MiniReview

Avian HSC emergence, migration, and commitment towardthe T cell lineage

Thierry Ja¡redo, Sandrine Alais, Karine Bollerot, Cecile Drevon, Rodolphe Gautier,Borhane Guezguez, Krisztina Minko, Pascale Vigneron, Dominique Dunon �

UMR CNRS 7622, Universite¤ Pierre et Marie Curie, 9, Quai St Bernard, 75005 Paris, France

Received 2 June 2003; received in revised form 27 August 2003; accepted 12 September 2003

First published online 28 October 2003

Abstract

To date three sites of emergence of hemopoietic cells have been identified during early avian development: the yolk sac, the intraaorticclusters and recently the allantois. However, the contributions of the hematopoietic stem cell (HSC) populations generated by thesedifferent sites to definitive hematopoiesis and their migration routes are not fully unraveled. Experimental embryology as well as theestablishment of the genetic cascades involved in HSC emergence help now to draw a better scheme of these processes.8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Yolk sac; Aorta; Allantois ; Avian embryo; Hemopoiesis ; Hemangioblast ; Hemopoietic stem cell ; Thymus; Runx-1; GATA; VE-cadherin;Hemopoietic stem cell adhesion molecule

1. Introduction

T cells originate from hemopoietic stem cells (HSC) de-¢ned by their multipotentiality and self-renewal capacity.In adult higher vertebrates, HSC settle in the bone marrowwhere they can di¡erentiate into progenitors with morerestricted lineage potential and generate all blood lineagesvia a cascade of commitment events. In contrast, the ori-gin of HSC during embryogenesis is more complex andtheir migration is required to ensure a harmonious hemo-poietic development. On the basis of observations on para-biosed avian embryos or chorioallantoic grafts of organrudiments, Moore and Owen established that hemopoieticorgan rudiments, such as the spleen and the bone marrow,are colonized by extrinsic HSC [1,2]. These conclusionswere con¢rmed and extended, with the quail/chick markerleading to the ¢ne-tuning dissection of thymic colonization[3^5]. Genetic technologies in the mouse have revealednumerous mutations that a¡ect hemopoiesis. Recently,several genetic resources were released for the avian spe-cies allowing rapid identi¢cation and cloning of many avi-

an homologues. Despite minor di¡erences (absence of fetalliver hemopoiesis in birds), the overall development of thehemopoietic system is very similar in birds and mammals.Taken together with the fact that chick embryos are oftenmore appropriate to study the earliest events of hemo-poietic development, work in the avian embryo has ledto several breakthroughs in this ¢eld. Here we will reviewthe di¡erent sites of HSC emergence, HSC migration tothe bone marrow and their commitment and migrationtoward the thymus.

2. The ¢rst generation of HSC originate from the yolk sacbut do not di¡erentiate into T cells

Moore and Owen ¢rst postulated a central role for theyolk sac (YS) in the production of HSC. According to theauthors, this appendage produced a stock of HSC, a partof which would di¡erentiate in situ while the other partwould colonize the hemopoietic organ rudiments [6]. Inorder to identify the relative contributions of the extraem-bryonic and intraembryonic compartments in HSC pro-duction, F. Dieterlen and co-workers associated a quailembryo onto a chick YS and analyzed the constitutionof the hemopoietic organs for quail or chick cells usingthe quail/chick marker [7]. Hemopoietic organs were

0928-8244 / 03 / $22.00 8 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.doi :10.1016/S0928-8244(03)00295-5

* Corresponding author. Tel. : +33 (1) 44 27 35 00;Fax: +33 (1) 44 27 34 97.E-mail address: dunon@ccr.jussieu.fr (D. Dunon).

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seeded exclusively by quail cells and at embryonic day (E)13, 70% of circulating blood cells were of quail origin [8].Thus, YS hemopoietic cells (HC) are incapable of long-term renewal whereas the source of ‘de¢nitive’ HSC colo-nizing the hemopoietic organs comes from the embryoproper. These experiments also revealed that the YS con-tributes to two distinct generations of red cells : a primitiveone (embryonic) that derives entirely from the YS and asecond one (fetal) that derives from both the embryo andthe YS, this latter being able to generate erythrocytes witha fetal globin make-up [8,9]. In addition, YS produced asmall population of HC with characteristics of primitivemacrophages which may represent the ¢rst microglial cells[10^13].

3. Intraembryonic HSC giving rise to de¢nitive hemopoiesisare produced in the aortic region

The aortic region was shown to harbor HSC in birds aswell as in mammals. In the avian embryo, the aorta ex-hibits two di¡erent aspects of hemopoiesis : one occurringbetween E3 and E4, called the intraaortic clusters, whichconsists in small groups of cells with hemopoietic charac-ters protruding into the aortic lumen (Fig. 1), a second,called the paraaortic foci, developing between E7 and E9

outside the aorta and consisting in large groups of cellsdistributed in a loose mesenchyme ventral to the vessel[14]. Isolated E4 chick or quail aortae grafted onto a re-verse host produced HC capable of seeding the hemo-poietic organs and acquired surface antigens characteris-tics of T or B lymphocytes and granulocytes [15]. Whendissociated into individual cells and seeded into a semi-solid medium supplemented with appropriate cytokines,chick aortic cells displayed erythroid, monocytic, myeloidor multipotential colonies [16^18].Intraaortic clusters develop exclusively on the ventral

side part of the aorta. Orthotopic exchanges of somitesand splanchnopleural endoderm between quail and chick-en embryos showed that two distinct endothelial aorticlineages become committed in the embryo proper, a dor-sal, angioblastic lineage originating from the somites, en-dowed uniquely with endothelial properties and a ventral,hemangioblastic, splanchnopleural derivative participatingin the building of the ventral aspect of the aorta [19]. Theaorta has thus a dual origin responsible for the restrictionof HC emergence in the aortic £oor.

4. Characterization of cells giving rise to HSC atgastrulation stages

The blood-forming system, i.e. endothelial cells (EC)and HC, di¡erentiate from the mesoderm during the gas-trulation process, i.e. the ingression of cells from thesuper¢cial epiblast. Blood islands from which the ¢rstHC are going to emerge in the YS di¡erentiate from theextraembryonic mesoderm. Blood islands are typicallyconstituted of an outer layer of EC and a core of HC.At the time blood islands di¡erentiate, the embryonic me-soderm does not participate in this early blood-formingphase; however, it contains structures very similar to theseblood island, the vascular islands, that never develop he-mopoiesis although this area was in continuity with theextraembryonic mesoderm. The blood-forming potentialof this area is detected 2 days later as the aortic regionundergoes hemopoiesis. We have probed these early eventsby examining the patterns of several molecules shown tobe involved in HC and EC emergence. The study includedavian transcription factors which participate in multimericcomplexes: GATA-1, -2, -3, SCL/Tal-1, Lmo2 and VEGF-R2, a regulator of hemopoietic and endothelial commit-ment. During gastrulation, GATA-2 and VEGF-R2 weredetected the earliest in the whole ingressing mesoderm.SCL/Tal-1 and Lmo2 tagged the mesodermal aggregatesthat will become fated to YS blood islands (Fig. 2) [20].Interestingly, VEGF-R2-positive cells sorted out from

the very early blastodisc (stage HH4) gave rise to hemo-poietic colonies in the absence of growth factors in a clo-nal di¡erentiation assay [21]. When VEGF was added,endothelial colonies di¡erentiated while the number of he-mopoietic colonies decreased substantially. Mixed colonies

Fig. 1. Localization of intraaortic clusters. Transverse section through a3-day embryo at the level of the aorta. In situ hybridization for Runx-1.The intraaortic clusters, restricted to the ventrolateral aspect of the ves-sel, strongly express Runx-1. Ao: aorta; N: notochord; NT: neuraltube; S: somite; V: cardinal vein.

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never developed precluding the demonstration of the exis-tence of the hemangioblast, the putative common precur-sor of HC and EC. At stage HH4, cells giving rise to YSmesoderm, thus mostly the blood islands, are VEGF-R2-positive and are located in the posterior region of theprimitive streak and, very likely, cells giving rise to aortaHSC would also express this marker with a short delayand would be located at a more anterior position. At thisstage, these two VEGF-R2-positive epiblast cell popula-tions might exhibit similar hemopoietic potential. Indeed,the restricted di¡erentiation capacities of YS HSC prob-ably re£ect the in£uence of the YS microenvironment thatmay trigger HSC di¡erentiation into erythroid and mye-loid lineages. Of note is the fact that if an extraembryonicarea of an E2 quail embryo is associated in vitro with anE6 chick thymus, quail HSC colonize the thymus rudimentand undergo lymphoid di¡erentiation [22], meaning thatthis potentiality is present but masked by the microenvi-ronment. The recent identi¢cation of Notch-1 and delta-1expression in the vicinity of VEGF-R2-positive epiblasticcells suggests that the Notch pathway might be involved inthe hemopoietic commitment of these cells [23].VEGF-R2 is the target of the VEGF expressed by en-

doderm at this stage, particularly the YS endoderm[24,25]. Since endoderm^mesoderm interaction was shown

to play a critical role in the speci¢cation of the blood-forming system, VEGF would belong to di¡usible signalssecreted by adjacent endoderm and be required for bloodformation [26^29].

5. Aortic HSC emerge from an hemogenic endothelium andingress into the mesenchyme to form the paraaortic foci

Generation of HC in the aortic region has been ana-lyzed in detail. In the E3 aorta, HC appear to delaminatefrom the aortic £oor endothelium. Before cluster emer-gence, aortic EC were entirely positive for VEGF-R2. Atthe time of cluster di¡erentiation, VEGF-R2 was down-regulated and CD45, the pan-leukocyte antigen, upregu-lated in ventral EC. As HC bulge in the aortic lumen,rounded cells become all CD45+ [30]. In order to obtaina dynamic approach of the developmental relationshipsbetween cluster cells and the aortic endothelium, we usedtwo vital tracers. The ¢rst, acetylated low density lipopro-tein (AcLDL), coupled to a £uorescent lipophilic marker(DiI), was inoculated in the E2 vascular tree (HH12) 1 daybefore the emergence of intraaortic clusters. This marker isspeci¢cally endocytosed by EC and macrophages (whichare not present in the embryo at that stage) via a speci¢c

Fig. 2. YS blood islands express Lmo2. A: Two-day-old (10 somite pairs) chick embryo showing the expression of the Lmo2 gene. The dotted patternreveals the distribution of the blood islands that will give rise to the ¢rst (primitive) generation of erythrocytes. B: Cross-section at the level indicatedabove showing the maturation steps of the blood islands (BI). Arrows indicate the hemangioblasts that will give rise to the EC and HC. 1: This imma-ture BI is full of HC and EC are already di¡erentiated. 2: The BI has matured, several cells have been freed creating a hole. 3: The BI is fully mature.HC are free and detached from one another.

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receptor whereas the other cell types never take up themolecule. As soon as 2 h after inoculation, the wholevascular tree was labelled. The aorta was entirely linedby LDL-positive EC. Twenty-four hours after inoculation,the intraaortic clusters and the rest of the EC were entirelyLDL-positive. Moreover, LDL-positive cells were found inthe mesenchyme ventral to the aorta which suggests that asubset of HC ingress into this region [30]. These datademonstrated that the intraaortic clusters derived fromcells that previously had an endothelial phenotype at thetime of inoculation and underwent lineage switch duringcluster emergence. Thus, clusters were generated throughan EC intermediate located in the aortic £oor.Contrary to the intraaortic clusters, paraaortic foci de-

velop in a mesenchyme ventral to the aorta and thus ap-pear to have no relationship with the aortic endothelium.Is there a developmental relationship between the two as-pects of aortic hemopoiesis or do they belong to two in-dependent generations of HSC? To approach this questionwe have used non-replicative lacZ-bearing retroviral vec-tors inoculated in the same conditions as for AcLDL^DiI.In this assay clones can be identi¢ed because integrationoccurs in a few dispersed cells of the endothelial tree andthe reporter gene is stably expressed in the progeny ofinfected cells making it possible to follow tagged cellsfor long periods of time. At E3 the labelled clones com-prised either EC or HC but never both. Labelled cells wereobserved to ingress into the mesentery ventral to the aortic£oor. At E7, the paraaortic foci contained numerous lacZ-positive cells [31]. This demonstrated that paraaortic fociare seeded by progenitors derived from the E3 aortic £oorthus demonstrating the existence of a unique event of HSCemergence from the aortic endothelium.Using molecular markers, several of which we have

cloned, we have been able to identify several steps in thetransition from EC to HC. When the aortae were stillpaired and no sign of hemopoiesis was visible, the wholeaortic EC expressed molecules of a typical endothelium(the surface molecules VEGF-R2, VE-cadherin, and lowexpression of the transcription factors Lmo2 and SCL/Tal-1). Then just before and during fusion of aortae, Runx-1and GATA-2 expression appeared and SCL/Tal-1 andLmo2 became upregulated in the ventral aspect of theaorta that also retains expression of the EC-speci¢c genesat that time. No morphological sign of HC emergence isvisible yet. Finally, when fusion has occurred there is athickening of ventral EC accompanied by the increasedexpression of HC-speci¢c genes and the decrease of EC(downgrading of VEGF-R2 and VE-cadherin, upgradingof Runx-1, SCL/Tal-1, Lmo2, GATA-2, and GATA-3).Runx-1, the avian orthologue of which we have cloned,is the earliest gene patterned to the ventral region of theaorta and the only gene harboring such a pattern. In theavian embryo Runx-1 is expressed as early as E2.5 in ECof the ventral aortic £oor (Fig. 1). Expression begins whenthe aortae are still paired and persists upon fusion of the

vessels. At the time of hemopoietic cluster production,Runx-1 becomes restricted to HC bulging into the lumenand ingressing into the mesentery.

6. The allantois, a later source of HSC colonizing the bonemarrow?

In birds, the timing of HSC production by intraaorticclusters and paraaortic foci is compatible with the seedingof the thymus and bursa of Fabricius (Fig. 3). Cells of theparaaortic foci have been shown to have B and T lym-phoid potential [32]. However, seeding of the bone mar-row begins by E10.5 in the chick embryo and continuesuntil birth. At this time, the activity of the paraaortic focihas ceased. The possibility that cells from the paraaorticfoci remained in an undi¡erentiated state somewhere intothe embryo appeared unlikely. We thus have looked foranother progenitor-producing site and identi¢ed the allan-

Fig. 3. Emergence of HSC and thymus colonization. The ¢rst HSC aregenerated in the YS and their progeny contribute to primitive hemopoi-esis. HSC giving rise to de¢nitive hemopoiesis originate from the aorta.We have shown that paraaortic foci derive from intraaortic clusters andthat allantois HSC colonize bone marrow and contribute to de¢nitivehemopoiesis. The other hemopoietic organs, such as bone marrow andthe thymus, are colonized by HSC or progenitors. T cell progenitors ofthe ¢rst wave of thymus colonization derive from paraaortic foci where-as progenitors of the second and third waves originate from the bonemarrow. Finally, a direct contribution of allantois HSC to thymus colo-nization cannot be excluded.

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tois. This organ has the appropriate tissue make-up toproduce hemopoiesis, i.e. endoderm associated with me-soderm. This extraembryonic membrane was shown to beinvolved in gas exchange, excretion product accumulation,shell Ca2þ resorption and bone construction. By usingIndia ink or AcLDL^DiI microangiography, we ¢rst es-tablished that the allantois became vascularized at 75^80 hof incubation. It displayed conspicuous red cells even be-fore this period indicating that hemopoiesis occurred in-dependently from the rest of the embryo in this site [33]. Afull cellular and molecular hemopoietic program, charac-terized by the expression of SCL/Tal-1, Lmo2, GATA-2,and GATA-1, and the emergence of red cells from thesecond generation occurred in the allantoic mesoderm.This is accompanied by the expression of GATA-2 andGATA-3 in the associated endoderm [34]. The quail allan-toic rudiment was dissected out prior to vascularizationand grafted heterotopically into the coelom of E3 chicks.The bone marrow of E13 chicks was found to harbor asigni¢cant amount of quail cells, up to 8% of the totalbone marrow cells. Analysis on sections also revealedthat QH1-positive cells were not only hemopoietic butalso endothelial in nature [33]. Although seeding of dis-tinct progenitors has already been shown, the possibility ofseeding by a common progenitor cannot be excluded.

7. Origin and migration of T cell progenitors

An interesting question was to determine which HSCemergence sites contribute to thymus colonizationthroughout development. In birds the thymus anlagen de-rive from the ectoderm of the third and fourth branchialclefts and the endoderm of the third and fourth pharyn-geal pouches [4]. During embryogenesis, T cell progenitorscolonize the thymic epithelium, which creates an optimalmicroenvironment for T cell di¡erentiation. Studies per-formed in chick^quail chimeras showed that the thymusof birds is colonized in three waves, two during the em-bryonic period and one just after hatching. These wavesstart at day 6, day 12, and day 18 respectively [3,35,36].The duration of these waves is around 2 days and they areseparated by periods described as refractory for thymuscolonization.At E6, thymus colonization starts with the accumula-

tion of hemopoietic progenitors in the jugular vein, and inthe mesenchyme surrounding the thymus [37,38]. Duringthe second and the third waves of thymus colonization, Tcell progenitors enter the thymus via the blood stream. Itis now generally accepted that T cell progenitors ¢rst orig-inate from paraaortic foci [4,17,19]. This probably formsthe ¢rst wave of thymic colonization [39,40]. We con-¢rmed this observation by T cell progenitor quanti¢cationduring ontogeny in peripheral blood, paraaortic foci, bonemarrow, and spleen. Embryonic T cell progenitors wereidenti¢ed by their ability to di¡erentiate into T cells after

intrathymic injection. Cells of di¡erent hemopoietic organswere injected into thymi of irradiated congenic animalsdi¡ering by the ov molecule, an antigen expressed in pro-genitors and the T cell lineage [40,41]. The degree of chi-merism of the host thymus was subsequently measuredand correlated with the number of donor progenitors ini-tially injected. During the second and the third waves ofcolonization, T cell progenitors were encountered in thebone marrow and spleen. However, the spleen, in contrastto the bone marrow, contained progenitors unable tohome to the thymus via the blood stream.Although the origin of T cell progenitors as well as their

migration through the blood vessels are well identi¢ed,very little is known about the internal clock which directsavian thymus colonization. Several studies showed that thethymus itself governed its own colonization [4,42]. Quan-ti¢cation of T cell progenitors in the blood established thateach wave of thymus colonization is correlated with apeak in the number of progenitors whereas they were al-most absent from the blood during periods de¢ned asrefractory for thymus colonization [40]. Moreover, intra-venous injection of T cell progenitors showed that theywere able to home to the thymus without delay even dur-ing the refractory periods. Although a regulatory role ex-erted by the thymus is very likely, these ¢ndings demon-strated that the blood delivery of T cell progenitors plays amajor role in the thymus colonization.

8. Molecular characterization of T cell progenitors

Intrathymic injection of sorted cells between congenicchicks also made it possible to characterize the T cell pro-genitors. Injection of sorted VEGF-R2-positive cells fromthe mesoderm of chick embryos at the gastrulation stage(HH4) never gave rise to mature T cells in this system [43].In contrast to above, when the extraembryonic area of anE2 quail embryo is associated with an E6 chick thymus,quail HC colonize the thymus rudiment and undergo lym-phoid di¡erentiation [22]. Thus, VEGF-R2 mesodermalcells require at least an additional maturation step sup-ported by the microenvironment to di¡erentiate intoT cells. This assay made it possible ¢nally to identify cellsurface molecules expressed by T cell progenitors in em-bryonic bone marrow. Some of these molecules are in-volved in adhesion and/or signal transduction. They in-clude c-kit, hemopoietic stem cell adhesion molecule(HEMCAM), BEN, KIIL3 integrin, ChT1, MHC classII, CD44, and thrombomucin. Data on these moleculeswere reviewed recently [43], and we will focus only ontwo of them, HEMCAM and MHC class II L chain.HEMCAM is an adhesion molecule belonging to the

immunoglobulin superfamily with a V-V-C2-C2-C2 Ig do-main structure [44]. HEMCAM-positive bone marrowcells coexpressing c-kit could di¡erentiate into T, myeloid,and erythroid cells in vitro, suggesting that multipotent

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HSC expressed this adhesion molecule. HEMCAM ex-pression was not restricted to cells of the hemopoieticlineages, since this molecule was also expressed at highlevels on EC in many tissues, on myocytes, and on epithe-lial cells of the bursa of Fabricius [44,45]. HEMCAM isidentical to the chicken gicerin, a molecule involved inneurite outgrowth [46], and is the human homologue ofMCAM involved in melanoma progression and metastasis[47,48]. HEMCAM promotes cell^cell adhesion probablythrough both homophilic and heterophilic binding andwould interact with NOF [45], an extracellular matrix mol-ecule. HEMCAM might also transduce a signal [49,50].We have recently shown that HEMCAM regulated celladhesion since transfection of L cells by HEMCAMdownregulated cell surface expression of L1 integrins[51]. Two MCAM isoforms di¡ering in their cytoplasmictail have been identi¢ed. These two isoforms were di¡er-entially targeted to the basolateral and the apical poles ofepithelial cells (Vigneron et al., in preparation). This dif-ferential addressing into EC suggests that each isoform isinvolved in di¡erent steps of T cell progenitor homing.In the c-kit population of the bone marrow, the T cell

progenitors are restricted to the cells coexpressing theMHC class II L chain molecule at their surface [52].This population is present in the embryo as well as inthe young adult, although at lower numbers in the latter.Their cyclin A and B expression level suggested that theywere in the mitotic phase of the cell cycle. Interestingly,injection of MHC class II/c-kit/KIIL3 integrin-positive cellsresulted in the selection of cells with an increased T cellpotential [53]. The expression of MHC class II L chain wasthen lost when the progenitors di¡erentiated in CD4-CD8double-positive cells in the thymic environment. In con-trast, erythroid progenitors are restricted to the MHCclass II^c-kit-positive cell population.

9. Conclusions

The emergence of HSC and their commitment towardthe T cell lineage are now well established during aviandevelopment (Fig. 3). The YS gives rise to the ¢rst butonly transient hemopoietic progenitors. These progenitorsare unable to di¡erentiate into lymphoid cells. In the em-bryo proper at gastrulation stages, cells giving rise to HSCare located at the posterior part of the primitive streakand express VEGF-R2. HSC are then encountered inaortic clusters where they emerged from a hemogenic en-dothelium characterized by Runx-1 expression and endo-thelial markers such as LDL receptors and VE-cadherin.In a following step, HSC ingress in the mesenchyme andform the paraaortic foci. The allantois appears as a latersource of HSC. Graft experiments established that HSCderiving from both aorta and allantois colonize the bonemarrow. Adoptive transfer experiments showed that thy-mic progenitors derive from the paraaortic foci for the ¢rst

wave of colonization and from the bone marrow for thesecond and third waves of colonization. Graft experimentsshowed that bone marrow T cell progenitors of the latterwaves derive from HSC generated in the paraaortic fociand the allantois. The bone marrow T cell progenitors arecharacterized by MHC class II/c-kit/KIIL3 integrin/HEM-CAM expression. Finally, we cannot exclude that T cellprogenitors generated in the allantois contribute directlyto the second wave of thymus colonization.The fact that the YS gives only transient progenitors

whereas the embryo proper and very likely allantois gen-erate HSC raises important questions. How these pro-grams are established is not known but they are likelyin£uenced by di¡erences in the microenvironments. Theprecise knowledge of the gene repertoire expressed bythe HSC of the di¡erent sites of emergence using a largenumber of probes will allow the genetic cascades respon-sible for HSC commitment to be compared between thesedi¡erent sites. To determine what are the roles of severalkey transcription factors (Runx-1, CBFL, SCL/Tal-1) andsurface molecules (VE-cadherin, VEGF-R2, HEMCAM)in HSC emergence and migration will be the next chal-lenges.

Acknowledgements

This work was supported by the Association pour laRecherche contre le Cancer (ARC-5656 and 5131), ACI22-2002-296 of the Ministe're de l’EŁ ducation Nationale,and the CNRS.

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