Download Initiation of Hematopoiesis and Vasculogenesis in

Initiation of Hematopoiesis and Vasculogenesis in Murine Yolk Sac Explants
By James Palis, Kathleen E. McGrath, and Paul D. Kingsley
The blood islands of the visceral yolk sac (VYS) are the initial
sites of hematopoiesis in mammals. We have developed a
yolk sac explant culture system t o study the
process of blood
cell and endothelial ceil development from extraembryonic
mesoderm cells. No benzidine-positive cells or PH1-globin
mRNA expression was detected at the primitivestreak or
neural plate stage of development (E7.5). However, when
isolated €7.5 dissected tissues were cultured for 36 t o 72
hours in serum-free medium, hundreds of hemoglobin-producing cells and embryonic globin gene expression were
identified in both intact yolk sac and W S mesoderm explants. Explanted E7.5 extraembryonic mesoderm tissues
thus recapitulate in vivo primitiveerythropoiesis and do not
require the presence of a vascular network or the
W S endoderm. Yolk sac blood islands also contain endothelial cells
that arise by vasculogenesis and express flk-l. We detected
flk-l mRNA as early as the primitivestreak stage of mouse
embryogenesis. Culture of embryo proper and intact VYS
explants, whichcontain both mesoderm and endoderm
cells, produced capillary networks and expressed flk-l. In
contrast, vascular networks were not
seen when VYS mesoderm was cultured
alone, although flk-l expression was similar to thatof intact VYS explants. The addition of vascular
endothelial growth factor t o W S mesoderm explants did
not induce vascular network formation. These results suggest that the VVS endoderm or its extracellular matrix is
necessaryfor thecoalescence of developing endothelial
cells
into capillary networks.
0 1995 by The American Society of Hematology.
Y
hemoglobin (Hb) until E14.5, when they contain a mean of
100 pg Hb per ceL5 These nucleated cells are lost from the
circulation between E15.5 and E16.5, and hematopoiesis is
then sustained by fetal liver-derived non-nucleated erythrocytes, containing a mean of 28 pg Hb per ceL5 Before birth,
erythropoiesis begins to shift to the bone marrow, where it
remains throughout postnatal life.
The in situ development of endothelial cells from precursor mesoderm cells (angioblasts) is known as vasculogenesis. Vasculogenesis shares the processes of endothelial cell
proliferation andlumen formation with angiogenesis, the
formation of vascular networks by endothelial cells sprouting
from existing vessels. The vascular network of the yolk sac
blood islands and the intraembryonic dorsal aortae both arise
by vasculogenesis. Between 7 and 10 somite stages (E9.0),
the primitive vascular network of the VYS connects with its
embryonic counterpart, and the yolksac blood cells circulate
into the embryo proper as the heart begins to beat.‘ A receptor for vascular endothelial growth factor (VEGF), flk-l, is
expressed exclusively in endothelial cells of both the yolk
sac and the embryo proper, whether they arise by vasculogenesis or angi~genesis.~.’
Expression of flk-l has been detected as early as E7.0, at the primitive streak stage of development.x
We have developed a yolk sac explant system to investigate the process by which mammalian blood cells and endothelial cells first arise from extraembryonic mesoderm cells.
The morphologic and molecular characterization of this explant system is presented here. We conclude that (1) explanted
E7.5
extraembryonic mesoderm independently
recapitulates in vivo primitive erythropoiesis, ( 2 ) the development of red blood cells from extraembryonic mesoderm
cells does not require the presence of a vascular network,
and (3) the visceral endoderm or its extracellular matrix
facilitates the development of the yolk sac blood island vascular network.
OLK SAC blood islands are the first morphologic evidence of hematopoietic development during mammalian embryogenesis. The yolk sac forms during the complex
process of gastrulation, which begins at E6.5 in the mouse.
Mesoderm cells, destined for extraembryonic sites, exit the
posterior primitive streak and subdivide the embryo into
three separate cavities by the neural plate stage (E7.5). The
central cavity, the exocoelom, becomes completely lined
with mesoderm cells. These mesoderm cells lie adjacent to
the embryonic ectoderm to foml the amnion, the extraembryonic ectoderm to form the chorion, and the visceral endoderm to form the visceral yolk sac (VYS). While the visceral
endoderm serves nutritive and metabolic functions,’ the VYS
mesoderm gives rise to the first embryonic blood cells within
a rich endothelial network.
More specifically, between E7 and E7.5, mesoderm cells
in the VYS begin to proliferate, forming the mesodermal cell
masses.*These morphologically indistinguishable mesoderm
cells are the precursors of blood islands (Fig l ) . By the
early somite stages (E8.5), the central cells are accumulating
hemoglobin and losing their intracellular attachments, while
the outer cells are flattening to form an endothelial network.’
Primitive hematopoiesis in murine yolk sac blood islands
consists almost exclusively of red blood cells and macrophages3 Erythroid-specific gene expression (embryonic globins) has been detected as early as the headfold stage of
de~elopment.~
Primitive erythroblasts continue to synthesize
From theDepartment of Pediatrics, University of Rochester Medical Center, Rochester, NY.
Submitted November IO, 1994; accepted February 16, 1995.
Supported by National lnstitutes of Health Grant No.
R29HLA5573 (J.P.)and American Cancer Society Institutional Research Grant No. 1836 (P.D.K.).
Address reprint requests ro James Palis, MD, Universiy of Rochester Medical Center, Department of Pediatrics, Box 777, 601 Elmwood Ave, Rochester, N Y 14642.
The publication costs of this arlicle were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 sofeiy to
indicate rhis fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8601-0004$3.00/0
156
MATERIALS AND METHODS
Embryonic tissue isolation. CD-I ;ICR mice (Charles River Laboratories, Wilmington, MA) were maintained in a 12-hour lighVl2hour dark cycle. Vaginal plugs were checked the morning after
natural overnight matings (E0.3). At specified times, micewere
killed by cervical dislocation, and the uteri were removed from the
Blood, Vol 86,No 1 (July l ) , 1995: pp 156-163
INITIATION OF HEMATOPOIESIS AND VASCULOGENESIS
L
Fig 1. Histologic sections ofdeveloping visceral yolk sacs at E7.5,
E8.5, and E9.5 of murine development. At E7.5, a neural plate stage
embryo is shown at thejunction between embryo proper and visceral
yolk sac. The mesoderm (Me) cells are apposed to embryonic ectoderm cells(Ec) forming the amnion, and apposed to the visceral
endoderm (En) to form the yolk sac wall. At E8.5, the proliferation
of VYS mesoderm cells into early blood islands isevident. The mesothelium lining the exocoelomic cavity is now a distinct layer of Eells
(arrow). The W S endoderm consists of a single layer of cells with
an apical-basal polarity and a brush borderfacing the yolk saccavity.
At E9.5, the blood islands are now filled with maturing erythroid cells
surrounded by a vascular network of endothelial cells (arrow). The
erythroid cells becomemore basophilic asthey accumulatehemoglobin. Photographs were taken of 1.5-pm sections stained with methylene bluelazure II and are all at the same magnification.
peritoneum. The embryos were dissected in ice-cold Pb-l solution9
withno. 5 watchmakers forceps (Dumoxal; Electron Microscopy
Science, Ft Washington, PA), using a Wild M5 stereomicroscope
(Leitz, Rockleigh, NJ) and epiillumination. For initial blood island
histologic studies, embryos from E7.5, E8.5, and E9.5 were fixed in
2.5% gluteraldehyde in cacodylate buffer and embedded in Immunobed plastic (Polysciences, Warrington, PA). Embryos were sec-
157
tioned at 1.5-pm thickness, stained with methylene bluelazure 11,
and photographed with a Nikon Optiphot microscope (Melville, NY).
To obtain embryonic tissues for explant culture, embryos were
dissected free of decidual tissues, Reichert's membrane, and all vestiges of maternal blood cells. Embryos were staged according to the
morphologic criteria of Downs and Davies."' Late primitive streak
and neural plate stage embryos (E7.5) were transferred to fresh icecold Pb-l and further dissected, as shown schematically in Fig 2.
Embryos were trisected into embryo proper, intact VYS, and ectoplacental cone tissues. The VYS was separated into VYS mesoderm
and VYS endoderm germ layers by incubation in 2.5% pancreasin,
0.5% trypsin (Sigma, St Louis, MO) for 5 to 10 minutes at 4"C,
followed by gentle pipetting." The VYS endoderm is distinctly more
refractile than the VYS mesoderm, ensuring that completely separated tissues could be routinely obtained. RNA isolated from these
tissues showed no detectable crosscontamination when probed with
VYS mesoderm- and VYS endoderm-specific markersN2For some
explant experiments, the embryo proper was transected into distal
and proximal halves before culture (Fig 2).
To examine the morphology of primitive erythroblasts, cultured
explants and E9.5 yolk sac tissues were physically dispersed in 1 0 0
pL of Excell-300 and spun onto cytocentrifuge slides (Shandon,
Pittsburgh, PA). The slides were stained with Wright's in a Midas
I1 automated slide stainer (E.M. Diagnostic Systems, Gibbstown,
NJ).
Explanr culrure conditions. Dissected tissues were immediately
placed in a 96-well plate of tissue culture-treated plastic (Becton
Dickenson Labware, Lincoln Park, NJ) containing 1 0 0 pL of either
Dulbecco's modified Eagle's medium (DMEM) with 10%fetal calf
serum (FCS; Hyclone Laboratories, Logan, UT) or Excell-300 medium (JRH Biosciences, Lenexa, KS). Media were supplemented
with 100pglmL streptomycin and 100 UlmL penicillin andpreequilibrated at 37°C in anatmosphere of 5% CO,, 95% air for several
hours before use. Explanted tissues were individually cultured for
24 to 72 hours. For some experiments, wells were coated with polyL-lysine (Collaborative Research Inc, Bedford, MA). Humanrecombinant VEGF (V-1833; Sigma) was dissolved in sterile Pbl and
diluted in medium before use.
Benzidine sraining. Benzidine dihydrochloride (Sigma) staining
was used to identify hemoglobin-containing cells." Five microliters
of 30% hydrogen peroxide (Sigma) was added to 1 mL of benzidine
immediately before use. Benzidine, hydrogen peroxide (100 pL) was
added to the cultures, and the explants were examined with a Nikon
THS inverted microscope 5 to 15 minutes later. Benzidine-positive
cells stained dark blue, while benzidine-negative cells were light
yellow.
RNase protection. Total RNA was prepared from E7.5 embryos
(day 0) and from tissue explants cultured for 32 to 48 hours. Tissues
were dissolved in 8 moln guanidine hydrochloride, 0.3 molk
NaOAc (pH 6) brought to 1% sarkosyl. After passing through a 26gauge needle IO times, debris was removed by centrifugation at
14,000g for 10 minutes. The supernatant and 3 pg of carrier Escherichia coli RNA were. precipitated with2.5 v01 ethanol. Three to
five explant equivalents of RNA were used per sample time point,
and the pooled RNA was divided among the different probes. The
PHI-globin cDNA clone was provided byA. McMahon (Harvard
University, Boston, MA).I4 The flk-l cDNA was provided by J.
Rossant (Mt Sinai Hospital, Toronto, Canada).' Mal-tubulin was
used as a control for RNA quantity. We have found by Northern
blots that a-tubulin expression in VYS mesoderm, VYS endoderm,
and embryo proper tissues correlates with total RNA levels in these
tissues at E7.5." Using standard techniques," 32P-deoxycytidinetriphosphate (dCTP)-labeled single-stranded antisense probes were
prepared to a specific activity of 1.3 X IO9 cpm/pg. The probes were
gel-purified on a 4% denaturing acrylamide gel and used in standard
PALIS,McGRATH,
158
AND KINGSLEY
'aws
MESODERM
\
&f
PROXIMAL
EMBRYO
PROPER
DISTAL.
EMBRYO
PROPER
RNaseassays,I6with 2 X 105 cpm of probe per reaction.Yeast
tRNA (10 pg) was added to each tube to normalize RNA amounts
before digestion with RNase A (Boehringer Mannheim, Indianapolis,
IN) and RNase T1 (GIBCO BRL, Gaithersburg,MD). The protected
fragments weresized on a 6%polyacrylamide gel, and autoradiography was performed for 1 to 8 days with two intensifying screens.
RESULTS
Histology of normal blood island development. In initial
experiments, the development of yolk sac blood islands in
CD-l mice was examined histologically. Because of the
large variation in early postimplantation inter- and intralitter
development," we examined the histology of yolk sac tissues
at specific developmental stages. Proliferating VYS mesoderm cells are morphologically indistinguishable at the neural plate stage of development (Fig 1, E7.5). Just 24 hours
later, blood islands containing early erythroid cells, as well
as a mesothelial lining, were evident by early somite stages
(Fig 1, E8.5). Endothelial cells surrounding differentiating
basophilic erythroid cells were easily identifiable byE9.5
(Fig 1, E9.5). During these early postimplantation times, the
murine VYS endoderm remains as a single layer of cells
with distinct apical-basal polarity and a brush border facing
the yolk sac cavity (Fig 1).
Morphology of explant cultures. To begin investigating
the processes of both blood cell and endothelial cell development, embryonic tissues were isolated from late primitive
streak and neural plate stage embryos, as shown schematically in Fig 2. Embryo proper, embryo proper-proximal, embryo proper-distal, intact yolk sac, VYS mesoderm, and VYS
endoderm tissues were individually cultured for 24 to 72
hours in vitro and examined in phase with aninverted microscope.
In embryo proper explants cultured in DMEM with 10%
FCS, mesoderm cells attached both to tissue culture-treated
plastic and to poly-L-lysine-coated wells, spreading out
from the explant. After 2 days in culture, the embryo proper
tissue also contained a disorganized vascular network, and
over half of the cultures contained rhythmically contracting
Fig 2. Diagram of the dissection strategy to isolate specifictissues from the neural plate stage
mouse embryo lE7.51. At this stage, the embryo contains three cavities: the amniotic cavity lac), the exocoelom (exo), and the ectoplacental cavity (ecl. The
exocoelomis completely lined byextraembryonic
mesoderm cells, which form the amnion with embryonic ectoderm cells, the chorion with extraembryonic ectoderm cells, and the VYS with visceral endoderm cells. The embryo was dissected from the
deciduum and trisected into embryoproper IEP),
VYS, and ectoplacental cone IEC) tissue fractions.
The embryo proper was transected into proximal
and distal halves, and the W S was enzymatically
separated into W S mesoderm and W S endoderm
tissues. IO) Endoderm;11. mesoderm; (kW ectoderm
(epiblast); (B) extraembryonic ectoderm.
myocardial tissue. In contrast to the mesoderm cells, the
endoderm cells showed a slow and progressive sloughing.
The use of a serum-free medium (Excell-300) produced similar overall results, except that there were fewer mesoderm
cells attached to the culture well and the explanted tissues
retained a more three-dimensional structure within the medium.
In cultures of intact VYS in DMEM with 10% FCS, the
inner mesoderm cells attached equally well to the tissue
culture-treated plastic and to the poly-L-lysine-coated plastic. By 48 hours of culture, the attached mesoderm cells had
spread over the culture dish away from the explanted tissue.
Many of the VYS cultures contained organizing vascular
channels, as seen in Fig 3A. There was progressive sloughing
of the outer endodermal cells over the 48- to 72-hour culture
period, which was more pronounced with the use of Excell300.
To determine if the culture of VYS mesoderm tissue alone
could lead to blood island development, the VYS was separated into mesoderm and endoderm germ layers, and these
tissues were cultured separately for 24 to 72 hours. VYS
mesoderm explants produced hundreds of round nucleated
cells overlying fibroblast-like cells attached to the poly-Llysine-coated or treated plastic (Fig 3B). These round cells
were often not attached to either each other or the fibroblast
stroma by the end of the 48- to 72-hour culture. The attached
cells were morphologically similar to the fibroblast-like cells
seen in both the embryo proper and the intact yolk sac explants. The use of the serum-free Excell-300 medium resulted in fewer attached mesoderm cells, but there was no
qualitative difference in the number of rounded nucleated
cells overlying the fibroblasts. However, no vascular channels were seen in any of the VYS mesoderm explants (n =
38), whether serum-containing or serum-free medium conditions were used. Culture of the VYS endoderm tissue alone
resulted in loose clumps of nonadherent cells that became
progressively more granular over the 48- to 72-hour culture
period. By 72 hours of culture (in serum-free medium), the
INITIATION OF HEMATOPOIESISANDVASCULOGENESIS
159
B
F
Fig 3. Photographs of E7.5 explants cultured for 48 hours in DMEM with 10% FCS at 37°C in 5% CO, (K through
andphotogra&s of
primitive erythroblasts cytocentrifugedfrom intact W S (E)and W S mesoderm (G) explants cultured in Excell-300 for 48 hours and from E9.5
embryos (F). (A) W S explant with a forming capillary network. (B) W S mesoderm explant with hundreds of rounded cells atop a mesodenn
stroma. (C) W S explant stained with benzidine dihydrochloride. There are several hundred dark blue benzidine-positive cells within the
explanted tissue. Scattered benzidine-positivecells are also overlying attached mesodermcdls, while others appear t o be contained within
forming vascular networks (arrow). (D) W S mesoderm explant stained with benzidine dihydrochloride. Hundreds of dark blue benzidine
positive cells arelying over an attached stroma. (E)A representative primitive erythroblast from explanted W S tissue. (F) Primitive erythroblasts from an E9.5 CD-l embryo. (G) A primitive erythroblast from explanted W S mesoderm tissue.
PALIS.McGRATH,ANDKINGSLEY
160
Table 1. Benzidine Staining of E7.5 Embryos at the Start of Culture
(Day 01 and After 36 t o 72 Hours in Culture (Day 2-31
No. Benzidine-Positive Cultures1
Total Cultures
Day 0 (E7.5)
Day 2-3Cultures
2
0120
ND
ND
413 5
417
019
2
0120
ND
ND
412 4
23/23
0131
Embryo proper
Embryo proper
Embryo proper-proximal
Embryo proper-distal
Yolk sac
Intact yolk sac
W S mesoderm
W S endoderm
Embryos and explanted tissues were stained with benzidine dihydrochloride and hydrogen peroxide and examined in phase with an
inverted microscope.
Abbreviation: ND. not determined.
viability of endoderm explants as measured by trypan blue
exclusion was less than 10%.
Erythropoiesis: Benzidine stain. E7.5 dissected embryos
and explants cultured for 32 to 72 hours were stained with
benzidine to identify hemoglobin-containing cells. The results are summarized in Table 1. There were no benzidinepositive cells inanyof the late primitive streak or neural
plate stage tissue fractions at the start of the explant incubation. However, after 2 days in culture, there were always
several hundred benzidine-positive cells in the intact VYS
and VYS mesoderm fractions (Fig 3C and D). In the intact
VYS cultures, the benzidine-positive cells were contained
mostly within the explanted VYS tissue. However, scattered
benzidine-positive cells were also seen overlying attached
mesoderm cells, as well as within forming vascular networks
(Fig 3C, arrow). In the VYS mesoderm cultures, the benzidine-positive cells consisted of the rounded cells overlying
the flattened cells attached to the plastic (Fig 3D). These
cells from both the intact VYS and the VYS mesoderm
explants cultured for 48 hours in Excell-300 had a high
nuclear-to-cytoplasmic ratio, a basophilic cytoplasm, and a
condensing chromatin pattern without nucleoli (Fig 3E and
G). Their morphology was similar to primitive erythroblasts
from E9.5 yolk sac tissues (Fig 3F).
Culture of the embryo proper always revealed less, than
150 benzidine-positive cells. The embryo proper was also
transected into proximal and distal tissue fractions (Fig 2)
that were individually cultured. The distal embryo proper
produced no benzidine-positive cells, while the proximal embryo proper produced a small number (20 to 100) of benzidine-positive cells in more than half of the cultures (Table
1). No benzidine-positive cells were evident when the VYS
endoderm was cultured alone (n = 31).
Gene expression in neural plate stage embryos and cultured E7.5 explants. RNase protection assays were performed with pooled RNA from tissue explants to assess the
expression of embryonic PHI-globin (erythroid differentiation), flk-I (endothelial cell development), and cy-tubulin
(RNA control). No PHI-globin mRNA was detected at the
start of the explant cultures, ie, atE7.5 (primitive streak
stage; Fig 4, lanes 2 and 3). However, after 32 hours in
culture, PH1-globin message was evident in both intact VYS
and VYSmesoderm explant cultures (Fig 4, lanes 5 and
6). The amount of globin mRNA accumulation in the VYS
mesoderm explants was similar to that in the intact VYS
explants. Some globin mRNA was also detected in embryo
proper cultures at 32 hours of culture, but was always less
than in the VYS cultures. This result is consistent with the
small number of benzidine-positive cells observed in many
of the embryo proper explants. Two repeat experiments with
PHI-globin produced similar results.
The mRNA of flk-l is expressed by developing endothelial cells and in presumptive angioblasts as early as the primitive streak stage of development.8 Therefore, we examined
flk-l expression in E7.5 explanted tissues. At the start of the
culture (primitive streak stage), there were higher levels of
flk-l message in the forming yolk sac compared with the
embryo proper (Fig 4,lanes 2 and 3). By 32 hours of culture,
embryo proper, intactVYS,andVYSmesoderm
explants
expressed similar amounts of flk-l mRNA (Fig 4, lanes 4
through 6).
Although intact VYS and VYS mesodermcultures express
flk-l mRNA, VYS mesoderm explants do not form vascular
channels. To determine if VEGF, the ligand for flk-l, is
capable of inducing vascular network formation in VYS
0n
hr.
t
32 hr.
-
EP YS EP YS Me
flk- 1
m
Maltubulin
Fig 4. RNase protection assay with PH1-globin, flk-l, and Maltubulin at 0 hours and 32 hours of culture. Tissues were explanted
at the neural plate stage into embryo proper, intact yolk sac, VYS
mesoderm, and W S endoderm fractions and cultured at 37°C. 5%
CO2 in Excell 300 medium. Time 0 consisted of five primitivestreak
stage embryo proper (EP, lane 2) or VYS (YS, lane 3) tissues. For the
32-hour timepoint (lanes 4, 5, and 61, five explant cultures were
pooled. Lane 4 contains embryo proper
(EPI cultures, lane 5 contains
intact VYS (YSI, and lane 6 contains W S mesoderm (Me) cultured
alone. Lane 1 contains 10 p g of yeast tRNA (tlas a negative control:
1046 of theRNA was hybridizedwith thepH1-globin probe, 10% was
hybridized with a-tubulin probes, and 80% was hybridizedwith the
flk-l probe.
INITIATION OF HEMATOPOIESIS AND VASCULOGENESIS
mesoderm explants, exogenous recombinant human VEGF
was added at final concentrations of 1,3, and l 0 ng/mL. No
evidence of vascular channels was observed whether serum
was present or absent (n = 15 total). The addition of VEGF
at the same concentrations to intact VYS explants (n = 12)
did not increase vascular network formation compared with
control explants (n = 6 ) .
DISCUSSION
Gastrulation in mammals gives rise to both embryonic
and extraembryonic mesoderm cells that establish the basic
body plan and form the visceral yolk sac, respectively. The
first embryonic hematopoietic cells and endothelial cells
arise in blood islands, which develop where extraembryonic
mesoderm is apposed to visceral endoderm. The size and
location of the gastrulating mammalian embryo makes observation and experimental manipulation problematic. Murine
teratocarcinoma cells and embryonic stem cells have served
as in vitro models of early embryonic development and
have been used to specifically study embryonic hematopoiesis17-19 and vasculogenesis.” These systems produce cystic
embryoid bodies, disorganized masses of cells, which make
the study of specific germ layer interactions impossible. We
have developed an explant culture system to study the onset
of hematopoiesis and vasculogenesis in the mammalian embryo. The dissection and separation of specific germ layers
at defined developmental stages permit the investigation of
cell-matrix interactions;’ tissue-tissue interactions,22and cell
Despite the small number of cells in these explanted
tissues, gene expression can be investigated by RNase protection assays as well as the more sensitive, but less quantitative, reverse transcription-polymerase chain reaction (RTPCR) methods.
The first morphologic evidence of yolk sac blood island
formation in CD-l mice is at the neural plate stage of development, when extraembryonic mesoderm cells begin to proliferate to form mesodermal cell masses (E7.5). We did not
detect either benzidine-positive cells or PH1-globin mRNA
accumulation in late primitive streak or neural plate stage
embryos. These results are consistent with the lack of morphologically identifiable erythroblasts at these stages of development (Fig l , E7.5). However, when neural plate stage
yolk sac tissues were explanted, hundreds of erythroid cells
developed over 32 to 72 hours in culture. That the benzidinepositive cells arise from the explanted tissues is proven by
three observations. First, no benzidine-positive cells were
found in the E7.5 embryos (Table 1). Second, the benzidinepositive cells that arise in the explanted VYS mesodermcontaining tissues are nucleated, while maternal (definitive)
red blood cells are not. Third, RNase protection assays detect
the presence of embryonic (PHI) globin mRNA, which is
not expressed during adult erythrop~iesis.’~
Although it is generally accepted that blood cells arise
from extraembryonic mesoderm cells, it has also been postulated that yolk sac blood islands originate from VYS endoderm ~ells.’~*~’
In our experiments, the onset of embryonic
erythropoiesis, evident both by benzidine staining and globin
mRNA synthesis, occurs in vitro when VYS mesoderm, but
not VYS endoderm, is cultured alone. This result supports
161
the hypothesis that blood cells originate from epiblast cells
migrating through the posterior primitive streak to form the
VYS mesoderm.
E7.5 VYS mesoderm explants give rise to primitive erythroblasts in a serum-free medium containing no hematopoietic
growth factors. As the VYS mesoderm is explanted intact,
hematopoietic growth factors necessary for primitive erythropoiesis may be provided by the VYS mesoderm microenvironment. The morphology of the VYS mesoderm explants
suggests that an intact vascular network is not necessary for
the development of blood cells and that some extraembryonic mesoderm cells serve the function of a hematopoietic
stroma. It is not known if this stroma is composed of mesothelial cells or developing endothelial cells (see below). The
possibility that pluripotent mesoderm cells might give rise
to hematopoietic cells, endothelial cells, and hematopoietic
stroma has received recent support.28Our explant experiments suggest that the visceral endoderm is not necessary
for the development of mammalian blood cells-results in
agreement with studies in the chick.22If the VYS endoderm
has an inductive role inblood cell development, it must
occur before the neural plate stage of development. These
experiments do not exclude a permissive effect of the visceral endoderm on hematop~iesis.’~
However, no evidence
of a permissive effect was detected in these explants experiments, as intact VYS explants did not produce more PH1globin mRNA compared with VYSmesoderm cultures alone
(Fig 4).
Explants of the embryo proper often produced a small
number of benzidine-positive cells and small amounts of
BH1-globin mRNA despite careful dissections specifically
avoiding contamination with yolk sac tissues and maternal
blood cells. There are at least two possible explanations
that could account for this finding. First, gastrulation is not
complete until well into early somite
Therefore,
mesoderm cells destined for the yolk sac and fated to become
blood cells may still be egressing from the posterior primitive streak atE7.5. Second, an intraembryonic source of
hematopoietic stem cells has been p~stulated,~’.~’
as blood
island-like clusters have been noted in the ventral aorta at
the level of the mesonephroi at midgestation in many species,
including the
Intraembryonic hematopoietic stem
cells might arise from precursor mesoderm cells that migrate
through the primitive streak to reach paraaortic sites. These
hematopoietic precursors would thus be located within the
embryo proper of the neural plate stage embryo. This explanation is consistent with the murine fate map, where the
region of the epiblast that gives rise to blood cells is adjacent
to cardiac anlage.35
The earliest known expressed molecular marker of endothelial cells and presumptive angioblasts is flk-l. Higher
levels of flk-l mRNA were detected in the forming yolk sac
compared with the embryo proper of the primitive streak
embryo (Fig 4,lanes 2 and 3). This result is consistent with
the formation of the yolk sac vasculature before that of the
intraembryonic dorsal aortae. We detected the formation of
vascular networks in embryo proper and intact VYS explants, but not in VYSmesoderm explants. Studies, primarily
in the avian system, have shown that organs derived from
162
endoderm, such as the VYS and the liver, are vascularized
by in situ mesoderm cells, while ectoderm-derived organs,
such as thebrain, are vascularized by angiogenesis? Explant
experiments performed withthe chick yolk sacz2 and during
quail cardiac tube formationz4 also suggest that
initial vascular network development requires a permissive or inductive
influence from endoderm cells.
Our explant datain the mouse support theconclusion that
thevisceral endoderm isnecessary for capillarynetwork
formation. It is possible that theVYS endoderm cells syntheas fibroblast growthfactor
size or transportfactors,such
(FGF) or VEGF, that induce or permitthedevelopment
and proliferation of endothelial cells and the formation of
vascular networks. It has been suggested that the VYS endoderm transports exogenously synthesizedbasic FGF from
the yolk sac space,” and FGF has beenshown to induce
endothelialcelldevelopment indissociated quailepiblast
cells.’* VEGF message is expressed by rat decidual cells and
gianttrophoblast cells at the neuralplate stage of rodent
development.”VEGF is a highly conserved protein, and
human VEGF is knowntoinduce rodentendothelial cell
proliferation.“’ However, the additionof recombinant human
VEGF did not induce vascular network formation in these
murine VYS mesoderm explants.
The lackof vascular network formation inVYS mesoderm
explantsmayalsobeduetothe
disruption of mesoderm
interactions with the extracellular matrix caused by either the
p h y ~ i c a lor~ the
~ . ~enzymatic
~
(this study) separation process.
These interactions, particularly with laminin and fibronectin,
may be necessary for vascular network f ~ r m a t i o n . When
~’
fibronectin or one of its receptors, a5 integrin, is absent from
theembryo,the yolk sacmesodermandendodermgerm
layers separate, andyolk sac vasculogenesis is disrupted.42,43
Interestingly, primitive blood cell development appears to
occur normally in both of these mutant mice, suggesting that
primitive erythropoiesis does not require the formation of
an intact vascular network.
The explant system described here allows
the study of the
molecules involved in the initial development of mammalian
yolk sac blood cells and vascular networks. The addition of
exogenous factorspotentiallyinvolved
in theseprocesses
can be studied. Furthermore, genes endogenously expressed
in these explanted tissues can be blocked by antisense oligonucleotides or neutralizing antibodies, and the resultant effect on both erythropoiesis and vasculogenesis can be investigated.
PALIS,MCGRATH,
AND KINGSLEY
2. Haar JL.AckermanCA:Aphaseandelectronmicroscopic
study of vasculogenesis and erythropoiesis
in the yolk sac of the
mouse. Anat Rec 170:199, 1971
3. MetcalfD,Moore M: Haemopoieticcells, in Neuberger A,
Tatum EL (eds): Frontiers in Biology, v01 24. London, UK, NorthHolland Publishing CO, 1971
4. Leder A, Kuo A, Shen M, Leder P: In situ hybridization reveals
co-expression of embryonic and adult (Y globin genes in the earliest
murine erythrocyte progenitors. Development l 16: I U4 I . 1992
5 . Steiner R, Vogel H: On the kinetics of erythroid cell differentiation in fetal mice. J Cell Physiol 81:323, 1973
6. Kaufman MH: The Atlas of Mouse Development. New York,
NY, Academic Press, 1992
S, Schnurch H, MartinezR,
7.MillauerB,Wizigmann-Voos
Moller NPH, Risau W, Ullrich A: High affinity VEGF binding and
developmental expression suggest flk-l as a major regulator of vasculogenesis and angiogenesis. Cell 72:835, 1993
8. Yamaguchi TP, Dumont DJ, Conion RA, Breitman ML, Rossant J: flk- I , an flt-related tyrosine kinase, is an early marker for
endothelial cell precursors. Development 1 I8:489, I993
9. Monk M: MammalianDevelopment:A
Practical Approach.
Oxford. UK, IRL Press, 1987
IO. Downs KM, Davies T: Staging
of gastrulating mouse embryos
by morphological landmarks in the dissecting microscope. Development I18:1255,1993
I I . Levak-SvajgerB,Svajger A, Screb N: Separation of germ
layers in presomite rat embryos. Experientia 25:131 I , 1969
12.PalisJ,
Kingsley PD: Differentialgeneexpressionduring
early murine yolk sac development. Mol Reprod Dev (in press)
13. Hicks DC, Ohlsson-Wilhelm BM, Farley BA, Kosciolek BA,
Rowley PT: K562 cell erythroid differentiation: Requirement for a
factor in fetal bovine serum. Exp Hematol
13:273, l985
14. Wilkinson DC, Bailes JA, Champion
JE, McMahon AP: A
molecular analysis of mouse development from 8 to I O days post
coitum detects changes only in embryonic globin expression. Development 99:493, 1987
15. Melton DA, Krieg PA, Rebagliati
MR. Maniatis T, Zinn K,
Green MR: Efficient in vitro synthesis of biologically active RNA
and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035, 1984
16. AusubelFM,BrentR,KingstonRE,MooreDD,Seidman
JG. Smith JA, Struhl
K: CurrentProtocols in MolecularBiology.
New York, NY, John Wiley & Sons, 1994
17. Martin G, Evans M: Differentiation of clonal lines of teratocarcinoma cells: Formation of embryoid bodies in vitro. Proc Natl
Acad Sci U S A 72:1441, 1975
18. DoetschmanTC,Eistetter H, KatzM,Schmidt W, Kemler
R: The in vitro development of blastocyst-derived embryonic stem
cell lines: Formation of visceral yolk sac, blood islands and myocardium. J Emb Exp Morph 87:27, 1985
19. Keller G, Kennedy M, Papayannopoulou T, Wiles MV: Hematopoietic commitment during embryonic stem cell differentiation
in culture. M o l Cell Biol 13:473, 1993
ACKNOWLEDGMENT
20. Wang R, Clark R, Bautch VL: Embryonic stem cell-derived
We thank Janet Rossant for initial guidance in embryo dissections.
cystic embryoid bodies form vascular channels:
An in vitro model
We also thank David Korones and Frank Pendola for technical
assisof blood vessel development. Development 114303, 1992
tance in initialexplantexperiments,LouiseSilver-Morseforher
2 1 . Burdsal CA, Damsky CH, Pedersen RA: The role of E-cadfor assistancewith
expertiseinanimalhusbandry,JudyMcGann
herin and integrins in mesoderm differentiation and migration at the
dissections, and David Penney and Kathleen Maltby
of the Cancer
mammalian primitive streak. Development I I8:829, 1993
Center Experimental Pathology and Ultrastructure Facility for assis22. Wilt FH: Erythropoiesis in thechickembryo:Therole
of
tance with the preparation of histologic sections and figures.
endoderm.Science147:1588, 1965
23. Dieterlen-Lievre F, Pardanaud L, Yassine F. Cormier F: Early
REFERENCES
haemopoietic stem cells in the avian embryo. J Cell Sci 10:29, 1988
24. Antin PB, Taylor RC, Yatskievych T: Precardiac mesoderm
l , JollieWP:Development,morphology,andfunctionofthe
is specified during gastrulation in quail. Dev Dyn 200:144, 1994
yolk-sac placenta of laboratory rodents. Teratology 41:361, 1990
INITIATION OF HEMATOPOIESIS AND VASCULOGENESIS
25. Whitelaw E, Tsai S-F, Hogben P, Orkin SH: Regulated expression of globin chains and the erythroid transcription factor
GATA-I during erythropoiesis in the developing mouse. Mol Cell
Biol 10:6596, 1990
26. Yoffey JM: The stem cell problem in the fetus. Isr J Med Sci
7:825, 1971
27. Takashina T: Haemopoiesis in the humanyolk sac. J Anat
151:125, 1987
28. Huang S, Terstappen LWMM: Formation of haematopoietic
microenvironment and haematopoietic stem cells from single human
bone marrow stem cells. Nature 360:745, 1992
29. Miura Y, Wilt FH: Tissue interaction and the formation of
the first erythroblasts of the chick embryo. Dev Biol 19:201, 1969
30. Hogan B, Costantini F, LacyE: Manipulating the Mouse
Embryo: A Laboratory Manual. Cold Spring Harbor, NY, Cold
Spring Harbor Laboratory, 1986
31. Godin IE, Garcia-Porrero JA, Coutinho A, Dieterlen-Lievre
F, Marcos MAR: Para-aortic splanchnopleura from early mouse embryos contains Bla cell progenitors. Nature 364:67, 1993
32. Medvinsky AL, Samoylina NL, Muller AM, Dzierzak EA:
An early pre-liver intra-embryonic source of CFU-S in the developing mouse. Nature 364:64, 1993
33. Jordan HE: Evidence of hemogenic capacity of endothelium.
Anat Rec 10:417, 1916
34. Smith RA, Glomski CA: “Hemogenic endothelium” of the
embryonic aorta: Does it exist? Dev Comp Immunol 6:359, 1982
35. Lawson KA, Meneses JJ, Pedersen RA: Clonal analysis of
epiblast fate during germ layer formation in the mouse embryo.
Development 113:891, 1991
163
36. Vandenbunder B, Pardenaud L, Jaffredo T, Mirabel MA,
Stehelin D: Complementary patterns of expression of c-ets 1, cmyb and c-myc in the blood forming system of the chick embryo.
Development 106:265, 1989
37. Yasuda Y, Nishi N, Takahashi JA, Konishi H, Ohara I, Fujita
H, Obta M, Itoh N, Hatanaka M, Tanimura T: Induction of avascular
yolk sac due toreduction of basic fibroblast growth factor by retinoic
acid in mice. Dev Biol 150:397, 1992
38. Flamme I, Risau W: Induction of vasculogenesis and hematopoiesis in vitro. Development 116:435, 1992
39. Jakeman LB, Armanini M, Philips HS, Ferrara N: Developmental expression of binding sites and messenger ribonucleic acid
for vascular endothelial growth factorsuggests a role for this
protein in vasculogenesis and angiogenesis.
Endocrinology
1332348, 1993
40. Yamane A, Seetharam L, Yamaguchi S, Gotoh N, Takahashi
T, Neufeld G, Shibuya M: A new communication system between
hepatocytes and sinusoidal endothelial cells in liver through vascular
endothelial growth factor andFlt tyrosine kinase receptor family
(Flt-l and KDR/Flk-1). Oncogene 9:2683, 1994
41. Ingber DE, Folkman J: How does extracellular matrix control
capillary morphogenesis? Cell 58:803, 1989
42. George EL, Georges-Labouesse EN, Patel-King RS, Rayburn
H, Hynes RO: Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development
119:1079, 1993
43. Yang RT, Rayburn H, Hynes RO: Embryonic mesoderm defects in a5 integrin-deficient mice. Development 119:1093, 1993