Embryonic origins of mammalian hematopoiesis

Experimental Hematology 31 (2003) 1160–1169

Embryonic origins of mammalian hematopoiesis Margaret H. Baron Department of Medicine, Molecular, Brookdale Department of Cell and Developmental Biology, Ruttenberg Cancer Center, and Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY, USA

Hematopoiesis and vasculogenesis in the mammalian embryo begin in the blood islands of the yolk sac and continue, somewhat later, within the embryo proper. A subset of the first endothelial and hematopoietic cells of the yolk sac arise in close spatial and temporal association, apparently from a common mesodermal progenitor, the “hemangioblast.” The mechanisms that control formation of hemangioblast and embryonic hematopoietic and endothelial (angioblastic) stem/ progenitor cells are still not well understood. Formation of these cell types from nascent mesoderm requires signals from an adjacent outer layer of primitive (visceral) endoderm. Indian hedgehog (Ihh), a member of the hedgehog family of extracellular morphogens, is secreted by visceral endoderm and alone is sufficient to induce hematopoiesis and vasculogenesis in explanted embryos. While gene targeting studies in mice support a role for hedgehog signaling in these processes in vivo, they also suggest that additional molecules (perhaps, for example, Wnt proteins) are required for induction and patterning of hematopoietic and vascular mesoderm. Indian hedgehog likely functions through upregulation of genes encoding other signaling molecules, such as bone morphogenetic protein (Bmp)-4, in the target tissue. This review will focus on hematopoietic and vascular development in the early mouse embryo and will discuss potential implications of recent studies for stem cell transplantation in humans. 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

The first differentiated cells to form in the mammalian embryo are those of the hematopoietic and endothelial lineages. These cell types are mesodermal in origin and emerge during gastrulation, when cells recruited from the epiblast (embryonic ectoderm) migrate through the primitive streak, a posterior midline structure, and become organized into mesodermal cell layers of the embryonic and extraembryonic regions. The process by which different populations of mesoderm are set aside and patterned to form various mesodermal tissues is not well understood [1]. The characterization and functional analysis of early mesodermal cell populations and their immediate progeny is now of particular interest in light of the increasing focus on regenerative medicine and the potential for development of stem cell-based therapies for human diseases [e.g., see refs. 2–5]. Embryonic beginnings of hematopoietic and vascular development in the yolk sac The earliest hematopoietic cells of the developing mammalian embryo form in the extraembryonic yolk sac. Embryonic

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or “primitive” hematopoiesis is morphologically detectable by about 7.5 days postcoitum (d.p.c.) in the mouse [6–9] and between the second and third week of human gestation [10]. Thereafter, hematopoiesis shifts to the fetal liver and again, around the time of birth, to the bone marrow (Fig. 1). Primitive hematopoiesis (Fig. 1) results in the production primarily of large, nucleated erythroblasts [11] as well as some megakaryocytes [12] and primitive macrophages [13,14]. Whether or not these various cells arise from one or more types of primitive progenitor is at present unclear. Quantitation of primitive erythroid progenitors at different stages of early embryogenesis has indicated that the primitive erythroid lineage is transient: formation of these progenitors is undetectable by day 9.0 [15]. The yolk sac surrounds the entire mouse embryo (Fig. 2A, B, C, and F) as a result of axial rotation or “turning,” a peculiarity of that species. In all other mammalian species, including humans, the yolk sac is a balloon-shaped structure that connects to the midgut via a long stalk (Fig. 2D). In both mouse [Fig. 2G and ref. 16] and other mammalian embryos [17], the yolk sac comprises adjacent mesodermal and primitive (visceral) endodermal (VE) cell layers. Blood and endothelial cells form within “blood islands” in the yolk sac mesoderm [Fig. 2A, B, C, G and ref. 18] by late gastrulation, around 7.5 d.p.c. [8,9]. Endothelial and hematopoietic cells arise in close spatio-temporal association in

쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.

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definitive counterparts [29]. Thus, primitive and definitive erythropoiesis are much more similar than previously believed. Though many of the same genes are expressed in the progenitors of both primitive and definitive erythroid cells [e.g., Gata-1, Gata-2, Fog-1, Lmo2, Cbfa2/Runx1/ Aml1, CD41, refs. 25,30–33], their functions in the two cell types may be distinct. For example, the primitive erythroid lineage apparently forms independently of the Runx1-pathway [31,34,35]. Definitive hematopoietic stem cells (HSCs) are produced both in the mature yolk sac [36,37] and within the para-aortic splanchnopleura (day 8.5–9.5) [38,39] and

Figure 1. Changes in the major hematopoiestic sites during mammalian development. The earliest hematopoietic (“primitive”) and endothelial cells form in the yolk sac (YS). Primitive hematopoiesis is almost exclusively erythropoietic; some macrophages and megakaryocytes are also produced. “Definitive” hematopoietic stem cells with the potential to give rise to all hematopoietic lineages form both in the aorta-gonad-mesonephros (AGM) region and in the yolk sac. Thus, there is temporal overlap between the primitive and definitive phases of hematopoietic development. The definitive hematopoietic stem cells formed in the yolk sac and AGM region do not mature in situ but instead are believed to migrate to and seed the fetal liver, where they undergo terminal differentiation. The fetal liver remains the major hematopoietic organ until around the time of birth. Thereafter, normal hematopoiesis occurs largely in the bone marrow. Thymus and spleen also contribute importantly to lymphoid cell maturation but are not shown here. The diamond shapes denote the approximate times during embryogenesis when each phase of hematopoiesis takes place; the highest point of the diamond indicates the peak.

the yolk sac and may arise from a common mesodermal progenitor, the “hemangioblast” [see below and refs. 19,20]. Once the heart has begun to function effectively as a pump, primitive erythroblasts begin to circulate from the vitelline vessels of the yolk sac to the embryo and back [e.g., see refs. 21,22].

The formation and maturation of definitive hematopoietic stem cells Primitive vs definitive hematopoiesis “Definitive” hematopoiesis results in the production of all hematopoietic lineages. Primitive and definitive erythroid cells differ in embryonic site of formation, size, morphology, and expression of globin and other genes [23–25]. They are currently believed to represent distinct lineages. However, some experimental evidence suggests that these cell types could potentially share a common progenitor [26–28]. The established dogma regarding the distinction between primitive and definitive erythroid cells has recently been challenged by the intriguing observation that primitive erythroblasts, like definitive erythroid cells, undergo nuclear condensation and enucleation. The enucleated primitive erythroid cells are found in peripheral blood along with their

Figure 2. Mammalian hemato-vascular development. (A): The mouse embryo is encased in the yolk sac, an extraembryonic membrane. This panel shows an embryo (∼8.0 d.p.c.) from a human ε-globin-lacZ transgenic mouse line [73] stained for expression of β-galactosidase in primitive erythroid cells. Blood islands coalesce to form a primitive vascular plexus in a broad, extraembryonic ring. The allantois (al) is visible and protrudes upward from the posterior aspect of each embryo. fg, foregut (anterior aspect of embryo). (B): Embryo (∼8.5 d.p.c.) from the same transgenic mouse line as the embryo in panel A. A more extensive vascular network is now apparent. The position of the developing heart (ht) is indicated. (C): An embryo at about the same stage as the one shown in panel B, but with the yolk sac peeled down to expose the embryo proper. hf, head folds; al, allantois; ys, yolk sac. (D): Photograph of a 5-week-old human embryo. Ao, dorsal aorta; AL, anterior limb rudiment; L, liver; H, heart; YS, yolk sac. Reproduced with permission [45]. (E): Photograph of a human εglobin-GFP transgenic embryo at around day 9.5 p.c. Fluorescent and bright field images have been superimposed. The dorsal aorta (Ao) is clearly seen and the position of the developing heart (ht) is indicated. (F): The yolk sac vasculature is easily seen in this photograph of an embryo at about 12.5 d.p.c. The placenta is to the left. Arrow indicates one of the vitelline vessels of the yolk sac. Panel taken from [73], with permission. (G): Section through the yolk sac of an ∼8.5 d.p.c. embryo showing extraembryonic mesoderm and visceral endoderm layers as well as endothelial and primitive erythroid (EryP) cells. The amnion, another extraembryonic membrane, is also visible in this section. Reproduced with permission [9].

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aorta-gonad-mesonephros region (AGM, day 10.5–11.5) [40,41] of the embryo. Thus, within the yolk sac, there is a temporal (if not also spatial) overlap between primitive and definitive hematopoiesis (Fig. 1). The AGM region: An intraembryonic source of hematopoietic stem cells Clusters of round cells expressing several markers of HSCs have been observed within major intra- (AGM) and extraembryonic (vitelline and umbilical) arteries in both the mouse [31,42,43] and human [44–46] embryo and are thought to represent stem cells of the definitive hematopoietic lineages. Definitive HSCs also form in the yolk sac. Although it was concluded from early studies of yolk sac vs AGM hematopoiesis that the definitive HSCs formed in the yolk sac contribute only to primitive hematopoiesis [47], these cells do have definitive hematopoietic potential. Yolk sac cells isolated around day 9 and later can engraft and repopulate recipient animals following transplantation into the livers of newborn mice [36,48,49]. Cells with high proliferative potential are present in the yolk sac in significantly higher numbers and at later times than within the AGM [37,50]. Yolk sac cells develop the ability to repopulate adult bone marrow following coculture on certain stromal cells [51] or when HOXB4 is ectopically expressed [52]. Differentiation of HSCs does not occur within the AGM region [53]. Yolk sac-derived definitive HSCs appear to circulate [37,54] and can presumably seed intraembryonic tissues, including not only the liver but also large arteries. Together, these studies suggest that maturation of definitive yolk sac HSCs (perhaps within the AGM and/or fetal liver) may be a prerequisite for their differentiation and/or for their ability to home to critical intraembryonic and postnatal hematopoietic tissues. The fetal liver remains the site of definitive hematopoiesis until around the time of birth, when the bone marrow becomes the major hematopoietic tissue, as indicated in Figure 1 [37,39,41,55]. Hematopoiesis continues throughout the life of the animal. The hemangioblast: A common progenitor for hematopoietic and endothelial cells In addition to their development together within mesodermal blood islands (see above and Fig. 2G), hematopoietic and endothelial progenitors share expression of a number of genes, including Flk1 [56–59], CD34 [60], Scl/tal-1 [61], Flt1 [62], Gata-2 [63], Cbfa2/Runx1/AML1 [31], and Pecam1 [64]. Among these genes, only mutation of Flk1 results in complete absence of the hematopoietic and endothelial cell lineages in vivo. Analysis of “knockin” embryos in which Tal1 is expressed under the control of the Flk1 locus suggests that Flk1 and Tal1 act in combination to regulate cell fate decisions for formation of endothelial, hematopoietic, and smooth-muscle cells in early development [65]. Identification of cells with the properties of the hemangioblast has been difficult [65,66]. The best evidence for

the existence of the hemangioblast comes from work in the mouse embryonic stem (ES) cell differentiation system. When plated in the absence of a feeder layer and leukemia inhibiting factor (LIF), ES cells aggregate to form “embryoid bodies” (EBs) which recapitulate a number of aspects of normal embryonic development, including primitive and definitive hematopoiesis [67]. Upon replating of cells from EBs formed between days 2 and 3 in culture, a colony type (“blast”) is found which has the potential to give rise to both adherent endothelial cells and nonadherent hematopoietic cells [20,68]. Blast colonies have recently been shown to give rise to a third lineage, the smooth-muscle cell [65]. Analyses of blast colony formation by ES cell lines in which Flk1 [65,69] or Runx1 [35] have been genetically ablated have demonstrated their importance in the formation and differentiation of the hemangioblast in vitro. In addition, expression in VE of one of the ligands of Flk1, VEGF-A, is required for blood island development in vivo [see below and ref. 70]. Blast colonies can be identified only during a narrow window of time in culture, following the appearance of mesodermal “transitional colonies” [68] and preceding the appearance of colonies of stem/progenitor cells with broad hematopoietic potential [20,26]. Blast colonies have been detected in small numbers in dissected mouse embryos [66], suggesting that hemangioblasts may form during normal mouse development. The technical difficulties encountered in identifying cells with the properties of the hemangioblast in mouse embryos suggests that these cells are produced in very small numbers and for a very short time. Perhaps the cells themselves are also short-lived, differentiating soon after their formation. Orthotopic transplantation experiments with cultured gastrulation-stage mouse embryos led to the conclusion that yolk sac erythroid progenitors emerge from the primitive streak at an earlier time than do the angioblastic progenitors of the vitelline endothelium [1]. Those studies suggested that the primitive erythroid and endothelial cell lineages may arise independently rather from a hemangioblastic progenitor [1]. However, they do not exclude the possibility that yolk sac development could have both hemangioblastic and angioblastic components. Thus, hemangioblasts might arise from a transient subpopulation of nascent mesoderm whose presence escaped detection in the transplantation assay. Resolution of this question may now be possible with the recent development of a culture system that permits dynamic imaging of embryonic hematopoietic and vascular development [21].

Endodermal signals involved in induction of embryonic hematopoiesis and vasculogenesis Signaling interactions between neighboring tissues are a common theme in embryonic development. The primitive or visceral endoderm of the mammalian embryo is a secretory

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epithelium that is strategically positioned to signal to the underlying ectoderm and, following the onset of gastrulation, to nascent mesoderm [see Fig. 2G and refs. 16,71–73]. The anterior VE (AVE) is a well-described signaling center that forms very early during postimplantation development and plays a decisive role in antero-posterior axis determination and in positioning of the primitive streak [74,75]. Patterning of the posterior epiblast (the embryonic ectoderm) is less well understood. Just prior to and during the initiation of gastrulation, bone morphogenetic protein (Bmp)-2 [76] and a mouse orthologue of the Xenopus and zebrafish Mix/Bix genes [77–79] are transiently expressed in the posterior VE and are later expressed in the epiblast. The primitive streak itself is thought to function as a posterior organizing center [74,75] whose signals are mediated by specific sets of transcription factors. Several members of the transforming growth factor (TGF)-β/BMP family of secreted signaling molecules, their extracellular agonists and antagonists, and a variety of transcription factors regulate the position and size of the streak as well as the formation and/or patterning of at least some populations of mesoderm [75,80–82]. By late gastrulation, mesodermal cells have moved into the extraembryonic region and are physically in contact with three different lineages (embryonic and extraembryonic ectoderm and VE). However, only those mesoderm cells adjacent to VE will form the first hematopoietic and endothelial cells of the yolk sac [9]. Strikingly, the first blood islands and the earliest vascular network form in an extraembryonic ring [Fig. 2G and refs. 9,73,83]. The close apposition of mesoderm and visceral endoderm in the gastrulating embryo and in the mature yolk sac [9,16] suggested that interactions between primitive endoderm and mesoderm might play a role in the initiation of embryonic hematopoiesis and vasculogenesis, as had been demonstrated previously in the chick [84–86]. To explore this possibility, an explant culture “induction” assay was devised [Fig. 3 and ref. 73]. Transgenic embryos carrying a lacZ reporter gene expressed in primitive erythroblasts [73] or endothelial cells [Flk1 or Cbfa2/Runx1/ AML1; see 31,87] were separated into ectodermal and endodermal components and cultured alone or as recombinants between transgenic and nontransgenic tissues [73,83]. These experiments led to the discovery that molecules secreted by the VE provide potent signals for activation of hematopoiesis and vasculogenesis. Therefore, although both hematopoietic and endothelial cells arise from yolk sac mesoderm, they do not develop autonomously in this tissue. In a modification of the explant culture assay, anterior epiblasts dissected from mid-streak embryos (∼6.75 d.p.c., Fig. 4) were used. The anterior epiblast of an embryo at this stage does not yet contain mesoderm [88] and is fated to form neurectoderm but not hematopoietic or endothelial cells. Epiblasts stripped of VE were dissected into mesoderm-free anterior pieces and posterior pieces with their associated mesoderm (Fig. 4). Transgenic anterior epiblast pieces were recombined with nontransgenic VE in collagen droplets

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Figure 3. Induction assay used to demonstrate function of visceral endodermal (VE) signaling in activation of embryonic hematopoietic and vascular development. Pre- or early-gastrulation-stage embryos are stripped of their outer layer of visceral endoderm (VE) and cultured alone or as recombinants with VEs from other embryos. RNA is then prepared and analyzed for expression of endogenous genes using RT-PCR or the explant is stained with Xgal to reveal expression of lacZ (β-galactosidase). Examples of transgenic mouse lines that have proven especially useful for these studies are human ε-globin/lacZ [73], Flk1/lacZ [87], and Cbfa2/Runx1/AML1 [31]. Panel to right shows reverse transcriptase–polymerase chain reaction (RT-PCR) analysis of separated VE and epiblast (E, embryonic ectoderm) tissues. Boxes around VE and E lables indicate that the tissues were taken from the same embryo. A control (lane “-”) in which cDNA was omitted from the PCR is shown. Afp, α-fetoprotein, a marker of VE; Fgf4, fibroblast growth factor-4, a marker of the primitive streak of the epiblast. Actin served as an internal control. Modified from [16], with permission. The lower panel shows an RT-PCR analysis of individual whole embryos, epiblasts stripped of VE, or recombinants between epiblast and VE. A yolk sac from a 10.5 d.p.c. embryo served as a positive control. Negative controls (NT, no cDNA template; ⫺RT, no RT added to cDNA synthesis reaction) are shown. Actin served as an internal control. εY, one of the mouse embryonic β-like globin genes, expressed only in primitive erythroblasts. Reproduced with permission [73].

and cultured in this “reprogramming” assay. Surprisingly, transgenic anterior epiblasts recombined with nontransgenic VE, but not anterior epiblasts cultured alone, produced large numbers of β-gal⫹ blood cells. This experiment confirmed that the erythroid cells present in cultured recombinants were derived from anterior epiblasts (transgenic) and not from VE (nontransgenic) [73]. Analysis by semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) of expression of a panel of endogenous hematopoietic and endothelial markers indicated that VE signaling results in activation not only of genes characteristic of differentiated erythroid and endothelial cells but also of genes expressed in progenitor cells, including Flk1 and CD34 [73]. Cells replated from epiblast-VE recombinants produced primitive erythroid colonies in clonogenic (methylcellulose) cultures [83]. Together with the observed downregulation of markers of prospective neurectoderm in anterior epiblast-VE recombinants, these findings suggested that the inductive signals from VE are instructive [see below and 73,83]. Transfilter assays and experiments with conditioned medium

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Figure 4. Photograph of a mid-gastrulation (midstreak)-stage mouse embryo (∼6.75 d.p.c.) used in the reprogramming assay. The epiblast is surrounded by a layer of visceral endoderm (VE). Anterior, a; posterior, p; epi, epiblast; exe, extraembryonic ectoderm; m, mesoderm beginning to form between epiblast and VE. Arrow indicates approximate position of extraembryonic-embryonic border. For the reprogramming assay, an anterior epiblast (region included within the dashed lines) stripped of VE is cultured alone, as a recombinant with VEs from other embryos, or with recombinant signaling molecules added directly to the medium or adsorbed to heparin-acrylic beads [73,83]. Explants are cultured and stained with Xgal or analyzed for expression of endogenous RNAs by RT-PCR.

[73,83] demonstrated that the respecification of anterior epiblast cells to a posterior fate is mediated by diffusible signal(s) from VE.

Hedgehog morphogens in hematopoietic and vascular development Later work implicated a member of the Hedgehog family of signaling molecules [89,90], Indian hedgehog (Ihh), in hematopoietic and vascular induction in the embryo. Ihh is expressed specifically in the VE surrounding the gastrulating epiblast as well as in the endodermal layer of the mature yolk sac at much later stages [16,83]. The genes encoding Ptch, Smo, and Gli are all expressed in the target epiblast adjacent to the VE, indicating that this tissue is competent to respond to Hh signaling [83]. The hedgehog signaling pathway Hedgehogs are morphogens and function in a diverse array of biological processes [reviewed comprehensively in ref. 90]. The hedgehog protein is initially translated as a larger peptide that comprises an amino-terminal domain, destined to become the biologically active molecule, and a carboxyterminal autocatalytic domain. The pro-peptide undergoes

proteolytic cleavage and esterification with a cholesterol moiety [89,91,92]. Cholesterol functions not only as an adduct for biologically active hedgehog protein, playing a role in trafficking and movement across cellular fields [93], but also as a cofactor in the autoprocessing reaction [94]. Hedgehog is modified by other lipids in addition to cholesterol [95]. The active form of hedgehog is the aminoterminal (HH-N) peptide covalently linked at its carboxyl terminus with cholesterol [96–99]. Patched (Ptch) is the membrane receptor for Hh but does not directly transduce the hedgehog signal. This function is performed by Smoothened (Smo), a relative of G-protein-coupled membrane receptors. Ptch is a tumor suppressor [90,100,101] and catalytically represses Smo activity in the absence of Hh ligand, possibly in association with a smallmolecule transport activity [102]. Binding of Hh to Ptch relieves this inhibition [103]. Members of the Gli family of zinc finger transcription factors are activated by Hh signaling. They may function as activators or repressors, as indicated in the highly simplified cartoon in Figure 5. Induction of hematopoietic and endothelial cells by hedgehog protein in vitro Recombinant human HH-N protein is sufficient to induce formation of hematopoietic and endothelial cells in both cultured early streak epiblasts (in the “induction” assay) and in mid-streak anterior ectoderm (in the “reprogramming” assay), suggesting a role for this protein in early hematovascular development in vivo [83]. Although the biologically relevant protein is presumably Ihh, recombinant forms of all three mammalian hedgehog proteins can substitute for VE tissue in vitro [83]. When cells from HH-treated anterior epiblasts are replated in secondary cultures in the presence of appropriate cytokines, both primitive [83] and definitive (unpublished) hematopoietic colonies form, indicating that, as expected, functional hematopoietic stem/progenitor cells as well as their differentiated progeny are produced in response to recombinant HH-N peptide. Antibody blocking of Ihh function in VE inhibits activation of hematopoiesis and vasculogenesis in the adjacent epiblast, indicating that Ihh is an endogenous VE signal [83]. These conclusions are supported by work with differentiating embryonal carcinoma (EC) and ES cells [104]. Interestingly, in ES cell–derived embryoid bodies formed in the presence of the hedgehog antagonists cAMP and forskolin, the outer endoderm layer underwent a transition to parietal (rather than visceral) endoderm and expression of hematopoietic and vascular markers was reduced [104]. Functional redundancy or compensation by other proteins Analysis of Ihh mutant mice suggests that there may be some functional redundancy in the hedgehog pathway during early embryonic development. About 50% of Ihh-null

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Figure 5. Simplified diagram of the hedgehog signaling pathway. Autocatalytic cleavage and addition of a cholesterol moiety to the amino-terminal peptide results in the production of the biologically active hedgehog ligand. Additional lipid modifications have been observed. Smoothened (Smo) is the signaling component of the Hh receptor complex and may be a Gprotein-coupled receptor. Its activity is constitutively inhibited by Patched (Ptch) in the absence of Hh ligand; the mechanism of inhibition may involve small molecule transport. The fates of Ptch and Smo in the cell appear to be intimately linked to the endocytosis-lysosome pathway; see [108] for a thoughtful review on this subject. Hh ligand availability is regulated by proteins such as HIP (Hh interacting protein) [126] and Dispatched (regulates availability of secreted Hh peptide to responding cells) [127–129], as well as proteoglycans [90]. Gli is a small family of zinc finger transcription factors [130]. In the absence of Hh, Gli is proteolytically cleaved to a form (Gli-Rin the figure) that lacks a transactivation domain but retains a DNAbinding domain. Gli-R is translocated to the nucleus, where it functions as a repressor [130]. Inhibition of signaling by Smo is relieved upon binding of Hh to Ptch and involves hyperphosphorylation primed by PKA. Subsequent phosphorylation of Gli (Gli-A in the figure) renders it resistant to proteolytic digestion. Gli-A can then move into the nucleus to activate Hh target genes. Target genes (direct or indirect) may include Bmp4, Vefg, and angiopoietins [118,131]. Expression of Ptch is also upregulated in response to Hh signaling, thereby providing negative feedback control by once again inhibiting Smo. Yet another level of complexity not indicated in this figure (and as yet still not well understood) is conferred by interactions between Gli, Costal2 (Cos), and Fused (Fu) within a complex that associates with microtubules. It is possible that protein kinase A (PKA), GSK3, CK1, or phosphatases are recruited into this complex [90,108], which is thought to regulate the phosphorylation state (and availability for proteolysis) of Gli.

mutant embryos are smaller than their wild-type or heterozygous littermates and die around mid-gestation, apparently due to cardiovascular defects [105,106]. Another Hh family member, Desert hedgehog (Dhh), is activated in the embryo during gastrulation [83] and is expressed in the mature yolk sac mesoderm [16]. Recombinant Dhh protein can substitute for VE in explant culture [our unpublished data and 83]. In Ihh-null embryos, therefore, Dhh might compensate, at least in part, for the function of Ihh in hematopoiesis and vasculogenesis. Indeed, there is evidence for redundancy of Ihh and Shh function in at least some processes [107].

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Even more severe vascular defects have been reported [106] in the yolk sacs of Smo mutant embryos in which the hedgehog signaling pathway cannot function [107]. Primitive erythroblasts do form in the absence of Ihh or Smo [106], but more subtle hematopoietic defects have not been excluded. In contrast with Ihh or Smo mutant embryos, blood islands apparently do not form in embryoid bodies formed from Ihh- or Smo-null mutant ES cells [106]. Hedgehog signaling alone is not essential for formation of primitive hematopoietic and endothelial cells in vivo. A second pathway that might prove to function in these developmental processes is the Wnt pathway. Similarities between these pathways (e.g., lipid modification, function of phosphorylation in target transcription factor activation, involvement of a G-protein-coupled receptor in signaling) have been discussed elsewhere [e.g., see refs. 89,101, 108,109]. Interestingly, hedgehog [110] and Wnt3A [109] protein can stimulate proliferation of human and mouse hematopoietic stem cells, respectively. Because exogenous hedgehog protein [83], like VE tissue [73], could reprogram anterior neurectoderm to hematopoietic and endothelial cell fates, it was proposed that the endogenous signal is instructive [83]. The broad array of biological functions of the hedgehog pathway [90,111] would seem to suggest that the role of Hh is normally permissive. Detailed analysis of mutations in different components of the Hh signaling pathway (singly or in combination) will be required to establish whether, in vivo, Ihh regulates one or more processes such as the formation and/or proliferation of hematopoietic/vascular stem/progenitor cells, specifies cell fate, and regulates vascular remodeling and morphogenesis. Possible mechanisms by which hedgehog might regulate hematopoietic and vascular development The mechanism by which hedgehog signaling regulates hematopoietic and vascular development remains to be established, but may involve bone morphogenetic protein-4 (Bmp-4) in the target embryonic ectoderm and/or extraembryonic mesoderm [83]. Bmps are often downstream from hedgehog signals [90,112], and Bmp-4 is upregulated in explanted anterior epiblasts in response to recombinant IHHN [83]. As predicted, BMP-4 protein can substitute for VE tissue or hedgehog protein in explant cultures [our unpublished data and ref. 9]. Gene targeting of mouse Bmp-4 results in defects in hematopoietic and vascular development on some genetic backgrounds [113,114]. Therefore, endogenous Bmp-4 protein could mediate the hedgehog signaling activities observed in the explant cultures [83]. Ex vivo treatment of HSCs in human cord blood with recombinant Sonic hedgehog protein stimulates their proliferation, apparently through activation of BMP-4 [110]. In the mouse explant culture system, upregulation of the mouse Bmp-4 gene by Hh signaling in the explant cultures is likely to be indirect,

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as good consensus binding sites for Gli [115,116] or COUPTFII [another transcription factor activated in response to Hh, ref. 117] are not present within the promoter and upstream sequence of the mouse Bmp-4 gene (our unpublished data). Another interesting possibility is that hedgehog signaling may activate expression of VEGF and angiopoietins, as observed in a mouse hindlimb ischemia model [118]. In differentiating ES cell–derived embryoid bodies, BMP4 and VEGF synergistically stimulate formation of hematopoietic cells [119]. BMPs have also been shown to stimulate angiogenesis in bone explants through osteoblast-derived VEGFA [120]. VEGF is, like Ihh, secreted by VE. Interestingly, recent analysis of a genetically engineered hypomorphic Vegf mutant indicated that VEGF-A activity is required in visceral endoderm for proper hemato-vascular development of the early embryo [70]. Embryos homozygous for the hypomorphic Vegf allele die at 9.0 d.p.c. as the result of a severe abnormalities in yolk sac vasculature and in development of the dorsal aorta. When wild-type (WT) VE was provided to the hypomorphic embryos in tetraploid-WT embryo aggregation experiments, normal differentiation of yolk sac hematopoietic and endothelial cells was observed, but the defects in the embryo proper were not rescued. However, in the reciprocal experiment in which Vegf hypomorphic VE was provided to a WT embryo, the yolk sac abnormalities were not rescued. VEGF-A secreted by VE may function as a chemoattractant for nascent mesoderm cells migrating out of the primitive streak [70].

cyclopamine, a teratogen and antitumor agent, inhibits Hh signaling by binding directly to Smo [121,122]. Small-molecule inhibitors [122,123] and activators [123] of hedgehog signaling have been identified which, like cyclopamine, bind directly to Smo. There is, then, good reason to hope that such compounds may find applications in (to name a few possibilities) therapy of leukemias and other malignancies (e.g., through inhibition of blast cell proliferation and/or tumor angiogenesis), expansion of HSCs for transplantation purposes, or treatment of a variety of human anemias. It is worth noting that, because hedgehog proteins may function by upregulating the expression of multiple secreted signals such as BMPs, VEGF, and angiopoietins (see above), therapeutic approaches aimed at stimulation or inhibition of the Hh pathway might be more effective than the anti-tumor and anti-angiogenic agents currently available [e.g., see 124,125]. Given the stimulatory effects of hedgehog and WNT proteins on hematopoietic stem cells, potential interactions between these pathways in regulation of hematopoietic and vascular development should be explored: combination therapies may be more powerful than approaches directed at a single pathway.

Acknowledgments I thank M. Tavian and B. Pe´ault for the photograph shown in Figure 2D. Work in my laboratory is supported by National Institutes of Health grants RO1 DK52191 and HL62248.

References The hedgehog pathway as a potential therapeutic target Whether or not hedgehog signaling functions in hematopoiesis or vascular development in the adult, this pathway might still represent a potential target for new therapies for human blood and vascular diseases. Constitutive activation of the hedgehog pathway results in the formation of a number of different cancers in humans [90,101], perhaps by redirecting cells toward a more stem cell-like fate [101]. Though the Hh pathway has not been implicated in hematopoietic malignancies, it is worth noting that leukemia is considered a disease of stem/progenitor cells and that recombinant hedgehog protein has been shown to stimulate proliferation of HSCs in human cord blood [110]. Small-molecule hedgehog agonists and antagonists may, therefore, provide clinically useful tools for pharmacologic regulation of the cellular response to hedgehog signals. In addition, it might be possible to modify the differentiation or self-renewal of stem and progenitor cells by targeting the hedgehog, WNT, and BMP pathways using the tools and approaches of gene therapy. That the Hh pathway might be subject to control by small molecules under normal physiological conditions was first suggested by the observation that the steroidal alkaloid

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