THE INTERNATIONAL JOURNAL OF

Int. J. Dev. Biol. 54: 1033-1043 (2010)

DEVELOPMENTAL

BIOLOGY

doi: 10.1387/ijdb.103105gs

www.intjdevbiol.com

Primitive and definitive erythropoiesis
in the yolk sac: a bird’s eye view
GUOJUN SHENG*
Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan

ABSTRACT The yolk sac is the sole niche and source of cells for primitive erythropoiesis from E1
to E5 of chicken development. It is also the main niche and source of cells for early definitive
erythropoiesis from E5 to E12. A transition occurs during late embryonic development, after which
the bone marrow becomes the major niche and intraembryonically-derived cells the major source.
How the yolk sac is involved in these three phases of erythropoiesis is discussed in this review.
Prior to the establishment of circulation at E2, specification of primitive erythrocytes is discussed
in relation to that of two other cell types formed in the extraembryonic mesoderm, namely thesmooth muscle and endothelial cells. Concepts of blood island, hemangioblast and hemogenic
endothelium are also discussed. It is concluded that the chick embryo remains a powerful model
for studying developmental hematopoiesis and erythropoiesis.

KEY WORDS: chicken, primitive erythropoiesis, definitive erythropoiesis, hematopoiesis, yolk sac

Introduction
Studies on chicken hematopoietic development have been
instrumental in the establishment of several key concepts in thefield, including B lymphocytes, hematopoietic stem cells,
hemangioblasts and hemogenic endothelium. Research focus inrecent years has shifted heavily toward genetic and molecular
based investigation, leading to a bias against the use of avian
models. The wealth of published literature on descriptive andexperimental analyses of chicken hematopoiesis, however, is still
unsurpassed by any other model organism. The completion ofchicken genome (International Chicken Genome Sequencing
Consortium, 2004) has made molecular analysis relatively straightforward and genetic analysis possible. In addition to superb
spatial and temporal resolution in developmental studies, thechick model serves as an evolutionary link between mammals
and lower vertebrates. Here I will provide an embryologicaloverview of chicken extraembryonic hematopoietic development,
and focus more detailed discussion on the involvement of the yolk
sac in the generation of primitive and definitive red blood cells.
Readers interested in other related aspects of chicken hematopoietic development may find relevant details in articles andmonographs for general description of hematopoiesis (DieterlenLievre and Le Douarin, 2004; Jaffredo et al., 2003; Le Douarin,
1978; Metcalf and Moore, 1971; Romanoff, 1960), molecularregulation of hematopoiesis (Bollerot et al., 2005; Minko et al.,

2003; Siatskas and Boyd, 2000), hematological description ofdifferent blood lineages (Lucas and Jamroz, 1961), early vascularmorphogenesis (Drake, 2003; Drake et al., 1998; Drake et al.,2000) and developmental and adult immune systems (Davison etal., 2008).

Yolk sac, amnion, chorion and allantois
Extraembryonic tissues in developing chick embryo containthe following four components: yolk sac, amnion, chorion andallantois (Fig. 1 A,B). The term “extraembryonic”, however, issomewhat a misnomer, as there is no clear anatomical or molecular demarcation to separate the embryonic from the extraembryonic tissues during the first few days of development, and at laterstages both are integral components of the developing embryo.
This is especially so for the hematopoietic system after theestablishment of circulation by the end of embryonic day 2 (E2).
Furthermore, the yolk sac, the most prominent component of theextraembryonic tissues, is withdrawn into the abdomen after
hatching and is technically embryonic. The allantois, formed near
the tail bud at the junction between embryonic and extraembryonic tissues, has contributions from both. Nevertheless, thisreview will follow the conventional view of these tissues as
extraembryonic.
Before formation of the amnion, chorion and allantois, the yolk
sac is the only extraembryonic tissue (Fig. 1 C,D). The amnion*Address correspondence to: Guojun Sheng. Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan.
Tel: +81-78-306-3132. Fax: +81-78-306-3146. e-mail: sheng@cdb.riken.jp

Final author corrected PDF published online: 21 May 2010.

ISSN: Online 1696-3547, Print 0214-6282© 2010 UBC Press
Printed in Spain

1034

G. Sheng

and chorion are derived from yolk sac somatopleure. Expansionof extraembryonic coelom separates these two somatopleuraltissues from the splanchnopleural tissue, which becomes thelater yolk sac. The amnion encloses the developing embryo by theend of E3 (Adamstone, 1948; Lillie, 1919; Romanoff, 1960; Wu etal., 2001). The remainder of the extraembryonic somatopleurebecomes the chorion. The allantois grows rapidly from a small
out-pocket of hindgut anlage during E3, and from E5 it starts tofuse with the chorion into chorioallantois (DeFouw et al., 1989;
Leeson and Leeson, 1963; Lillie, 1919; Melkonian et al., 2002;
Romanoff, 1960; Steinmetz, 1930). The formation of chorioallantois involves only the distal wall of the allantois. Its proximal wall
starts to fuse with the amnion from E7 (Romanoff, 1960; Steinmetz,
1930) and with the yolk sac during the last few days of embryonicdevelopment (Romanoff, 1960). From the perspective of germlayer contribution, the allantois contains endoderm and mesoderm cells, the amnion and chorion contain ectoderm and mesoderm cells, and the yolk sac contains all three germ layers at earlystages and mesoderm and endoderm cells after the expansion of
the extraembryonic coelom.

Area pellucida, area opaca and area vasculosa
For the convenience of embryological description and manipulation, early chick blastoderm is separated into area pellucida andarea opaca based on the opaque appearance of endoderm cells
in the outer region with abundant intracellular yolk granules andattached acellular yolk (Bellairs, 1958; Bellairs, 1963; Bellairs,1964) (Fig. 1 C,D). This does not, however, correspond to the
traditional division of intraembryonic and extraembryonic tissues,
because a significant part of the area pellucida contributes also to
extraembryonic tissues (Fig. 1D). Depending on the extent of
mesoderm cell invasion, the area opaca is further divided into thearea vasculosa, area vitellina and margin of overgrowth (Bellairs,
1963). The margin of overgrowth, located at the extreme peripheral region, contains a special type of ectoderm cells responsible
for maintaining epiblast tension and for the epiboly process thateventually encloses the entire yolk (Bellairs, 1963; Bellairs et al.,
1967; New, 1959). The area vitellina is the region of the yolk sacthat does not yet contain mesoderm cells. The area vasculosa,the place where yolk sac vasculogenesis and hematopoiesis takeplace, is marked laterally by the extent of extraembryonic mesoderm invasion (Fig. 1 B,D). Medially, the area vasculosa is
continuous with vascular regions in the area pellucida. The areavasculosa expands laterally to replace the area vitellina (Bellairs,
1963; Romanoff, 1960). When circulation starts at E2 (Fig. 1 C,D),
the area vasculosa occupies only a very small part of the entire
yolk sac, whereas the margin of overgrowth has already expanded to the equatorial region. By E5 (Fig. 1 A,B), the areavasculosa reaches the equator and the area vitellina has covered
most of the yolk. By E15, the area vasculosa covers
the entire yolk and the area vitellina disappears. The
expansion of the area vitellina and area vasculosa
shows noticeable variations in relationship both toeach other and to the growth of the embryo proper
based on the Hamburger and Hamilton staging criteria(Hamburger and Hamilton, 1992), suggesting a needin the future to establish an independent staging system for the extraembryonic tissues. As yolk sac erythropoiesis takes place exclusively in the mesoderm
lineage, only the area vasculosa part of the yolk sac
will be discussed in this review. Early yolk sac erythropoiesis occurs in the extraembryonic mesoderm before the expansion of the extraembryonic coelom andthe invasion of the allantois, and thus in the context of
all three germ layers. Late yolk sac erythropoiesis
takes place in splanchnic mesoderm-derived tissues
adjacent to the yolk sac endoderm.

Extraembryonic mesoderm and ventral mesoderm

Fig. 1. General organization of chicken extraembryonic tissues. (A,B) E5 embryo.
(C,D) E2 embryo. (A) Top view of an E5 embryo before breaking the vitelline membrane
and chorion. (B) Top-side view after partial breaking of the vitelline membrane andchorion to reveal the embryo proper (with the amnion) and allantois. At this stage, thearea vasculosa has reached the equator and the area vitellina has reached the opposite
pole (not seen here). (C) Top view of an E2 (HH13) embryo, showing the area pellucida
and area vasculosa. Most of the area vitellina is not included here. (D) Schematic
diagram of (C).

All mesoderm cells are generated from the primitive
streak in the area pellucida (timeline in Figure 4). The
most lateral extraembryonic mesoderm cells are derived from early mesoderm cells generated from stage
HH2 onward in the elongating primitive streak. Fatesof mesoderm precursors in the primitive streak varydepending on the stages. Generally speaking, posterior part (1/2 to 1/3) of the primitive streak continues togive rise to extraembryonic fated mesoderm from HH2
to at least HH9 (Murray, 1932; Nakazawa et al., 2006;
Nocolet, 1970; Peebles, 1898; Psychoyos and Stern,
1996; Rawles, 1936; Rosenquist, 1966; Rudnick, 1938;

Primitive and definitive erythropoiesis in chicken yolk sac 1035
Sawada and Aoyama, 1999;
Schoenwolf et al., 1992; Spratt, 1942;
Spratt, 1946; Spratt, 1947; Spratt andHaas, 1965). Relative to more axiallylocated mesoderm lineages (the axial,
paraxial, intermediate and lateral plate
mesoderm), extraembryonic mesoderm precursors are of the ventralmost fate and populate the lateralmost regions of the developing embryo. The extraembryonic mesoderm
covers a wide area and has its own
dorsal/ventral and anterior/posterior
patterning processes. The chick embryo during first few days of its development takes the shape of a relatively
flat sheet (Fig. 2), and the dorsal/
ventral axis of the extraembryonicmesoderm is reflected in the medial/
lateral positional difference relative tothe embryo proper. Its anterior/posterior axis is similar to that in the embryo
proper and reflects the extent of extraembryonic mesoderm migrationfrom the posterior end of the primitive
streak to the anterior-most position
rostral to the embryonic forebrain. This
aspect has not received much investigation so far, although early work by
Sabin provided some descriptions ofanterior/posterior positional difference Fig. 2. Primitive erythropoiesis in the yolk sac. (A) rho globin expression at HH10, revealed using a rho
in early phases of primitive hemato- intron-specific probe. (B) scl expression at HH10, marking the blood precursors at this stage. (C) lmo2
poiesis (Sabin, 1920). Within the ex- expression at HH10, marking both the developing vasculature and blood cells. (D) Schematic diagram ofan E2 embryo section, showing primitive blood cells in relationship to the ectoderm, endoderm and othertraembryonic mesoderm, the axis fromextraembryonic mesoderm cell types.
the extraembryonic ectoderm to theextraembryonic endoderm is sometimes also referred to as dorthe separation of blood and endothelial cells within the bloodsal/ventral. To avoid confusion, mesoderm positional difference
island cell population (Nakazawa et al., 2006). Sabin reported asimilar observation that the cells contributing to exocoelomic
along this axis is discussed here with specific reference to thewalls separate first from the angioblasts (blood island cells)
ectoderm, endoderm or coelomic cavity.
(Sabin, 1917; Sabin, 1920). The splanchnic wall of the extraemVentral mesoderm lineages
bryonic coelom is the source of cells for the vascular smoothmuscle layer during vasculogenesis, and after formation of theDuring and after the migration to populate extraembryonicvascular network, vascular smooth muscle layer is in direct
territories, mesoderm cells differentiate into three main lineages:
contact with the extraembryonic coelomic cavity. This generalblood, endothelial and smooth muscle cells (Fig. 2) (Shin et al.,organization of somatopleural and splanchnopleural tissues in2009). Yolk sac splanchnic mesoderm contains all three lineages
the yolk sac is supported by ultrastructure studies (Kessel and(Fig. 2D). Yolk sac somatic mesoderm contains mainly smooth
Fabian, 1985; Murphy and Carlson, 1978).
muscle cells that line the extraembryonic ectoderm (Adamstone,
Blood island
1948; Oppenheim, 1966; Pierce, 1933; Romanoff, 1960; Shin etal., 2009; Wu et al., 2001) (Fig. 2D). Small and infrequent blood
islands with differentiated blood and endothelial cells have also
Progenitor cells for blood and endothelial cells form aggrebeen observed in the extraembryonic somatopleure in our analygates. This is the earliest morphological sign of differentiation inses (unpublished data) (Alev et al., 2010), suggesting that blood
the extraembryonic mesoderm. Different terms have been usedand endothelial lineages do not segregate completely into theto describe these aggregates, including blood islands, angioblasts
splanchnopleure during early extraembryonic mesoderm patternand hemangioblasts. Wolff (1764 Theorie von der generation) is
ing. Among these three lineages, progenitors for the smooth
generally credited for the earliest description of the blood islands.
muscle cells, contributing to both the somatopleure and splanchInitially called “substanzinseln” by Wolff and “Island of Wolff” innopleure, separate first from the other two soon after their ingresEnglish literature, some confusion regarding the connotation ofsion through the primitive streak (Shin et al., 2009), followed by
the term “blood island” arose in early 20th century [summarized in

1036

G. Sheng(Adelmann, 1966; Ruckert, 1906)]. This confusion, still outstandoften form from small blood islands with no accompanying erything today, is due to three main observations already apparent toropoiesis.
embryologists a century ago (Ruckert, 1906): 1) prior to circulaPrimitive erythrocyte differentiation
tion, aggregates can be observed in the area vasculosa that areassociated with both vasculogenesis and hematopoiesis; 2) agBlood islands first appear at around stage HH6 in a U or V
gregates can be observed both before and after the appearance
shaped area marking the posterior and lateral extents of extraemof vascular endothelial cells; and 3) aggregates observed before
bryonic mesoderm expansion. The earliest erythropoietic differthe appearance of vascular endothelial cells are associated more
entiation marked by hemoglobin gene expression occurs at stage
with the territory of vasculogenesis than with that of hematopoieHH7 (0-1 somite stage) (Nakazawa et al., 2006). Molecularly,sis. These seemingly contradictory observations, although not sohowever, extraembryonic mesoderm differentiation starts as soonwith current knowledge, led Sabin to call early blood islands prioras they are generated from the posterior primitive streak. The
to the appearance of vascular endothelial cells as angioblasts andearliest appearance of extraembryonic coelom varies from HH6
later blood islands inside the vasculature as true blood islands
to HH8. Hemoglobin expression appears initially only in the most
(Sabin, 1920). Early blood islands, Sabin’s angioblasts, werereferred to as hemangioblasts by Murray (Murray,1932). Each hemangioblast, capable of generating
both blood and endothelial cells but not necessarily
giving rise to both, was defined by Murray as one
blood island aggregate, containing many cells. Thisdefinition is different from the current use ofhemangioblast as a single cell capable of generating
blood and endothelial cells after division(s) (Choi etal., 1998; Huber et al., 2004; Park et al., 2005; Ueno
and Weissman, 2006; Vogeli et al., 2006). Angioblastsof Sabin are not often used in current literature andhemangioblasts of Murray currently have a strong
connotation of cell differentiation potentials. This
review uses the term blood island(s) to describe theaggregate(s) prior to the formation of vasculature,
blood cell aggregate(s) to describe Sabin’s blood
island(s) inside vessel lumen prior to their separation into individual blood cells, and hemangioblasts,in the sense that each is a single cell, only whenreferring to the bi-potential nature of these early
blood island cells.
Larger blood islands, constituting more cells in agiven blood island and with more cells percentagewise contributing to the blood lineage, are present inmore lateral regions of the area vasculosa. Progressively smaller blood islands are observed medially(Fig. 2). Blood islands formed in extraembryonic partof the area pellucida and in the intraembryoniclateral plate are very small, containing a few cells
and only giving rise to endothelial cells. Thus during
Fig. 3. Definitive erythropoiesis in the yolk sac. (A) View of a region of E5 yolk sac fromprimitive erythropoiesis, the area vasculosa can be the ectoderm side. (B,C) Section of E5 (B) or E4 (C) yolk sac after in situ using a betaA
divided into hemogenic and non-hemogenic regions. globin-specific locked nucleic acid (LNA)-based probe, showing active erythropoiesis in
Blood islands in the hemogenic region generate association with venous vessels. Black arrow: artery. Red arrows: extra-vascular clusters.
both primitive blood and endothelial cells and those Red arrowheads: intra-vascular clusters. (D) View of a region of E10 yolk sac from thein the non-hemogenic region generate only endot- endoderm side. (E,F) Section of E10 yolk sac after in situ using the betaA LNA probe,
helial cells (Nakazawa et al., 2006; Ruckert, 1906; showing active erythropoiesis in association with venous vessels. More intra-vascular
Sabin, 1920). The transition between hemogenic clusters are seen at this stage. Arrows: artery. (G) Schematic drawing of E5 yolk sac
and non-hemogenic regions is gradual and not clearly section. (H) A model for how early definitive erythrocytes are generated in the yolk sac.
demarcated morphologically or molecularly. This is Two possible sources, as discussed in the text, are combined here in this model. At E3,
both primitive erythrocytes and vascular endothelial cells retain some hematopoietic
also true for the transition between the nonpotential. At E3.5, this potential is lost in most primitive erythrocytes and endothelial cells,
hemogenic extraembryonic region and the intra- but is retained or enhanced in a small percentage of both cell types. At E4, dedifferentiaembryonic lateral plate vessels. During primitive tion of these hematopoietic cells occurs, leading to formation of hematopoietic cell
hematopoiesis, erythrocyte formation has always clusters, and at slightly later stage (E4.5-5), of erythropoietic clusters. Some of thesebeen observed in association with endothelial cell hematopoietic cells may give rise to other blood lineages or stem/progenitor cells (marked
formation in our studies, whereas endothelial cells with orange nucleus in E4 and E5).

Primitive and definitive erythropoiesis in chicken yolk sac 1037
peripheral blood islands, reflecting progressive phases of differentiation (Fig. 2 A-C). Although it is considered that more centrally
located cells in a given blood island differentiate into blood
whereas outer cells into endothelial cells, details of morphologicaldifferentiation at the cellular level are so far unclear. Within a given
blood island, hemoglobin gene expression precedes morphological distinction between blood and endothelial cells (Nakazawa etal., 2006). Although cell divisions occur in undifferentiated extraembryonic mesoderm cells and subsequently in blood island
cells and later differentiated mesoderm lineages, the cell fatechoice between blood and endothelial lineages among blood
island cells is made largely in the context of cell population,
instead of progenies of cell divisions (Weng et al., 2007), suggesting that hemangioblasts as currently defined do not play a majorrole during primitive blood and vascular formation. After thedifferentiation of blood island cells into blood and endothelial
lineages, endothelial cells from individual blood islands anastomose to form primary vascular plexus. By stage HH10 (10somites), before the initiation of circulation at stage HH12-13, awell-formed vascular network is recognizable in the area vasculosa(Fig. 1C). In the hemogenic region of the area vasculosa, blood
cells at stage HH10 are still observed as clusters, the blood
islands by Sabin’s definition. Individual blood cells have smoothersurface than at earlier stages, yet they still adhere to each otherand to the endothelial wall. By stage HH12-13, when the circulation starts, most blood cells in well established vascular channels
have lost contacts to each other and to the endothelium, andbecome free circulating cells (Fig. 4). More medially located blood
islands at this stage still have an “immature” morphology and are
not connected to the circulation, reflecting the fact that they are
generated later than laterally located ones.

Primitive vs. definitive erythrocytes
The shift from primitive to definitive erythropoiesis is considered to start at E5 of chicken development. This is based onstudies of dynamic changes in hemoglobin composition anderythrocyte morphology in circulating blood. Physiological andmolecular mechanisms regulating the timing of this shift are notclear, although change in tissue oxygen levels has been hypothesized as one possible cause (Baumann and Meuer, 1992;
Baumann et al., 1983). Since the first report of partial beta globin
peptide sequences from normal and sickle human blood cells(Ingram, 1956), efforts were made throughout the sixties andseventies to understand developmental changes in chicken hemoglobin heterogeneity at the protein level (Beaupain et al., 1979;
Brown and Ingram, 1974; Bruns and Ingram, 1973; Fraser, 1961;
Hashimoto and Wilt, 1966; Manwell et al., 1966; Manwell et al.,
1963; Saha, 1964; Saha and Ghosh, 1965; Shimizu, 1972;
Simons, 1966; van der Helm and Huisman, 1958; Wilt, 1962; Wilt,
1967). This was followed by the elucidation of chicken hemoglobin genes and genomic organization in the early eighties (Dodgson
and Engel, 1983; Dodgson et al., 1981; Dodgson et al., 1983;
Dodgson et al., 1979; Dolan et al., 1983; Dolan et al., 1981; Engeland Dodgson, 1980; Engel et al., 1983; Reitman et al., 1993;
Villeponteau et al., 1982; Villeponteau and Martinson, 1981) andof developmental changes in hemoglobin transcript profiles in theninties (Mason et al., 1995; Minie et al., 1992). These studies,
together with more recent genomic analysis, can be summarized

as follows. There are seven hemoglobin genes in chickens, threeof alpha type (pi-alphaD-alphaA) and four of beta type (rhobetaH-betaA-epsilon) in alpha and beta loci, respectively. Bothchicken alpha and beta loci are orthologous to eutherian (truemammals) counterparts (Patel et al., 2008). pi is orthologous tohuman zeta/pseudo-zeta, alphaD to human mu (also called
pseudo-alpha2 or alphaD) and alphaA to human pseudo-alpha1/alpha2/alpha1/theta. Individual beta hemoglobin genes in chickens and mammals arose by independent duplications and do notform orthologous groups, although their ancestral beta genes
before duplication are considered orthologous (Alev et al., 2009;
Cooper et al., 2006; Goh et al., 2005; Patel et al., 2008). Primitive
erythrocytes in chickens have high pi, rho and epsilon hemoglobin
proteins, and definitive erythrocytes have high alphaD, alphaA
and betaA. All three alpha globins, however, are present in bothprimitive and definitive erythrocytes. betaA, in addition to being
the major beta globin in definitive erythrocytes, is also weakly
present in primitive erythrocytes (Alev et al., 2008). betaH is notdetected in primitive erythrocytes and is very low in definitive
erythrocytes (Alev et al., 2008; Mason et al., 1995). Analyses ondevelopmental changes of hemoglobin heterogeneity suggestthat definitive erythrocytes appear in circulation at E5 and start toconstitute the majority of circulating erythrocytes by the end of E7(Fig. 4). Changes in hemoglobin subtypes have so far only been
analyzed with relatively large numbers of blood cells. It is therefore unclear whether changes in hemoglobin transcript and protein heterogeneity can occur at single cell level, either in differentiated erythrocytes or in erythropoietic cells of either lineage.
Both primitive and definitive erythrocytes are nucleated. Mature primitive and definitive erythrocytes have distinct oval shapes
measuring on average 8 by 12.7 microns for the former and 7 by
10.3 microns for the latter (Romanoff, 1960). Both lineages
progress through several maturation stages, from the most immature megaloblast/hemocytoblast, through proerythroblast, basophilic erythroblast, polychromatophilic erythroblast and reticulocyte, to the final mature erythrocyte (Bruns and Ingram, 1973;
Dantschakoff, 1908; Dawson, 1936; Edmonds, 1966; Forkner,
1929; Lemez, 1964; Lucas and Jamroz, 1961; Romanoff, 1960;
Small and Davies, 1972; Sugiyama, 1926). These distinctions
have been made based on a number of criteria, including generalcell and nuclear morphology, histological staining, ratio of nuclei
and cell sizes and intracellular organelle and microtubule abundance and distribution. They are all referred to here as erythrocytes based on the fact that even the most immature megalobalsts/
hemocytoblasts have initiated hemoglobin expression. Thesestudies on erythrocyte morphological changes revealed a similar
picture to that derived from hemoglobin heterogeneity studies.
Definitive lineage cells appear in circulation at E5 and overtake
primitive lineage cells percentage-wise by the end of E7. Morphological studies also showed that by the time circulation starts at
E2, primitive erythrocytes are still at the “proerythroblast” stage
with large and round shape. By the end of E3, oval morphology ofpolychromatophilic erythroblasts takes over, which last until E6,
when they develop first into reticulocytes and finally into matureerythrocytes at the beginning of E9 with a complete loss ofcytoplasmic ribosomes. Therefore, by the time definitive lineage
appears in circulation in the middle of E5 and takes over as the
major lineage by the end of E7, the primitive erythrocytes have notcompleted its maturation process (Fig. 4). Maturation of definitive

1038

G. Sheng

erythrocytes follows similar progression of stages to that of theprimitive lineage, with a notable difference in that the definitive
lineage cells do not mature synchronously, due to the fact thatnew definitive cells are continuously being generated as theembryo grows.

Transition from primitive to definitive wave
Before definitive erythrocytes enter circulation in the middle ofE5, their progenitor cells have been reported to be associated withvenous vessels in the yolk sac from the beginning of E4(Dantschakoff, 1908; Dantschakoff, 1909; Sabin, 1917). Thisobservation is supported by our recent molecular studies (Nagai
and Sheng, 2008). Chick/quail and chick/chick chimera studies(Beaupain et al., 1979; Dieterlen-Lievre, 1975; Dieterlen-Lievre etal., 1976; Lassila et al., 1978; Lassila et al., 1982; Martin et al.,1978) and chick/chick parabiotic studies (Metcalf and Moore,
1971; Moore and Owen, 1965; Moore and Owen, 1967) indicatedthat, at least during the first few days of definitive erythrocyte
generation, the source of cells is mainly yolk sac-derived. At about
the same time, if not earlier, intraembryonic hematopoietic cells
have been observed in association with the dorsal aorta (DieterlenLievre and Martin, 1981; Jaffredo et al., 2003; Jaffredo et al.,2005; Jaffredo et al., 2000; Jaffredo et al., 1998; Jordan, 1916;
Nagai and Sheng, 2008; Olah et al., 1988; Pardanaud et al.,1996). A number of questions can be asked, for instance, about
whether dorsal aorta-derived hematopoietic cells undergo definitive erythropoiesis either in circulation or in the yolk sac niche;
whether yolk sac-derived hematopoietic cells can find their way tothe dorsal aorta; and what is the exact type of cells from the yolksac that contribute to early definitive erythropoiesis. Chimera
studies suggested that even at E5-6, though making minor
contributions, intraembryonically-derived cells can generate definitive erythrocytes. Dorsal aorta-derived hematopoietic cells,
currently considered to be the only intraembryonic hematopoietic
source, may be involved in this process. Our molecular analyses
with hematopoietic markers support the notion that hematopoietic
cells associated with dorsal aorta are derived from dorsal aorta
endothelium (Nagai and Sheng, 2008). This is in agreement withdata from cell labeling and EM analyses (Jaffredo et al., 1998).
Erythropoiesis, however, is not observed at this early period ineither intra-vascular or peri-vascular hematopoietic cells associated with the dorsal aorta (Geerts et al., 1993) (H. Nagai and G.
Sheng, unpublished data). Our observations also suggest thatduring this period, most yolk sac vasculature-associated hematopoietic cells undergo definitive erythropoiesis in situ (Nagai andSheng, 2008). Dantschakoff distinguished two hematopoietic
populations in the yolk sac, the intra-vascular erythropoietic cells
and extra-vascular leucopoietic cells (Dantschakoff, 1908;
Dantschakoff, 1909). Our observations, however, suggest that atleast for erythropoiesis, it can occur both intra- and extra-vascularly(Nagai and Sheng, 2008) (Fig. 3 A-C,G,H). This is supported bya recent EM study (Niimi et al., 2008).

Sources of early definitive erythrocytes
The source of yolk sac erythropoietic cells is not clearly
resolved. As mentioned above, dorsal aorta-derived cells havebeen suggested to be able to take up the yolk sac niche andundergo erythropoiesis (Beaupain et al., 1979; Dieterlen-Lievre,
1975; Lassila et al., 1978; Lassila et al., 1982; Martin et al., 1978).
This in principle may happen, but currently with no strong evidence either for or against it. Judging from the relative timing andscales of hematopoietic cell generation in the yolk sac and dorsal
aorta, it seems reasonable to assume that yolk sac erythropoietic
cells during early definitive erythropoiesis are primarily yolk sacderived. It is another matter, however, to pinpoint which yolk saccell lineage(s) is involved in early definitive erythropoiesis. Circulating hematopoietic stem cells in E2 and E3, generated from theprimitive wave, could be one possible source, although histological and EM studies argue against such a scenario (Bruns andIngram, 1973; Dantschakoff, 1908; Dawson, 1936; Edmonds,
1966; Lemez, 1964; Lucas and Jamroz, 1961; Romanoff, 1960;
Small and Davies, 1972; Sugiyama, 1926). Cells with hematopoietic stem cell potential, residing outside the vasculature after the
initiation of circulation at E2, have also been suggested as apossible source for definitive erythrocytes (Dardick and Setterfield,
1978). Our analyses on mitotic activities and timing of the appearance of definitive hematopoietic and erythropoietic clusters in theyolk sac, however, argue against such a stem cell-based hypothesis, at least during the early stages of definitive erythropoiesis.
This leaves two types of yolk sac-derived cells as possible
main sources, the vascular endothelial cells and primitive erythrocytes (Fig. 3H). Dantschakoff suggested the venous endothelium as a likely source for definitive erythropoietic cells(Dantschakoff, 1908; Dantschakoff, 1909). This hypothesis is
supported by our molecular marker study (Nagai and Sheng,2008) (Fig. 3 A-C,G). From E2 to E5, yolk sac vasculature
undergoes at least two phases of remodeling. Hemodynamicchanges initiated by heart pulsation remodels early vascular
plexus into arterial and venous trees between E2 and E3, followedby the remodeling from two-dimensional apposing arterial/venous
trees to a three-dimensional paired arterial/venous network between E3 and E5 (Eichmann et al., 2005; Gonzalez-Crussi, 1971;
le Noble et al., 2004; Nagai and Sheng, 2008; Van Mierop andBertuch, 1967) (Fig. 4). This second remodeling phase happensin the context of the sinking of major arterial vessels into the yolk,
together with associated yolk sac endoderm (Fig. 3G). Smaller
venous vessels fuse and come to lie above sunken arteries, andform main paired venous vessel for each artery. Yolk sac definitive erythropoiesis is initiated during this second phase of yolk sac
vascular remodeling (Nagai and Sheng, 2008). Hematopoietic
foci are observed in venous vessels adjacent to large arteries at
E3.5-4, with globin positive definitive erythropoietic cells appearing at E4-4.5 and entering the circulation at E4.5-5. Early definitive erythropoiesis in the yolk sac takes place both intra-vascularlyand extra-vascularly in association with remodeling venous vessels adjacent to major arteries and in association with smaller
vessels (Fig. 3 B,C). These observations suggest that yolk sac
endothelial cells can give rise to erythropoietic cells, either bydedifferentiation first into a hemangioblast type cells or by directtransdifferentiation (Fig. 3H). Such a hypothesis, that hematopoietic cells can be generated from morphologically endothelial
shaped cells, is in a sense in agreement with the recent finding of
direct generation of hematopoietic cells from the endothelium of
dorsal aorta (Bertrand et al., 2010; Boisset et al., 2010; Kissa andHerbomel, 2010). Dantschakoff went one step further and commented that all early vascular endothelial cells possess hemato-Primitive and definitive erythropoiesis in chicken yolk sac 1039
poietic potency, which is exhibited or restricted differentially in
different locations (Dantschakoff, 1909).
The other possibility, that primitive erythrocytes may dedifferentiate into definitive erythrocytes, was also mentioned by
Dantschakoff (Dantschakoff, 1908) and others (Lemez, 1964;
Romanoff, 1960; Sugiyama, 1926) (Fig. 3H). Our analyses indicate that by the end of E3, circulating primitive erythrocytes stillretain prominent hematopoietic marker expression (Nagai andSheng, 2008). These cells are in the process of primitive erythrocyte maturation as discussed earlier, and exhibit primitive erythrocyte hemoglobin profile. During the period of E4, hematopoietic
markers are rapidly downregulated in circulating primitive erythrocytes, and definitive hematopoietic/erythropoietic clusters appear in the yolk sac venous vessels and small capillary vessels(Nagai and Sheng, 2008). Interestingly, it is also during this periodthat most primitive erythrocytes have been reported to undergo
the last mitotic division among on average six divisions from theearliest megaloblast/hemocytobalst stage, and enter post-mitotic
stage (Campbell et al., 1971; Hagopian and Ingram, 1971;
Weintraub et al., 1971) (Fig. 4). It may therefore be interesting totest in future experiments whether at least part of definitive

clusters is formed by the dedifferentiation of primitive erythrocytes
which do not enter the post-mitotic phase (Fig. 3H). Althoughcurrently without direct evidence in the context of chicken yolk sac
definitive erythropoiesis, similar hypotheses with regard to differentiation plasticity and potentials have been proposed in othersystems (Dzierzak, 2002; Graf, 2002; Kingsley et al., 2006;
McNagny and Graf, 2003; Orkin and Zon, 2002; Prindull andFibach, 2007).

Late definitive erythropoiesis

The picture becomes more complex as embryonic development proceeds. Chimera studies suggest that contribution fromintraembryonically-derived definitive erythrocytes, at least byorigin, gradually increases, and by E12-13 surpasses the contribution from extraembryonically-derived cells. The major embryonic erythropoietic niche, the bone marrow, has just started active
generation of definitive erythrocytes by this time (Dantschakoff,
1909; Metcalf and Moore, 1971). It is possible that someintraebmryonically-derived hematopoietic cells find their erythropoietic niche during mid-incubation period in the yolk sac (Beaupain
et al., 1979; DieterlenLievre, 1975; Lassila etal., 1978; Lassila et al.,
1982; Martin et al. ,1978). Some reportssuggested that the yolk
sac remains as the major niche for definitive
erythropoiesis until E20(Bruns and Ingram,
1973; Dantschakoff,1908). A recent histological and EM study suggested this process lasts
until E19 (Niimi et al.,2008). In our analysis
using molecular markers, prominent erythropoiesis in the yolk sac
continues at least until
E10 (Fig. 3 D-F), whichwanes significantly by
E15 (Nagai and Sheng,2008). Combining these
data, it is likely that sometime between E12 andE15, the bone marrowtakes over as the major
erythropoietic organ, although the yolk sac continues to contribute tothis process until hatching (Fig. 4).
These studies, although showing that theyolk sac remains a maFig. 4. A timeline of primitive and definitive erythropoiesis in the chick embryo. Key events of erythropoiesis andjor niche even for latesome of developmental landmarks are listed, with primitive line-related events marked by circles and definitive linerelated events by squares.
definitive erythropoiesis,

1040

G. Sheng

did not address the source of erythropoietic cells at late stages.
Similar to the situation in early definitive erythropoiesis, thesource of cells for later embryonic and post-hatching erythropoiesis remains contested. Erythropoietic cells in the bone marrow areseeded exogenously. Hematopoietic stem/progenitor cells that
do not undergoing erythropoiesis, derived from both the dorsal
aorta intraembryonically and the yolk sac extraembryonically, arein circulation at least from late E4 (McIntyre et al., 2008; Nagai andSheng, 2008). It is unclear whether they remain as dormant stem/
progenitor cells in circulation or in a transitory niche before thebone marrow niche starts to form at about E9 and becomes
receptive for seeding by circulatory stem cells at about E12 (Bruns
and Ingram, 1973; Dantschakoff, 1909; Metcalf and Moore, 1971;
Romanoff, 1960).
Other intraembryonic erythropoietic organs, including the liverand spleen, make relatively minor contributions to definitive
erythropoiesis during embryonic development. The liver, a major
erythropoietic organ in mammalian embryos, is not considered tocontribute significantly to definitive erythrocyte generation inchickens. It is noteworthy that development of chicken embryonicliver coincides with the initiation of yolk sac erythropoiesis. Largebilateral yolk sac veins, also known as omphalomesenteric veinsor vitelline veins, merge as ductus venosus before reaching thesinus venosus part of the heart. Hepatic vessels are derived fromout-branching of ductus venosus endothelium. At E3.5, a verysmall percentage of extraembryonic blood entering the heartgoes through the liver. By E7, however, all yolk sac-derived bloodneeds to pass through liver tissue as the ductus venosus breaks
down after hepatic vessel maturation. Indeed, erythropoietic cells
are detected in the liver at E7 both intravascularly andextravascularly (Haff, 1914; Karrer, 1961; Romanoff, 1960; Wongand Cavey, 1993). Erythropoiesis in the liver, however, never
becomes very active and stops by the end of E9 (Haff, 1914;
Romanoff, 1960). Similarly, the spleen serves as a minor erythropoietic organ for a few days during mid-incubation period(Romanoff, 1960; Yassine et al., 1989). The relatively minor roleof liver and spleen as embryonic erythropoietic organs in chickens, however, may not be a general phenomenon in birds, as inpasserine birds, such as starling, these two organs were reported
to participate in erythropoiesis throughout embryogenesis and fora few weeks after hatching (Romanoff, 1960).
A strong argument for yolk sac origin of hematopoietic cells
seeding the bone marrow came from parabiosed and twinned
chick embryos of opposite sex (Metcalf and Moore, 1971; Moore
and Owen, 1965; Moore and Owen, 1967), in which approximately one fourth to a half of hematopoietic cells in the bonemarrow at late embryonic stages are reciprocally derived from
opposite sexes. This hypothesis was supported by experiments inwhich yolk sac-derived cells were injected into irradiated chickembryos, showing that yolk sac cells are capable of contributing
to post-hatching bone marrow hematopoietic cell population(Lassila et al., 1978; Metcalf and Moore, 1971; Moore and Owen,
1967). Interpretation of these experiments, however, has to take
into consideration the techniques used. For instance, scoring of
mitosis at metaphase is not necessarily indicative of hematopoietic cell fate. More importantly, the timing of parabiosis (ranging
from E4 to E8) would not distinguish yolk sac-generated fromintraembryonically-generated hematopoietic cells in circulation.
The age of yolk sac donor (E7) for irradiated embryos may include

intraembryonically-generated hematopoietic cells either in circulation or temporarily taking up the yolk sac niche. Studies using
chick/quail (Beaupain et al., 1979; Dieterlen-Lievre, 1975;
Dieterlen-Lievre, 2005; Martin et al., 1978) and chick/chick (Lassila
et al., 1978; Lassila et al., 1982) chimeras presented an opposite
picture. Quail or chick embryos ranging from HH9 to HH16 weregrafted to chick yolk sacs, and relative contributions of graft andhost cells to circulating blood or bone marrow were analyzed.
Increasing percentage of donor (embryonic) cells was detected incirculation as chimeras developed and after E17 made up the
majority of cells in the bone marrow. These experiments overcame some of the problems in parabiotic/twin analyses, but werealso limited by the relatively small sample number, reliability in
identifying donor/host cells as hematopoietic cells and variationsin the age and graft size of donor embryos, all of which could
influence the interpretation. Nevertheless, the latter set of observations suggested that intraembryonically-derived hematopoietic
cells make an increasingly significant, and possibly exclusive,
contribution to the late embryonic and post-hatching definitive
erythrocyte population. It is still unclear, however, whether hematopoietic cells detected in the dorsal aorta during early embryogenesis represent the hematopoietic stem cells seeded in thebone marrow.
An additional possible source of definitive hematopoietic stem
cells, which so far has not received careful investigation, is the
blood islands around regressed primitive streak near the tail bud.
As discussed earlier, the distinction between embryonic andextraembryonic tissues is not clear-cut, especially in the posterior
part of the developing embryo. New blood islands are continuously being formed in this area even at E3 when circulation has
been well established. Whether to consider these blood islands asembryonic or extraembryonic may be a semantic issue. Molecularly, however, these blood islands can be distinguished fromthose generated earlier and located more peripherally (unpublished data) (Minko et al., 2003). It will be interesting to test
whether these hematopoietic cells may have different differentiation potentials from more conventionally-defined and yolk saclocated hematopoietic cells.

Conclusion
The chick embryo has been and remains to be a superb model
system for studying developmental hematopoiesis. It has played
important historical roles in providing descriptions and conceptual
frameworks fundamental to the field. With diverse model systems
and rich resources of molecular and genomic information, elucidation of many currently unresolved questions concerning vertebrate hematopoietic development can be anticipated in the near
future. The chick model can continue to make important contributions to this endeavor.

Acknowledgements
I would like to thank Ms. Kanako Ota and RIKEN CDB library staff forhelp in obtaining articles not accessible electronically.

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G. Sheng

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5 yr ISI Impact Factor (2009) = 3.253