Embryonic origins of mammalian hematopoiesis Margaret H Baron  Experimental Hematology  Volume 31, Issue 12, Pages 1160-1169 (December 2003) DOI: 10.1016/j.exphem.2003.08.019

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. Experimental Hematology 2003 31, 1160-1169DOI: (10.1016/j.exphem.2003.08.019)

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 ϵ-globin-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]. Experimental Hematology 2003 31, 1160-1169DOI: (10.1016/j.exphem.2003.08.019)

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]. Experimental Hematology 2003 31, 1160-1169DOI: (10.1016/j.exphem.2003.08.019)

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. Experimental Hematology 2003 31, 1160-1169DOI: (10.1016/j.exphem.2003.08.019)

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 G-protein-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 DNA-binding 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. Experimental Hematology 2003 31, 1160-1169DOI: (10.1016/j.exphem.2003.08.019)