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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. REVIEW ARTICLE Developmental Biology of Hematopoiesis By Leonard I. Zon T HE MYSTERIOUS GENESIS of hematopoietic stem cells during early vertebrate development has intrigued investigators for several centuries. The molecular steps that lead to embryonic hematopoiesis remain to be determined, although recent studies have begun to define early events and migration patterns that regulate the hematopoietic program. This review will provide a historical background of the developmental biology of vertebrate hematopoiesis and highlight the systems and methods that are being used to study the regulation of the hematopoietic stem cell during embryogenesis. THE ONTOGENY OFPRIMITIVE AND DEFINITIVE HEMATOPOIETIC CELLS During vertebrate ontogeny, hematopoietic sites change as new populations of stem cells emerge. In mammalian development, stem cells sequentially occupy the embryonic yolk sac, fetal liver, spleen, and adult bone marrow (BM). The factors that regulate homing to hematopoietic sites have not been identified, yet lower vertebrates provides some information about the process. The BM is first used in evolution as a site of adult hematopoiesis by certain amphibians (Rana species but not Xenopus).’ Seasonal changes can cause a shift in sites of hematopoiesis in some amphibians (between the spleen and BM), possibly because of alterations in circulating steroid levels.’ Although the yolk sac, fetal liver, spleen, and BM are common sites of hematopoiesis in many vertebrates, their use is not required, and some organisms have developed unique characteristics of these hematopoietic sites. The chicken does not use the fetal liver as a major erythropoietic organ but instead maintains blood formation on the yolk sac and in the mesenteric regions until BM hematopoiesis is established. In some organisms, hematopoiesis occurs in unusual places such as the ovary, testes, adrenal glands, and endocardium. Therefore, hematopoietic stem cells possess the ability to migrate within the organism to a region that will support their existence and allow subsequent differentiation. In early studies of hematopoiesis, a model was suggested in which fetal and adult hematopoietic stem cells are derived from the original embryonic blood stem cells that occupy the early yolk sac (Fig 1, top). In this model, a yolk-sac stem cell migrates or “metastasizes” to a fetal hematopoietic site, such as the liver, where it expresses a fetal program on From the Division of Hematology, Children’s Hospital, Boston, MA. Submitted January 20, 1995; accepted June 6, 1995. Supported by National Institutes of Health Grant No. HL48801. L1.Z. is an Assistant Investigator of the Howard Hughes Medical Institute. Address reprint requests to Leonard I. Zon, MD, Division of Hematology, Children’s Hospital 300 Longwood Ave, Boston, MA 02115. 0 1995 by The American Society of Hematology. oooS-4971/95/8608-0011$3.00/0 2876 differentiati~n.~ Therefore, the activation of the fetal or adult program relies predominantly on transcriptional regulation, which could be triggered by environmental cues. Although this modelhad certain appeal, other studies showed that embryonic blood cells represent a distinct population of cells from the fetal hematopoietic cells (Table 1). For instance, embryonic erythrocytes morphologically are larger in size, are nucleated, and have a more generous cytoplasm than fetal-adult blood cells. Embryonic hematopoietic cells are mostly restricted to the erythroid lineage in vivo, although this maypartly be due to characteristics of the hematopoietic microenvironment. Embryonic cells are constantly cycling during development and do not pause for very long periods of time to differentiate, whereas fetal and adult hematopoietic stem cells remain in Go for extensive periods of time and are only activated occasionally, when needed. Finally, embryonic, fetal, and adult hematopoietic cells each synthesize distinct globins. These differences suggest a model4-*(Fig 1, bottom) in which the embryonic (“primitive”) and fetauadult (“definitive”) hematopoietic stem cells are distinctly derived early in development. Primitive cells undergo programmed cell death during fetal life, and only defintive progenitors give rise to fetal and adult hematopoiesis (Fig l, bottom). A corollary to the primitivedefinitive model suggests that globin switching is largely governed by changes in cell populations rather than transcriptional events. INDUCTION AND MIGRATION OF HEMATOPOIETIC STEM CELLS AND PROGENITORS Hematopoietic Stem Cells Are Derived f r o m Ventral Mesoderm To comprehend the early events that lead to embryonic hematopoiesis, it is necessary to understand pattern formation of the early vertebrate embryo. Classical studies in developmental biology have used amphibians to examine embryogenesis (see Figs 2, 3, and 6), and the general principles of embryonic development are maintained in higher organisms. The unfertilized Xenopus egg contains an animal pole (top hemisphere by convention) and a vegetal (bottom hemisphere) p0le.’3’~ After fertilization, the dorsal axis of the embryo will arise opposite the sperm entry point, and, by the 32-ce11 stage, a single ventral cell (the C4 blastomere, Fig 2) is fated to become blood. The early events of embryogenesis are driven solely by maternal determinants deposited in the egg during oogenesis. The mid-blastula transition defines a developmental period, approximately 7 hours after fertilization, when maternal mRNAs degrade and zygotic transcription intiates. An early vertebrate embryo at the blastula stage consists of two germ layers, ( l ) ectoderm, which is located in the animal pole and is fated to become skin and neural tissues, and (2) endoderm, which is located in the vegetal pole and later becomes the gut. During gastrulation, morphogenetic movements (invagination just below the equator of the embryo at the blastopore lip) brings ectoderm into apposition with endoderm. By the elaboration of Biood, Vol 86,No 8 (October 15), 1995: pp 2876-2891 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2077 DEVELOPMENTALBIOLOGY OF HEMATOPOIESIS Hemo lobin Swilch )D P Embryonic Site Table 1. Comparison of Embryonic, Fetal, and Adult Erythropoiesis Hemo lobin Swilch Fetal Site )D Adulb Site ( b ! . ....).iYI 4 --- - - - - - ---- Embryonic Site Fetal Site Embryonic All Lineages Stem cell Erythroid site H M C X F Adult Site Fig 1. Two developmental models for hematopoiesis. In the top model, embryonic hematopoietic stem cells (P) migrate from the yolk sac t o the fetalliver, where they differentiateinto fetal hematopoietic stem cells (D). Later, the second switch o w n when the fetal liver stem cells migrate t o t hBM. e In the bottommodel, embryonic hematopoietic stem cells (P)exit the yolksac and undergo programmed cell death. Another distinct populationof cells (D) migrates from the yolk sac t o the intra-embryonic dorsalmesentery or f e t a l liver and becomes the definitive pool. Some definitive cells from the fetal source can also migrate t o t h eembryonic site. P, primitive cells; D, definitive cells. soluble peptide growth factors or by cell-cell interactions, the endoderm induces ectoderm to form a third germ layer known as mesoderm. This layer is initidly located between the two other germ layers (ectoderm and endoderm) in a middle layer called the marginal zone. Mesoderm is the undifferentiated, self-renewing cells of the embryo that will provide stem cells for the following tissues (from ventral to dorsal across the marginal zone): blood, mesenchyme, kidney, muscle, and notochord. merefore, blood development involves the induction, proliferation, and differentiation Of ventral mesoderm. Yolk Sac Hematopoiesis In most vertebrate organisms, hematopoietic stem cells and vascular progenitors migrate to an extra-embryonic position on the yolk sac (which is considered ventral by embyologists) to form the blood islands. Later, these cells enter the embryo to form the primitive and definitive hematopoietic lineages and a subset of the vasculature, respectively (Fig 4).5.11"5A blood island consists of at least three layers: an endodermal layer that supports growth, a central core of Nucleated RBC H Erythroid Cycling All GO Yolk sac Yolk sac Yolk sac Ventral island Liver Liver Yolk sac, diffuse Liver, (kidney) IM Pronephros, liver Adult Go BM BM, spleen BM Spleen, liver, (BM) Kidney, liver, spleen Yes Yes Yes Yes Yes No No NO No Yes Yes Yes Yes Yes Yes M C P. OH, P A PA X PT1 PT1 PA M C X F a Globin H M C X p Globin H Only major globins are listed; eg, murine Pm globin is a low-abundance variant of Ph1globin. The values shown in parentheses indicate site(s) used by Some amphibian Abbreviations: H, human; M, mice; C, chicken; X, Xenopus; F, fish fteleostst; RBC, red blood ce,ls hematopoietic stem cells that ultimately differentiates into erythroblasts, and an endothelial layer that surrounds the blood island as it is developing (Fig 4,bottom).16 Mammals. In the mouse, on day 7 'h, blood island formation begins in a central area of the egg cylinder. At day 7 there are regions of thickening of the inner mesodermal layer in contact with the endoderm, which is thought to support the growth of the hematopoietic tissue. Endothelial cells subsequently differentiate and encompass the hematopoietic progenitors, which then budoff of the endodermal base, forming a blood island. By day 9, there is a capillary network, and mature erythroid cells enter circulation, but yolk sacblood formation proceeds through day 12. In hu- animalpole Fig 2. A 32-cell fate map of the Xenopus embryo. This was constructed by injection of various dyes into a single cell (also called blastomere) of each tier of the 32csll embryo and following dye the as develpredomiopment proceeds.The C4 blastomere is the nant cellfated to form blood (Reprinted with permission.'") Fetal/Prehatching/ Larval dor sal ventral vegetalpole From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2878 LEONARD I. ZON Animal Pole e$(+-) MK W Vegetal Pole Blastula Gastrula mans, the process is similar to that for the mouse; hematopoiesis occurs initially at day 15 and continues for 6 weeks of gestation (Fig 4). Mammalian embryonic erythroid cells are nucleated, similar to the erythroid cells of lower vertebrates including fish, frogs, reptiles, and chickens. Although most progenitor populations are present in the yolk sac, the terminal differentiation occurs primarily to the erythroid lineage.'"19 Occasionally, monocytes and megakaryocytes have been identified in blood islands. Birds. The early embryology of blood formation has been well-defined in the chicken (Fig 5A, B, and C)." The boundary of the embryo is not distinct until after primitive steak development, but the area opaca is considered extraembryonic even from early stages (Fig 5A). Cells originating from the epiblast (the upper layer) invaginate through the primitive streak and enter the area pellucida between the epiblast and hypoblast (the lower layer) by 15 to 16 hours. The cells are later separated into somatic and splanchnic layers. Extra-embryonic splanchnoderm consists of two different layers, an endodermal layer and a mesodermal layer that will ultimatelygive rise to blood islands in the yolk sac. The vascular area invades the vitelline area, the two layers become indistinct, and, eventually, the entire yolk sacis vascularized. The initial blood islands are seen in the posterior area opaca (Fig 5B) at the head-process or early somite stages, at which time the posterior area pellucida becomes vascularized. Hemoglobin can be detected as early as the 24 somite stage." As the yolk sac spreads over the yolk surface (Fig 5C), the central vascular area consists of blood vessels, sinus marginalis, and blood islands. The yolk sac in chickens will remain hematopoietic for most of embryonic development. Amphibians. Amphibians form blood in the ventral region of the embryo (Fig 6A). Brauns" described the prospective blood island region at early neurula stage as a monolayered or bilayered sheets of cells extending along the entire ventral region of the embryo. At first, these cells migrate slowly in a ventral direction, then quickly migrate to form the characteristic hematopoietic cords of blood island in a Dorsal Fig 3. Formation of the mesodermgermcell layer. Signals from endoderm (arrows1 located the in vegetal pole induce mesoderm, which is derived enfrom tirely the animal ectoderm. pole The mesoderm ."" layer isalso called themarginal zone, whose cell fate is organized in a ventral-to-dorsal pattern of blood (B), mesenchyme (MI, kidney (K),somites (SI,and notochord (NI. "V" shape (Fig 6B). As ventral migration occurs, the lateral plate mesoderm converges between the blood cells and skin and yields the endothelium necessary for circulation. Excision of this region during neurula stages leads to tadpoles with no circulating blood within a normal circulatory system.22.23Bloodless tadpoles can live for over l month, indicating that blood is not critical to early embryonic development in amphibians. In Xenopus, hemoglobinization occurs in an anterior-toposterior directionz4 starting at 36 hours after fertilization. The island is anteriorly bounded by hepatic endoderm, which is thought to secrete erythropoietin-like activity that accounts for the anterior-to-posterior wave of hemoglobinization. The heart starts beating at stage 33/34 (45 hours), the anterior cells of the blood island enter the heart from the primitive vasculature, and circulation is fully established at stage 351 36 (50 hours). The immune system begins to develop between stages 42-45 (80 hours), when the thymus becomes histologically visible. Fish. Bony fish (teleosts) form embryonic erythroid cells in a distinct dorsal-lateral compartment of the embryo known as the intercellular mass (IM) of Oellachef' (described in 1872; see Fig 7), and are, therefore, an exception to the general vertebrate rule of embryonic hematopoiesis on the yolk ~ a c . * ~The - ~ *IM is derived from lateral mesoderm at the posterior border of the embryo and is similar in position to the initial yolk sac-derived hematopoietic mesoderm in higher vertebrate^.^' Some Teleosts such as the angelfish, killifish, and cartilaginous fish form embryonic blood both on the yolk sac and in the IM.33 Dorsal Hematopoiesis In addition to ventral embryonic (or yolk sac) hematopoiesis, most vertebrates have an additional intra-embryonic stem cell population in the dorsal mesentery (also called the AGM region for aorta, gonad, mesonephros region) that colonizes later fetal (larval) sites of blood formation. In higher vertebrates, this dorsal population predominantly gives rise to * Fig 4. Mammalian embryonic blood formation. Human embfyo at various stages. By day 16, cdls from the opiM.rt begin to invaginate. in intra- and extra-embryonkIocdons. At day 'IS, the expmQd picture on and form the m d e r m a l layer. By day 18, mesoderm is pr-nt the islrnd is adb-nt the left demonstrates the typical appearonce of a blood island. Erythroblasts are surrounded by endothelial cells, and to the endoderm. (Adapted and reprintedwith permiuion'"1 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS 2879 Day I Day2 Day3 Fertilization 2 Cell Stage Morula Duy4 Blastocyst Early _.* ._. :_. ., .._. + _ DuyS . ,Maternal and Late Blastocyst Trophot)last Vessels piblast ,Amnion -Ectoderm Endoderm - Hypoblast - - Yolk sac Duy 16 Duy 18 Primitive Streak" Ectoderm Mesoderm (invaginattngl Endoderm. Yo1k sac Duyf9 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LEONARD 1. ZON 2880 40-42 HOUKS 7-8Hours 7 Hensen's Node --. ..,.L%Y -Area Pellucida B -A Area Pellucida Opaca Islands Fig 5. Avian embryonicblood formation. (A) Early mesoderm inductionuntil 15 to 16 hours. The invagination of mesoderm through the primitive streak is depicted in a cross-section. (B)Chicken embryonic hematopoiesisat the 7-somite stage. (Adapted and reprinted with permission.') Note that theinitial hematopoietic progenitors arise in the area opaca. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. DEVELOPMENTALBIOLOGY 2881 OF HEMATOPOIESIS C /, Area pellucida l \ Vascular area inus terminalis Fig 5. (Cont'd) (C) Expansion of yolk sac hematopoiesis from day 1 (top embryo), day 2 (second), day 3 (third), and day 4 (bottom embryo). (Adapted and reprinted with permission.") definitive hematopoiesis, but in lower vertebrates both primitive and definitive cells arise from these cells. Studies using an in vitro embryo culture assay show that the yolk sac is required for both primitive and definitive hematopoiesis in mammals. Culture of day-7 (early 1-4 somite) mouse embryos in tissue-culture plates showed normal somite development, yolk sac hematopoiesis, and a beating h e a t 3 Culture of day-7 yolk sac alone yielded abundant hematopoietic colonies, but culture of day-7 embryos from Yolk ' which the yolk sac has been removed develops without blood in a normal circulatory system. In some of these embryos, there was an absence of fetal liver hematopoietic progenitors, suggesting that the genesis of the definitive hematopoietic program requires the yolk sac structure. Therefore, the primitiveand definitive lineages are likely to be derived from common progenitor cells colocalized in the yolk sac (IM or ventral blood island) region very early during embryogeneSIS. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LEONARD I. ZON 2882 (3hours,32cellsl B/ustu/u (6hours;5000 cellsl Mid Blastula .. - ""_"""" ~ ~ " " " " " " " Gusfru/u (70 hours;3Q 000 cellsl Neuru/u (20hours; 90,000 celisl Epidermis System 7"Stuge (40hours; 200,000ceils Notocord Endoderm Lateral Plate (Mesenchyme) 7udpo/e Fig 6. Xenopus embryonic blood formation. (A) Schematic view of hematopoietic induction and development (Adapted and reprinted with permis~ion.'~') The Xenopus egg contains an animal and a vegetal pole that are distinguishable by pigment. The blastomere of the embryo that will become blood is determined as early as the 32cellembryo (shaded). Presumably, the vegetal blastomere under this shaded region is responsible fortheinductionof ventral type mesoderm. Mesoderm is derived from the ectodermal animal pole. T w o p r e dominant signals have been postulated t o affect the process of mesoderm induction, the ventral signal (V) and the dorsal signal (D). During gpstrula stages, the Spemann organizer (arrow) functions t o pattern mesoderm across themarginal zone (the centralregion of the embryo). The commitment t o form blood occurs in the ventral region of the marginal zone. Later in neurula and tailbudstages, blood is the most ventral mesoderm, and is locatedadjacent t o the epidermal layer. (B) Xenopus embryo at stage 28 (about 34 hours after fertilization). This embryo is stained for embryonic @-globin usingwholeembryoimmunohistochemistry analysis. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2883 DEVELOPMENTALBIOLOGY OF HEMATOPOIESIS 18 somite Intermediate Cell Mass hematopoietic ~undifferentiated =vasculogenic 0 Intermediate Fig 7. Zebrafish embryonic hematopoiesis, a 24 schematic view. Blood is formed in the IM, a dorsal somite intra-embryonic location. At 18 somites, the mass consists of intermittent hematopoietic progenitors. Undifferentiated cells will become vascular by the 24-somite stage. The posterior region of the mass remains undifferentiated and may represent a stem cell pool. (Adapted and reprinted with p e r m i s ~ i o n . ~ ~ l The migration of hematopoietic progenitors during and after neurula has been extensively studied in amphibians. By transplanting a cytogenetically distinct ventral blood island tissue into wild-type hosts, a small ventral portion of the Xenopus neurula embryo has been shown to contain primitive and definitive erythroid and T-cell/myeloid progenitors (Fig gA).h.7.34-42This region in Rana pipiens contributes to primitive erythropoiesis but does not contribute to T-cells or myeloid cell populations>14h In Rana cutesbeiana, the definitive progenitors of the dorsal mesentery colonize the larval liver, whereas the primitive progenitors of this region colonize the larval pronephro~."~ Thus, the distribution of progenitor cells in the ventral or dorsal compartment of the embyro is not conserved during vertebrate evolution, even in closely related species. Cell transplantation studies have also shown that T-cell progenitors are derived from the posterior region of the embryo during neurula stages (Fig 8B), and these cells migrate anteriorly across tissue planes to eventually colonize the dorsal aorta region, the postcardinal veins, and the thymus. The thymus may also be colonized by circulating cells. The migratory pattern of hematopoietic cells throughout amphibian development is schematically drawn in Fig 8C. The embryonic region to which the stem cells are transplanted influences the frequency of contribution to definitive cell lineages (Fig 8D).3hLymphoid contribution is significantly increased when cells are transplanted to a peripheral location in the embryo, whereas erythroid contribution is increased when cells are placed in a central region. Hence, environment apparently exerts a directive effect on stem cells of the embryo to form particular hematopoietic tissues. The embryonic location of definitive hematopoietic progenitors in birds has been studied with interspeciesgrafts between chickens and quails, so-called "yolk sac chimera^."^.' In these experiments, age-matched chicken and quail eggs are incubated for 30 to 36 hours (between8-12 somites), and the quail embryonic body is grafted into the analogous position of a chicken blastoderm. The quail embryo develops on a chick yolk sac, and, at varying developmental stages, quail cells are recognized by morphological differences or by markers such as immunofluorescencewiththequail-specificmonoclonal antibodies, QH 14x or MB I >9 QHl and MB 1 recognize glycoproteins on quail hematopoietic, vascular, and germ cell lineages, but not on chicken cells. Between d3 and d5, chickens and quails have hematopoietic foci on the ventral wall of the a ~ r t a . ~ .These ~ . ~ " foci may be induced by endoderm that physically associates with the ventral surface of the two dorsal aortae before they fuse." By day 6-8, the definitive hematopoietic progenitors in chicken and quail embryos are distributed differently in the mesenchyme (Fig 9A). In the chicken, definitive hematopoietic foci are found throughout themesenteryand around visceral organs, including the dorsal region that will become the thoracic duct. In the quail embryo, hematopoietic tissue is found associated with the anterior cardinal vein system, at the angle of the duct of Cuvier. These dorsal hematopoietic cells are only separated from the vascular lumen by endothelium and can infiltrate the wall oflarge venous vessels. Using the QHI marker, cells from the dorsal regions can be followed as they colonize the yolk sac or thymus by direct migration or as they differentiate in situ. The dorsal hematopoietic program of avians is regulated by local environment. At day 3-4, mesodermal cells surrounding the quail dorsal aorta can be reciprocally transplantedto the analogous position in the chick embryo." The donor quail cells contribute to blood-forming foci and, occasionally, to endothelial structures based on QHI staining. When day 3-4 mesodermal cells are cultured with day 6.5 chick thymic rudiment, the cells stain with QHl and also react with the CTl antibody (a T-cell marker). This suggests that the mesoderm adopts a T-cell program in the environment of the thymus. Recently, similar dorsal hematopoietic cell populations have been shown for mammals (Fig 9).s2-s4 Spleen colonyforming unit activity was shown in the region of the aorta, gonad, and mesonephros from day-8 to day- 1 1 embryos. By grafting intra-embryonic splanchnopleura from IO- to18somite embryos in SCID mice, IgM-secreting plasma cells and the B 1a cell subset were reconstituted. Thus, the dorsal Compartment in mice is ultimately capable of hematopoietic development as documented for early spleen colony-forming unit and B-cell development. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LEONARD I. ZON A General Hypothesis for Migration Pathways of Hematopoietic Cells During Vertebrate Embryogenesis During early gastrulation, induced ventral mesoderm migrates to a lateral position on the yolk sac. As gastrulation proceeds, these cells either migrate further onto the yolk sac or return to an intra-embryonic location in the dorsal mesentery. Most cells on the early yolk sac are primitive progenitors that will form blood islands and eventually enter the circulation. As the fetus develops, these primitive cells undergo programmed cell death. Fetal-adult blood cells are derived either from definitive progenitors that colonized the yolk sac at the same time as the primitive progenitors or from the dorsal mesenteric hematopoietic cells. The dorsal mesenteric cells either migrate to the yolk sac or thymus or can enter the circulation and colonize hematopoietic sites such as the fetal liver. Thus, the prominent waves (from early to late) of hematopoiesis in the embryo include (l) primitive cells on the yolk sac, (2) definitive cells on the yolk sac (which are likely derived from ventral mesoderm that migrated with the primitive cells), and (3) early intraaortic and later para-aortic hematopoietic cells. Some of the dorsal hematopoietic cells migrate to the yolk sac and form a later wave of hematopoiesis on the yolk sac. These migratory patterns of hematopoietic cells in the vertebrate embryo are generally conserved throughout evolution. genitor includes the finding of antigens (such as QH1)“~4X~‘’’ or molecular markers (such as GATA-2 and several tyrosine kinases) that are common to early hematopoietic progenitors and vascular progenitors. Recently, we have studied a zebrafish mutant with deficiencies of hematopoietic and endocardial progenitors, potentially representing a defect in a common blood-vascular p r ~ g e n i t o r .Despite ~~ the above data supporting the existence of a hemangioblast, the vasculature and heart develop normally in bloodless amphibian embryos from which the presumptive ventral blood island region has been explanted during the neurula stage. It is possible that, by early neurula, fates have been determined and the vascular lineage becomes distinct from the hematopoietic lineage; most ventral mesoderm becomes hematopoietic, whereas the lateral mesodermal cells become the vascular progenitors. STUDIES ON LOWERVERTEBRATES We have used the fish and frog as model systems to study the induction of the hematopoietic stem cell during ontogeny. The embryos from these species are more accessible than higher organisms, and their early development has been wellcharacterized. The study of their hematopoietic program, which may be more simple, will provide general principles regarding hematopoiesis in vertebrates. Zebrajish Hematopoiesis Hematopoietic and Endothelial Development The vascular and hematopoietic compartments are coordinately regulated so that blood cells enter the circulation as soon as terminal differentiation has occurred. A common progenitor called either a “hemangiocytoblast,” ‘‘hemangioblast,” or “angioblast”55~58 has been postulated to exist during embryogenesis and has the potential to form either vascular endothelial cells or hematopoietic cells in the blood island region. Morphological data derived from experiments with cultured chicken blastoderms support the hypothesis that these bipotential cells can form either tissue. In mice and humans, support for this concept is less apparent. Support for the existence of a common bloodvascular pro- We have characterized the expression of the zebrafish GATA- 1 and GATA-2 during early development. These transcription factors are initially induced during gastrulation and delineate cells that will form the hematopoietic IM (Fig 7). The posterior region of the IM contains early hematopoietic progenitors (or “stem” cells) that express GATA-2, but not GATA-l. Thus, the visibility of the zebrafish embryo allows easy examination of early and late hematopoietic populations. Studies of genetic mutants for blood formation in the zebrafish system may provide insight into the induction events that effect hematopoiesis. Using GATA-l and GATA-2 as markers, we have studied three zebrafish mutants of blood ’* Fig 8. Studies of definitive hematopoiesis in amphibians. (A) Species-specificdifferences highlighted by transplantation experiments. Reciprocal transplantation of ventral (V) or dorsal (D) regions of diploid embryos (Zn) into triploid1311) embryos at neurula stages(A, anterior; P, posterior).The transplantedembryos areallowed to reach a premetamorphic stage to evaluate the primitive lineege anda postmetamorphic stage when definitive hematopoiesis is occurring. Blood or thymic cells are analyzed by DNA flow cytometry to cletermine the contribution of the grafted tissue to particular blood lineages. +, contribution to the lineage; -,does not contribute to the lineage. Myeloid blood cells are derived from the same regions as T cells. Note the difference in T-cell and myeloid contribution in Rana and Xenopus; in Rana, the vantral region only contributes to the red blood cell lineage previousto metamorphosis andnot to the early T-cell ormyeloid populations. (B) Studies of thymus development by transplantation of diploid (2n) regions into triploid (3nl embryos during early neurula. The T-cell population is derived from the posterior compartment of the embryo (top), but not from the posterior third ( m n d embryo). Anterior migration occurs from the posterior region to the dorsal mesentery and, later, to the dewloping thymus. Grafting experiments ofthe pronephros and praumptive ventral blood island regions showed that the thymus is derived from bothdorsal and ventral regions. (C) Cumulative summary of migratory events of hematopoietic callsduring development in amphibians. The migratory pattern is thought to be similar for most vertebrate wecies. The ventral blood island (VBI) contributes to the development of all hematopoietic lineages. Primitive cells form the blood island and enter the circulation (1). During neurula stages, mesoderm aroundthe pronephric duct (PD) migrates anteriorly (2) to the region of the dorsel aorta (DA; 3) and ducts of Cuvier(DC; 31 and the pronephros (P;4). These cells either colonizethe thymus (5) or enter circulation through the aorta or ducts of Cuvier 16). Definitive celh within the VBI enter circulation also and eventually colonizethe thymus (T; 71 and the larval liver (LL; 81. Definitive cells also enter the circulation from the larval liver (9). In chickens and mice, the circulation is establiied before the yolk sac structure is disrupted. Some definitive cells in the dorsal mesentery or in the circulation colonize the yolk sac. The chicken does not form blood in the fetal liver (FLI, but has extensive hematopoiesis throughout the mesentery. The axis of the embryo is marked as follows: A, anterior; P, posterior; D, dorsal; V, ventral. (D) Environmental influences ofdefmitive hematopoietic commitment. When the ventral t i i u e is transplanted to a ventral central location. the donor tissue becomes erythroid, whereas transplant a to a lateral location becomes T cells. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS 2885 Neurula C A J p D A l P V '-cell INeurula I Primitive I Definitive 1 1 -+ 1 ;I ;1 ;1 Xenopus Dorsal Rana + + + - Dorsal Xenopus Ventra I Rana B Neurula .".L"I_l I Ventral Ventral View - + ? ? (20 hrs) Thymus D A p V + 60 hrs V From www.bloodjournal.org by guest on June 18, 2017. For personal use only. LEONARD I. ZON 2886 A .. Chick Quail B Fig 9. Schematic diagram of hematopoiesis in the dorsalparaaortic focusof birds (A) and mammals (B). This region has been shown to bea site of hematopoiesisby morphologicalcriteria, benzidene staining, and immunolocalizationwith QH1 antisera (in birds) and by spleen colony assays and transplantation experiments. The chicken has diffusedefinitive hematopoiesis, particularly around the aorta (dark shaded region), whereas the quail has a more restricted distribution of hematopoietic cells surrounding the Ducts of Cuvier (darkshaded region). The hematopoietic progenitor cells in the mouse have been studied functionally (see text) but have not been morphologicallyorhistologically defined. DC,ductsofCuvier;A, aorta; E, esophagus; B, bronchi. (Adapted and reprinted with permission.‘) formation: bloodless, spadetail, and ~ I o c h e . ’ *The ~~~ bloodless mutation only affects hematopoiesis, whereas the spadetail mutation affects muscle andblood induction. Whole embryo in situ analysis shows that bloodless (Fig 10) and spadetail mutant embryos each lack GATA- 1 and GATA-2 expression, except for wild-type levels of GATA-2 expression in the posterior “stem” cells. Despite their similarities, complementation analysis has shown that the bloodless and spadetail mutations are in distinct genes. Thus, the selfrenewal or differentiation of early hematopoietic cells is affected in the mutants. The cloche mutation lacks the endocardial layer of the heart and the blood cells in the IM region. Interestingly, cloche mutants do not express GATA-2 or GATA- 1, even in the posterior stem cells. Thus, the cloche mutation affects vascular cells and hematopoietic cells and, therefore, supports the existence of a bipotential “hemangioblast” during development. Fate mapping experiments in wild-type embryos at the 500-cell stage have shown that single ventral blastomeres give rise to blood and blood vessel progenitors, further suggesting a common origin. Therefore, the defect in cloche effects the genesis of hematopoietic (and endothelial) stem cells. Two independent laboratories, Driever (Boston, MA) and Nusslein-Volhard (Tubingen, Germany), have produced over 10,OOO zebrafish mutations using chemical mutagenesis.m.6’ In this method, fish were treated with ethyl nitroso urea (ENU), a chemical that causes point mutations in the sperm genome. These sperm were used to fertilize eggs, the progeny were mated individually to wild-type fish or to the parent, and a family was derived. Each family is then individually screened for mutant phenotypes, and over 30 mutants affecting the blood system have been characterized. The mutants can be used to genetically order a cascade of steps for embryonic blood formation. This is a similar to drosophila or yeast genetic epistasis analysis, which involves paired matings of different mutants, thereby generating fish that are mutant at two (or three) genetic loci. Assessment of the phenotype of these double mutants allows a positioning in a cascade of developmental events. These studies are coupled with the examination of gene expression with specific markers in mutant embryos andwith the analysis of the effect of forced gene expression. A zebrafish genome map consisting of over 400 random amplified polymorphic DNA (RAPD)markers distributed throughout the genome has recently been and we have started to map the location of the genes affected in the mutant zebrafish. A position-cloning strategy to isolate genes that are affected in the mutants is then possible. Xenopus Hematopoiesis The induction of hematopoietic stem cells during embryogenesis is likely to be regulated by signals that affect mesoderm induction and patterning. These signals may not function in the same manner as hematopoietic cytokines; mesoderm-inducing factors are thought to induce distinct tissues in a gradient rather than threshold mechanism. It is possible that low doses of particular inducing factors would stimulate blood formtion, but higher doses may inhibit hematopoiesis. Mesoderm induction has been so well-characterizedin Xenopus that this species is ideal to characterize factors that regulate the early events in hematopoiesis. Assays in the frog for the induction of hematopoietc stem cells. Transplantation studies have shown that Xenopus animal-pole ectodermal cells can contribute to almost any tissue of a developing embryo. As such, animal pole cells (also called animal caps) are similar to murine embryonic stem cells. Through the use of animal pole-vegetal pole “recombinants” (see Fig 1 I), Nieuwkoop and colleague^^"^' have shown that the dorsal-ventral organization of mesoderm is determined by the vegetal pole. Culture of animal pole alone in a simple salt solution results in ciliated epidermis (skin), whereas culture of the vegetal pole yields undifferentiated cells and gut (Fig 1 A recombinant of animal and vegetal pole yields mesodermal tissues, including some From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2007 DEVELOPMENTAL BIOLOGY OF HEMATOPOIESIS blood cells. During blastula stages, two predominant mesoderm-inducing signals are thought to act on overlying equatorial cells. The ventral vegetal signal induces ventral mesoderm such as blood and mesothelium and the dorsal vegetal signal induces dorsal mesoderm including muscle and notochord. Subsequently, during gastrulation, a dorsal region called the Spemann organizer patterns mesoderm across the marginal zone.- These data suggest that the endoderm elaborates a signal required for the induction of blood, and that the program is modulated by cell-cell interactions. Surgical excision and culture of the dorsal marginal zone (DMZ) yields mostly notochord and muscle, whereas ventral marginal zone (VMZ) yields predominantly blood and mesenchyme (Fig 11, bottom).70 VMZs express globin after 40 hours in culture, and, thus, the inducers of blood are available or programmed in this ventral region. A VMZ culture can be viewed as equivalent to hematopoietic progenitor assays with all of the authentic growth factors present. Interspecies grafts between two different amphibian species (Axolotl and Xenopus) have been used to characterize the ventral and dorsal signals. Culture of Xenopus VMZ with Axolotl DMZ yielded mostly Xenopus muscle and kidney differentiation, showing the respecification of ventral mesoderm by the dorsal signal. If the organizer region of the DMZ is grafted onto the ventral region of an intact embryo (a classic “Spemann Organizer” graft), a mirror-image duplication of the embryo is created.” Neither the primary nor the secondary embryo is actually complete because they both a ”1 Skh Aninal Cao STME 8 vegetal P& L Gut 8- m a l Cap RECOMBINANT Mesoderm hdwm Vegetal Pole Aninal Cap - + Activin (ie. A) Mesoderm hductDn ”” Ventral Marginal Bbod, Zone c Mesenchyme Dorsal M a r m l Zone i k s c l e , Notocord Fig 11. Mesoderm induction assays. (Top panel) The animal cap of a blastula embryo (about8 hours] can be removed, cultured in a satt solution, andwill form skin (ectoderml. The vegetal pole can be removed andwill form gut [endoderm). Recombination of the animal cap (ectoderm) and vegetal pola (endodorm) leadsto the induction of mesoderm entirely from the ectoderm. Animal caps can also be incubated with purified growth factors that induce mesoderm. AcWin and FGF induce mesoderm such as muscle, but blood is not formed. (Bottom penel) The marginal zone of the gastrula embryo type of mesodermis spacontains the mesodermal proganitors. The tially localized acrossthe marginal zone. For example, explant the of VMZ leads the induction of hematopoietic cells, whereas tha DMZ does not form blood. lack blood islands. Thus, it is possible that the organizer can reprogram ventral mesoderm and, thereby, functionally repress blood formation. The ventral and dorsal signals have been shown to be soluble factors based on their ability to cross a millipore filter placed between embryonic ex plant^.^'-^^ Very low levels of globin expression are detected in animal-vegetal recombinants, but not to the level in a VMZ e ~ p l a n t ? This ~ . ~ ~suggests that globin production requires signals in the ventralmost endoderm of the marginal zone?6 Studies of mesodermal inducing factors in Xenopus. Activins are members of the transforming growth factor (TGF- p) superfamily and are dimers of inhibin p chains.77 Activin is a very potent inducer of mesoderm in animal cap assay^^^.^^" and can also induce differentiation of erythroleukemia cells.8sWhereas recombinants between animal and vegetal poles yield all mesodermal components, activin A (either from Xenous or mammalian sources) induces mostly notochord, muscle, and kidney tubules. At low concentrations of 0.1 to 1ng/mL, activin induces some blood-like ~ e l l s . ~However, ~ , ~ ’ the blood cells do not express hemoglobin based on immunoflorescence with an anti-Xenopus globin antisera. Fibroblast growth factor (FGF) treatment of animal pole explants at low concentrations mimics the effects of the ventral vegetal signal in V ~ V O . ~ &This ~ ’ consists of a concentric arrangement of mesenchyme and mesothelium. Some blood-like cells are present that do not express hemoglobin but have a characteristic m ~ r p h o l o g y . ’Other ~ ~ ~ ~growth ~~~ factors effect the induction of hematopoietic mesoderm and ventral patterning. BMP-4 is a member of the TGF-p superfamily and is capable of ventralizing whole embryos and WNT-8, increasing the expression of g l ~ b i n ? ~ - ~ ~ a member of the wnt family, also can ventralize tissues when expressed correctly after the onset of zygotic transcription96s97; however, if expressed earlier, WNT-8 predominantly dorsalizes tissues. More research is needed to determine the exact effect these proteins have on hematopoietic induction, but it is clear that dose and timing of exposure to stem cells will be critical varibles to explore. There are also several dorsalizing factors including TGF-P, wnr, and noggin98”* polypeptides that have been described in Xenopus embryos that may function to suppress hematopoiesis. Studies of embryonic hematopoiesis in Xenopus. Using whole embryo in situ analysis for GATA-1 and SCL RNA expression, we have shown that the hematopoietic program initiates at least as early as 11 hours after fertilization (during gastrulation).“’ GATA-l RNA was not initially detected in uninduced animal cap cells, but, after 17 hours of culture, GATA-I was surprisingly expressed.”’ The level continued to increase until 25 hours and then decreased. Later in CUIture, embryonic a-globin was also expressed at a low level in the animal cap cells. The expression of these genes is in accord with the normal temporal pattern of expression. MyoD, amuscle-specific transcription factor, is also expressed in the uninduced animal caps early in the culture and is downregulated so that caps at 48 hours do not express MyoD.’” Thus, the mesodermal programs (such as blood and muscle) are evident in the animal cap cultures, but, in the absence of induction, the programs are not maintained. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. I l Activin or FGF induction of animal pole explants leads to the maintenance of MyoD expression and formation of muscle; however, neither factor rescues the blood program or maintains GATA-1 expression. As discussed above, the ventral vegetal signal(s) present in VMZ explants is capable of inducing and maintaining the blood program as defined by GATA-1 and embryonic a-globin expression (Fig 12). The animal cap itself has also been postulated to affect hematopoietic differentiation." When animal pole tissue is recombined with isolated VMZ cells, an increase in hemoglobin is detected, indicating that the animal pole contains signals that enhance globin expression. A focus of our laboratory is to use these assays to isolate and define factors that participate in ventral axis patterning or mesoderm induction. SUMMARY The cellular and environmental regulation of hematopoiesis has been generally conserved throughout vertebrate evolution, although subtle species differences exist. The factors that regulate hematopoietic stem cell homeostasis may closely resemble the inducers of embryonic patterning, rather than the factors that stimulate hematopoietic cell proliferationand differentiation. Comparative study of embryonic hematopoiesis in lower vertebrates can generate testable hypotheses that similar mechanisms occur during hematopoiesis in higher species. ACKNOWLEDGMENT I appreciate thehelpful discussionswith Jim Turpen (Universityof Nebraska, Lincoln,NB) and Bob Broyles (Universityof Oklahoma, Norman, OK). I thank Todd Evans (University of Pittsburgh, Pittsburgh, PA), Gordon Keller (National Jewish Hospital, Denver, CO), and Ramesh Shivdasani (Dana-Farber Cancer Institute, Boston, MA) and Merlin Crossley, Mitch Weiss,and Andrew Perkins (Children's Fig 12. Localizationofhematopoieticmesoderm in the VMZ as Hospital of Boston) for their critical review of the manuscript. determined by globin expression. Three VMZs were explanted during gastula stages and allowedto develop for 36 hours. These explants (bottom)and the whole embryo(top) were subjectedto in situ analysis with an antisense digoxigenin-labeled probe to embryonic a-globin. The expression is detected by an alkaline phosphatase-coupled digoxigenin-labeled antibody. 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