Handbook of Stem Cells || Ontogeny of the Hematopoietic System

Chapter 47

Ontogeny of the Hematopoietic System

Malcolm A.S. MooreMemorial Sloan-Kettering Cancer Center, New York, NY, USA

Ha

Co

Chapter Outline

Historical Perspective 533

Sites of Initiation of Primitive and Definitive

Hematopoiesis and Vasculogenesis 534

Yolk Sac Development 534

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pyright �

Molecular Pathways Involved in Yolk Sac

Hematopoietic Development
534
Onset of Primitive and Definitive Hematopoiesis in

the Yolk Sac
536
Macrophage and Microglial Ontogeny 537

Ontogeny of the Vasculature 537

Initiation of Hematopoiesis in the AortaeGonade

Mesonephros Region 537

Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00047-0

2013 Elsevier Inc. All rights reserved.

Avian Intra-aortic and Para-aortic Hematopoiesis
537
Cell Migration to Later Sites of Hematopoiesis 541

Ontogeny of Liver Hematopoiesis 541

Ontogeny of Bone Marrow 542

Ontogeny of the Spleen 543

Cell Migration to Primary Lymphoid Organs 543

Ontogeny of the Thymus 543

Origin and Commitment of Thymic-Colonizing Cells
544
Ontogeny of B-Lymphocytes 545

Avian Bursa of Fabricius
545
Mammalian B-Cell Ontogeny
545
References 546

HISTORICAL PERSPECTIVE

The origin and potentiality of the hematopoietic stem cellhas been debated for more than 100 years between twomainconcepts: the monophyletic hypothesis recognizinga common stem cell for all lymphomyeloid lineages and thepolyphyletic hypothesis recognizing a variety of distinctstem cells (Danchakoff, 1916;Maximow, 1924). The currentconsensus recognizes a pluripotential lymphomyeloid stemcell and a hierarchy of progressively lineage-restrictedprogenitor cells with a major bifurcation at the level of thecommon lymphoid progenitor (CLP) and the commonmyeloid progenitor (CMP) (Moore, 2003; Akashi et al.,1999). One early assumption had been that each developinghematopoietic or lymphoid organ produces its owncomplement of stem cells, with morphological studiesvariously proposing an in situ origin in hematopoietic tissuesfrom undifferentiated mesenchyme or endothelium and, inprimary lymphoid organs such as the thymus or the avianbursa of Fabricius, a derivation from epithelium (Maximow,1924). More than 35 years ago, this view was challenged byMoore and Owen (Moore and Owen, 1965, 1966a, 1966b,1967a. 1967b; Metcalf and Moore, 1971) in a series ofchromosome marker studies in avian embryos that showed

developing hematopoietic and lymphoid organs were colo-nized from the outset by an inflow of stem cells from theblood stream. Since the yolk sac (YS) was identified at thattime as the first site of hematopoiesis as early as 35e48hours of incubation, it was proposed as the site of origin ofstem cells that colonized all subsequent hematopoietic andlymphoid organs (Moore and Owen, 1967b). This hypoth-esis was modified following marker studies in chickequailand chickechick chimeras established by reciprocal graft-ing of the early (35e48 hours) embryo and YS, whichdemonstrated the embryo origin of hematopoietic orlymphoid cells that later appeared in the spleen, marrow,thymus, and bursa of Fabricius (Dieterlen-Lievre, 1975;Lassila et al., 1978; Lassila et al., 1982; Dieterlen-Lievreet al., 1997). Nevertheless, injection of YS cells into irra-diated embryos demonstrated that the 7-day YS was a richsource of stem cells capable of repopulating all hemato-poietic and lymphoid organs (Moore and Owen, 1966a;Metcalf andMoore, 1971). This suggested that the avian YSserved a role analogous to the fetal liver in mammals,expanding a small initial stem cell population that hadmigrated from the intraembryonic para-aortic region andsecondarily seeded stem and progenitor cells to other sites.

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534 VOLUME | 2 Adult and Fetal Stem Cells

Inmammalian systems, hematopoiesis is clearly initiatedin the YS (Moore andMetcalf, 1970). It subsequently occursin the aortaegonademesonephros (AGM) region in associ-ation with the dorsal aorta and vitelline veins, where the firststem cells arise with the capacity to engraft adult recipients(Medvinsky et al., 1993; Medvinsky and Dzierzak, 1996;Kumaravelu et al., 2002; Tavian et al., 1999). There has beenextensive debate about the role of the YS in mammalianhematopoietic ontogeny. On the one hand, the site is viewedas the initial source of stem cells that then migrated into theembryo (Moore andMetcalf, 1970); the other extreme viewsthe YS as a transient source of primitive erythroid progeni-tors with definitive generation stem cells arising exclusivelyin the AGM region (Tavian et al., 1999, 2001). There isincreasing evidence that stem cells arise independently inboth sites and contribute to the subsequent colonization ofthe fetal liver (Kumaravelu et al., 2002; Matsuoka et al.,2001; Yoder et al., 1997).

SITES OF INITIATION OF PRIMITIVEAND DEFINITIVE HEMATOPOIESISAND VASCULOGENESIS

The sites of hematopoiesis, and the primary sites of lym-phopoiesis, with times of initiation of activity in develop-ment, are show in Table 47.1. Although there have beenextensive developmental studies in Xenopus and zebra fish,space will limit this review to studies in avian, murine, andhuman systems.

Yolk Sac Development

The YS forms during gastrulation, which begins in themouse embryo at embryonic day 6.5 (E6.5). Mesodermal

TABLE 47.1 Comparative Chronology of

Hematopoietic Development

Initiation of hematopoiesis

or lymphopoiesis (days)

Mouse Man Chicken

Yolk sac 7.5 18 2

AorticeAGM region 9.5e10 27 3

Liver 11 42 No

Spleen 13 48 8

Bone marrow 15 77 11

Thymus 11 40 7

Bursa of Fabricius No No 14

Onset of circulation 8.2 24 2.5

cells destined for extraembryonic sites exit the posteriorprimitive streak and subdivide the embryo into three cavi-ties by the neural plate stage (E7.5). The central cavity, theexocoelom, becomes completely lined with mesoderm;where this is adjacent to visceral endoderm, visceral YSforms. Between E7 and 7.5, mesodermal cells in thevisceral YS proliferate and form mesodermal cell massesthat are the precursor of the blood islands. Central cellsaccumulate hemoglobin, and the outer cells flatten andform endothelium. Lineage tracing experiments show thatthe hematopoietic mesoderm arises from posterior primi-tive streak mesoderm (Belaoussoff et al., 1998). Usinga Lac reporter gene linked to embryonic globin regulatorysequences, Baron (2001a,b) showed that as early asE6.5e6.75, nascent mesoderm had already receiveda blood-inducing signal from the visceral endoderm. Tissuerecombination studies in the chick embryo also indicatedthat YS hematopoiesis requires diffusible signals fromextraembryonic endoderm (hypoblast), which is analogousto visceral endoderm of the mouse. Indian hedgehog (Ihh)and smoothened (Smo), a receptor component essential forall Hedgehog signaling, are required for YS development(Byrd et al., 2002). Mice with null mutations of Ihh or Smodie at midgestation because of vascular defects in the YS(Byrd et al., 2002). Ihh appears to be the endodermalsignal-inducing hematopoietic and vascular specification ofYS mesoderm, acting through the induction of bonemorphogenic protein-4 (BMP4) in mesoderm that in turninduces hematovascular development (Belaoussoff et al.,1998; Baron, 2001a,b) (Figure 47.1).

Molecular Pathways Involved in Yolk SacHematopoietic Development

Blood islands express: the vascular endothelial growthfactor (VEGF) receptors Flk1 and Flt1 and the cytokinereceptors cKit and cMpl; the potential adhesion moleculesCD34, CD41, CD44, PECAM-1, and VE-cadherin; thetranscription factors SCL/Tal-1, GATA1, GATA2, andAML1/Runx1; and transforming growth factor (TGF)family members TGFb, BMP4, and their downstreamsignaling components e SMADs (Baron 2001b; Liu et al.,2003; Cantor and Orkin, 2002; Dickson et al., 1995). SCLis a transcription factor whose inactivation leads toembryonic death at E9.5 because of the absence of red cells(Gering et al., 1998). CD41, possibly a direct target of SCL,is normally restricted to the megakaryocytic lineage inthe adult, but in the YS it is expressed on multilineagedefinitive hematopoietic cells (but not on primitiveerythroid lineage) and precedes the expression of the pan-lympho-hematopoietic marker CD45 (Mikkola et al., 2003).AML1-deficient mice die between E12e13 and fail to formdefinitive lineage hematopoiesis in the liver, but they havenormal primitive YS erythropoiesis (North et al., 2002;

Mesoderm

BMP4

Mesoderm

Ihh

BMP4

IhhBMP4

Visceralendoderm

BMP4

Blood islands

Visceral endoderm

Mesoderm

Endothelial cell

Erythroblast

Visceralendoderm

FIGURE 47.1 Activation of primitive hematopoiesis and vasculogenesis in the developing yolk sac. Ihh is secreted from the visceral endoderm to

the target extraembryonic (YS) mesoderm, where it activates expression of bmp4. The BMP4 protein feeds back to extraembryonic mesoderm (in an

autocrine or paracrine manner), activating genes such as Flk1, CD34, SCL/tal-1, and AML1. This is associated with the formation of hematopoietic and

vasculature stem and progenitor cells, possibly through a common precursor, the hemangioblast. Reproduced with permission from Baron (2001a).

535Chapter | 47 Ontogeny of the Hematopoietic System

Mikkola and Orkin, 2002). VEGFA plays a pivotal role inthe first step of endothelial and hematopoietic developmentin the YS and at the onset of gastrulation is expressed in theYS visceral endoderm and mesothelium (Damert et al.,2002). Its receptors, Flk1 and Flt1, are expressed on thehemangioblasts and angioblast, and Flt1 is expressed on thehematopoietic stem cells (HSCs) developing in the bloodisland (Kabrun et al., 1997; Nishikawa et al., 1997; Choi,2002). YS multilineage progenitors are also regulated bya hypoxia-mediated signaling pathway that requireshypoxia-inducible factor 1 (Adelman et al., 1999). Micedefective in this pathway exhibit decreased YS hemato-poiesis and vessel formation, accompanied by decrease inVEGF production. Core-binding protein (CBP), a coac-tivator of several transcription factors, also plays a criticalrole in establishing YS vasculature, and mice with mutatedCBP die between E9.5e10.5 with lack of a vascularnetwork with secondarily decreased YS hematopoiesis(Oike et al., 1999). The cytokine receptors cKit and cMplplay an essential role in HSC proliferation and act as earlyas the hemangioblast stage (Xu et al., 2001; Xie et al., 2003;

Perlingeiro et al., 2003). The Mpl ligand, thrombopoietin(Tpo), is expressed in the early YS and is responsible formaturation of primitive megakaryocyte progenitors to low-ploidy megakaryocytes by E8.5 with subsequent plateletrelease (Xu et al., 2001). Tpo-responsive, definitivemegakaryocyte and mixed erythroidemegakaryocyte-progenitors appear by E8.25 (Xie et al., 2003). GATA1,a zinc-finger transcription factor, is critical for erythroiddevelopment, and GATA1 null mice die at E10.5 of anemiawith erythroid maturation arrest (Cantor and Orkin, 2002).TGFb is expressed by E7.5 in YS blood islands, and TGFb-null embryos have defective YS vascularization andreduced erythroid cell production (Dickson et al., 1995).SMAD5 transduces both BMP4 and TGFb receptor signals,and its targeted disruption results in multiple defects anddeath between E10.5e11.5 (Liu et al., 2003). However, theYS of these mice at E9.5 shows reduced proliferation ofprimitive erythroid precursors but elevated numbers ofdefinitive, high-proliferative potential multipotent colony-forming cells (HPP-CFCs) with enhanced replatingpotential.


Onset of Primitive and DefinitiveHematopoiesis in the Yolk Sac

The YS is the site of primitive erythropoiesis and macro-phage production and of definitive multilineage, myeloidlineage-restricted, and erythroid lineage-restricted progen-itor cell production (Moore and Metcalf, 1970; Yoder andHiatt, 1997; Palis et al., 2001). The wave of primitiveerythropoiesis begins in the YS at E7.5 with nucleated redcells producing embryonic globin. It predominates in thecirculation through E14.5 with eventual replacement byfetal liver-derived red blood cells (RBCs) expressing adultglobins between E15.5e16.6. In humans, b-like embryonicglobin (hemoglobin ε) is expressed first in embryonicnucleated RBCs in YS blood islands. Subsequently, fetalglobins (hemoglobin Ag and Gg) are expressed in defini-tive RBCs developing in the fetal liver. Finally, adult d- andb-globins are expressed around the time of birth withinbone-marrow-derived RBCs (Cantor and Orkin, 2002).A zinc-finger transcription factor, erythroid Kruppel-likefactor, plays a role in coordinating erythroid cell prolifer-ation and hemoglobinization, participating in the switchfrom embryonic to fetal or from fetal to adult b-globinexpression (Cantor and Orkin, 2002).

A population of HPP-CFCs was detected at E8 (1e8somites), exclusively in the YS. This remained thepredominant site of expansion (>100-fold) of these mul-tipotent precursors until between E10.5e11.5, when therewas a dramatic increase of HPP-CFCs in the circulation andliver with a concomitant drop in the YS (Palis et al., 2001).Upon secondary replating, these HPP-CFCs exhibited thestem cell features of self-renewal and potential to generatedefinitive erythroid and macrophage progeny (Yoder, 2001;Yoder and Hiatt, 1991). Separation of YS and embryo at theearly E8 stage, before a common circulation is established,showed that HPP-CFCs were found exclusively in the YS,and examination of the blood at E8.5 suggested that theyentered the circulation from YS with the first erythroblasts.Definitive erythroid (BFU-E) and myeloid (CFU-GM)progenitors and mast cell precursors are detected in the YSat E8 (Moore and Metcalf, 1970; Wong et al., 1986), butthey do not differentiate there; they migrate to the fetal liver,where BFU-EeCFU-E initiate definitive erythropoiesis(Johnson and Barker, 1985).

The YS does not contain cells capable of long-termmultilineage engraftment of adult irradiated mice untilafter such cells have appeared in the AGM region atE10.5, but they are present by E11 and until E13 (Mooreand Metcalf, 1970; Kumaravelu et al., 2002; Huang andAuerbach, 1993). Although it has been argued that theAGM region is the sole site of origin of “definitive” HSCscapable of engrafting adult recipients and that the pres-ence of these cells in the YS at later stages represents animmigrant population (Tavian et al., 1999, 2001; Cumano

et al., 2000, 2001), total body quantitation and tissuedistribution analysis of competitive repopulating HSCsdetected by limiting dilution analysis argues for twoindependent sources of “definitive” HSCs, both partici-pating in seeding the fetal liver (Kumaravelu et al., 2002).Support for this view was obtained by Matsuoka et al.,(2001), who generated both colony-forming unitespleen(CFU-S) and HSCs that engrafted adult mice by cocultureof either E8e8.5 YS or intraembryonic splanchnopleuricmesoderm (para-aortic splanchnopleura, or P-Sp) ona stromal cell line generated from the AGM region.Theconventional definition of an HSC is that it must possessextensive self-renewal potential and multilineage differ-entiation potential. By this criteria, the YS contains HSCsas early as E9, as demonstrated in several studies showingthat CD34þ, cKitþ, and CD38þ YS cells at this stage,when injected directly into the liver of busulphan-condi-tioned neonatal mice, gave long-term lymphomyeloidreconstitution (Yoder and Hiatt, 1997; Yoder, 2001;Dagher et al., 1998). Secondary engraftment in sublethallyirradiated mice demonstrated that the YS-derived HSCsacquired normal marrow homing properties. The argumentthat the “true” stem cells must have the ability to repo-pulate the adult mouse after intravenous injection is notrelevant to normal ontogeny since the only requirement ofthe initial HSC population is that it be capable of colo-nizing the liver. Within that environment, it may thenacquire marrow homing features that permit it to becomean “adult” stem cell. The molecular basis for thedistinction between the embryonic and the adult-type stemcell is unknown but may involve adhesion receptors,including CD34, CD44, selectins, and a- and b-integrins.In this context, b1-integrin-deficient HSCs fail to seedboth fetal and adult hematopoietic organs (Potocnik et al.,2000).

The CXCR4 receptor and its ligand, the chemokineSDF-1, mediates HSC chemotaxis (Jo et al., 2000) and iscritical for HSC homing to marrow but not for fetal livercolonization (Nagasawa et al., 1996). Retroviral-mediatedtransduction of HOXB4 into cells isolated from pre-circulation YS (E8.25) endowed them with long-termlymphomyeloid engraftment capacity in adult irradiatedmice (Kyba et al., 2002). HOXB4 overexpressionenhances HSC self-renewal and suppressed primitiveerythroid potential. The report that coculture of YS onAGM stroma confers adult engraftment potential(Matsuoka et al., 2001) suggests an inducible function forstromal cells in this region, particularly mesenchymal cellson the floor of the dorsal aorta. As currently envisaged,this inductive influence confers HSC potential in locallygenerated hematopoietic clusters, but it is conceivable thatearly YS HSC traffic into the dorsal aorta comes under theinfluence of the AGM stroma and acquires engraftmentpotential.


Macrophage and Microglial Ontogeny

In murine ontogeny, primitive macrophage progenitorsappear in the proximal region of the egg cylinder associatedwith expression of SCL/tal-1 and GATA1 at E7. Theyincrease until the early somite stage (E8.25) and thendecline sharply to undetectable levels by the 20-somitestage (Moore and Metcalf, 1970; Palis et al., 1999).Definitive lineage multipotent precursors with macrophagepotential (HPP-CFCs) as well as more restricted definitiveCFU-GM and CFU-M appear at E8.25 (Moore andMetcalf, 1970; Palis et al., 2001). Separation of YS andembryo at E8.25 (before the circulation) showed that HPP-CFCs were found exclusively in YS; once the circulation isestablished, HPP-CFCs can be found in the circulation,indicating migration into the embryo (Palis et al., 2001).

In avian studies, chickequail chimeras established asearly as E2.5 show numerous YS-derived cells, includingmacrophages, within the embryo vasculature and withinmesenchyme (Cuadros et al., 1992). At late E2, numerousscattered CD45þ cells appear in YS and in blood, exitingthe circulation through the endothelium and rapidlyinvading the whole embryo (Jaffredo et al., 1998). Thesecells express high levels of CD45 and correspond to cellsidentified as monocyteemacrophages. YS-derived macro-phages showing marked acid phosphatase activity andphagocytic capacity appear within the neural tube, liveranlage, and nephric rudiments, beginning even beforecirculation is established (Cuadros et al., 1992; Kurz andChrist, 1998). In the mouse, the macrophage progenitorsappearing in the YS between E7.5e8 migrate into themesenchyme surrounding the brain rudiment. Initialmigration occurs before a circulation is established and isinterstitial, but after E8.2, seeding occurs through thevasculature (Kurz and Christ, 1998). These YS-derivedmacrophages continue to proliferate and generate the brainmicroglia, which ultimately comprises 10% of the brain(Alliot et al., 1999). The primitive-generation YS-derivedmacrophages develop between E7e8 in the absence ofa monocyte or promonocyte intermediary stage and differfrom adult macrophages in the pattern of enzymesproduced (Naito et al., 1996). Peroxidase-positive prom-onocytes of the definitive lineage appear in YS at E10(Cline and Moore, 1972).

Ontogeny of the Vasculature

Morphogenesis of blood vessels is defined by a sequentialpattern of gene expression in which SCL/Tal1 and Flk1 areexpressed first at the angioblast stage followed by PECAM,CD34, VE-cadherin, and later Tie2, and SCL expression isdownregulated in the endothelium of mature vessels (Drakeand Fleming, 2000). As somatogenesis begins, vasculardevelopment is asymmetric, centered in YS blood islands

and in the embryo proper, where angioblasts begin tocoalesce into the aorta. By the murine 3-somite stage,vascular development has spread through the YS, andextended aortic tubes are visible (McGrath et al., 2003).Beginning at the 4-somite stage, erythroblasts dispersethrough the distal YS and a few erythroblasts appear in thehead and tail region of the embryo where the embryonicand YS vasculature meet. The circulation onset is definedas erythroblast movement within vessels and is initiated atthe murine 4- to 6-somite stage during E8.25.

Initiation of Hematopoiesis in theAortaeGonadeMesonephros Region

Avian Intra-aortic and Para-aorticHematopoiesis

In birds, the first intraembryonic hematopoiesis occurs atE3 in endothelium-associated intra-aortic clusters ofmarkedly basophilic cells with prominent nucleoli; itsubsequently occurs (E6e8) in diffuse hematopoietic“para-aortic foci” located in the dorsal mesentery ventral tothe aorta (Jaffredo et al., 1998, 2000). Hematopoieticprogenitors (CFU-E, BFU-E, CFU-GM, and CFU-Mix) aredetected in the aortic wall between E3e4 (Cormier andDieterlen-Lievre, 1988). At E2, prior to the appearance ofthe intra-aortic clusters of CD45þVEGFR-2�ve hemato-poietic cells, the aortic endothelium consisted entirely ofCD45�veVEGFR-2þ flat endothelial cells. Chickequailmarker studies showed that the intra-aortic clusters devel-oped in situ and not by migration from the YS (Martinet al., 1980). Acetylated low-density lipoprotein (Dil-Ac-LDL) labeling indicated that the hematopoietic clustersoriginated from aortic endothelium, and retroviral vectormarking indicated that the para-aortic foci were derivedfrom intra-aortic clusters (Jaffredo et al., 1998, 2000).Mesodermal subdivision transplantation showed that theendothelium of the roof and sides of the dorsal aorta arosefrom somite mesoderm that generates “pure” angioblasts;the floor develops from splanchnopleural mesoderm thatgenerates progenitors with dual hematopoietic and angio-genic potential (Pardanaud and Dieterlen-Lievre, 1999).

In the human embryo, hematopoietic clusters contain-ing up to 800 CD34þ cells appear in the aorta at 27 daysgestation, 8e9 days after initiation of YS hematopoiesisand 6 days after the onset of the circulation (Tavian et al.,1996, 1999, 2001) (Figure 47.2). The precursors of thehematopoietic clusters in the periaortic region wereconsidered “endothelial” based on expression of CD34 andCD31 (PECAM-1), absence of CD45, and uptake of Dil-Ac-LDL (Tavian et al., 2001; Oberlin et al., 2002). In mice,hematopoietic clusters appear at E10.5 in the ventral floorof the dorsal aorta. They appear to be in direct contact withunderlying mesenchyme where the endothelium is

Endothelialcell

Sub-aorticmesenchyme

Somitemesoderm

Splanchnopleuralmesoderm

Hematopoieticintra-aorticcluster

c

CD34, CD31, VE-cadherin,Flt-1, Flk-1/KDR, Tie2, AA4.1,-kit, vWF, SCL, AML1 ,

GATA2, VEGF

-1, -

BMP4, GATA3, AA4.1,tenascin C, CD166,smooth muscle-α actin, Sca-1,AML1, GATA2, SDF1, VEGF,c-Kit ligand, IL-6, OSM, Tpo

Red bloodcell

CD45, CD164, CD43, CD44,CD41, CD34, CD31, AA4.1,VE-cadherin, c-kit, c-Mpl, gp130,G-CSFR, TIE2, CXCr4, Flt-1,Flk-1/KDR?, cMyb, LMO2,SCL, AML1, GATA2, GATA3

Dorsal

Ventral

FIGURE 47.2 Intra-aortic hematopoietic clusters formation in the floor of the dorsal aorta. Based on data from E10.5 murine (Marshall et al.,

1999; Marshall and Thrasher, 2001) and E27 human (Tavian et al., 1996, 1999, 2001; Oberlin et al., 2002) studies. The dorsal endothelium of the aorta

derive from angioblasts developing from somite mesoderm, whereas the ventral “hemogenic endothelium” or hemangioblast develops from splanch-

nopleural mesoderm (Pardanaud and Dieterlen-Lievre, 1999). The dorsal endothelium expresses markers associated with differentiated endothelium

(Drake and Fleming, 2000). The hematopoietic intra-aortic cluster expresses surface antigens and cytoadhesion molecules found on hematopoietic stem

and progenitor cells, hematopoiesis-related transcription factors, and receptors for hematopoietic and angiogenic growth factors (Ziegler et al., 2002;

Delassus et al., 1999; Mitjavila-Gracia et al., 2002). The expression of Flk-1 on intra-aortic clusters is controversial (Dieterlen-Lievre, 1975; North et al.,

2002; Labastie et al., 1998). The subaortic mesenchyme expresses cytokines, chemokines, and cytoadhesion molecules and has molecular features of

vascular smooth muscle (Anstrom and Tucker, 1996; Xu et al., 1998; Marshall et al., 1999, 2000; Teyssier-Le Discorde et al., 1999; De Bruijn et al., 2000;

Marshall and Thrasher, 2001).


interrupted; however, ultrastructural studies in humansshow that the endothelial basal lamina is intact where itinteracts with the cells in the hematopoietic clusters andthat the hematopoietic and endothelial cells are inter-connected by tight junctions (Marshall et al., 1999;Marshall and Thrasher, 2001) (Figure 47.2). VE-cadherin-expressing cells in the AGM have hematopoietic potential,and VE-cadherin is expressed on the luminal aspect ofvascular endothelium but not in CD45þ aorta-associatedhematopoietic clusters (Mikkola and Orkin, 2002; Oberlinet al., 2002; Nishikawa et al., 1998; Fraser et al., 2002). VE-cadherin is essential in stabilizing tight junctions betweenendothelial cells, suggesting the endothelial origin ofhematopoiesis (Fraser et al., 2002). Similar populations ofCD34þCD45�ve cells in the human para-aortic region labelwith the endothelial marker Ulex Europus and generateboth von Willebrand (vW)þ endothelium and hematopoi-etic cells (Oberlin et al., 2002). This blood-forming“endothelium” is in the human embryo AGM region at 28days, with a peak frequency at 31 days, and is absent by 44days (Oberlin et al., 2002). Culture of the P-Sp from 21- to26-day human embryos on marrow stromal cells generatedhematopoietic cells, demonstrating the hematogenicpotential of P-Sp mesoderm that precedes the appearanceof “hemogenic endothelium” (Tavian et al., 2001). The pre-“endothelial” mesodermal precursors are CD34�ve,CD45�ve (Tavian et al., 2001), and VEGFR2/KDRþ

(Cortes et al., 1999a).

Since cells in the human embryo AGM prior to 28 dayslacked direct hematopoietic potential, it was postulated thatthey must receive some inductive signal from surroundingtissue to induce this potential. A discreet region of denselypacked mesenchymal cells lies beneath the ventral floor ofthe dorsal aorta; it is 3e4 layers in the mouse and 5e7layers in the human (Tavian et al., 1999; Marshall et al.,1999; Marshall and Thrasher, 2001) (Figure 47.2). Thesemesenchymal cells are interconnected by tight junctions,express smooth muscle a-actin (SMa-A), and may bea precursor of vascular smooth muscle. BMP4 may playa crucial role in the induction of hematopoietic potential inthe AGM region, much as it does in the YS (Baron, 2001b;Marshall et al., 2000). BMP4 is polarized to the ventral wallof the dorsal aorta in the mesenchymal layer underlying theintra-aortic hematopoietic clusters, where it could induceP-Sp mesoderm to become hemangioblasts and HSCs(Marshall et al., 2000) (Figure 47.2). The mesenchymeprobably also expresses the activated leukocyte cell adhe-sion molecule ALCAM/CD166 or its human equivalent,HSC antigen (Marshall and Thrasher, 2001). CD166 isexpressed on embryonic (but not adult) aortic endotheliumand has been implicated in capillary tube formation,hemangioblast differentiation, and homophilic adhesion ofprimitive CD166þ HSCs (Cortes et al., 1999b; Ohnedaet al., 2001). High levels of expression of the extracellularmatrix glycoprotein tenascin-C have been observed inmesenchyme adjacent to the ventral half of the dorsal aorta


in association with hematopoietic buds, and it is expressedon the basal surface of the hematopoietic cells budding intothe lumen (Marshall et al., 1999; Anstrom and Tucker,1996). During development, tenascin influences cell shapeand promotes motility of many cell types, possibly byinterfering with cellefibronectin interactions (Anstrom andTucker, 1996). It may also function by immobilizinggrowth factors such as BMPs and TGFb, favoring receptorbinding. Stroma from tenascin-null mice have significantlyreduced capacity to support hematopoiesis (Ohta et al.,1998).

The mesenchymal-inductive influence extends beyondthe induction of multilineage hematopoietic precursors; itpotentially includes the induction of development of B- andT-lineage-restricted progenitors (see the section “CellMigration to Primary Lymphoid Organs”) and of HSCscapable of long-term repopulation of adult mice. Medvin-sky et al. (1993). identified the mouse AGM region at E10.5as a source of CFU-S, preceding their appearance in YS.CFU-S precursors were in both AGM and YS by E9 sincecultures of either tissue at this stage generated CFU-S(Medvinsky and Dzierzak, 1996). In vivo engraftment wasobtained with cells from the E10.5 dorsal aorta and vitel-lineeumbilical artery (De Bruijn et al., 2000a,b). Quanti-tation of long-term repopulating stem cell numbers bylimiting dilution assay has shown that they first appear inthe AGM at E10.5 and are present in equal numbers inAGM, YS, circulation, and early liver rudiment by E11(Kumaravelu et al., 2002) (Figure 47.3). The colonizationof the fetal liver by stem cells is initiated by a wave ofmigration from the AGM peaking at E11; this is followedby a second wave from the YS (Figures 47.4 and 47.5).Cumano et al. (2001) reported that cells capable of long-term lymphomyeloid engraftment could be generated inorgan culture of P-Sp isolated prior to the onset of thecirculation (E7.5e8), whereas cultured YS failed togenerate such HSCs. However, Matsuoka et al. (2001) were

YS

AGM

Circulation

Liver

Embryo age (days) 11 12 13

Total HSC 40 600 2675

FIGURE 47.3 Origin and distribution of definitive hematopoietic

stem cells. The numbers of CRUs are detected by limiting dilution,

competitive, long-term repopulation assay in adult irradiated mice,

adjusted for a calculated seeding efficiency of 10%. The figure shows

absolute numbers in each tissue at daily intervals from E11 to E13 in the

mouse embryo. Modified from Kumaravelu et al. (2002).

able to generate repopulating HSCs in coculture of equallyearly P-Sp and YS with a stromal line (AGM-S3) derivedfrom the E10.5 AGM region. This line may have someunique properties since, in a study of 100 stromal linesgenerated from AGM subregions, most were supportive ofadult HSC expansion but none were able to induce devel-opment of engraftable HSCs when cocultured withCD34þcKitþ cells from E10 AGM or YS (Oostendorpet al., 2002). The AGM-S3 stromal line was VECAM-1þ,CD13þ, and Sca-1þ; it produced IL-6 and oncostatin M,cytokines that stimulate HSC proliferation using the gp130signaling pathway (Xu et al., 1998; Mukouyama et al.,1998). Gp130-null mouse embryos have dramaticallyreduced numbers of hematopoietic progenitor cells in theAGM region and fetal liver (Takizawa et al., 2003).The proliferative potential of hematopoietic cells in thegp130-null AGM could be restored by retroviral vectortransduction of either wild-type gp130 or mutants capableof activating STAT3.

The various inductive influences of the subaorticmesenchyme could cause bipotential precursors located inthe floor of the aorta to preferentially adopt a hematopoieticfate. Alternatively, endothelial cells may dedifferentiateand switch to a hematopoietic fate in response to local ortransient signals. A third possibility is that hemangioblastsor their precursors may migrate secondarily into the aorticwall, either from the underlying mesenchyme or from thecirculation, in response to a chemokine gradient. Endo-thelial cells, angioblasts, and HSCs express CXCR4, andmesenchyme adjacent to the dorsal aorta expresses highlevels of its ligand SDF-1, indicating a role for this che-mokine pathway in both vasculogenesis and migration ofhemangioblastic precursors to the floor of the aorta(McGrath et al., 1998).

Studies using embryonic stem (ES) cells differentiatingfor 2.5e3.5 days have demonstrated the transient devel-opment of cells capable of forming blast cell colonies (BL-CFCs) in the presence of VEGF and the cKit ligandestemcell factor (SCF), that precede the appearance of hemato-poietic colony-forming cells (Choi, 2002; Kennedy et al.,1997; Choi et al., 1998). Cells in the blast colony expressseveral genes common to hematopoietic and endotheliallineages (CD34, SCL, and Flk1) and, on replating, generateendothelial progenitors and both definitive and primitivehematopoietic progenitors. The Tpo receptor cMpl wasdetected by day 3 of embryoid body formation whenhemangioblasts first arise (Perlingeiro et al., 2003). Tpoalone supported BL-CFC formation and nearly doubled thenumber of BL-CFCs when combined with VEGF and SCF.Since hematopoietic and endothelial development are notextinguished by targeted inactivation of cMpl or Tpo genes,there must be some redundancy of cytokine pathwaysacting on hemangioblasts. Other overlapping pathwaysactive on hemangioblasts include VEGF/Flk1 (Shalaby

pBpT

Yolk sac

Liver

Dorsal aorta

Sinusoid

Epithelium

Mesenchyme

Capillary

pB

pT

Thymus

pT

HSCpMIX

CFU-GMBFU-ECFU-E

pT

FIGURE 47.4 Vascular migration streams of hematopoietic stem cells and myeloid (CFU-GM, BFU-E, CFU-E, pMix) and lymphoid (pT, pB)

progenitor cells. Migration between the dorsal aortaeAGM, the yolk sac blood islands, the embryonic liver, and the thymus occur between E10.5 and

E11.5 in the mouse embryo. Immigrant cells (shown as basophilic blast cells), detach from the YS blood islands and from the aortic hematopoietic

clusters, enter the circulation, egress from the microvessels within the hepatic rudiment or within the perithymic mesenchyme, and accumulate and

proliferate within these developing organs.


et al., 1995), cKit/SCF (Choi, 2002), BMP4 (Baron, 2001b;Johansson and Wiles 1995), and TGFb(Dickson et al.,1995). The ES cell results support the concept ofa hemangioblast as the precursor of both endothelium andhematopoiesis. Tpo and cMpl transcripts are present in theYS between E6.5e7.5 prior to appearance of the first blood

0

600

d11 d12 d13

Liver

AGM

450

300

150

HSC

s/Ti

ssue

FIGURE 47.5 Colonization of the embryonic liver of the mouse

embryo with hematopoietic stem cells. Determined by CRU assay in

adult irradiated mice and adjusted for seeding efficiency. These numbers

are based on the numbers that the AGM and YS are able to generate

in vitro. In vivo, the high cumulative activity of the AGM region and the YS

may provide the liver with a high proportion of definitive HSCs. The data

suggests consecutive colonization of the embryonic liver with HSCs

from the AGM region and the YS. Reproduced with permission from

Kumaravelu et al (2002).

islands (Xie et al., 2003); from between E8.5e9.5, cMpl isexpressed on embryonic blood vessels and aorta (Ziegleret al., 2002). Other cytokines stimulating hemangioblastsare expressed in the AGM region, including VEGF, SCF,and BMP4 (Teyssier-Le Discorde et al., 1999; Marshallet al., 2000; Marshall and Thrasher, 2001).

Most cells in the AGM region that express CD34,CD31, and Flk1 also express podocalyxin-like protein 1(PCLP1), which has some sequence homology with CD34and is a ligand for L-selectin (Hara et al., 1999). Thispopulation differentiates into both endothelium andhematopoiesis, leading to the suggestion that PCLP1 maybe a surface marker for hemangioblasts. The tunica internaendothelial cell kinase (TEK) that has a similar domainstructure to Tie also appears to be a hemangioblast marker(Hamaguchi et al., 1999). Mice lacking TEK die betweenE9.5e10.5 with defects in angiogenesis and vascularmodeling, and cKitþ, CD34þ, and Sca-1� AGM cells thatcould generate both endothelium and hematopoiesisexpress TEK (Hamaguchi et al., 1999).

The respective roles of hemangioblast and “hemogenicendothelium” for initiating hematopoiesis in the AGM orYS is debated (Nishikawa et al., 1998; Xu et al., 1998;Jaffredo et al., 2000; Marshall and Thrasher, 2001; Choi,2002; Fraser et al., 2002; Mikkola and Orkin, 2002;Oberlin et al., 2002). Several studies identify endotheliumas the source of hematopoiesis based upon isolationof cells expressing markers associated with endothelium(VE-cadherin, CD31/PECAM, Flk1 Flt1, Tie2, Ulex


Europus binding, and Dil-Ac-LDL uptake). None of thesemarkers is unique to endothelium and possibly could beexpressed on hemangioblasts or prehematopoietic meso-derm; some are expressed on HSCs. Direct derivation ofhematopoietic cells from mature, already differentiatedendothelium has been reported in Dil-Ac-LDL labelingstudies (Jaffredo et al., 1998, 2000; Sugiyama et al., 2003).Following intra-cardiac injection ofE10mouse embryoswithDil-Ac-LDL, staining was found within 1 hour and wasconfined toCD31þ, CD34þ, andCD45�ve endotheliumalongthe entire vascular tree (Sugiyama et al., 2003). At 12 hoursafter injection, 1.4% of circulating cells were DiI-Ac-LDL,and of these, 43% expressed the erythroidmarker Ter 119 andadult globin, characteristic of definitive erythropoiesis. Therest of theDiI-Ac-LDLcells are committedwhite blood cells,lineage-restricted and multipotent progenitors as revealed bycolony formation, and HSCs. The remarkable feature of thisobservation is the rapidity with which the endotheliumdifferentiates to multipotent HSCs and lineage-restrictederythroid progenitors (BFU-E) that in turn differentiate to theextent that they express globin genesematuration steps thatnormally require at least 4 to 6 cell divisions. It is also likelythat the transition fromhemangioblast to endotheliumorHSCis not abrupt but is a progressive process of increasingrestriction, not unlike B- and T-cell commitment (see thesection “Cell Migration to Primary Lymphoid Organs”).

Ly-6A/Sca-1 is a stem cell marker. Using a transgenicapproach with an Sca-1-GFP marker gene, it was shownthat green fluorescent protein (GFP) was expressed in allfunctional HSCs in the midgestation aorta and was local-ized to cells residing in the endothelial layer lining theventral wall of the dorsal aorta but not to the adjacentmesenchyme (De Bruijn et al., 2002). At E11, there were1,600 Sca-1-GFPþ (also cKitþ, CD31þ, CD34þ, and VE-cadherinþ) cells in the AGM, and they provided multi-lineage engraftment in adult mice. Note, however, thatSca-1 is probably not expressed on hemangioblasts orhemogenic endothelium in the AGM (Hamaguchi et al.,1999), and its expression is not restricted to HSCs since it isexpressed on AGM-derived stromal cell lines with hema-topoietic support capacity (Xu et al., 1998; Charbord et al.,2002). Molecular analysis of single cKitþCD34þ cells fromthe E11 mouse AGM region, when populations ofengraftable HSCs as well as lymphoid- and myeloid-committed progenitors are present, showed that mostexpressed hematopoietic-specific transcription factorsAML1, GATA-2, PU-1, and LMO2 (Delassus et al., 1999).The majority of cells also express the G-CSF receptor, anda minority express the erythropoietin receptor. Themyeloid-specific geneMPO was expressed in 90% of cells,and the erythroid-specific b-globin was expressed in 50%,indicating that genes of mutually exclusive differentiationlineages may be expressed simultaneously in a singleprimitive cell prior to lineage commitment.

AML1 plays a critical role in the establishment ofdefinitive hematopoiesis in the AGM. AML1 expression isinitiated in mesenchymal cells at the distal tip of theallantois, in endothelial cells in the ventral portion of paireddorsal aorta at E8.5, in both endothelium and mesenchymeof the ventral AGM, and in the intra-aortic hematopoieticclusters between E9.5e11.5 (North et al., 2002). In AML1/LacZ mice, some 30% of the cells recovered from the AGMand the vitelline and umbilical arteries at E11.5 wereAML1/LacZþ; this population contained all HSCs capableof engrafting irradiated recipients (North et al., 2002).These HSCs were initially CD45�ve at E10.5, but by E11.5,HSC activity was present in both CD45þ and CD45�ve

fractions. Most HSCs were CD41þ and were found in boththe positive and the negative fraction of AGM stained withCD31, Flk1, or VE-cadherin (North et al., 2002; Mitjavila-Gracia et al., 2002). The expression of Flk1 on these HSCpopulations is controversial, with some studies showingexpression of the receptor in the hematopoietic clusters(Labastie et al., 1998), others showing that it is barelydetectable in hematopoietic clusters but is expressed onhemangioblastic precursors (Cortes et al., 1999a), and stillothers providing data suggesting that some HSCs that lackFlk1 did engraft, indicating downregulation of Flk1 onCD45þ intra-aortic clusters (North et al., 2002).

In AML1-deficient mice, HSCs and intra-aortic clusterswere absent; in AML1 hemizygous mice, hematopoieticclusters were reduced in number and size and HSCnumbers and distribution were altered, indicating a genedosage effect (North et al., 2002). Haploinsufficiency ofAML1 results in an earlier appearance of engraftable HSCsin the YS, with E10 YS cells reconstituting the hemato-poietic system of irradiated adult mice (Cai et al., 2000). Atthe same time, there is premature termination of HSCactivity in the AGM explants consistent with a change inthe balance of HSC emergence, migration, maintenance, ora combination of these. With hemozygous levels of AML1,the YS may autonomously generate HSCs simultaneous totheir generation in the AGM; the AGM may autonomouslygenerate abundant HSCs that immediately and rapidlymigrate to the YS, where they are detected in abundance; orAML1 insufficiency may block the emigration of HSCsfrom the YS, resulting in an accumulation there anda deficiency in the AGM.

CELL MIGRATION TO LATER SITESOF HEMATOPOIESIS

Ontogeny of Liver Hematopoiesis

Lineage tracing studies support a derivation of the liverfrom ventral foregut endoderm induced by signals frompericardium and septum transversum mesenchyme toproliferate and adopt a hepatic state (Guladi et al., 1996).


The fetal liver stroma consists of cells that express featuresof epithelium (cytokeratin-8), mesenchyme (vimentin andosteopontin), and vascular smooth muscle (SMa-A) (Cha-graoui et al., 2003). These cells are supportive of long-termHSC proliferation, and their presence in the liver coincideswith the duration of hepatic hematopoiesis. At late gestationwhen hematopoiesis declines, these cells are replaced byepithelial cells resembling mature hepatocytes and by aminority of myofibroblasts. Oncostatin M, produced byhematopoietic cells within the liver, has been implicated ininducing hepatic maturation of the epithelialemesenchymalstromal cells with loss of hematopoietic support capacity(Chagraoui et al., 2003).

Fetal liver isolated before the 28-somite stage (E9.5)and grafted beneath the kidney capsule of adult miceresulted in the survival of hepatic tissue, but no hemato-poietic elements were present (Johnson and Moore, 1975).Administration of hematopoietic cells into the circulationof recipient mice resulted in multilineage hematopoieticengraftment of the implanted fetal liver. Grafts of liverisolated later than the 28-somite stage showed autonomoushematopoiesis, defining the time of initiation of HSC entryinto the liver between E9.5e10. In humans, the number ofBFU-E drops abruptly in the YS at 35 days; simultaneously,they appear in the liver, reflecting oriented migration(Peault, 1996). The definitive BFU-E and CFU-E generatedin the YS do not differentiate in that site; most likely, theyseed the fetal liver and rapidly establish definitive eryth-ropoiesis. This is supported by the observations that largenumbers of CFU-E appear simultaneously with BFU-E atthe onset of hepatic hematopoiesis and that embryonicblood contains significant numbers of definitive hemato-poietic progenitors immediately prior to liver development(Johnson and Barker, 1985).

There is a daily logarithmic increase in CFU-GM andBFU-E in the liver between 10e13 days paralleled by anextensive expansion of CFU-S (Moore and Metcalf, 1970;Metcalf and Moore, 1971). HSCs detected in long-termcompetitive repopulation assays were reported at E12 in theliver, increasing 38-fold to E16 and decreasing thereafter(Ema and Nakauchi, 2000). These investigators failed tofind either short- or long-term repopulating HSCs in E11liver. However, Kumaravelu et al. (2002) used limitingdilution long-term repopulation assay to demonstrate atleast one competitive repopulating unit (CRU) in the liver,circulation, AGM region, and YS at E11, with an increasein the liver to 53 CRU at E12 and 260 CRU at E13(Figure 47.3). The 24-hour seeding efficiency of murinefetal liver HSCs into adult marrow as measured by limitingdilution competitive repopulation assays is ~10%, essen-tially identical to seeding of adult marrow HSCs (Szilvassyet al., 2001). Thus ~10 HSCs initiate hematopoiesis byseeding the hepatic rudiment. These numbers could beexplained by colonization from the AGM, but at E12, the

YS makes a significant contribution; thus, these HSCs maynot require processing in the AGM but may mature in situ(Kumaravelu et al., 2002). Relative to adult bone marrow,fetal liver HSCs provide long-term lymphomyeloid repo-pulation of adult mice fivefold more efficiently than adultmarrow (Harrison et al., 1997), explained by their seven-fold higher concentration and the observation that fetalHSC clones generated ~threefold more cells than marrowHSCs (Szilvassy et al., 2001).

In conclusion, the liver appears to be colonized by anearly wave of committed and multipotent progenitors andmacrophages that may be predominantly of YS origin andby two waves of HSCs. The initial HSC wave from theAGM appears to arrive at E10, reach a maximum at E11,and disappear by E13; on E12, a second wave arrives fromthe YS (Figures 47.3 through 47.5). It is possible thatangioblasts also migrate from the YS into the liver toinitiate hepatic vasculogenesis. There is no evidence ofhemangioblasts or hematogenic endothelium in fetal liver,and sorted hepatic endothelial cells isolated immediatelyprior to onset of human hepatic hematopoiesis (E27) weredevoid of hematopoietic potential (Oberlin, 2002).

Ontogeny of Bone Marrow

The primordium of the bone marrow cavity developsfollowing penetration by perichondrial mesenchymal cellsand blood vessels into the zone of calcified cartilage in thecentral region of the long bones (Moore, 2003). Hypertro-phic chondrocytes secrete VEGF that recruits vascular cellsto penetrate the perichondrium and bring osteoblastprecursors and circulating hematopoietic cells, includingprimitive macrophages (Blazsek et al., 2000). Followingresorption of the cartilaginous matrix, the developingmarrow cavity appears as a network of connective tissueand a plexus of widely dilated veins. Marrow hematopoi-esis is initiated by accumulation of large numbers ofundifferentiated basophilic blast cells within the dilatemarrow capillaries, beginning between E11e12 in thechick, at E17 in the mouse, and between E70e77 in thehuman (Metcalf and Moore, 1971). Subsequently, separateand distinct areas of erythropoiesis and granulopoiesisdevelop. Avian parabiosis studies demonstrated thatcirculating stem cells colonize the developing marrowbeginning between E11e12 (Moore and Owen, 1965).Following intravenous injection of tritiated thymidine-labeled embryonic YS or spleen cells into chick embryos,significant numbers of labeled blast cells localized in themarrow at E11 (Metcalf and Moore, 1971). Cells fromthese tissues also repopulated the marrow when injectedinto irradiated embryos (Moore and Owen, 1966a). In themouse, CFU-GM/BFU-E, CFU-S and HSCs appear in thefemoral marrow at E17; the populations double every 34hours. The marrow progressively expands in the first 2


months of postnatal life, associated with a decline inhepatic hematopoiesis in the first week of life and a declineof splenic hematopoiesis after the third week.

Proof that fetal liver stem-progenitor cells wereresponsible for colonizing the marrow was provided instudies in which rats were injected in utero at E16 witha retroviral vector (Clapp et al., 1995). Clonal identificationof viral integration sites showed that fetal liver-derivedclones appeared in the marrow and circulated throughoutthe life of the animals. The role of SDF-1 produced bymarrow stromal cells in the chemoattraction of CXCR4þ

HSCs is well established in the adult (Jo et al., 2000). Thefailure of development of marrow hematopoiesis in micewith inactivation of the CXCR4/SDF-1 pathway (Naga-sawa et al., 1996) strongly suggests that the initial wave ofHSC migration is also SDF-1 dependent.

Ontogeny of the Spleen

The splenic primordium appears as a dense syncytial-likemesenchymal thickening in the dorsal mesogastrium.Themesenchymal condensation is interspersed with vascularspaces where circulating blood comes into direct contactwith mesenchymal reticulum cells. At the earliest stage,corresponding to E8 in chick embryos and E13 in mouse,large immature cells characterized by intense cytoplasmicbasophil and prominent nucleoli are observed both in thevascular spaces and in the perivascular mesenchyme of thespleen (Metcalf and Moore, 1971). Within 24 hours, thesebasophilic cells appear scattered throughout the mesen-chyme and frequently extend long tails of cytoplasmbetween the reticulum cells for some distance from themain body of the cells. By 72 hours, granulopoiesis isextensive with erythropoietic foci. Lymphopoiesis is initi-ated around birth, and myelopoietic activity progressivelydeclines thereafter. Sex-chromosome marker studies inparabiosed and twin embryos demonstrated extensivechimerism in the splenic rudiment of the chick embryo asearly as E12 (Moore and Owen, 1965). Cell-labelingstudies confirmed that circulating cells colonized the aviansplenic rudiment as early as E8 and, with embryonichematopoietic cell reconstitution of irradiated embryos,indicated that splenic colonizing cells were in the YS andcirculation at the initiation of splenic hematopoiesis(Metcalf and Moore, 1971). Labeled thymic lymphocytesbegan to localize in the spleen with high efficiency by E17,coinciding with the initiation of lymphopoiesis.

The origin of cells initially colonizing the mammalianspleen rudiment is most likely the fetal liver, since theAGM and YS are no longer hematopoietic at this stage andthe marrow has not yet developed. CFU-GM/BFU-E, CFU-S, and HSCs are detected by E15 in the splenic rudimentand increase in absolute numbers through the third week of

postnatal life, then progressively decline as the spleenceases to be a myelopoietic organ.

CELL MIGRATION TO PRIMARYLYMPHOID ORGANS

Ontogeny of the Thymus

The thymus is an example of interaction between mesen-chymal (neural-crest-derived) and epithelial (endodermal)tissues. Notch signaling may play a role in the induction ofthymic epithelium. In the mouse embryo at E9.5, Notchreceptors and the Notch ligand, Jagged1, are expressed inthe third pharyngeal pouch at the initiation of thymicorganogenesis (Parreira et al., 2003). The thymic anlage atE11 consists of stratified epithelium that over the following24 hours changes to clustered epithelium and begins toexpress high levels of MHC class II antigen and of Delta1,the Notch ligand that plays a critical role in T-cell devel-opment (Parreira et al., 2003). Comparable development ofthe thymic rudiment is seen ~35 days in the human embryoand 7e8 days in the chick embryo. Chromosome markerstudies in parabiotic chick embryos demonstrated very highlevels of thymic lymphoid chimerism following the estab-lishment of a YS vascular union between 6e7 days ofincubation but not following a later-established chorioal-lantoic vascular anastomosis, indicating that circulatingstem cells colonized the rudiment between 6e7 days(Moore and Owen, 1967a). Subsequent studies (Cotleyet al., 1989) showed the avian thymus is colonized in threewaves during embryogenesis at E6, E12, and E18.Progenitors in the first wave came partly from para-aorticfoci, with the second and subsequent waves coming frommarrow and, to a minor extent, from spleen.

The role of the YS as a source of thymic immigrant cellswas suggested by studies in which 7-day YS cells wereinjected into irradiated chick embryos and colonized thethymus (Moore and Owen, 1966a; Lassila et al., 1978).However, sex-mismatched YS and embryo chimerasgenerated between 33e55 hours of incubation showed thatthe thymus as well as the bursa, spleen, and marrow werepopulated by cells of the sex of embryo and not by YS(Lassila et al., 1978). Studies in chickequail chimeras alsoshowed that the primary lymphoid organs were colonizedby cells that originate in the para-aortic region (Dieterlen-Lievre, 1975; Dieterlen-Lievre et al., 1997). It appears thatin the avian system the YS is colonized at a very early stageby cells originating in the para-aortic region and, like themammalian fetal liver, may serve as a site for expansion oflymphoid progenitors that secondarily migrate to thethymus and bursa of Fabricius. During initial thymiccolonization (beginning E11 in the mouse and E7 in thechick), which precedes the onset of vascularization by 48hours, blood-borne precursors must leave adjacent


pharyngeal vessels then traverse perithymic mesenchymeand basement membrane surrounding the epithelial rudi-ment to enter the thymus (Figure 47.4). Approximately 20T-cell precursors enter the thymus between E11e12, 300between E12e13, and 3,000 between E13e14 (Douagiet al., 2002).

Evidence for thymic chemotactic factors has beenprovided in functional transfilter cell migration studies thathave shown that alymphoid thymic lobes attract cells fromfetal liver fragments (Wilkinson et al., 1999). MHC classIIþ epithelial cells are a source of chemoattractant factors,and response is dependent on G-coupled receptors. Severalchemokines and their receptors are expressed either onimmigrant cells or epithelium; one example, TECK, ischemotactic for thymocytes (Wilkinson et al., 1999).Chemokine signaling can induce expression of metal-loproteinases (MMP9) on immigrant cells that digestextracellular matrix and basement membrane material andaid penetration of the avascular epithelium. The failure ofthymic development in the Nude mouse is caused by a lossof function mutation in the Foxn1 transcription factoressential for thymic epithelial development and results infailure of lymphoid precursors to enter the epithelium fromthe perithymic mesenchyme (Nehls et al., 1996).

The lymphopoietic path of intrathymic developmentoccurs over 2 weeks with four phenotypically and geneti-cally distinct phases (Lind et al., 2001). The first stage(lineage double-negative stage 1) is CD4�ve, CD8�ve,CD25�ve, and CD44hi, and it generates T-cells, B-cells,dendritic cells (DCs) and NK cells. The second stage isCD4�ve, CD8�ve, CD25þ, and CD44hi T-cells or DCs only.Stage 3 is CD4�ve, CD8�ve, CD25þ, and CD44lo and iscommitted to T-cells. The final stage is pre-CD4þ, CD8þ,and CD25�. A 4000-fold expansion occurs in the first threephases, and a 250-fold expansion takes place in the earlypre-CD4þ and CD8þ double-positive stage (Lind et al.,2001).

Origin and Commitment of Thymic-ColonizingCells

The YS and AGM region and the early stage of fetal liverare all sources of lymphomyeloid stem cells (HSCs) atthymic colonization, yet extensive studies using in vitrothymic lobe cultures to identify thymic-colonizing cellssuggest that they are not HSCs and are to some degreeT-committed prior to entry into the thymus. Ohmura andKawamoto and colleagues (Kawamoto et al., 1997, 1998,1999, 2000; Nishikawa et al., 1998; Ohmura et al., 1999;Itoi et al., 2001) report that lymphoid commitment coin-cides with the first appearance of hematopoietic clusters inthe vascular endothelium of the AGM region betweenE9.5e10. At this stage, the CD45þ, cKitþ, and CD34þ

population contains multilineage progenitors as well as

progenitors restricted to T (pT), T-NK plus macrophage(pTm), B (pB), or B plus macrophage (pBm) but not topBT. A CLP identified in adult mouse marrow asa precursor of both B- and T-lineage can be distinguishedfrom the HSC and CMP by expression of IL-7Ra, inaddition to being Sca-1þ, cKitþ, and Lin�ve (Akashi et al.,1999; Kondo et al., 1997). It appears that in fetal devel-opment, a CLP stage comparable to the adult does not exist(Akashi et al., 1999; Kawamoto et al., 2000; Ohmura et al.,2001). The fetal pT cells have T-cell receptor rearrange-ments and appear in the fetal blood and liver betweenE11e12, where they greatly outnumber pB (Ohmura et al.,2001). This coincides with the period of initial thymiccolonization, when pT in the liver initially increase thenrapidly decline in number and pB, initially rare, rapidlyincrease in number (Figure 47.4).

Cells isolated from the perithymic mesenchyme duringinitial colonization were IL-7Raþ and Lin�ve (Harmanet al., 2003). Consistent with the paucity of Notch ligand inthe fetal liver environment, expression of Notch targetgenes Hes-1 and pre-Ta were not detectable in either fetalliver or perithymic IL-7Raþ and Lin�ve populations, indi-cating that Notch signaling was not activated in these cellsprior to thymic entry (Harman et al., 2003). In contrast,Notch target genes were detected in precursors that hadentered the epithelial environment where Jagged and Deltaare readily detected. This suggests that a Notch influenceon the T-lineage does not occur until cells enter the thymus.However, it is also possible that prethymic commitment tothe T-lineage is induced by factors other than Notch but isonly revealed as a result of Notch signaling in the thymus.The importance of Delta-Notch signaling in T-cell devel-opment has been shown by culturing fetal liver Sca-1hi,cKithi, CD25lo, and Lin�vecells on the OP9 marrow stromalcell line with IL-7 and c-Kit ligand (Schmitt and Zuniga-Pflucker, 2002). Ectopic expression of Delta ligandinhibited B-lymphoid differentiation and induced a normalprogram of T-cell differentiation through to functionallymature CD4 and CD8 populations. The expression ofIL-7Ra on the immigrant pT and the role of IL-7 on in vitroT-cell development contrasts with the reported lack of IL-7Ra on the earliest T-lineage progenitor within the thymus(Allman et al., 2003) and with their loss of thymic homingcapacity. Mouse IL-7Ra chain mutants show profounddefects in thymopoiesis in the adult, but fetal thymopoiesisis relatively intact, suggesting alternate cytokine pathwayscan operate during ontogeny (Kincade et al., 2002). Furtherdifferences between adult and fetal thymopoiesis arerevealed in Ikaros-null mutants that have defective fetalthymopoiesis even though the adult thymus is intact(Kincade et al., 2002).

To understand lymphoid-lineage specification from theHSC, it is best to think of it as a progressive bias alongtheT-, B-, or myeloid pathway without abrupt transition


steps. The first cell to enter the thymus is predominantly Tdirected but retains some myeloid and B potential. Thelatter has been revealed by the ability of these cells toundergo delayed B-cell development in culture and theappearance of B-cell development in the thymus of micewith null mutations of Delta (Radtke et al., 1999). The pBand the adult CLP are more biased toward B than toward Tdifferentiation, and the former retain some myeloidpotential. The expression of the Pax5 transcription factorhas been correlated with B-cell differentiation andprogressive downregulation of myeloid and T-specificgenes (Rolink et al., 1999). The existence of an embryonicprecursor of both the T-biased and B-biased progenitors,with equal capacity to generate T or B, has been proposed(Montecino-Rodriquez and Dorshkind, 2003).

Ontogeny of B-Lymphocytes

Avian Bursa of Fabricius

In the avian system, the bursa of Fabricius primordiumappears by E5 as an epithelial thickening at the ven-trocaudal contact of the cloaca with external ectodermalepithelium. As both bursal and thymic anlagen develop atsites of endodermaleectodermal interaction, this originmay be of some fundamental importance in the earlyinduction of primary lymphoid organs (Moore and Owen,1966b; Metcalf and Moore, 1971). By E10e11, longitu-dinal folds of lining epithelium project into the lumen,and by E12e13, epithelial buds project into the under-lying mesenchyme. Bursal lymphopoiesis begins by E14,and at this stage, many large basophilic cells are observedin the blood vessels of the mesenchyme adjacent to theepithelial buds. These cells pass through the epithelialbasement membrane and localize within the epitheliumfollicle where they proliferate extensively, differentiatinginto typical bursal B-lymphocytes. Chromosome markerstudies in parabiosed embryos and embryonic grafts ofbursal rudiments demonstrated that the bursa was colo-nized by circulating lymphoid precursors that enteredbetween E13e14 (Moore and Owen, 1966a,b). Injectionof embryos between E10e14 with tritiated thymidine-labeled embryonic YS, spleen marrow, and blood cells(but not thymocytes) showed rapid localization of labeledbasophilic cells in the bursal epithelium with subsequentproliferation and lymphoid differentiation (Metcalf andMoore, 1971). The “receptivity” of the bursal epitheliumfor labeled cells declined abruptly at E15. Parallelinjection studies into E14 irradiated embryos using sex-chromosome marking confirmed that the precedingembryonic hematopoietic tissues, but not the thymus,contained bursal lymphoid precursors (Moore and Owen,1966a). Using specific markers expressed on restricted B-and T-precursors, it was shown that cells on the E14

spleen or marrow that colonized the bursa upon injectionwere already B committed with VJ recombination anddistinct from T-lineage precursors (Houssaint et al.,1991). Since such B-restricted precursors appear to be inthe circulation and in all hematopoietic organs at the timeof bursal colonization, the precise site of B-lineagecommitment is unclear. What is known, as a result of sex-mismatched and chickequail YSeembryo chimeras, isthat the B-precursors ultimately derive from the intra-embryonic para-aortic region, possibly as early as E2(Lassila et al., 1978, 1982) e although their subsequentexpansion and B-commitment may occur within the YSand then the spleen.

Mammalian B-Cell Ontogeny

The fetal liver has long been considered the initial site ofB-cell commitment beginning E14 in the mouse; fromthen on, the B-progenitors expand in a synchronous wave-like pattern, reaching a peak in the perinatal stage(Strasser et al., 1989). It was also generally accepted thatearly B-cell progenitors expressed CD45R/B220 prior toCD19 expression. However, it has become recognized thatB-lineage gene expression (DJH rearrangements and Iggerm line transcripts) could be detected in P-SpeAGMand liver before E12.5 (Cumano and Paige, 1992).Furthermore, Rag-2, Rag-1, and CD19 gene expressionhas been found at E9 in YS and P-Sp (Marcos et al., 1997;De Andres et al., 2002). By functional and phenotypicanalysis, AA4.1þ and FcgRþ or AA4.1þ, Sca-1þ, andB220�ve cells with B-macrophage potential were detectedin E12.5e13 fetal liver (Cumano and Paige, 1992; Lacaudet al., 1998; Mebius et al., 2001). A novel population ofcKitþ, AA4.1þ, CD34þ, Sca-1�ve, CD45þ, and CD19þ

cells (70% expressing IL-7Ra but negative for CD45R/B220) were detected in E11 P-SpeAGM and liver thatdifferentiate exclusively into B-cells (De Andres et al.,2002). Numerically, at E11, these very early pro-B-progenitors comprised ~100 in the AGM region, ~500 inthe liver, and smaller numbers in the blood and YS(Figure 42.4).

Coculture on marrow stromal lines such as S17 or ST2,often with the addition of cytokines such as IL-7 and cKitligand, has been used to assay for B-lymphoid potential(Ogawa et al., 1988; Huang et al., 1994; Kawamoto et al.,1997; Lu and Auerbach, 1998). Such studies have identifiedthe emergence of B- or B-macrophage-restricted progeni-tors in the AGM region at E10, coinciding with thedevelopment of the aortic hematopoietic clusters (Ohmuraet al., 1999). The number of progenitors capable ofgenerating B-cells in the AGM at E10 (30e35 somite pairs)was 10 and increased 400-fold by E12, when they werelocated in the fetal liver within the CD45þ, Kitþ, Sca-1lo,and IL-7Raþ population (Ohmura et al., 1999, 2001;


Kawamoto et al., 2000). The numbers and location of theseB-progenitors correspond to the CD19þ pro-B-cellsdescribed previously (De Andres et al., 2002).

There has been a long, and as yet unresolved, debateon the role of the YS in generating pB. Early studies withIg allotype markers showed that YS cells transplanted intoadult mice generated B-cells (Tyan and Herzenberg,1968), and in utero injection of E8e10 YS resulted inpostnatal B and T chimerism (Weissman et al., 1978).Chromosome-marked E11 YS cells were shown to providelong-term lymphoid (T and B) and myeloid engraftment inmice (Moore and Metcalf, 1970). In a series of studies,Auerbach and coworkers (Huang et al., 1994; Lu et al.,1996; Lu and Auerbach, 1998) showed that AA4.1þ cellsfrom YS as early as E8eE9, when on marrow stroma orYS endothelial lines, could generate cells with B and Tpotential as measured in vitro and when transferred in vivointo immunodeficient mice. Cumano et al. (1993) initiallyreported that AA4þ, Sca-1� cells in both YS and embryoat E8.5 were able to generate both T- and B-cells in an S17stromal coculture with IL-7 and cKit ligand. In a subse-quent study, Cumano et al. (1996) separately culturedmouse embryo and YS at the presomite, E7.5e8 stage for48 hours and observed B-cell only in the embryo. Theyonly observed lymphoid potential in cultured YS after thecirculation was established, favoring their view that theYS is colonized by P-Sp-derived lymphoid progenitors.The absence of definitive hematopoiesis (CFC-mix orBFU-E) in their cultured YS, despite the presence of thesecells in the YS in situ, suggests that the culture conditionswere deficient for maintenance of a normal pattern oflymphohematopoiesis, rendering a negative result incon-clusive. Ogawa et al. (1988) reported B-cell progenitorsdeveloping in E9.5 embryos but not of YS, althoughexcessive mast cells in the latter cultures may haveinhibited lymphoid. Nishikawa et al. (1998) isolated VE-þ

and CD45�ve “endothelial” cells from an E8.5e9.5embryo and YS and showed B-cell differentiation on anOP9 stroma with IL-7. The variable and often conflictingdata can be attributed partly to culture variables such aswhole-tissue versus dispersed cells, the use of differentstromal lines, and particularly the use of supplementalcytokines such as IL-7 and cKit ligand. Certain of theseconditions may provide an environment capable of sup-porting lymphoid differentiation from hemogenic endo-thelium or hemangioblasts, others may differentiationfrom lymphomyeloid stem cells and pMix, and otherscan expand only those cells that have undergone B-cellcommitment (pB or pBm). By restricting a committedB-cell progenitor to cells expressing CD45, andprobably CD19, IL-7Ra, and cKit, they first appear withinthe E10 AGM region where they may traffic to the YS aswell as the early liver. It is unknown whether the moreprimitive cells in the YS that have B-potential can

migrate into the embryo and undergo pB commitment inthe AGM region.

The myeloid suppressing transcription factor Pax5plays a critical role in B-cell commitment, and Pax5-defi-cient mice display B-maturation arrest at the pro-B level(Rolink et al., 1999). The B-macrophage progenitors in thefetal liver and AGM express Pax5 but at a significantlylower level than the adult CLP (Mebius et al., 2001). Thisobservation may account for the retention of macrophagepotential in the fetal progenitor and the block of thispathway in the adult CLP. In the absence of Pax5, the pB,when transferred to RAG2-deficient mice, provide long-term reconstitution of the thymus and generate matureT-cells; in vitro with appropriate cytokines, they canproduce T-cells, macrophages, osteoclasts, DCs, gran-ulocytes, and NK cells (Rolink et al., 1999). Migration ofB-progenitors from the AGM to the liver begins E10 withsubsequent increase because of continued immigration andintrahepatic expansion (Figure 47.4). Since HSCs are alsocolonizing the liver at this time, the possibility of intra-hepatic generation of additional pB from these moreprimitive cells cannot be excluded.

The role of the chemokine SDF-1 in promoting themigration of pB from the AGM region to the liver is sug-gested by the demonstration that its receptor, CXCR4, isexpressed on IL-7Raþ pB and that SDF-1 induceschemotaxis of these cells (Egawa et al., 2001). Mutant micewith targeted disruption of the SDF-1 or CXCR4 genesdisplay defects in B-lymphocyte development in the fetalliver (Nagasawa et al., 1996; Ma et al., 1998) that could beaccounted for by defective colonization of the liver bypB as well as by impairment in B-cell proliferation, sinceSCF-1, acting in synergy with cKit and Flt-3 ligands,promotes proliferation of pB cells (Egawa et al., 2001).

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