelium haematopoietic stem cells) Intermediate aorta and is equivalent to zebrafish cell mass Pronephros lateral plate mesoderm and 18 hours 32 hours Dorsal aorta HSC Posterior mammalian splanchnic post fertilization post fertilization mesoderm. cardinal vein Figure 1 | Haematopoietic stem cells. a | Haematopoietic stem cell (HSC) specification is preceded by earlier waves of Mid-neurula embryonic haematopoiesis. Primitive macrophages and erythrocytes arise first. A transient definitive erythromyeloid In Xenopus laevis, a time in the progenitor (EMP) population also precedes HSCs. b | Primitive blood in mouse derives from extra-embryonicNature Reviews tissue | Immunology of middle of formation of the the yolk sac in blood islands. In zebrafish, primitive red blood cells derive from mesoderm of the intermediate cell mass neural plate that occurs and primitive myeloid cells derive from anterior lateral plate mesoderm. EMPs are specified in the mouse yolk sac and between the end of gastrulation (formation of the zebrafish caudal haematopoietic tissue. c,d | HSCs (red) appear at embryonic day (E)10.5 in mouse and 32 hours post germ layers) and the initiation fertilization in zebrafish from haemogenic endothelium of the dorsal aorta, from where they bud into the arterial of somitogenesis. circulation (mouse; c) or the venous circulation (zebrafish; d). Haemogenic endothelium Endothelial cells found most notably in the ventral floor of by the specification of HSCs that self-renew for the life of Some lineage tracing studies suggest that HSC specifica- 24,25 the dorsal aorta that give rise the individual and are capable of producing all adult line- tion also occurs in the yolk sac . However, the signal- to haematopoietic stem cells. ages, including lymphocytes. That EMPs produce myeloid ling events involved in HSC specification in the dorsal and erythroid lineages from a single progenitor and that aorta are currently the best understood, and this site of Yolk sac An extra-embryonic tissue murine EMPs produce erythrocytes expressing adult emergence for HSCs is conserved in vertebrate models derived from the fertilized egg globins has caused some confusion, especially in in vitro such as frog and fish, which has helped to illuminate the in mammals that ultimately assays seeking to generate HSCs, because it is impossible native processes controlling specification. It is likely that surrounds the embryo and at to distinguish EMPs and HSCs on the basis of assays with many (but probably not all26,27) mammalian HSCs arise early times functions as a only an erythromyeloid readout. Furthermore, EMPs and in the intrae­mbryonic dorsal aorta20,28,29. In chick, graft- signalling centre. The yolk sac is also the site of primitive HSCs cannot be distinguished by cell surface phenotype. ing experiments have shown that adult blood is entirely 30 and transient definitive These issues highlight the need to test haematopoietic pre- embryo derived , and in X. laevis, grafting experiments haematopoiesis. cursors for their long-term reconstitution and lymphoid have established that adult blood comes from the tissue potential side‑by‑side with bone marrow HSCs when fated to form dorsal aorta10. Furthermore, in zebrafish31,32 Dorsal aorta 33 The earliest trunk vessel determining the success of in vitro HSC specification. and mouse explants the emergence of HSCs from and precursor to the adult endothelium in the ventral floor of the embryonic dorsal descending aorta. Endothelium Anatomy of HSC specification aorta has been directly visualized, despite differences in of the dorsal aorta forms from Across vertebrate phyla, HSCs arise from an endothe- the directionality of budding (into the venous circulation splanchnic mesoderm and lial precursor found specifically in arterial haemogenic in zebrafish compared with