Postępy Biochemii 59 (4) 2013

365

Patrycja Sroczynska

*

Biotech Research and Innovation Centre

(BRIC), University of Copenhagen, Copenha-

gen, Denmark

*

Biotech Research and Innovation Centre (BRIC),

University of Copenhagen, Ole Maaløes Vej 5,

2200 Copenhagen, Denmark; e-mail: patrycja.

[e-mail]

Received: September 25, 2013

Accepted: October 14, 2013

Key words: hematopoiesis, hemogenic endo-

thelium, hemangioblast, embryo

Abbreviation: HSCs — hematopoietic stem

cells; ES cells — embryonic stem cells; YS —

yolk sac; P-Sp — paraaortic splanchnopleura;

AGM — aorta-gonad-mesonephros; E — em-

bryonic day; BL-CFC — blast colony-forming

cell; Bry — Brachyury; Flk-1 — fetal liver ki-

nase 1; AcLDL — acetylated low-density lipo-

protein; EHT — endothelial to hematopoietic

transition; Ncx1 — sodium-calcium exchan-

ger-1; Scl — stem cell leukemia

Acknowledgements: I would like to thank

Georges Lacaud and Valerie Kouskoff, who

introduced me to the field of embryonic hema-

topoiesis.

Hemogenic endothelium — ontogenesis and role in blood production

ABSTRACT

E

ndothelial and hematopoietic lineages have long been thought to develop from a com-

mon ancestor, the hemangioblast. Alternatively, clusters of hematopoietic cells in the

dorsal aorta were observed to form in a close association with endothelial wall of the aorta,

leading to the hypothesis that a special subset of endothelial cells, called the hemogenic en-

dothelium, generates hematopoietic cells. Recent advances in time-lapse imaging, condition-

al labeling of cells in vivo and embryonic stem cell differentiation provided new evidence for

the existence of both, the hemangioblast and hemogenic endothelium. Importantly, these

seemingly contradictory theories can be merged into one model of hematopoietic differentia-

tion from mesoderm.

INTRODUCTION

Human hematopoietic system produces between 10

11

and 10

12

new blood cells

per day during steady state and even more during infection or after injury. This

can be achieved thanks to the life-long presence of hematopoietic stem cells

(HSCs), which are defined by two properties: the potential to differentiate into

all types of blood cells (multipotency) and the ability to generate progeny with

the same potential (self-renewal). This initial pool of HSCs, that supports blood

production throughout the whole life, is generated during embryonic develop-

ment. It is therefore of outmost importance to understand the process of hemat-

opoietic cell commitment from mesoderm in the embryo.

Experiments leading to the current view of hematopoietic development star-

ted at the beginning of the 20

th

century and were performed over decades in se-

veral different vertebrate models, among others: zebrafish, chicken, mouse and

human embryos. In addition to these in vivo studies, the in vitro differentiation

of embryonic stem (ES) cells provided an excellent experimental system to study

the earliest steps of hematopoiesis. Data described in this review refer to mouse

studies, unless otherwise stated.

EMBRYONIC HEMATOPOIESIS

The initial steps of hematopoietic commitment in the adult take place in the

bone marrow, which provides a niche for the HSCs. In the growing embryo the

need for oxygen supply and specialized blood cell production precedes the for-

mation of the bone marrow. Embryonic blood production occurs therefore in

several waves and utilizes distinct anatomical sites: yolk sac (YS), paraaortic

splanchnopleura (P-Sp), aorta-gonad-mesonephros (AGM) that forms in the pla-

ce of P-Sp, placenta, allantois, chorion and fetal liver (Fig. 1).

PRIMITIVE HEMATOPOIESIS

All hematopoietic cells are derived from mesoderm, which forms in the pro-

cess known as gastrulation starting in the mouse embryo from embryonic day

(E) 6.5. The first restricted hematopoietic cells are of erythroid lineage and are

called primitive erythrocytes, due to several unique features distinguishing

them from adult-type, definitive erythrocytes. These primitive erythrocytes de-

velop in the blood islands, which are clusters of primitive erythroid cells surro-

unded by endothelial cells in the YS. In the mouse embryo blood islands emerge

around E8.25 [1].

Progenitors for the primitive erythrocytes can be detected by colony-forming

assay already at the primitive streak stage (E7.0) and disappear by E9.0 [2]. These

transient erythroid cells, unlike definitive erythrocytes, enter circulation while

still containing nuclei. They mature in the bloodstream and in the fetal liver,

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enucleate between E12.5 and E17.5, and continue to circu-

late until 5 days after birth [3,4]. Primitive erythrocytes are

also bigger than definitive erythrocytes, contain higher le-

vels of hemoglobin and express unique globin genes: βH1,

εy and ζ [5].

DEFINITIVE HEMATOPOIESIS

In this review term “definitive hematopoiesis” refers to

all hematopoietic lineages other than primitive erythroid

cells, i.e. myeloid, lymphoid and definitive erythroid. Ho-

wever, different definitions of definitive hematopoiesis can

be met in other reviews, e.g. hematopoiesis restricted to the

HSCs able to reconstitute adult recipient and the progeny of

such HSCs [6]. Alternatively, definitive hematopoietic cells

can be divided into several classes, based on the develop-

mental potential of the cells [7].

The short-time window of the generation of primitive

erythroid progenitors (E7.0-E9.0) is overlapped and follo-

wed by the generation of macrophage and definitive ery-

throid progenitors starting from E8.25 [2]. This first wave

of definitive hematopoiesis initiates in the YS and coincides

with the onset of circulation (Fig. 1). Since the circulation is

not fully functional for the first two days, the first defini-

tive progenitors are thought to be of YS origin [8]. Howe-

ver, other parts of the pre-circulation embryo, intraembry-

onic P-Sp and extraembryonic allantois and chorion, when

isolated and pre-cultured in vitro, are also able to generate

myeloid and definitive erythroid cells [9-11]. The emerging

hematopoietic progenitors, of both primitive and definitive

lineages, express the αIIb integrin (also known as CD41),

which is the earliest hematopoietic extracellular marker [12-

14].

In the second wave of definitive hematopoiesis, lympho-

id progenitors are formed in parallel with the progenitors

of erythro-myeloid potential. The origin of the first lym-

phoid progenitors was a matter

of a long-standing controversy.

Pre-circulation P-Sp, but not YS,

when isolated from the embryo

and cultured in vitro, gave rise to

lymphoid progenitors [15]. Ho-

wever, more recent studies with

the sodium-calcium exchanger-1

(Ncx1), deficient mouse embry-

os that lack cardiac contractions

and circulation, provided evi-

dence that not only P-Sp but also

YS and placenta autonomously

generate lymphoid progenitors

[16,17].

HSCS

In the final wave of definitive

hematopoiesis, the embryo ge-

nerates HSCs, which, unlike he-

matopoietic progenitors, possess

long-term multilineage reconsti-

tution potential of adult hemato-

poietic system upon transplanta-

tion. The first HSCs can be isola-

ted from the AGM region of the embryo at E10.5 [18,19].

Within AGM, the emerging HSCs are specifically associated

with the endothelial wall of the dorsal aorta [20]. HSCs can

be also found at E10.5 in the major arteries of the embryo:

the vitelline artery, that connects embryo proper and YS,

and the umbilical artery, that connects the embryo proper

and placenta [20], and at E11.0 in the placenta [21].

The hematopoietic progenitors and HSCs colonize fetal

liver starting from E10.0 and E11.5, respectively. Thereafter

liver becomes the major hematopoietic organ of the embryo,

where HSCs expand reaching their highest number of about

1600 HSCs per liver at E16 [22]. Subsequently HSCs migrate

to the developing bone marrow, which becomes the hema-

topoietic center of the organism.

HEMANGIOBLAST

Based on the observation that primitive erythroid cells

and endothelial cells develop in close association to each

other in the blood islands, it was suggested that the hema-

topoietic and endothelial lineages derive from a common

precursor, named the hemangioblast [23,24]. The first direct

evidence for the existence of the hemangioblast was provi-

ded by studies of hematopoietic commitment using mouse

ES cell differentiation. They led to identification of an in vi-

tro equivalent of the hemangioblast, called the blast colony-

-forming cell (BL-CFC). Single BL-CFC contains endothelial,

hematopoietic (definitive and primitive) and smooth musc-

le potential [25,26]. It is defined by co-expression of the me-

sodermal marker Brachyury (Bry) and fetal liver kinase 1

(Flk-1, known also as vascular endothelial growth factor

receptor type 2, VEGFR-2) [27].

The existence of the hemangioblast in vivo was first do-

cumented in 2004, when the group of Gordon Keller iden-

Figure 1. Milestones of hematopoietic development in the mouse embryo.

Postępy Biochemii 59 (4) 2013

367

tified a clonal precursor to hematopoietic, endothelial and

vascular smooth muscle cells in the posterior primitive

streak of gastrulating mouse embryos [28]. A cell with he-

matopoietic and endothelial potential was also identified in

zebrafish [29], fruit fly [30] and chicken [31]. In each case the

hemangioblast was found to be a rare precursor cell (fre-

quency lower than 1% within Bry

+

Flk-1

+

cell population

of E7.5 mouse embryo), and present only during a narrow

window of development (in the mouse embryo between

E7.0 and E7.5). In addition to its infrequency and transience,

the hemangioblast has no single specific marker, making its

tracking in vivo a very challenging task. It remains therefore

unknown whether all blood and endothelial cells, or only a

special subset of them, are generated through this common

precursor. It has been however shown by lineage tracing

experiments that essentially all embryonic and adult hema-

topoietic cells are progeny of Flk-1-expressing cells [32]. In

addition, Flk-1-deficient mouse embryos die between E8.5

and E9.5 lacking both endothelial and hematopoietic types

of cells [33].

Paradoxically, the blood islands, that originally triggered

the hemangiobast hypothesis, were found to be of a non-clo-

nal origin and hematopoietic and endothelial cells in sin-

gle blood islands were shown to be derived from different

precursors [34]. This observation does not however invali-

date the hemangioblast theory, but rather suggests that the

hemangioblast’s progeny reach the blood islands already

after their commitment to endothelial and hematopoietic

lineages [35] (Fig. 2).

HEMOGENIC ENDOTHELIUM

In parallel to the hemangioblast hypothesis, a seemingly

contradictory theory developed, according to which hema-

topoietic cells are generated by a special subset of endothe-

lial cells, called the hemogenic endothelium. This theory

originated from the observations that hematopoietic cells

form characteristic clusters that are closely associated with

endothelial cells in the wall of the dorsal aorta [36-39]. When

endothelial cells were isolated from mouse embryo at dif-

ferent stages between E8.5 and E10.5, and cultured in vitro,

they differentiated into definitive erythroid, myeloid and

lymphoid progenitors [40-42]. Importantly, long-term adult

repopulating HSCs were also shown to emerge in the endo-

thelial layer of the main vessels of the embryo: the aorta and

the vitelline and umbilical arteries [20,43]. These early HSCs

express a whole panel of endothelial cell surface markers,

such as VE-cadherin, Tie2, CD31 and CD34 [43-46] together

with hematopoietic markers, CD41 (early hematopoietic

marker) and CD45 (late hematopoietic marker).

Based on the above findings it was suggested that the

definitive hematopoietic cells derive directly from hemo-

genic endothelial cells. This theory had been however lac-

king unequivocal evidence. It was also speculated that the

hematopoietic cells of the intra-aortic clusters might in fact

derive from the mesenchyme underlying the endothelium

[47]. The first direct evidence of a bona fide endothelial ori-

gin of hematopoietic cells came from studies performed in

chicken embryos. Jaffredo et al. injected acetylated low-den-

sity lipoprotein (AcLDL), a marker specific for endothelial

cells and macrophages, labeled with fluorescent dye, into

chicken embryos at the stage of development when macro-

phages are not yet detected. Newly generated CD45

+

hema-

topoietic cells in the aorta contained AcLDL, proving their

endothelial origin [48]. The same question was addressed

in a mouse study using an inducible reporter system allo-

wing a timed labeling of VE-cadherin-expressing cells [49].

Endothelial (VE-cadherin

+

) cells were labeled in vivo in mo-

use embryos for about 48 h starting from E9.5. The proge-

ny of labeled cells contributed to a pool of hematopoietic

cells both in the fetal liver and in the bone marrow. Since

only a portion of adult hematopoietic cells in this study was

derived from the labeled endothelial precursors, it remains

uncertain whether all blood cells arise from the hemogenic

endothelium.

It is now widely accepted that hematopoietic cells have

an endothelial ancestor. Little is known however about the

mechanism through which the endothelial cells lose their

endothelial-specific properties and become blood cells. Re-

cent live imaging studies provided an opportunity to follow

endothelial to hematopoietic transition (EHT) in a real time.

Zebra fish embryos, due to their transparency, provided an

ideal model to follow the emergence of hematopoietic cells

[50-52]. It was observed that the hematopoietic cells form

specifically at the ventral wall of the dorsal aorta. Impor-

tantly, the hematopoietic cells do not emerge from endothe-

lial cells as a result of cell division. Instead, endothelial cells

bend in a characteristic stretched manner, round up and fi-

nally detach from neighboring cells [52].

A direct observation of the emergence of hematopoietic

cells in the aorta inside mouse embryo is hindered by the

opaque nature of the embryo. Biosset et al. used slices of mo-

use embryos to overcome this problem and visualize aorta

[53]. They observed cells expressing CD31 (endothelial mar-

Figure 2. Schematic view of hemangioblast commitment to endothelial, blood

and vascular smooth muscle lineages in gastrulating mouse embryo. H — he-

mangioblast; E — endothelium; HE — hemogenic endothelium; VSM — vascular

smooth muscle; D — definitive hematopoietic progenitor; EryP — primitive ery-

throid progenitor.

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ker) turning on the expression of CD41 (early hematopoietic

marker) concurrently with budding of these cells from the

ventral wall into the lumen of the aorta. It is still unclear

if hematopoietic cells in the mouse embryo are formed in

an EHT similar to the one described in the zebrafish. Ano-

ther intriguing question is whether the CD41

+

cells budding

from the aortal wall are HSCs or rather an intermediate sta-

ge between endothelium and HSCs, called pre-HSCs. It is

evident that within intra-aortic clusters the majority of cells

are not HSCs, since e.g. at E11.5 there are more than 400 he-

matopoietic cells but only 2 HSCs in the aorta [54].

HEMANGIOBLAST

VS. HEMOGENIC ENDOTHELIUM

Are hemangioblast and hemogenic endothelium just two

different names for the same cell population? Or could they

be two distinct blood precursors, each giving rise to a spe-

cific subset of hematopoietic cells? Two studies, in which

cells falling into hemangioblast or hemogenic endothelium

category were distinguished and analyzed in the same set

of experiments, shed light on this puzzle [55,56]. Time-lapse

imaging and single cell sorting led to the conclusion that a

single mesodermal Flk1

+

cell without an endothelial pheno-

type can give rise to an endothelial cell, which can subse-

quently generate hematopoietic cells. Thus, the hemangio-

blast does not generate hematopoietic and endothelial cells

simultaneously, but gives rise to blood cells through an en-

dothelial intermediate stage. Both primitive and definitive

hematopoietic lineages were generated through this sequ-

ence of events. It is however impossible to say if HSCs are

also formed in the hemangioblast–hemogenic endothelium–

hematopoietic progenitor sequence of transitions, as HSCs

cannot be efficiently generated in vitro using current ES cell

differentiation protocols. It is likely that in the embryo the

short-lived hemangioblast from the E7.0-E7.5 primitive stre-

ak [28] gives rise to hemogenic endothelium (along with

non-hemogenic endothelium and vascular smooth muscle

cells), which can be found in the yolk sac starting from E7.5

[40,41,55] and later in the AGM, the vitelline and umbilical

arteries and the placenta [49]. Anatomical sites with docu-

mented presence of hemogenic endothelium are schemati-

cally shown in figure 3.

TRANSCRIPTIONAL REGULATION OF

HEMOGENIC ENDOTHELIUM

Evidence for role of specific transcription factors in the

development of hemogenic endothelium and hematopoie-

tic progenitors was obtained mostly through observation of

developmental defects in knockout mice and during in vitro

differentiation of knockout ES cells.

HEMANGIOBLAST — HEMOGENIC

ENDOTHELIUM TRANSITION

The activity of the transcription factor stem cell leukemia

(Scl), known also as T-cell acute lymphoblastic leukemia 1

(Tal1), is critical for the formation of both endothelial and

hematopoietic types of cells. Scl

-/-

mouse embryos die by

E9.5 lacking any type of blood cells [57]. However, deletion

of Scl specifically in cells expressing Tie2 (endothelial mar-

ker) did not have a major impact of hematopoiesis [58], in-

dicating that Scl is critically needed for blood development

before the hemogenic endothelium stage. Indeed, dissecting

hematopietic commitment in vitro using differentiation of

ES cells showed that Scl is necessary for the generation of

hemogenic and non-hemogenic endothelial cells from the

hemangioblast, but does not appear to be necessary for the

development of hemangioblast from mesoderm or its diffe-

rentiation into smooth muscle cells [55,59] (Fig. 4).

The expression of Scl was shown to be controlled by Etv2

[60]. Etv2

-/-

mouse embryos lack vasculature and blood cells

and die around E9.5 [61]. Detailed analysis of hematopoie-

tic commitment in Etv2

-/-

embryos and ES cells showed that

Etv2 is not necessary for the development of Flk1

+

meso-

derm, but is absolutely critical for the formation of endothe-

lial cells, including hemogenic endothelium [62,63].

HEMOGENIC ENDOTHELIUM — HEMATOPOIETIC

PROGENITORS TRANSITION

Runx1 (known also as acute myeloid leukemia 1, Aml1)

has long been known as a master regulator of hematopo-

iesis. Runx1

-/-

mouse embryos develop hemorrhages in the

Figure 3. Anatomical sites with reported hemogenic endothelium activity. (A)

Gastrulating mouse embryo. YS — Yolk sac. (B) Midgestation mouse embryo.

DA — dorsal aorta; UA — umbilical artery; VA — vitelline artery; P — placenta.

Drawings are not to scale.

Figure 4. Transcriptional control of hemogenic endothelium development and

differentiation into hematopoietic progenitors. H — hemangioblast; E — endo-

thelium; HE — hemogenic endothelium; VSM — vascular smooth muscle; D —

definitive hematopoietic progenitor; EryP — primitive erythroid progenitor.

Postępy Biochemii 59 (4) 2013

369

central nervous system and die by E12.5 [64,65]. The acti-

vity of Runx1 is necessary for the generation of definitive

hematopoietic progenitors both in vivo and in vitro [65,66].

Primitive erythropoietic progenitors still develop in the ab-

sence of Runx1 with only minor defects [67]. Importantly,

Runx1 activity is specifically required in endothelial cells, as

shown in mice with Runx1 deletion in Tie2

+

or VE-cadherin

+

endothelial cells [68,69]. Moreover, re-expression of Runx1

in Runx1

-/-

endothelial cells rescues definitive hematopoiesis

[55,70]. Together, these data define the window of Runx1

function in the hematopoietic development specifically at

the transition from hemogenic endothelium to definitive he-

matopoietic progenitors (Fig. 4).

A recent study showed that Runx1 and Hoxa3 are expres-

sed in a mutually exclusive manner and act antagonistical-

ly. High Hoxa3 levels maintain endothelial character of the

cells and repress hematopoiesis. In contrast, Runx1 expres-

sion overrides endothelial gene expression program establi-

shed by Hoxa3 and promotes hematopoiesis [71].

Factors involved in the regulation of primitive hemato-

poiesis are largely unknown and it remains unclear how

and at which stage of hematopoietic commitment the choice

between definitive and primitive developmental programs

is made. It has been however suggested that this choice is

determined by the interplay between Wnt and Notch signa-

ling pathways [72].

FINAL REMARKS

•

Given their indispensable role in the production of

HSCs and hematopoietic progenitors, still relatively little is

known about hemogenic endothelial cells:

•

What markers distinguish hemogenic endothelium

from other endothelial cells?

•

Do all endothelial cells have the potential to beco-

me hemogenic?

•

What extracellular signals determine hemogenic

vs. non-hemogenic status of endothelial cells?

•

Does hemogenic endothelium generate both endo-

thelial and hematopoietic cells, or is its potential restricted

to hematopoietic lineage?

•

Is hemogenic endothelium specific only for embry-

onic development or is it present also in adults?

Several studies suggest that adult HSCs posses endothe-

lial potential (reviewed in [35,73]), however hemogenic en-

dothelial activity in the steady-state adult hematopoiesis is

yet to be shown. Further studies are needed to fully under-

stand the transition from endothelium to blood cells. In vitro

ES cell differentiation has proven to be a powerful tool in

dissecting the earliest stages of hematopoietic commitment.

Not only it faithfully resembles hematopoietic development

in the embryo, but also it gives access to rare cell popula-

tions that are otherwise extremely difficult, if not impossi-

ble, to isolate or track in vivo.

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Postępy Biochemii 59 (4) 2013

371

Śródbłonek krwiotwórczy — ontogeneza i rola w hematopoezie

Patrycja Sroczyńska

*

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Kopenhaga, Dania

*

e-mail: [e-mail]

Słowa kluczowe: hematopoeza, śródbłonek, hemangioblast, zarodek

STRESZCZENIE

Przez długi czas uważano, że linie komórek śródbłonkowych i hematopoetyczych rozwijają się ze wspólnej komórki prekursorowej, heman-

gioblastu. Z drugiej strony, obserwowano skupiska komórek hematopoetycznych w aorcie grzbietowej zarodka tworzące się w bezpośrednim

sąsiedztwie komórek śródbłonka ściany aorty. Doprowadziło to do sformułowania hipotezy, że subpopulacja komórek śródbłonkowych,

zwana śródbłonkiem krwiotwórczym, jest źródłem komórek hematopoetycznych. Ostatnie badania z wykorzystaniem obrazowania poklat-

kowego, warunkowego znakowania komórek in vivo oraz różnicowania zarodkowych komórek macierzystych dostarczyły nowych dowodów

na istnienie zarówno hemangioblastów jak i śródbłonka krwiotwórczego. Co ważne, te pozornie sprzeczne teorie mogą zostać połączone w

jeden model różnicowania komórek linii hematopoetycznej i śródbłonkowej z mezodermy.

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