Development of the hematopoietic system in the mouse

Transcription

1 Experimental Hematology 27 (1999) Development of the hematopoietic system in the mouse Gordon Keller a,b, Georges Lacaud a, and Scott Robertson a a National Jewish Medical and Research Center, Denver, CO; b Department of Immunology, University of Colorado Health Sciences Center, Denver, CO (Received 20 January 1999; accepted 2 February 1999) Offprint requests to: Gordon Keller, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206; kellerg@njc.org Introduction The hematopoietic system is established early in embryonic development and functions throughout fetal and adult life to provide a continuous supply of mature blood cells to the embryo, the fetus, and the adult. Maturation of the hematopoietic system in ontogeny represents a succession of developmental programs beginning in the yolk sac and progressing to intra-embryonic sites, initially to the region defined as the para-aortic splanchnopleura (P-Sp)/aorta-gonadmesonephros (AGM) and then to the fetal liver, which assumes the predominant hematopoietic role until birth [1 4]. Late in gestation, hematopoietic precursors seed the bone marrow that, shortly after birth, becomes the principal site of hematopoietic activity. Our understanding of lineage relationships, growth regulation, and control of differentiation within the hematopoietic system is largely derived from studies on adult bone marrow and fetal liver. While there are some differences between the fetal and adult hematopoietic systems, in general they share many similarities including the development of multiple lineages from a common precursor known as the multipotential stem cell [5 8]. Both fetal and adult stem cells are able to provide long-term hematopoietic repopulation following transplantation into adult recipient animals, a characteristic that distinguishes them from all other cells in the hematopoietic system [9,10]. In contrast to multilineage hematopoiesis found in the fetal liver and adult marrow, the yolk sac produces predominantly a single mature erythroid population [2]. While recognized for many years as the first hematopoietic cells to develop in the embryo, little is known about the yolk sac erythrocytes including their relationship to other hematopoietic lineages and the mechanisms regulating their development, growth, and maturation. This review will focus on the events leading to the development of the early yolk sac lineages and the transition to multilineage hematopoiesis in the mouse. Many other aspects of hematopoietic development in the mouse as well as in other species have been covered in recent reviews [11 15]. The primitive hematopoietic system The first visible sign of hematopoietic activity in the mouse embryo is the appearance of blood islands in the developing yolk sac at approximately Day 7.5 of gestation [2]. In situ studies of the early embryo have demonstrated that genes known to play a role in the onset of hematopoietic development (e.g., GATA-2, scl/tal-1, rbtn2), are expressed prior to the appearance of the blood islands [16], suggesting that the molecular program that leads to hematopoietic commitment begins shortly after grastrulation, at approximately Day 7.0 of gestation. The yolk sac blood islands consist of two lineages, a population of erythroid cells surrounded by a layer of angioblasts that eventually form the developing vasculature. The parallel development of these lineages in close association in the blood islands of the yolk sac provided the basis for the hypothesis that they arise from a common precursor, a cell called the hemangioblast [17 20]. The erythroid cells within the blood islands are known as embryonic or primitive erythrocytes and differ from those found in the fetal liver and adult bone marrow in that they are large, nucleated, and produce the embryonic forms of globin [2,21,22]. As the vascular system develops and the heart begins to beat at approximately Day 8.5 of gestation (8 somite pairs), the primitive erythrocytes circulate throughout the embryo and persist in the circulation until mid to late gestation [22]. The primitive erythroid population represents the major mature hematopoietic component of the yolk sac and is only produced during this stage of development. As primitive precursors are difficult to grow in culture, the duration of active erythropoiesis within the yolk sac has not been well defined. The only study to address this question suggests that these precursors are present in the yolk sac for a relatively short time, possibly no more than 48 hours, indicating that the entire primitive erythroid population could be produced in one rapid and synchronous wave of development [23]. Given the predominance of primitive erythropoiesis in the yolk sac, this stage of hematopoietic development is known as primitive hematopoiesis X/99 $ see front matter. Copyright 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S X(99)

2 778 G. Keller et al./experimental Hematology 27 (1999) The early yolk sac-restricted development of the primitive erythroid lineage suggests that it represents a distinct lineage, separate from all other hematopoietic lineages. Support for this interpretation is provided by gene-targeting studies that have identified transcription factors essential for the development of all hematopoietic lineages, except primitive erythrocytes [24,25]. The only other mature hematopoietic cells present in the early yolk sac are macrophages [26]. Their early development has led to speculation that they also represent a primitive population. The observation that they mature rapidly, possibly bypassing the monocyte stage of development, together with the finding that they express lower levels of certain genes than later stage macrophages suggest that they could represent a unique population [27,28]. However, the distinction is not as clear with the primitive erythroid lineage, because it is unknown if the molecular events leading to their development differ significantly from other types of macrophages. Therefore, at the present time, it is unclear if this population should be considered as part of the primitive or part of the definitive hematopoietic system. The definitive hematopoietic system All lineages other than primitive erythroid and early macrophage will be considered as belonging to the definitive hematopoietic system. While the yolk sac origin of primitive hematopoiesis is well accepted, identification of the initial site of definitive hematopoiesis has been the subject of intense investigation and controversy over the past three decades. Historical perspective Given the transition of hematopoietic activity from the extraembryonic to intraembryonic sites, it was not unreasonable to assume that multilineage definitive hematopoiesis develops in the fetal liver from blood-born precursors of yolk sac origin. Support for this interpretation was provided by early studies of Moore and Owen [29], which demonstrated that the chick yolk sac contained cells capable of repopulating the hematopoietic system of recipient embryos. Additional support for this hypothesis came from experiments of Moore and Metcalf [30], which indicated that yolk sac, but not the embryo proper, from precirculation embryos generated hematopoietic precursors when maintained in culture for several days. The yolk sac origin of definitive hematopoiesis was, however, seriously challenged by chick/ quail embryo grafting experiments, which provided the first evidence that hematopoiesis initiates at two independent sites; the yolk sac, which gives rise predominantly to primitive erythropoiesis and the embryo proper that generates definitive hematopoiesis [31,32]. Studies on amphibian embryos also provided evidence for the existence of independent hematopoietic sites with different potentials [33,34]. In recent years, several groups have identified a putative intraembryonic hematopoietic region in the mouse embryo, prior to the development of the fetal liver, and have suggested that this could be the site of origin of the definitive hematopoietic system [3,4,35,36]. While these studies support the interpretation that there are two sites of hematopoietic development, the full potential of both as well as their relative contributions to the normal developing embryo are not well understood. In addition, the findings from some of these studies are complicated by the assays used and the lineages analyzed. In an attempt to clarify some of these issues, we will review the development of the definitive hematopoietic system by analyzing the establishment of specific lineages. Definitive erythroid and myeloid precursors Definitive erythroid cells are distinguished from primitive cells by the fact that they are small, they enucleate, and they produce the adult forms of globin [2,21,22]. Analyses of the early stages of embryonic development have shown that precursors of the definitive erythroid and myeloid lineages can be detected in the yolk sac at approximately Day 8.0 of gestation, shortly after the initiation of primitive erythropoiesis [23,37]. As no details were provided on the types of myeloid lineages analyzed, it is unclear if more than macrophage precursors were present. No precursors were detected in the embryo proper at this early (precirculation) stage of development, suggesting that this phase of definitive erythroid and myeloid commitment initiated in the yolk sac. The development of these populations in the yolk sac demonstrates that this extraembryonic site is not restricted to primitive hematopoiesis. Although these definitive hematopoietic precursors can be detected early in the yolk sac, they do not mature in this extraembryonic environment, suggesting that they are produced for purposes other than contributing to hematopoiesis at this site. One possibility is they develop to seed the fetal liver and rapidly establish the earliest wave of intraembryonic definitive hematopoiesis. Two observations support this interpretation. First, the embryonic blood contains significant numbers of definitive hematopoietic precursors prior to the development of the fetal liver [37]. Second, if one compares the developmental progression of the definitive erythroid lineage in the yolk sac and fetal liver, there are striking differences. In the yolk sac, early stage precursors (BFU-E) can be detected before those representing later stages of development (CFU-E), a sequence that suggests that the lineage is establishing from an immature cell ([37] and Palis J, Keller G, et al. (1999), unpublished observations). In contrast, large numbers of CFU-E together with BFU-E and myeloid precursors can be detected at the earliest stages of fetal liver development, a finding more consistent with migration than de novo generation ([37] and Palis J, Keller G, et al. (1999), unpublished observations). Further studies will be required to determine if this interpretation is correct.

3 G. Keller et al./experimental Hematology 27 (1999) Lymphoid precursors Although considerable effort has been invested into defining the embryonic/fetal origin of the lymphoid lineages, there is presently little consensus among the various reports as to where and at what stage of development these precursors arise. In some studies precursors with lymphoid potential were detected in the yolk sac prior to the embryo proper and found as early as Day 8 of gestation (2 9 somites) [38,39], whereas others place the lymphoid precursors in the embryo proper before the yolk sac [40]. Finally, a third group of experiments has detected these precursors in both sites at the same developmental stage [41,42]. As the lymphoid program does not initiate until early fetal liver and thymus development, it is quite possible that these analyses of early embryonic populations detect lymphoid progeny generated from multipotential rather than committed precursors. If such multipotential cells are relatively rare in both the yolk sac and embryo proper, subtle differences in culture conditions, methods of tissue harvest, and assays could easily account for the reported differences. Support for this interpretation is provided by the studies of Godin et al. [36] in which clonal assays were used to demonstrate that B- and T-cell precursors found early in embryonic development derive from multipotential cells. The precise stage of lymphoid development and the characteristics of the earliest precursors committed to these lineages remain to be determined. We have recently identified a precursor in early fetal liver that is restricted to the B-cell, T-cell, and macrophage lineages [43]. Given this developmental potential and its early appearance in the fetal liver, this precursor could represent one of the earliest developmental stages within the lymphoid program. Multipotential precursors and long-term repopulating stem cells By Day of gestation, yolk sac hematopoiesis begins to decline and intraembryonic sites become active [1]. While the liver has traditionally been recognized as the predominant hematopoietic tissue in the fetus, a number of studies in recent years have identified an intraembryonic site of hematopoietic development, prior to the establishment of the fetal liver. This region, situated in the caudal portion of the embryo, is known as the para-arotic splanchneopleura (P-Sp) at Days [3] of gestation and the aorta-gonad-mesonephros (AGM) at days [4]. To investigate the origins of definitive hematopoiesis in the mouse, Godin et al. [36] analyzed, in detail, the development of multipotential cells in the P-Sp and yolk sac of the early embryo. In this study, multipotential cells were identified by their capacity to generate erythroid, myeloid, and lymphoid progeny in culture. Cells with this potential could be detected in both the P-Sp region of the embryo and the yolk sac as early as Day 8.5 (10 somites) of gestation, albeit at low numbers. The number of precursors increased in these tissues over the next 24 hours of development, and although their frequency was higher in the P-Sp, the total number in both regions was similar. Given that most of the analyses were carried out when circulation was already established, it was not possible to determine the origin of the multipotential cells in this study. To further investigate this question, this group used the same approach as Moore and Metcalf [30] and cultured isolated yolk sac and splanchnopleura (Sp) from precirculation embryos (from 0 to 6 somites) to allow their respective hematopoietic potential to develop [44]. In contrast to the findings of Moore and Metcalf [30], they observed that both the yolk sac and Sp could develop hematopoietic cells, although the potential differed. The yolk sac gave rise to erythroid and myeloid cells, while the embryo proper generated lymphoid in addition to erythroid and myeloid cells. The different outcome between this study and that of Moore and Metcalf [30] could reflect differences in culture conditions. The findings from this recent study provide strong support for the concept of two independent hematopoietic sites in the early embryo. The extraembryonic yolk sac shows a somewhat restricted potential being able to generate the primitive erythroid, definitive erythroid, and myeloid lineages. The embryo proper, by comparison, displays true multipotentiality in being able to give rise to definitive erythroid, myeloid, and lymphoid progeny. While this interpretation may be correct, a concern with the study is that the culture conditions might not be optimal for the development of yolk sac multipotential cells. Further studies using a variety of different culture conditions will be required to define the full potential of the yolk sac in culture. Although these investigators demonstrated multilineage hematopoiesis and the presence of multipotential cells at these early stages of development, they were unable to detect long-term repopulating stem cells (LTRSCs) in embryos at the 2 to 8 somite stage (Day 8) [44]. Kinetic studies have shown that stem cells able to provide multilineage engraftment of adult animals are not detectable until Day 10.5 of gestation (30 35 somites) and are found at low frequency only in the AGM region at this time [35]. By Day 11.5, both the AGM and yolk sac contain LTRSCs. Their presence in the AGM before the yolk sac is used as further evidence that definitive hematopoiesis originates within the embryo proper. The fact that cells displaying multipotentiality in culture can be detected earlier than the LTRSC in the embryo/fetus raises some important questions about the type of cells measured by different assays. The LTRSC assay is clearly more stringent than in vitro assays, as in addition to multipotentiality, it requires that a cell express appropriate surface markers enabling it to home appropriately and function in an adult environment. The early embryonic multipotential cells appear to have developmental potentials similar to the LTRSC, but may not yet have developed these critical surface markers and, as a consequence, are unable to function in the repopulation assay. As such, they may represent a

4 780 G. Keller et al./experimental Hematology 27 (1999) pre-ltrsc. To determine if tissues from stages of development earlier than Day 10.5 have the potential to generate LTRSCs, Medvinsky and Dzierzak [45] isolated P-Sp/AGM and yolk sac from embryos at Day 10 of gestation and maintained them in organ culture for 48 hours. Following this culture period, the P-Sp/AGM, but not the yolk sac, contained LTRSCs. There are two possible interpretations of these findings. The first is that the Day 10 P-Sp/AGM does contain pre-ltrscs that mature into functional repopulating cells over the 2-day culture period. The second is that low numbers of LTRSCs are already present in the Day 10 P-Sp/AGM, and their numbers increase to detectable levels in the organ culture. At the present time it is not possible to distinguish between these two interpretations. The failure of the yolk sac explants to generate LTRSCs may reflect the fact that the extraembryonic environment does not support the maturation of these cells or that this region does not contain LTRSCs or pre-ltrscs at this stage of development. If one of the functions of the P-Sp/AGM is to provide an environment for maturation of LTRSCs, it is possible that cells with pre-ltrsc characteristics are present in other tissues such as the yolk sac, and need to be placed in an appropriate environment to demonstrate this potential. A recent study by Yoder et al. [46] provides evidence to support this interpretation. These investigators transplanted Day 9 yolk sac-derived CD34 /c-kit cells into the livers of newborn pups, reasoning that this environment, being more fetal-like, might be better suited to support the growth and development of these embryonic cells. Using this model, they were able to demonstrate that this yolk sac population, as well as a CD34 /c-kit P-Sp population isolated at the same stage of development, contained LTRSCs. When the same cells were transplanted into adult animals they failed to show any repopulating potential, confirming the findings of Muller et al. [35]. The fact that significantly higher numbers of CD34 /c-kit cells were present in the yolk sac compared to the P-Sp suggests that they are of yolk sac origin. However, to formally demonstrate this, tissues isolated from precirculation embryos need to be analyzed. While the ultimate origin of these repopulating cells may not yet be defined, these studies underscore the importance of the microenvironment when analyzing the potential of embryonic precursors. The findings from these different studies strongly suggest that the difference in potential between the yolk sac and the P-Sp/AGM is not as simple as primitive versus definitive. Rather, the yolk sac appears to have significant potential that includes primitive and definitive hematopoietic precursors, and quite possibly LTRSCs or pre-ltrscs (Fig. 1). The P-Sp/AGM appears to represent an intraembryonic site of definitive hematopoiesis (Fig. 1). Given this overlapping potential, what is the contribution of each to the developing embryo and fetus? It is clear that the initial function of the yolk sac is to generate primitive erythrocytes. However, the early fetal liver phase of definitive hematopoiesis may also be of yolk sac origin as there is little evidence that the P-Sp/AGM region generates committed precursors ([23] and Palis J, Keller G, et al. (1999), unpublished observations). Finally, given the findings of Yoder et al. [46] we must consider the possibility that, in the mouse, the yolk sac is the ultimate source of the definitive LTRSC. If LTRSCs are of yolk sac origin, the function of the P-Sp/AGM may be to provide an environment for the maturation of these cells. Alternatively, the P-Sp/AGM may represent a new site of hematopoietic development, which generates a population of LTRSCs that contributes to later stages of fetal liver development and ultimately gives rise to the adult hematopoietic system. At the present time it is not possible to distinguish between these different interpretations as all of these studies demonstrate what can happen following experimental manipulation but may not necessarily reflect the situation in the developing mouse embryo. The resolution of this issue may require new experimental approaches that enable one to track the migration of cells in the mouse embryo in situ. In vitro differentiation of embryonic stem cells: a model for embryonic hematopoiesis Although the origins of definitive hematopoiesis are not yet fully understood, it is well accepted that the yolk sac represents the earliest site of hematopoietic and endothelial development and that both lineages are derivatives of the extraembryonic mesoderm. The developing yolk sac should, therefore, be a good model for studying the early events regulating the commitment, growth, and maturation of these lineages. To address these questions, it is important to focus on the stage of development immediately following gastrulation but prior to the establishment of the blood islands. However, such studies are extremely difficult as it is not easy to access the mouse embryo at this stage of development and the amount of tissue available for study is very limited. To overcome these problems, a number of groups have focused on the in vitro differentiation potential of embryonic stem (ES) cells as a possible model for early hematopoietic and endothelial development [47 55]. When removed from conditions that maintain them in an undifferentiated state, ES cells will spontaneously differentiate and form colonies or embryoid bodies (EBs) that contain many precursors including, among others, those of the hematopoietic and endothelial lineages [56,57]. With appropriate culture systems it is now possible to routinely generate the primitive and definitive erythroid lineages, most myeloid lineages, and endothelial cells in a predictable pattern from developing EBs (Fig. 2). Although the ES/EB model provides access to large numbers of early cell populations, does it recapitulate the developmental programs found in the normal embryo? While all aspects of this issue have not been addressed, the

5 G. Keller et al./experimental Hematology 27 (1999) Figure 1. Scheme of embryonic hematopoietic development. E p, E d, Myeloid, etc. refer to committed precursor cells. Multipotential precursors: cells with erythroid, myeloid, and lymphoid potential. LTRSC long-term repopulating stem cells as assayed either in the newborn pup or the adult. Crossbars indicate blocks in development as determined by gene knock-out studies. following observations support the interpretation that the early events in hematopoietic and endothelial commitment in the EBs are similar to those found in the embryo. First, analysis of hematopoietic precursor development within the EBs showed that the primitive erythroid and macrophage lineages appear before those of the definitive erythroid and other myeloid lineages; a pattern reminiscent of the early yolk sac [51]. In addition, as in the yolk sac, primitive erythropoiesis within the EBs is a transient developmental program. Second, gene expression studies on the developing EBs demonstrated that markers that define mesoderm are expressed earlier than those found in hematopoietic and endothelial precursors, which in turn precede those that define specific hematopoietic lineages [51]. These patterns are consistent with the well-established dogma that the hematopoietic and endothelial lineages develop from mesoderm. Third, kinetic analysis of endothelial commitment within the EBs indicates that genes involved in the early stages of lineage development are expressed before those that define more advanced stages [55]. Fourth, gene-targeting studies have clearly demonstrated that the same molecular programs regulate hematopoietic development in the embryo and EBs [58 61]. Taken together, the findings from these different studies strongly suggest that the early events of hematopoietic and endothelial commitment in the ES/EB model are comparable, if not identical, to that of the early embryo. In contrast to the erythroid and myeloid lineages, lymphopoiesis has been much more difficult to demonstrate in a reproducible fashion within EBs. Recent studies suggest that oxygen tension may affect the development of these lineages, as lymphoid precursors were found consistently in EBs cultured in a low oxygen environment [62]. As with the lymphoid lineages, LTRSCs are not easily detected in developing EBs and to date only a few studies have documented any repopulation following transplantation of ES cell-derived precursors. In several different studies, precursors from differentiated ES cells were able to generate lymphoid progeny following transplantation into either SCID or RAG-1 / recipients [63 65], and low levels of multilineage repopulation were detected in mice transplanted with the entire cell population from Day 4 EBs [66]. Although

6 782 G. Keller et al./experimental Hematology 27 (1999) Figure 2. Scheme of hematopoietic differentiation in developing embryoid bodies. Time lines for the appearance of precursor populations from Keller et al. (51) and Potocnik et al. (62). these findings are encouraging, the fact that relatively few studies have demonstrated extensive multilineage repopulation with cells of ES origin suggest that the generation of LTRSC from differentiated EBs remains problematic. It is possible that this difficulty simply reflects the fact that early embryonic hematopoietic cells are unable to function efficiently in an adult environment, as observed with the yolk sac-derived stem cells. If this is true, EB-derived repopulating cells might be more routinely detected following their transplantation into newborn pups. Although all aspects of lymphoid and LTRSC development within EBs are not fully understood, the fact that the early stages of erythroid, myeloid, and endothelial development are efficient, reproducible, and similar to those found in the normal embryo, makes the ES differentiation system a powerful model for studying the early events in hematopoiesis and vasculogenesis. Relationship of the primitive and definitive hematopoietic programs The observation that the primitive erythroid lineage develops before definitive erythroid, myeloid, and multipotential precursors, and the LTRSCs in the yolk sac is an unexpected sequence of events. In most schemes of fetal and adult hematopoiesis, the LTRSC is considered the most immature cell and the one from which all other lineages derive. The unusual pattern of lineage development observed in the yolk sac raises the following question: what is the relationship of the primitive erythroid lineage to the definitive populations? Several different studies have addressed this question. Using the ES/EB differentiation model, we identified a precursor that, in response to vascular endothelial growth factor (VEGF), generates colonies in methylcellulose cultures containing precursors of both the primitive and definitive hematopoietic lineages [67]. These VEGF-responsive precursors represent a transient population that arise in the EBs within 72 hours of development, prior to the appearance of any other hematopoietic lineage; a finding consistent with the interpretation that they represent one of the earliest stages of hematopoietic commitment (Fig. 2). Although the complete developmental potential of this precursor remains to be fully determined, its identification clearly demonstrates that, at least in this model system, primitive and definitive hematopoiesis can develop from a single cell. In a more recent study, we have isolated EB-derived clonal cell lines displaying both primitive and definitive hematopoietic potential, a finding that further supports the notion that these lineages share a common precursor [68]. In the third study, Turpen et al. [69] found that the mesoderm of the ventral blood islands, and the dorsal lateral plate of the Xenopus embryo have both primitive and definitive hematopoietic potential. Although clonal origins of these lineages were not demonstrated in this study, transplantation of these different sources of mesoderm to different regions of the embryo strongly suggest that the decision to commit to either the primitive or definitive programs can be influenced by the microenvironment. The influence of microenvironment on regulation of primitive and definitive hematopoiesis was recently investigated by Geiger et al. [70], who introduced adult bone marrow-derived stem cells back into an embryonic environment by injecting them into preimplantation blastocysts. Analysis of the developing embryos and fetuses demonstrated that progeny from the injected stem cells could be detected in the yolk sac, fetal liver, and peripheral blood. The stem cells used in this study were derived from a transgenic mouse carrying the complete human -globin gene locus, enabling these investigators to analyze the status of the human embryonic, fetal, and adult globin gene expression patterns in donor-derived cells in the resulting embryos. Expression of the human embryonic specific -globin gene was detected in different tissues of both Day 11.5 and 12.5 embryos, suggesting the reactivation of at least some embryonic programs in the adult marrow stem cells in this environment. While this study represents an important first step, the next

7 G. Keller et al./experimental Hematology 27 (1999) critical experiment will be to examine the reactivation of the mouse embryonic globin genes in the progeny of the injected stem cells and to determine the potential of these cells to contribute to the primitive erythroid lineage. In recent years, findings from different gene targeting experiments have provided evidence to support the concept that the primitive and definitive hematopoietic lineages develop from a common precursor by distinct molecular programs, and that the respective cell populations are regulated by different growth factors. The helix-loop-helix transcription factors scl/tal-1 [71,72] and the LIM-domain protein rbtn2 [73] are utilized by both lineages and presumably by the common precursor as mice lacking functional alleles of either gene develop no hematopoietic cells. In contrast, AML-1 (CBFA2) clearly distinguishes the two hematopoietic programs as AML-1 / knock out mice show normal yolk sac erythropoiesis but display no fetal liver definitive hematopoiesis [24,25]. C-myb / mice also show normal yolk sac development and again almost no definitive hematopoiesis with the exception of megakaryocyte development [74]. Other genes including jumonji [75] and LH2 [76] also appear to be utilized preferentially by the definitive hematopoietic system although the defects in the definitive lineages in the respective mutant animals are not as complete as observed in the AML-1 / and c-myb / mice. With respect to the regulation of growth and development of these populations, naturally occurring W and Sl mutations clearly demonstrate that the interaction of c-kit/ SLF is essential for full development of the definitive erythroid lineage in the fetal liver but not for yolk sac primitive erythropoiesis [2,77]. Gene-targeting studies have shown that the early stages of primitive erythropoiesis appear to progress in the absence of Epo while definitive erythropoiesis is Epo-dependent [78,79]. Finally, the gp130-signaling molecule appears to be essential for the proper development of the definitive program, but not for primitive erythropoiesis [80]. Together, these studies have identified transcription factors and growth regulatory molecules that are essential for definitive hematopoiesis but dispensable for primitive erythropoiesis. While they clearly demonstrate differences in the two programs, they also highlight our lack of understanding of the molecular mechanisms that specify the primitive erythroid lineage and regulate its development and growth. Analysis of mice with targeted alleles of essential primitive erythroid-specific genes could be complicated by early embryonic death, due to the lack of primitive hematopoiesis. The ES/EB system offers an alternative approach for such studies as the development of an EB is probably not dependent on functional primitive erythropoiesis. The hemangioblast: a common precursor for the hematopoietic and endothelial lineages The hypothesis that the early hematopoietic and endothelial lineages develop from a common precursor arose from histological studies of yolk sac, which showed that both populations matured rapidly and simultaneously from what was considered to be a common group of precursor cells [17,18]. Although this hypothesis was put forward many years ago, the identification, isolation, and characterization of a precursor with these characteristics has eluded developmental biologists for most of this time. While isolation of this precursor has been difficult, the concept of the hemangioblast has gained support from a variety of different experiments. First, many studies have demonstrated that developing hematopoietic and endothelial cells express a number of genes in common, including CD34 [81], flk-1 [82 85], flt-1 [86], TIE2 [87], scl/tal-1 [88], GATA-2 [89], and PECAM-1 [90]. Second, gene-targeting studies have shown a complete absence of hematopoietic and endothelial development in the yolk sac and P-Sp/AGM of embryos lacking a functional flk-1 receptor tyrosine kinase [91,92] and a marked defect in the growth and development of both lineages in the yolk sac of embryos lacking TGF 1 [93]. Third, studies on zebrafish have identified a mutation known as cloche that affects the development of hematopoietic lineages and endocardium in the embryo [94]. Fourth, overexpression of scl/tal-1 in zebrafish embryos leads to the overproduction of blood and vascular cells [95]. While these findings are consistent with the concept of a hemangioblast, they do not provide unequivocal proof for its existence. During the past 18 months, four different studies using clonal analysis, cell purification, and cell tracking approaches have provided new evidence supporting the concept of the hemangioblast. In the first study, we further analyzed the potential of the VEGF-responsive EB-derived precursor with primitive and definitive hematopoietic potential and found that many of these cells also demonstrated the capacity to generate adherent cells with endothelial characteristics [96] (Fig. 2). The VEGF responsiveness of these precursors is consistent with the above gene-targeting studies, demonstrating that a functional VEGF receptor (Flk-1) is required for the development of both the hematopoietic and endothelial cells in vivo. The fact that these EB-derived precursors display primitive and definitive hematopoietic potential suggests that they might represent the equivalent of the yolk sac hemangioblast. In the second study, Nishikawa et al. [97] demonstrated the presence of precursors with hematopoietic and endothelial potential in Flk-1 populations isolated from differentiating ES cells. Given that they express Flk-1, these precursors are likely similar, if not identical, to the VEGF-responsive precursors we have identified. In a third study, the same group isolated VE-cadherin cells from either the yolk sac or P-Sp/AGM region of the embryo proper and demonstrated multilineage hematopoietic potential in these populations [98]. As VE-cadherin is thought to be endothelial specific, these investigators concluded that these hematopoietic cells develop from a population of endothelial cells they referred to as hematogenic endothelium, a population that is presumably functionally equivalent to the hemangioblast. While these findings are consistent with this interpre-

8 784 G. Keller et al./experimental Hematology 27 (1999) tation, similar results would be obtained if a subpopulation of hematopoietic-restricted precursors also expressed VEcadherin. This possiblilty can and needs to be addressed by clonal analysis of the VE-cadherin precursors under conditions that support the development of both hematopoietic and endothelial cells. Finally, Jaffredo et al. [99] have recently provided evidence suggesting that intra-aortic hematopoietic cells are derived from endothelial cells in the chick embryo. To demonstrate this relationship, they used uptake of aceytlated low-density lipoprotein (AcLDL) to mark endothelial cells in chick embryos, and then followed the fate of the marker over a 24-hour period. AcLDL uptake is a characteristic of endothelial cells and macrophages [100,101]. Expression of CD45 was used as a marker of the hematopoietic lineage and the appearance of CD45 LDL cells was taken as evidence that hematopoietic cells developed from the marked endothelial cells. Although these findings support these interpretations, it is difficult to exclude the possibility that CD45 or CD45 lo immature macrophages or macrophage precursors took up the AcLDL independent of the endothelial cells and contributed to the CD45 LDL population. Formal proof of a precursor/progeny relationship between these lineages will require clonal analysis. Together, these most recent studies add further evidence in support of the hemangioblast. However, it is clear they only represent the beginning and that much more work needs to be done to isolate and characterize this elusive population. The studies using the ES/EB system provide the most direct evidence for the presence of a hemangioblast, as isolated clonal populations were shown to give rise to both hematopoietic and endothelial progeny. However, given that it is a model system, cells with similar potential need to be identified in the early embryo. The other two studies have provided evidence that cells with endothelial characteristic can generate hematopoietic progeny, and have demonstrated that these cells exist in the P-Sp/AGM of the embryo proper. Isolation of these cells and further characterization of their potential will be required to formally demonstrate that they represent the hemangioblast. Conclusions and future directions The large number of studies published in the last 5 years on different aspects of developmental hematopoiesis are a clear indication of the interest focused on understanding the events that regulate the commitment, growth, and maturation of the embryonic/fetal hematopoietic system. While there are many important questions that remain unanswered, we feel the following three areas are of particular interest. Development and maturation of the long-term repopulating stem cell As discussed, recent studies have provided evidence for the existence of subpopulations of LTRSC representing different stages of maturation. Specifically, those found in the Day 9.0 yolk sac/p-sp appear to require a fetal environment, whereas those present in Day AGM and fetal liver are able to function in the adult. Understanding the differences between these populations and identifying the mechanisms that enable a LTRSC to home, proliferate, and differentiate in an adult environment is an important issue. This will have obvious implications for the derivation of large numbers of LTRSCs from EBs. Development and growth regulation of the primitive erythroid lineage Gene-targeting studies have identified transcription factors that play a role in both primitive and definitive hematopoiesis, as well as those that are specific for the definitive lineages. However, primitive erythroid-specific factors have not yet been identified. Similarly, cytokines such as SLF and Epo are essential for definitive, but not primitive, erythropoiesis. The factors that specifically regulate the growth and maturation of the primitive erythroid lineage are unknown. Characterization of the molecular events involved in the commitment of the primitive erythroid lineage and the identification of the cytokines that regulate its growth are important aspects of developmental hematopoiesis that need to be addressed. An additional issue that needs to be resolved, with respect to primitive hematopoiesis, is that of the yolk sac macrophage population. Does this population represent a primitive lineage and if so, what is its relationship to the primitive erythroid lineage as well as to laterstage macrophages? Development and differentiation of the hemangioblast Recent studies have provided new evidence that further supports the concept of the hemangioblast. Formally demonstrating that this precursor exists in the early embryo is the goal of ongoing studies in many different labs. Assuming these studies are successful, the next important challenge will be to define the molecular events and cellular interactions that regulate the development of the hematopoietic and endothelial lineages from this precursor as well those specifying its commitment from mesoderm. Scl/tal-1 appears to be one of the important players in the early stages of hematopoietic commitment from the hemangioblast as, in its absence, no hematopoietic lineages develop. Identification of genes involved in the development of the endothelial lineages as well as others involved in the earliest stages of hematopoietic commitment are clearly important areas for future studies. With respect to commitment of mesoderm to the hemangioblast, a number of different studies have provided evidence that BMP-4 [ ], TGF 1 [93], and flk-1 [92] could play a role at this stage of development. In addition, transcription factors such as Mix. 1, thought to be involved in early hematopoietic committment [106], may be involved in the specification of the hemangioblast. Further characterization of these early stages of development will most certainly lead to the identification of additional impor-

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