# Extraembryonic hematopoietic lineages-to macrophages and beyond

Alina Sommer, Elisa Gomez Perdiguero

► To cite this version:

Alina Sommer, Elisa Gomez Perdiguero. Extraembryonic hematopoietic lineages-to macrophages and beyond. Experimental Hematology, 2024, 136, pp.104285. 10.1016/j.exphem.2024.104285. hal-05238095

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# Extraembryonic hematopoietic lineages—to macrophages and beyond

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# Extraembryonic hematopoietic lineages—to macrophages and beyond

Alina Sommer<sup>a,b</sup>, and Elisa Gomez Perdiguero<sup>a*</sup>

<sup>a</sup>Macrophages and Endothelial Cells Unit, Department of Developmental and Stem Cell Biology, Institut Pasteur, Université Paris Cité, Paris, France; <sup>b</sup>Sorbonne Université, Collège Doctoral, Paris, France

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The first blood and immune cells in vertebrates emerge in the extraembryonic yolk sac. Throughout the last century, it has become evident that this extraembryonic tissue gives rise to transient primitive and definitive hematopoiesis but not hematopoietic stem cells. More recently, studies have elucidated that yolk sac-derived blood and immune cells are present far longer than originally expected. These cells take over essential roles for the survival and proper organogenesis of the developing fetus up until birth. In this review, we discuss the most recent findings and views on extraembryonic hematopoiesis in mice and humans. © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## HIGHLIGHTS

* Extraembryonic tissues give rise to the first hematopoietic cells in mice and humans.

* Primitive hematopoiesis mainly gives rise to erythroblasts and megakaryocytes.

* Erythro-myeloid progenitors differentiate into erythroid and myeloid cells in the yolk sac and the fetal liver.

* Yolk sac contribution to blood and immune cells over time is cell type specific.

* Lineage tracing shows the maintenance of yolk sac-derived macrophages into adulthood.

Extraembryonic membranes, such as the amnion, chorion, yolk sac, and allantois, are essential for the survival of developing amniotic embryos [1]. These membranes take over functions such as absorption of macro- and micronutrients, gas exchange, excretion of metabolic waste, and hematopoiesis, which is the production of blood cells [2].

In this review, we discuss the most important and recent findings on the contribution of extraembryonic hematopoiesis to blood and immune cell production in the mouse model, and we summarize the most recent advances in extraembryonic hematopoiesis in humans.

## DEVELOPMENT AND FUNCTION OF EXTRAEMBRYONIC TISSUES

Phylogenetically, the yolk sac is the oldest of the four extraembryonic membranes and evolved in fish over 500 million years ago [3]. It consists of an outer endodermal and an inner mesodermal lining

(Figure 1). The endodermal layer of the yolk sac develops from the extraembryonic visceral endoderm (originating from the primitive endoderm). The mesodermal layer on the other hand emerges from the posterior epiblast after epithelial-mesenchymal migration (EMT) at the primitive streak during gastrulation (at embryonic day (E) 6.5) [4]. Around 24 hours later, the emergence of the first blood cells from this mesoderm layer will mark the start of hematopoiesis.

## HISTORICAL AND CURRENT VIEW ON DEVELOPMENTAL HEMATOPOIESIS

Initially, direct observation of avian embryo development showed that the first red blood cells of the embryo arise in the yolk sac, within vascular structures termed blood islands [5,6], before the onset of circulation [7]. Subsequent advances in histology at the beginning of the 20th century enabled pioneering scientists to observe the development of red blood cells, megakaryocytes, and macrophages in the mammalian yolk sac in cats, rabbits, mice, and rats [8] as well as in chickens [9,10] and humans [11]. We would like to highlight the illuminating work performed by embryologists Vera Dantschakoff and Florence Sabin that noticed the close relationship between hematopoietic cells and endothelial cells [9,10]. It was hypothesized that hematopoietic stem cells might emerge during this developmental period.

Work in the chicken embryo demonstrated that hematopoietic stem cells arise intraembryonically and not from the yolk sac, contrary to what was initially proposed [12,13]. This was later confirmed in the mouse model by several research groups. They identified the para-aortic splanchnopleura (P-Sp), the precursor of the aorta-gonad mesonephros (AGM), in the embryo proper as the site of hematopoietic stem cell emergence [14–17]. Within the AGM region, hematopoietic cells emerge from the endothelium of the dorsal aorta
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**Figure 1** Yolk sac hematopoiesis. Primitive potential for erythroblasts, megakaryocytes, and macrophages is first detected around embryonic day (E)7.5 in the yolk sac. These primitive cells directly emerge from the mesoderm in the blood islands of the yolk sac. Around E8.5 transient definitive erythro-myeloid progenitors (EMPs) arise from the hemogenic endothelium in the yolk sac. *In situ* EMPs differentiate into megakaryocytes and macrophages in a *Myb*-independent manner. *Bona fide* EMPs and EMP-derived committed progenitors migrate to the fetal liver where they differentiate into definitive red blood cells, megakaryocytes, mast cells, neutrophils. and macrophages in a *Myb*-dependent manner.

through a *Runx1*-dependent endothelial-to-hematopoietic transition (EHT) [18–22], confirming the initial notion of a close relationship between hematopoietic and endothelial cells. The EHT process has been extensively reviewed [23,24].

Thanks to morphological and histological observations, transplantation and colony-forming assays, lineage tracing, bar-coding, and single-cell omics, it is now widely accepted that the blood and immune

system develops in several consecutive waves. These progenitor waves are not only spatiotemporally distinct; analysis of mutant embryos for key transcription factors (such as *Runx1* and *Myb*) also revealed that they are controlled by different molecular mechanisms.

In mice, the extraembryonic yolk sac first gives rise to (1) a primitive wave of blood cells, followed by (2) a transient definitive wave consisting mainly of erythro-myeloid progenitors (EMPs) [8,12,25–27].
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**Figure 2** Hematopoietic waves build the layered immune system. (A) The extraembryonic yolk sac is the first site of hematopoiesis giving rise to primitive hematopoiesis between E7.5 and E8.5. These are followed by transient definitive erythro-myeloid progenitors (EMPs) that produce blood and immune cells in the yolk sac between E8.5 and E10.5 and in the fetal liver between E11.5 and E13.5. Afterward, embryonic multipotent progenitors (eMPPs) and hematopoietic stem cells (HSCs) emerge in the AGM intraembryonically around E10.5. In the fetal liver, eMPPs start producing mature hematopoietic cells around E12.5, and HSCs around E14.5 both retain their hematopoietic activity into adulthood. (B) The first EMP-derived cells found in circulation are primitive erythroblasts, followed by EMP-derived neutrophils and platelets, and finally EMP-derived erythrocytes. (C) In the brain, EMPs are the only source of microglia throughout life. EMPs also contribute to other tissue-resident macrophages such as Langerhans cells in the skin or Kupffer cells in the liver. The skin harbors EMP-derived mast cells, which are however replaced after birth.

Hematopoiesis then switches to the embryo proper, wherein (3) embryonic multipotent progenitors and (4) hematopoietic stem cells emerge from the dorsal aorta in the AGM. Intra- but not extraembryonic hematopoiesis can sustain life-long hematopoiesis [14,15,17,28,29] (Figure 2A).

Although it was long thought that yolk sac-derived hematopoiesis is simply a transient wave important for the survival of the embryo until hematopoietic stem cells are mature enough to supersede blood and immune cell production, research of the last decades has provided ample evidence that yolk sac-derived cells remain in the organism long after the end of yolk sac hematopoiesis and even into adulthood [26,30–33] (Figure 2B, C).

## YOLK SAC-DERIVED PRIMITIVE HEMATOPOIESIS

Here, we define primitive hematopoiesis as the production of mature blood cells from progenitors that arise directly from the mesoderm, without undergoing an endothelial-to-hematopoietic process. In the mouse, they emerge directly from the mesoderm in the blood islands between E7.5 and E8.5 [8,25,34] (Figure 1). Primitive cells are independent of *c-Myb* [31,35] and *Runx1* [36,37], which means that they can develop in the absence of these factors regardless of whether they express them (Figure 1). The term primitive can refer to different features and is further discussed in Box 1.
Box 1 Primitive versus definitive hematopoiesis

In the context of hematopoiesis, the terms “primitive” and “definitive” have been used inconsistently in the literature.

Originally, the term “primitive” was introduced at the beginning of the 20th century to distinguish transient nucleated erythroblasts from the enucleated erythroblasts that replace the first [8,9,38,39]. “Primitive” in this case refers to the resemblance to red blood cells of lower vertebrates in which only large, nucleated red blood cells are found [40]. The term also reflects the presence of embryonic hemoglobin. In this school of thought, blood cells emerging from the same ancestor as nucleated red blood cells will also be called primitive cells. Cells related to enucleated red blood cells without embryonic hemoglobin would be termed “definitive.” Accordingly, EMPs and their progeny should be termed “definitive.”

However, the term “primitive” can also refer to the absence of full reconstitution potential in transplantation assays. By this definition, only bona fide stem cells and multipotent progenitors are “definitive” [30,41].

A third use of the term “primitive” is to describe cells not following the adult differentiation pathway. A well-known example of this is the differentiation of EMP-derived macrophages that develop without passing through a GMP or monocyte intermediate [26,27,42].

Lastly, “primitive” can also refer to immature morphology or distinct functions in comparison to adult cells, which would be the case for most embryonic mature immune cells. This is the case in regards to primitive macrophages as termed by Naito et al [43]. Yolk sac macrophages at E10.5 lacked the classical ultrastructural features of fetal liver and adult macrophages (hence “primitive” morphology) and they were observed before any monocytes were detected (“primitive” development that did not follow the classical adult differentiation cascade from monocytes) [43].

We propose that progenitors arising from an endothelial-to hematopoietic transistion (EHT) should be termed as "definitive", irrespectively of the "primitive" or immature features of the mature cells they give rise to. In addition, the potential of primitive progenitors is restricted to a single lineage (either erythroid or myeloid). Recently, some papers have distinguished between primitive EMP and definitive EMP. EMP are defined as having erythroid and myeloid potential at the single-cell level (and they arise from an EHT), therefore they should not be referred to as primitive. To distinguish EMPs from bona fide HSPCs the terms "pro-definitive" [44] or "transient-definitive" [45] have been introduced.

PRIMITIVE ERYTHROBLASTS

Primitive erythroblasts are the first hematopoietic cells that develop in the vertebrate yolk sac and are essential for embryonic survival in mammals [46]. The development of primitive erythroblasts within the yolk sac blood islands is the best studied. Blood islands are mesodermal cell masses proliferating between the visceral endoderm and mesothelial cell layer. Although the outer cells differentiate into endothelial cells, mesodermal cells in the center of the cluster differentiate into primitive erythroblasts [47,48]. These are larger than their definitive counterpart and contain both embryonic ζ-hemoglobin (εγ- and βH1-globin) and adult α- and β-hemoglobin (α1-, α2-, β1- and β2-globin) [49] and mature in circulation while retaining their nucleus until E12.5 [50]. The primitive wave is the only source of red blood cells until E12.5 and can sustain the embryo until E15.5 [50,51]. By E17.5, most primitive erythroblasts are gone, but few are still detectable as late as 5 days after birth (Figure 2B) [50].

MEGAKARYOCYTES

Concomitantly with primitive erythroblasts, megakaryocyte poten-
tial emerges in the yolk sac around E7.5, peaks around E8.5, and
is no longer detectable after E13.5 [8,52,53]. Primitive megakar-
yocyte progenitors are more proliferative and require fewer cyto-
kines than their adult counterparts [52]. Compared with
primitive erythroblast progenitors, primitive megakaryocyte pro-
genitors are however low in number. Furthermore, these first
megakaryocytes are smaller and have lower polyploidy when
compared with their fetal liver or adult counterparts [37]. Primi-
tive platelet formation is faster [52], does not require thrombo-
poietin [54] and gives rise to larger platelets that contain more
RNA, and show reduced clot-forming capacity than their adult
counterparts [55].

MACROPHAGES

Macrophage potential is first detected at E7.5 (on average 4 pro-
genitors/yolk sac compared with 42 erythroid progenitors/yolk
sac at the same stage) [25], whereas the first zygotic macrophages
(defined as F4/80+) only emerge in the yolk sac around E9.5. It
is generally assumed that the first macrophages detected in the
yolk sac are primitive, in accordance with findings in the zebrafish
model [56]. They were initially described as primitive based on
their ultrastructural organization and detection before monocyte
emergence (Box 1) [43,57]. Further, macrophage potential is
more abundant than erythro-myeloid potential at E8 in limiting
dilution assays [58]. Nevertheless, several findings are in support
of a definitive (EMP) origin for yolk sac macrophages. First, mac-
rophage potential in the yolk sac coincides with definitive ery-
throid potential and not with primitive erythroid potential [251].
Second, macrophage numbers peak at E10, after definitive EMP
progenitors emerge in the yolk sac [26]. Finally, yolk sac macro-
phages and microglia, the brain macrophage considered the pro-
totype of primitive macrophages, are efficiently labeled with an
endothelial inducible Cre driver [32], whereas primitive erythro-
cytes are not labeled (unpublished data from our laboratory), in
line with an origin from a progenitor that underwent an endothe-
lial-to-hematopoietic transition. Recently, a new mouse strain was
proposed to specifically label the primitive wave but it also lead
to very efficient labeling (80%) of definitive EMPs at E10.5 [59],
limiting the scope of the conclusions. The lack of a robust primi-
tive macrophage population in the mammalian embryo when
compared with the zebrafish could be in part explained by the
difference in "lifestyle" (in utero versus external development) and
the presence of maternally derived macrophages in the early
stages of mammalian development. Maternally derived macro-
phages are predominant in the yolk sac until E9, with their num-
bers peaking at E7.5/E8 (40 cells/yolk sac) [58]. Indeed, to date,
a faithful tool to trace the primitive wave is not available in the
mouse and is most necessary to clarify the quantitative contribu-
tion of primitive hematopoiesis to macrophage pools, and micro-
glia in particular.

YOLK SAC-DERIVED DEFINITIVE HEMATOPOIESIS—
ERYTHRO-MYELOID PROGENITORS

EMPs are transient definitive multipotent progenitors that give rise to
definitive red blood cells, megakaryocytes, macrophages, mast cells,
and granulocytes (Figure 1) [25,60]. They emerge in the yolk sac
between E8.5 and E10.5 [25,58] from endothelial cells in the blood
islands and vascular plexus through a *Runx1*-dependent EHT [61
–63]. Unlike intraembryonic hematopoiesis [18,64] EMP emergence
is independent of blood flow [62,65].

EMPs are defined by the expression of Kit, CD41, CD16/32, and
AA4.1 (CD93) [26,65,66]. Interestingly, EMP commitment and differ-
entiation into mature cells is niche dependent. Although all EMPs
express *Myb*, a transcription factor important for the maintenance of
proliferating hematopoietic stem and progenitor cells [67], EMPs in the
YS differentiate *in situ* directly into macrophages and megakaryocytes
without the requirement of *Myb* [26,31,66]. Additionally, they commit
to more restricted erythroid and myeloid progenitors [66]. *Bona fide* mul-
tipotent EMPs, as well as downstream committed progenitors, seed the
fetal liver around E9.0-E11.0 [65,68]. Here, they unfold their full poten-
tial and differentiate in a *Myb*-dependent manner into definitive red
blood cells, megakaryocytes, mast cells, and granulocytes and mono-
cyte-like cells (Figure 1) [65,66,69,70]. For some lineages, such as mast
cells and macrophages, final maturation and tissue specialization will
continue after seeding peripheral organs [71].

Red Blood Cells

EMPs are the main source of circulating red blood cells from E12.5
onwards [25,26,50], sustaining the embryo until birth (Figure 2B)
[33,51,71]. Accordingly, EMPs are skewed toward red blood cell pro-
duction in the fetal liver [65,70]. During differentiation, EMP-derived
red blood cells pass through a megakaryocyte-erythrocyte progenitor
(MEP) stage that is dependent on *Myb* (Figure 1) [51,66]. In contrast
to primitive erythroblasts, EMP-derived red blood cells enucleate in
the fetal liver before entering circulation [50]. Further, they only
express adult and not embryonic hemoglobin [25,49,50]. Due to
these differences compared with primitive erythroblasts, EMP-
derived red blood cells are defined as “definitive” (Box 1).

Megakaryocytes

EMPs give rise to megakaryocytes through two distinct development
trajectories in a time- and niche-specific manner: (1) through a direct
*Myb*-independent differentiation path in the yolk sac and (2) through
a *Myb*-dependent MEP-intermediate in the fetal liver [66] (Figure 1).
The earlier differentiation pathway is reminiscent of the novel subset
of Mk-primed HSCs that directly produce Mk in the adult bone mar-
row [72–74]. EMP-derived *Myb*-independent megakaryocytes super-
sede primitive megakaryocytes in the yolk sac at E10.5. EMP-derived
megakaryocytes then enter circulation, where they peak around
E11.5 and are detectable until at least E14.5 (Figure 2B) [66]. Whether
primitive megakaryocytes and *Myb*-independent EMP-derived mega-
karyocytes in the yolk sac are functionally different needs to be fur-
ther investigated. However, the two waves of EMP-derived
megakaryocytes likely have distinct traits because it has been shown

that megakaryocytes in the yolk sac and the fetal liver reach different
polyploidy and have a distinct dependency on thrombopoietin [54].

Mast Cells

The first mast cells in the embryo are derived from EMPs. Although mast cell commitment from EMP can be detected *in vitro* [25] and at the transcriptomic level [66] in the yolk sac, mast cell differentiation only starts in the liver from E11 onwards, when the number of mast cell progenitors peaks [69]. Mast cell progenitors then enter the circulation and seed tissues, such as the skin, spleen, lung, brain, heart, and peritoneal cavity, where they complete their maturation [32,69]. EMP-derived tissue mast cells persist beyond birth, and it was shown that ontogeny was linked to transcriptomic and immunophenotypic differences (Figure 2C) [32,75].

Macrophages

The most famous EMP-derived cells are tissue-resident macrophages [26,30,31,76–78]. In the yolk sac, EMPs directly differentiate into macrophages via a macrophage precursor, without passing through a mono-cyte-intermediate stage independently of *c-Myb* [26,31,57,42]. Once the heart starts beating, EMP-derived macrophages seed the entire embryo proper via the circulation [30,78]. There, they adapt tissue-specific signatures over time [79]. EMP-derived macrophages are often termed tissue-resident macrophages, as they self-maintain throughout life independently of bone marrow hematopoiesis (Figure 2C) [26,30,31,76,77]. This is evident for microglia in the brain [30], red-pulp macrophages in the spleen, alveolar macrophages in the lung, Langerhans cells in the epidermis, Kupffer cells in the liver [26,77], and macrophages in the heart [80] and testis [81]. The enhanced capability to give rise to tissue-resident macrophages is a distinct feature of EMPs and not a matter of window of opportunity [82]. It was proposed that EMPs can be distinguished as early and late EMPs, the latter presumably giving rise to monocyte-like cells in the fetal liver and proposed to contribute to tissue-resident macrophage pools [42]. The distinction of “early” and “late” is largely based on genetic labeling by tamoxifen (or 4-hydroxytamoxifen, its active metabolite)-induced Cre recombination at two time points that are 24 hours apart from each other [66,69,42]. More experimental evidence is required to support the existence of two biologically distinct EMP populations (and not just time- and niche-dependent effects) and to provide a quantitative assessment of the contribution of EMP-derived monocytes to adult macrophages.

Neutrophils

EMP-derived production of neutrophils begins around E11.5 in the
fetal liver. These first neutrophils enter the blood around E12.5 where
they circulate until at least E14.5 (Figure 2B) [65,70]. Ontogenetic dif-
ferences in neutrophils have been studied in zebrafish [83] and mice
[84]. In the latter, there was a dramatic change in the ability of neutro-
phils to roll and adhere to inflamed yolk sac vessels, correlating with
the change on neutrophil ontogeny. Before E15, when most blood
neutrophils are EMP derived [70], rolling and adhesion were essen-
tially absent. Later (at E17), fetal neutrophils acquire the ability to roll
and adhere as surface expression of CXCR2 begins to increase late in
fetal life. Similarly, the rolling and adhesive capacities of fetal human
neutrophils in vitro correlate with gestational age [85]. Thus, func-
tional differences between EMP- and intraembryonic-derived neutro-
phils exist and remain to be further investigated in the mouse model.
## Lymphoid Cells

Whether the yolk sac is endowed with lymphoid potential has long been under discussion. It has become evident that previous to the emergence of hematopoietic stem cells (HSCs), a pool of progenitors with lymphoid potential appear around E9.5 [86]. Their anatomical origin however has remained unclear. Several studies have associated lymphoid potential with the yolk sac [86–88]. The main source of debate is whether this extraembryonic lymphoid potential originates *in situ* in the yolk sac or rather derives from the embryo proper, as it is always detected after circulation starts. While studies in embryos lacking circulation due to impaired cardiac contraction still identify lymphoid potential in the yolk sac [87], this is in conflict with earlier culture and transplantation assays demonstrating that only the intraembryonic AGM and not the yolk sac has B, T, or natural killer (NK) cell potential [17,89], in particular before circulation starts. In line with the latter, most recent fate-mapping studies suggested that the first lymphoid progenitors emerge in the AGM, around 24 hours before bona fide HSCs are born [28,29].

In regards to EMPs, most studies did not find lymphoid potential. For instance, when McGrath et al [65] cultured EMPs (Kit⁺ CD41⁺ CD16/32⁺) from E9.5 yolk sacs they did not detect any B cell output. Furthermore, fate-mapping experiments using Csf1r<sup>MeriCreMer</sup> pulsed at E8.5 or E9.5 came to the same conclusion that EMPs did not have B or T cell potential in the mouse [32,90]. Intriguingly, a recent study based on fate mapping (Csf1r<sup>MeriCreMer</sup> Rosa26<sup>tdTomato</sup> pulsed at E9.5) claimed that EMPs produce the first NK cells in the fetal liver at E14.5 and E15.5, even though at low numbers [91]. However, pulse-labeling at E9.5 in this fate-mapping model leads also to low labeling of intraembryonic hematopoietic stem and progenitor cells (HSPCs) [90]. Therefore, more studies are required to investigate this further.

## INTERPLAY BETWEEN YOLK SAC-DERIVED AND INTRAEMBRYONIC-DERIVED DEFINITIVE HEMATOPOIESIS

### Role of Yolk Sac-Derived Macrophages in HSC Establishment

EMP-derived macrophages can be found in the AGM and are crucial for the establishment of the adult hematopoietic system. They interact with hematopoietic stem and progenitor cells (HSPCs) in the AGM and support their generation and maturation [92]. Once HSCs seed the fetal liver, yolk sac-derived macrophages are crucial for HSCs to enter their normal maturation and commitment process. Depletion of EMP-derived macrophages leads to alterations of the fetal liver extracellular matrix (ECM) and precocious commitment of HSCs toward erythroid and myeloid fates, leading to a transient increase in circulating monocytes and neutrophils until the postnatal period. Importantly, this dysregulation of HSC commitment in utero has long-lasting consequences into adulthood, with a reduced pool of lymphoid-primed multipotent progenitors (MPP4) [82] (Figure 3).

### Competition Between Extra- and Intraembryonic Blood Cells for Cytokines and Growth Factors

In the fetal liver, extra- and intraembryonic-derived hematopoietic progenitors mature and differentiate side by side. Since they share the same niche, they compete for cytokines and growth factors that are mainly produced by the fetal liver stroma [93]. Bona fide EMPs and EMP-derived committed progenitors however outcompete HSPCs

in their proliferation and differentiation potential [65]. EMPs require less erythropoietin (EPO) for red blood cell production in vitro [33] and produce neutrophils faster in competitive transplantation assays than intraembryonic HSPCs [70]. Furthermore, EMPs and intraembryonic HSPCs depend on distinct sources of Kit ligand [64].

### Switch From Extra- to Intraembryonic Hematopoiesis

Both extra- and intraembryonic hematopoietic progenitors commit to immunophenotypic common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cells (summarized as Lin⁻ Kit⁺ Sca-1⁻ cells (LK)), which are progenitors with increased commitment toward erythroid and myeloid cells. The switch from extra- to intraembryonic contribution to these progenitor populations occurs around E14.5 [29,70]. Intraembryonic hematopoiesis then contributes to mature blood and immune cells in the fetal liver (neutrophils, mast cells, red blood cells, and megakaryocytes) another 48 hours later. Surprisingly, intraembryonic-derived mature blood and immune cells are not found in circulation until birth (neutrophils, red blood cells) [29,33,70]. In tissues, EMP-derived mature cells can persist until adulthood. This is the case for mast cells in the tongue, adipose tissue, and peritoneal cavity [32,69,75], and tissue-resident macrophages in the brain, liver, heart, lung, or skin [26, 30, 76, 77]. Tissue-resident macrophages are capable of self-maintenance throughout life, and the rate and timing of contribution of adult HSCs to their pools is organ-specific. At one end of the spectrum, brain parenchyma microglia are almost never replaced by HSC-derived cells in homeostatic conditions; on the other end of the spectrum, macrophages in the lamina propria are constantly renewed from bone marrow-derived monocytes [30,94].

Collectively, these findings suggest that blood and immune cells from the yolk sac are present in the fetus for much longer than previously thought. This indicates that extraembryonic hematopoiesis is not only important for early embryonic development but plays a much more far-reaching role for the fetus and the newborn. Whether defects in yolk sac hematopoiesis cause diseases beyond birth requires further investigation.

## CONTRIBUTION OF YOLK SAC-DERIVED BLOOD AND IMMUNE CELLS TO FETAL DEVELOPMENT

Blood formation in the yolk sac is of crucial importance for the survival of the embryo and fetus, as it is the main source of red blood cells until birth ensuring oxygen supply (Figure 3) [33,46,51].

Moreover, YS-derived megakaryocytes and macrophages are involved in several processes that are important for proper organogenesis. Megakaryocytes and platelets govern the development of the lymphatic and blood vasculatures in the brain (Figure 3) [95,96]. Proper brain development is further secured through the synaptic pruning activity of yolk sac-derived macrophages (Figure 3) [97]. In addition, these macrophages are involved in branching morphogenesis via cytokine secretion and remodeling of the ECM in organs such as the testis [98], the lung [99], the kidney [100], the mammary gland [101], and the submandibular gland [102]. Macrophages also support bone development [103–105], angiogenesis [106–108], and clearance of dead cells by phagocytosis throughout the developing organism [109]. Even though dispensable for survival, yolk sac-derived mast cells support brain and cornea development by mediating vascular and nerve branching [110,111] (Figure 3). Furthermore, they may take over immune-modulatory functions prenatally and might be primed in utero
<figure><img src="image_6.png" /></figure>

**Figure 3** Functions of EMP-derived blood and immune cells. EMP-derived red blood cells sustain the embryo and fetus by supplying oxygen. Platelets from yolk sac-derived megakaryocytes are crucial for proper blood-lymph separation. Mast cells provide instructive cues that mediate vascular and nerve branching. Macrophages perform efferocytosis during organogenesis, synaptic pruning in the central nervous system, and erythroblast enucleation in the fetal liver thanks to their phagocytic activity. They assist hematopoietic stem and progenitor cell (HSPC) emergence in the dorsal aorta, modulate hematopoietic stem cell (HSC) commitment in the fetal liver, and support branching morphogenesis due to their extracellular matrix (ECM) remodeling traits. Macrophage instructive cues also support angiogenesis and bone development.

for stronger immune protection after birth [111,112]. The functions of neutrophils in development remain to be discovered, but they likely play critical roles at birth as neonatal neutropenia is correlated with increased susceptibility to bacterial infections and early-onset sepsis [113]. Altogether, the function of yolk sac-derived cells impacts tissues beyond the prenatal period, potentially affecting also postnatal life.

## OTHER EXTRAEMBRYONIC SITES OF HEMATOPOIESIS IN THE MOUSE

All extraembryonic tissues develop almost simultaneously from the mesoderm and are physically closely connected. Hence, it is natural to wonder whether (1) the allantois, (2) the chorion, (3) the placenta, (4) the vitelline vessels, and (5) the umbilical cord have hematopoietic potential.

### Development of Other Extraembryonic Tissues

The allantois buds from the extraembryonic mesoderm around E7.5 and grows toward the chorion. The latter is a bilayered tissue of

extraembryonic ectoderm (derived from the trophoblast) and extraembryonic mesoderm. Allantois and chorion fuse and become the placenta [114,115]. The umbilical artery links the dorsal aorta to the placenta, and the umbilical vein the placenta to the fetal liver. Similarly, the vitelline artery connects the dorsal aorta with the yolk sac, and the vitelline vein connects the yolk sac with the fetal liver [116,117]. In this manner, the dorsal aorta, placenta, yolk sac, and fetal liver are all connected building one closely linked circuit.

### Hematopoietic Potential

Several studies have found little or no hematopoietic potential in the allantois [25,118–120].

However, long-term reconstitution potential has been detected in the vitelline vessels and umbilical artery starting around E10.5 with increasing potential until at least E12.5 [19,121] concomitantly with the onset of AGM-hematopoiesis. High HSPC potential was also found in the placenta vasculature 24 hours before the fetal liver was seeded, indicating that HSPCs pass through and maturate in the placenta during migration to the fetal liver [116,122]. However, since all
these tissues are part of one circuit of blood flow, it is difficult to deci-
pher whether the hematopoietic cells are circulating or emerging de
novo. Experiments with embryos lacking circulation addressed this
issue and demonstrated de novo budding of CD41⁺ hematopoietic
progenitors from endothelial cells in the placenta [123].

HUMAN EXTRAEMBRYONIC HEMATOPOIESIS

Human pregnancies last on average 266 days or 38 weeks post con-
ception (280 days/40 weeks post gestation) instead of 19 days (post
gestation) in mice. Human embryos first develop a primary yolk sac
(Carnegie stage (CS) 4, 1 post conception week (pcw)). A secondary
replaces the primary yolk sac around 2.5 pcw (CS6) and persists until
~8 weeks (CS23) [124]. The secondary yolk sac, a balloon-like
appendage inside the amnion, is the site where hematopoiesis begins
in humans [45].

As in mice, the first blood cells in the human embryo are primitive
erythroblasts that emerge in blood islands in the yolk sac between
16 and 18.5 days after fertilization (CS7-8) [11,125,126]. These primi-
tive erythroblasts, contain embryonic hemoglobin (ζ/ε globins) and
enter the circulation as nucleated cells around 3 pcw (~4 ssp) [127].
Human primitive erythroblasts enucleate starting around 5 pcw
in the placenta through interaction with macrophages (Hofbauer
cells) [128].

Next to primitive red blood cells, the human yolk sac gives rise to
transient definitive EMPs that emerge from endothelial cells [125].
EMP5 (also called early HSPCs in some studies on human develop-
mental hematopoiesis) appear around 2-3 pcw and are detectable
until 8 pcw at the most [27]. They commit to myeloid progenitors
and megakaryocyte-erythrocyte progenitors and eventually differen-
tiate into mainly red blood cells, megakaryocytes, and macrophages
in the yolk sac and fetal liver [27,125,126,129]. One study also
attributed mast cell potential to human EMPs [129]. Unlike mice,
EMP5 in humans do not produce neutrophils. Granulopoiesis only
takes place in the bone marrow at 14 pcw [129,130]. Similar to find-
ings in the mouse model [26,31,42], human macrophages most
likely differentiate in the yolk sac without passing through a mono-
cyte-stage [27].

Early experiments on human embryoid bodies led to the conclusion
that yolk sac-derived EMPs have de novo T cell potential [131,132]. By
contrast, previous culture experiments and recent findings from single-
cell RNA sequencing of human hematopoietic tissues support the
notion that the EMPs in the yolk sac have no lymphoid potential, as
lymphoid cells in the yolk sac were detected only after the onset of
AGM-hematopoiesis [27,125,133]. Nevertheless, this highlights that
AGM-derived cells colonize the yolk sac before reaching the fetal liver
[134].

As in mice, the human placenta is a site of hematopoiesis. Whether
only maturation or also de novo generation occurs in the placenta is
still under investigation [134–137].

One of the biggest differences between murine and human devel-
opmental hematopoiesis is the onset of bone marrow colonization by
hematopoietic progenitors. Whereas in the mouse bone marrow
hematopoiesis only starts after birth, in human fetuses, HSCs seed
the bone marrow already around 12 pcw [116,138,139]. Thus,
although EMPs are the main source of mature blood cells until birth
in mice, yolk sac-derived cells most likely have different kinetics in
human development.

CONCLUSION

Among extraembryonic tissues, the yolk sac is essential during the
development of the hematopoietic system in all vertebrates. Line-
age tracing highlighted that yolk sac-derived cells, especially
erythro-myeloid progenitor-derived cells, remain in the developing
organism long after the end of yolk sac hematopoiesis and take
over essential roles in development, such as oxygenation and
organogenesis.

More research is now necessary to further understand the func-
tional differences between extra- and intraembryonic-derived blood
and immune cells and their role beyond development and in disease.

Conflicts of Interest Disclosure

The authors have nothing to disclose.

Acknowledgments

This work was supported by recurrent funding from Institut Pasteur,
CNRS, and Revive (Investissement d'Avenir; ANR-10-LABX-73). A.S.
was funded by a fellowship from FRM (FDT202304016740).

Author Contributions

A.S.: Conceptualization, Writing—Original Draft, Writing—Review & Editing.

E.G.P.: Conceptualization, Writing—Review & Editing,
Supervision.

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