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Development of Day-8 Colony-Forming Unit-Spleen Hematopoietic
Progenitors During Early Murine Embryogenesis: Spatial and
Temporal Mapping
By Alexander L. Medvinsky, Olga 1. Gan, Maria L. Sernenova, and Nina L. Sarnoylina
The ontogeny of the hematopoietic system in mammalian
embryos occurs during theyolk sac (YS) and the fetal liver
(FL) stages. Events leading to theestablishment of hematopoiesis in the FL remain obscure. The appearanceof colonyforming units-spleen (CFU-S) in the FL is preceded by a gradual increase of CFU-S in the YS and a more rapid increase
in the AGM region (area comprising dorsal aorta, gonads,
and mesonephros) during day 10 of development (Medvinsky et al. Nature 364:64, 1993). By this time, the AGM CFUS attain a high frequency equivalent t o that found in the
adult bone marrow. The analogous area gives rise t o adult
hematopoiesis in amphibians and probably in birds. We
present here a more complete pictureof CFU-S development
during transition from the
pre-liver t o liver stage of hematopoiesis. ( l )Dissectional analysis of the mouse AGM region
shows the presence of CFU-S both around the dorsal aorta
and in the uro-genital ridges. (2) The embryonic gut also
shows low but distinctive CFU-S activity. This initial intrabody patternof CFU-S distribution in murine embryogenesis
parallels that found for primordial
germ cells. (3) The beginning of definitive liver hematopoiesis is accompanied by
wide dissemination of CFU-S in the embryonic tissues. (4)
Comparision of spleen colonies arising from the AGM and
YS has shown morphologic differences. In contrast t o simple
erythroid constitution of theYS colonies, a broader variety
of cells are found within the AGM-derived colonies that are
similar t o those derived from 1l-day FL. These data suggest
a lineage relationship for hematopoieticprogenitors between the AGM region and the FL.
0 1996 by The American Societyof Hematology.
D
Despite the initial report indicating the presence of low
colony-forming unit-spleen (CFU-S) numbers in the YS beginning 8 dpc,' further investigations did not show any significant number of CFU-S giving rise to spleen macrocolonies in the YS on 8 to 9 dpc.".25-28A more detailed analysis
has shown that CFU-S appear in the YS and embryo body
simultaneously at 27 SP stage (late 9 d p ~ ) . ~This
' raises the
question as to whether CFU-S in the body are distributed
evenly or concentrated in some tissue(s). Therefore, we examined the localization of CFU-S within the developing
mouse embryo. We first tested the axial area comprising the
dorsal aorta, gonads, and mesonephroi (AGM),25the most
probable candidate for hematopoietic activity based on previous observations using amphibian and avian embryo^.^^"^
The CFU-S frequency in murine AGM region by the end of
10 dpc before their appearance in the liver increased to a
value comparable with that found in adult bone marrow. We
have previously shown that the AGM-derived cells at the
pre-liver stage of hematopoiesis are capable of complete
hematopoietic multilineage reconstitution of an irradiated
adult recipient.20 We have therefore proposed a two-wave
model of fetal liver (FL) colonization first from the YS and
subsequently from the AGM r e g i ~ n .However,
~ ~ . ~ ~ although
CFU-S frequency and total amount were verylowin the
body outside the AGM region, these data do not preclude
URING VERTEBRATE embryogenesis, diverse anatomical regions of the developing embryo show successive hematopoietic activity. In mammals, hematopoietic
events beginning in the yolk sac (YS) shift to the embryonic
liver, the main fetal hematopoietic source, and later to bone
marrow and spleen.' It has been generally accepted that this
picture reflects the consecutive migration of the pluripotential hematopoietic stem cells (HSCs) from the YS to definitive hematopoietic territories.2 Fromday 7.5 postcoitum
(dpc) in the mouse, the YS exhibits erythroid activity and,
under experimental conditions, gives rise to various hematopoietic lineages. In vitro cultures show erythroid as well as
macrophage-granulocyte hematopoietic precursors in 8 dpc
YS, but not in the embryo
The YS is able to produce
adult-type erythrocytes in the in vitro colony-forming
assay4.'.* or in organ culture under the influence of embryo
body- or liver-derived soluble factor^.^*'^ However, the day7.5 mouse embryo body does not initiate committed myeloid
precursors in vitro in the absence of the YS.2 At the preliver stage of hematopoiesis, YS cells are able to differentiate
into mast cells" and various subsets of the T-cell lineage
when cultured with lymphocyte-depleted fetal t h y m ~ s . ' ~It" ~
has been recently shown that, under certain culturing conditions or upon transplantation, 8- to 8.5-day YS but not embryo body gives rise to mature T and B
The
search for more primitive hematopoietic progenitors in the
YS capable of repopulating of adult hematopoietic system
led to a number of controversial report^."^^^-^^ Careful analysis has shown that there are no cells with repopulating ability
in the YS or body before late 10 dpc." In general, other
early experiments were interpreted as suggesting that all
hematopoiesis derived from the YS and that no hematopoietic activity could be detected in the embryo body. However,
several recent studies have, in fact, found significant hematopoietic activity in the embryo body. Day-8 and day-9 embryo
body tissues as well as the YS when cultured on S17 stromal
cells are capable of differentiating into B cells.21*22
Paraaortic splanchnopleura from day-9 embryo was also shown
to contain the Bla progenitor cells.23Recent data showed
that, beginning from the 10 to 25 somite pair (SP) stage,
this area in parallel with the YS harbors cells capable in
vitro to generate B and T lymphocytes and myeloid cells.24
Blood, Vol 87, No 2 (January 15), 1996: pp 557-566
From the Laboratory of Physiology of Hematopoiesis, National
Research Center for Hematology; and the Department of Embryology, Facultyfor Biology, Moscow State University, Moscow, Russia.
Submitted May 15, 1995; accepted August 30, 1995.
Supported in part by the Biotechnology Scient$c council of the
Russian Academy of Science.
Address reprint requests to Alexander L. Medvinsky, PhD, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 I A A , UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
OOW-4971/96/8702-0029$3.00/0
557
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MEDVINSKY ET AL
558
Table 1. Distribution of CFU-SI in the Tissues of the Developing Mouse Embryo (Total Number per Tissue)
Age of Embryos
10 d
Tissues
31-33SP
Viscera
AGM region
Gastrointestinal tract
Mesentery
Liver
Spleen
Lungs
Heart
Uro-genital sinus
Carcass
Head
Somites/anterior/t
Somites/posterior/t
Lateral wall/anterior/
Lateral walllposteriori
YS
Blood
No. of experiments (no. of embryos)
0.8 (0.8-0.8)
0
0
0
-
34-35SP
36-37 SP
0.8 (0.4-1.4)
1.8 (0.9-2.7)
0.6 (0.5-0.6)
0.2 (0-0.4)
0.03 (0-0.2) 0
0
0
0
0
0.4 (0.08-0.8)
0
0.2 (0.1-0.3)
0
0.06*
0.04*
0
0.01*
0
0.3 (0.1-0.6)
0.01*
0.1*
1.o
0.2
0.04*
0
0.9 (0.6-1.6)
2 (18)
7 (57)
0
0
0
0
0
0.2*
11 d
12 d
13 d
7.3 (6.5-8.0)
0.4 (0.1-0.7)
0.1* (0-0.1)
0.3
0.6 (0.3-1.01
0.5 (0-1.3)
0.1 (0-0.2)
16.4
0
0.1
0.03*
0.1 (0-0.2)
0.7
0.7 (0.3-0.9)
0
342
0
0.1*
0.2
0
-
-
0
0.01*
0.3 (0-1.O)
0
38-40SP
0
0
0
ND
0.6
ND
822
0
0.4
0
ND
0.02*
0.5
0
0.1*
0.1"
0.4
3.1 ( 1.O-5.0)
0.2
1.3
0.5
0.3
0.5
0.4
3.2 (2.0-5.2)
1.7
3.5
0.5
1.3
1.7
0.8
3.0 (1.6-5.8)
6.5
0.8
0.6
0.3*
0.5
0.5
1.9
4.2
3 (42)
2 (17)
5 (54)
2 (16)
1 (8)
For each experiment, the tissues indicated were pooledfrom 8 to 14 embryos of variousages and cell suspensions were injected into lethally
irradiated recipients. The mean number of CFU-S8 per donor embryonic tissue is shown and the range is indicated in parentheses.
Abbreviation: ND, not determined.
*The difference between experimental and control groups is statistically insignificant.
t The region also includes spinal cord with surrounding tissues.
One mouse survived in twoexperiments, and two colonies were shown.
Table 2. CFU-Ss Frequency in the Tissues of the Developing Mouse Embryo (per lo6 Viable Cells)
Age of Embryos
10 d
31-33 SP
Tissues
Viscera
AGM region
Gastro-intestinal tract
Mesentery
Liver
Spleen
Lungs
Heart
Uro-genital sinus
Carcass
Head
Somitestanterior/*
Somites/posterior/*
Lateral wall/anterior/
Lateral wall/posterior/
YS
Blood
No.experiments
of
embryos)
(no.
of
83 (70-94)
0
0
0
-
0
0
0
0
0
0
11.7
0
1.6 (0.7-2.8)
0
2 (18)
34-35 SP
49 (16-138)
15 (0-61
18
0
36-37SP
38-40SP
11 d
12 d
188 (46-331)
44 (32-53)
375 (368-384)
48 (20-76)
0.2
8.5
10 (5.6-17.8)
9 (0-24)
27
44
14
3.2 (0.3-0.9)
0
137
0
0
-
-
0
1.1
31 (0-76)
0.6
0.4
0
0
0
3.1 (1.0-6.1)
0.1
7 (57)
0
0
36 (6.5-66)
0.4
0.9
7.1
0.3
0
5.9 (3.3-15)
0.1
3 (42)
-
0
0
0
1.1
0
2.0
0.3
3.0
22 (7-38)
0.4
2 (17)
13 d
0
12
ND
119
0
0
0
3.4
0.5
1.1 (0-2.3)
0.9
3.6
4.8
1.1
1.5
1.7
1.4
1.8
16 (11-28)
2.3
5 (54)
ND
0.7
0.9
2.8
3.0
1.4
7.4 (2.7-18)
1.7
2 (16)
0
ND
0.5
1.o
0.5
0.6
1.o
6.1
1.8
1 (8)
The frequency of CFU-Ss calculated as the number of spleen colonies seen in lethally irradiated recipients per10' viable cells transplanted.
1.
This table was generated from the same experimental data used to produce Table
Abbreviation: ND, not determined.
*The region also includes spinal cord with surrounding tissues.
periments
of
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CFU-Se DEVELOPMENT IN THE MOUSE EMBRYO
559
Fig l. PCR analysis of spleen colonies derived from late 10 dpc
gut and circulation with primers that amplified fragments of Y-specific gene (Y353/B, 342 bp) and myogenin (245 bp). The mixture of
cells from male and female embryos was transplanted into irradiated
recipients. The lanes that are indicated by gut and circulation correspond to spleen colonies from the animals receiving transplants of
these tissues. As a control, the intercolonial tissue (i.coIJ was also
tested. No positive PCR amplificationof the 342-bp Y-specific product
was detected in this tissue.
the possibility of some other low CFU-S-generating tissues
within the embryo body.
We have now performed a detailed analysis on a variety of
embryonic tissues during the crucial period of FL definitive
hematopoietic establishment (10 to l 1 dpc). On 10 dpc, we
show that, in addition to the AGM region, the murine embryonic gut also develops CFU-S activity. No other embryo
body tissue or primordia contain significant numbers of
CFU-S up to the end of I O dpc. However, beginning at 1 I
dpc, when CFU-S activity appears in the FL, CFU-S can be
detected in various other embryonic tissues. Finer dissection
of the AGM region has not shown significant differences in
CFU-S generation between dorsal aorta and uro-genital
ridges. Finally, we performed comparative morphologic
analysis of spleen colonies derived from the YS,AGM region, body remnants, and liver. The morphology of AGMderived colonies more closely resembled that of FL-derived
colonies than those derived from other embryonic tissues.
MATERIALS AND METHODS
Animals andtissues.
Adult mice were all obtained from the
Stolbovaja animal breeding unit. Females C57BV6j were mated with
Fig 2. Scheme of fine dissection of AGM region. Ao, dorsal aorta;
UGR, uro-genital ridges.
CBA males. The day on which the plug was found was designated
as day 0 of gestation. All further manipulations were performed
under a dissecting microscope in Leibovitz L-l5 medium supplementedwith 5% fetal bovine serum (Flow Labs,Irvine, CA) and
antibiotics, 1 0 0 UlmL penicillin, and 0.05 mglmL streptomycin sulfate. Embryos were removed at I O to 13 dpc and embryos within
narrow age limits were pooled for CFU-S assay. After the preparation of tissues and cell suspensions, transplantations were performed
as described previously." Embryonic circulation was harvested directly after separation of the YS from the embryo body. Viability
counts were performed using the trypan blue exclusion test.
CFU-S ussoy. (CBA X C57BI/6)FI female mice were exposed
to 13.6 Gy of total irradiation from four '"CS sources at a dose rate
of 0. I76 Gy/min. Two irradiation procedures were separated by a
3-hour interval. Cell suspensions were injected viathe tail vein.
Eight days after the injection, recipients were killed, their spleens
were fixed in Bouin's solution, and macroscopic colonies were
counted under a dissecting microscope. Experimental groups were
compared with corresponding control groups (irradiation without
cell injection) using the homogeneity criterion for two samples according to Smirnov's criterion." No morethan I colony per 55
spleens were found in irradiated control group of mice. Donor origin
of CFU-S colonies was tested by polymerase chain reaction (FCR)
analysis using oligos designed byA. Rattigan and P. Burgoyne
(personal communication, 1995) amplifiying the 342-bp fragment of
Y353/B gene on the mouse Y-chromosome" in combination with
myogenin specific oligos (the size of amplified product is 245 bp)."'
The reactions for the combination Y353/B-myogenin were initially
heated at 95°C for 5 minutes, followed by 30 cycles of 94°C for IO
seconds, 60°C for 30 seconds, and 72°C for 35 seconds, followed
Table 3. Distribution of CFU-Ssin the AGMRegion Dissected Into Aorta Area and Uro-Genital Ridges of 10-Day Mouse Embryo
Age of Embryos
32-33SP CFU-Sn
Tissues
Aorta
Urogenital ridges
Liver
No.
Total
Per
loa
(183-340)
240
(0.4-0.9)
0.50.5
(433-509)
522
471
(0.3-0.9)
0.7
(58-413)
(0.6-2.3)
235 1.4 99 0.6
0
0
0
(no. of embryos)
1 (13)
36-37 SP CFU-Sn
Total
38-40 SP CFU-Sn
Total
Per 10s
(0-6.1 0
2 (18)
Total Number of CFU-Seand their frequency in tissues tested presented as in Tables 1 and 2.
3.8
(178-227)
2.0 (1.6-2.2)
209
0.2 (0-0.3)
2 (22)
Per 10s
1
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Fig 3. AGM region of the mouse embryo at stage 40 SP (the end
of 10 dpc). The presenceof erythropoietic focus with mitotic cells is
noted (arrows). The clusters of celhwith picnotic nuclei (open arrow
Stainedwith hematoxheads) areinthe area of mesonephric tubules.
ylin and eosin. Bar = 16 pm.
Fig 4. The liver of Il-day embryo. Proliferatingfoci of YSderived
erythroid cells showing the typical high cytoplasm/nucleus ratio and
eosinophilic cytoplasm (arrows). Stained with hematoxylin and BOsin. Bar = 16 pm.
10minutesat 37°C. In additiontospleencolonies,intercolonial
splenic tissue were used as controls. DNAwaspreparedaspreviously described",
Histology. The preparation of sections and histochemical staining for alkaline phosphatase was performedas described.= Ten and
11 dpc embryos were fixed in cold (4°C) formaldehyde-phos4%
phate-buffered saline (PBS) (pH 7.4).Four hours later, fixed specimens were transferred to cold PBS. Overnight washing with three
changes of PBS was followed by dehydration through graded alcohol. Specimens were infiltrated and embeddedin Historesin (Leica)
at 4°C according to the manufacturer's instructions. The blocks were
serially sectioned with a steel knife with a tungsten carbide edge
(Leica, Vetzlar, Germany) on a Reichert-Young 2030 microtome at
2 to 3 pm. Sections at 60 pm distances were transferred to slides,
stained for alkaline phosphatase (ALP) for 1.5 hours at 37"C,and
counterstained with neutral red. The ALP detecting kit including
Naphtol AS-B1 phosphate as a substrate and fast blue BB base
as
a capture agent
was used(catalogueno. 86-C, Sigma, St
Louis, MO).
Some sections were stained with hematoxylin and eosin. Photos were
taken with Axiophot microscope (Carl Zeiss Gmbh, Jena, Germany).
The morphology of spleen colonies was evaluated on 6-pm serial
*
Fig 5. Distribution of PGCs in the mouse embryo at stage 34 SP
( I O dpc). PGS (arrows)are presentin genital ridges andin the tissue
of the gut. Themost part of mesentery is lacking in PGC. Ao, dorsal
aorta; Mn, mesonephros; Me, mesentery;G, gut. Stainedfor ALP and
counterstained with neutral red. Bar = 40 pm.
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CFU-S,DEVELOPMENT IN THE MOUSE EMBRYO
paraffin sections of spleens taken at 60 pm distance and stained with
hematoxilyn and eosin.
Elecrron microscopy (EM). Portions of embryos including AGM
region were fixed in cold (4°C) 2.5% glutaraldehyde-PBS (pH 7.4)
overnight. Specimens were then washed in PBS and immersed into
1% OsO, for 4 hours. After dehydratation, theywere. dried for scanning EM or embedded into Epon (Fluka, Buchs, Switzerland) and
sectioned for transmission EM. For scanning EM, a Hitachi C-405A
electron microscope (Hitachi, Tokyo, Japan) was used.
RESULTS
Localization of CFU-S8 in the IO dpc mouse embryo. To
ascertain whether CFU-S were located in other 10 dpc embryonic tissues in addition to AGM region, the following
visceral structures were dissected and assayed, ie, the lung
buds, the heart, the gastrointestinal tract, the dorsal mesentery, the liver, and AGM region. When the dorsal mesentery
was dissected, the thick upper part located between genital
ridges remained with the AGM region. The embryo carcass
was dissected into the head; the anterior and posterior somites, with the adjoining tissues that included the neural
tube; the anterior and posterior lateral walls, which included
the limb buds; and the uro-genital sinus. Although the first
CFU-S appearing in 10-day-old embryos are predominantly
concentrated in the AGM region, lower numbers and frequencies of CFU-S were shown in the gastrointestinal tract,
beginning at the 34 to 35 SP stage (Tables 1 and 2). Many
fewer CFU-S were found in other structures of the 10 dpc
body. Although the mesentery and the uro-genital sinus
sometimes showed CFU-S activity, the irregularity of CFUS within these regions indicates that this was the result of
occasional contamination with the cells of the AGM region,
which is axial and contiguous to most of tested embryonic
tissues. As previously found:' the first CFU-S were observed
in the embryonic circulation on 10 dpc shortly before their
appearance in the FL at the 38 to 40 SP stage (Tables 1 and
2). The donor origin of colonies derived from the gut and
circulation was confirmed by PCR analysis (Fig 1)
To more precisely localize the site of CFU-S activity
within the 10 dpc AGM region, we dissected the dorsal aorta
from uro-genital ridges (Fig 2) and separately assayed these
tissues. The data in Table 3 show that both tested components of the AGM region harbor CFU-S. However, the total
number of CFU-S in the uro-genital ridges is higher, although the frequency is lower than in the aortic area.
Localization of CFU-S in 11- to 13-day mouse embryos.
At11 to 13 dpc, the low number of CFU-S found in the
embryo outside the liver was distributed uniformly throughout the body, as was observed for the tissues of the carcass
(0.5 to 2.8 per 10" cells; Table 2). This is most likely due
to blood in dissected embryonic tissues, because at this period the CFU-S concentration in circulating blood reaches
significant levels (1 .7to 2.3 per 10" cells). During this time,
the FL showed a dramatic increase of CFU-S number (from
16 to 822 CFU-S per liver). In the gastrointestinal tract, the
numbers of CFU-S remain the same as at 10.5 dpc, although
the frequency decreased significantly (Tables 1 and 2). Other
primordia (thymus, spleen, pancreas, metanephroi, and adrenal glands) in addition to those listed in Tables 1 and 2 were
also tested as they became available in the embryo. None of
the extra-liver visceral tissues excluding gastrointestinal tract
561
showed highand regular CFU-S activity at 1 1 to 13 dpc
(Tables 1 and 2 and data not shown)
Histologic changesin the mouse embryo during the
transition from the YS/AGM region to FL hematopoiesis. As we
reported earlier, although the AGM region contained a high
frequency of CFU-S, no visible hematopoietic foci were
found extravasculary in loose mesenchyme in this area on
IO dpc at stage 34 to 35 SP.*' However, certain visible
changes of hematopoietic tissues are observed parallel to the
appearance of the first CFU-S within the embryonic liver at
40 SP stage. By this time, mesenchyme becomes more dense
and some nucleated erythropoietic foci of the YS phenotype
inside small blood vessels can be shown in various tissues
of embryo and occasionally in the AGM region (Fig 3). In
the liver, these foci become big and numerous and are easily
distinguished from FL-derived nucleated erythroid cells by
Fig 6. Scanning electron micrographs. (A) A cluster of nonerythroid cells (arrow) on the inner surface of a dorsal aorta is distinguished from embryonic erythroid cells (ER) by microvilli. (B) An analogous cell with a philopodium suggesting an active motion. Bar = 2
pm.
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MEDVINSKY ET AL
562
Table 4. Morphology of Spleen Colonies Produced by CFU-S. Derived From Embryonic and Adult Hematopoietic Sources
Source of CFU-S
No. of
Colonies
Analyzed
Erythroid
Granulocytic
Megacaryocytic
Mixed
Undifferentiated
10-Day YS
10-Day AGM region
10-Day body remnants
1l-Day liver rudiment
17-Day FL
Adult bone marrow
40
81
19
44
64
62
98
81
100
80
67
60
0
2
0
2
3
10
0
0
0
2
14
11
0
4
0
5
14
8
12
0
11
2
11
Type of Colonies (%I
strong cytoplasmic eosinophily characteristic of YS-derived
nucleated erythrocytes (Fig 4). Primordial germ cells (PGCs)
can be identified by their strong ALP-positive staining of
cytoplasm as they migrate from the allantoic base.3944' In
the
middle of 10 dpc, PGCs are observed in the uro-genital
ridges. They can also be found around the gut, but not in
the intermediate part of the dorsal mesentery (Fig 5). This
is not surprising, because the migration of PGC from allantoic base is mainly over by this time and those found around
the gut are probably retarded." It is interesting that this area
is also deficient in CFU-S (Tables 1 and 2). Thus, PGCs and
CFU-S are confined to the AGM region and gut in the body
of the mouse embryo on 10 dpc, showing even stronger
colocalization than we reported earlier.z5
Certain changes of potential importance occur in the dorsal aorta wall between 10 and 1 1 dpc. On 1 1 dpc, many
undifferentiated cells, presumably of hematopoietic origin,
appear attached to the inner surface of the endothelium lumen (Fig 6A). Occasionally they form clusters. These cells
are distinguished from embryonic erythrocytes by microvillis on the cell surface. They have round or lobular nuclei
with prominent nucleolis and cytoplasm with scanty organelles (data not shown). These undifferentiated cells form
specialized contacts with endothelial cells (tight junctions),
suggesting that they may use endothelial cells as a substrate
for motion (data not shown). In agreement with this finding,
some polarized cells with philopodia can be observed on the
endothelial surface (Fig 6B).
Morphology of spleen colonies. The spleen colonies derived from various tissues of 34 to 39 SP (10 dpc) embryos
were analyzed histologically for predominance of erythroid,
granulocytic, megakaryocytic, mixed, or undifferentiated
cell types. The colonies were considered to be of a certain
cell type when this cell type conprised more than 90% of
the colony. Of the 40 colonies derived from the YS, only
one was undifferentiated, whereas others were completely
erythroid (Table 4). In contrast, the AGM region gave rise
to a greater variety of colonies (2%granulocytic, 4% mixed,
and 12%undifferentiated) that were very similar to the percentages found for 11 dpc liver-derived colonies (Table 4).
2
However, both 10 dpc AGM region and 1l dpc liver colonies
showed less variability than those originating from 17 dpc
FT. and adult bone marrow in which many fewer colonies
were erythroid. No spleen colonies other than erythroid were
found after transplantation of cells from extra AGM body
tissues at 10 dpc.
DISCUSSION
CFU-S progenitor^^^ are rather immature cells and represent a nonhomogenous population"-46 within the hematopoietic hierarchy of the adult mouse. They possess high proliferative potential, giving rise to colonies composed of hundreds
of thousands of cells in the spleen of the irradiated recipient.
Some of them also possess self-renewal potential.'" CFU-S
are absent from the hematopoietic hierarchy of day 8 to
middle of day 9 mouse embryos and, therefore, are characteristic of developmentally advanced stages of hematopoietic
o n t ~ g e n y . ~Although
~ . ~ ~ . ~precursors
~
of erythroid, myeloid,
and lymphoid lineages have been shown in the YS,2-5,'z"6.
21,22,48
the contribution of the YS to definitive hematopoiesis
remains unknown. Moreover, recent data suggesting the appearance of pluripotent HSCs simultaneously in the YS and
embryo bodyz4do not clarify the question of whether these
cells possess a self-renewal and expanding potential sufficient for the long-term population of the adult hematopoietic
system. CFU-S are not pluripotential HSCs because they can
be separated from long-term repopulating cell^.^^.'^ However,
the development of the CFU-S compartment during ontogeny does reflect crucial steps in the maturation of the definitive hematopoietic system and perhaps could be used to
define the onset of definitive hematopoiesis. Accurate dating
of CFU-S appearance showed that extra-liver body tissues
beginto generate CFU-S at the same timeandin larger
quantities than did the YS. At the pre-liver stage (10 dpc),
CFU-S are concentrated in the AGM region, witha frequency 10 to 30 times more than the YS, and are comparable
to the frequency found in adult bone
In the present study, we have attempted to more precisely
localize CFU-S in the AGM region. No significant difference
in CFU-S activity was found between the areas comprising
b
Fig 7. Schematically represented distributionof CFUS in various tissues of 10-day (A) and 11-day(B) embryos. The density of open circles
semiquantitatively rdects CFU-S frequency in embryo tissues. The left wall of the embryo is not shown. Carcass (outlined with thick line);
body wall; viscera (outlinedwith thin
Hd, head; AS, anterior somites; PS, posterior somites; AL. anterior lateral body wall; PL, posterior lateral
L, liver; Ht, heart; YS,
line); UGR, uro-genital ridgeswith dorsal aorta between them; Ms. mesentery; G, gut; Lu, lungs; UGS, uro-genital sinus;
yolk sac.
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MEDVINSKY ET AL
564
the aorta with the adjoining tissues and uro-genital ridges.
The difficulty in obtaining pure tissues from the AGM region
has made it difficult to follow distribution of CFU-S inside
this region at earlier stages; thus, the route of HSC migration
and their site of origin remains unclear.
We also examined other intra-body tissues for CFU-S
activity. Although less numerous than in the AGM, CFU-S
were found in the gastrointestinal tract. It is interesting that,
in some invertebrate species, the intestinal submucosa displays hematopoietic a~tivity.~'
None of the other tissues in
the embryo body showed significant quantities of CFU-S up
to the end of 10 dpc. Solitary CFU-S found in other tissues
may reflect contamination with cells from the AGM region
because it is contiguous to all tissues tested due to its axial
position in the body.
The pattern of CFU-S development obtained in 9- to l 1day mouse embryo (Fig 7) supports our hypothesis that the
liver rudiment may be colonized sequentially by the YScommitted progenitors and more immature population from
the AGM region.25s34
In fact, initially committed precursors
in the embryo can be detected in the YS beginning from 8
d p ~and
~ ,in~the circulation6 populating the liver rudiment
near the stage 28 SP (9 dpc).9,10,38,52
Only 1 day later (36 to
37 SP), when CFU-S attain peak numbers in the AGM region, do they appear in the circulation. This immediately
precedes the beginning of CFU-S activity in the liver rudiment on the end of 10 dpc/early l 1 dpc. Although presented
within the YS and AGM region at earlier stages,25CFU-S
do not enter the circulation and can be rescued from the
embryonic tissues only by enzyme digestion (compare Medvinsky et alZ5and Samoylina et a13'). Thus, the data suggest
that the second wave of immature hematopoietic progenitors
may come into the liver mainly from the AGM region and
also via circulation.
On day l 1 of gestation, CFU-S began to be observed in
the carcass tissues. Again, the most probable explanation
for the appearance of CFU-S in the body tissues is their
dissemination from hematopoietic sources (AGM, YS, and
FL)into nonhematopoietic tissues via the circulation, where
they reach significant values by this time. From the beginning of 11 dpc, the dorsal aorta had undifferentiated cells
attached to the inner surface of the aortic wall through the
specialized contacts and tight junctions. Smith and Glomsky53had proposed earlier hematopoietic activity of aortic
epithelium. However, in the light of our new data, one can
speculate that this picture may reflect transit of hematopoietic progenitors from the AGM region to blood stream. However, the migration can be conclusively proved only by experiments in which these cells are marked in some way.
The successive waves of liver colonization may be morphologically indistinguishable inside the liver rudiment, because the YS in vitro is able to produce erythroblasts that
which synthesize adult-type hemoglobin and extrude their
nuclei in response to factors derived from the liver rudiment.'
However, it has been shown that erythroblasts in 10-day
liver rudiment can be distinguished antigenically from those
of 1l-day liver rudiment with recently developed rat monoclonal antibodiess4 This result suggests there are two different erythrocyte populations in the developing FL. This finding is consistent with the proposed two-wave colonization
model.
Additional evidence in favor of the AGM role in definitive
hematopoiesis provides the morphologic analysis of spleen
colonies derived from various embryonic tissues. The YS
gives rise predominantly to pure erythroid colonies, whereas
greater morphologic variety was observed in AGM-derived
colonies. Twelve percent of AGM-derived colonies were
undifferentiated. The morphologic composition of spleen
colonies originating from 1l-day liver and10-dayAGM
region wasvery similar, providing further evidence of a
relationship between 10-day AGM region and 1l-day FL.
Spleen colonies derived from 10- to 1l-day embryonic tissues were less heterogeneous than those from mature fetal
liver or adult bone marrow, possibly reflecting the immaturity of early hematopoietic tissues.
It is interesting that, on day 10 of development, the localization of CFU-S and PGC is more similar than we have
previously rep~rted.'~
Both types of progenitors are found
in the AGM and gut regions but are absent from the intermediate area of the dorsal mesenterium. This suggests that (I)
a common factor(s) influences both cell types in the developing mouse embryo.' One possible candidate is stem cell
factor (SCF), which is involved in the development of both
hematopoietic and reproductive tissues in the mose emb r y ~ . ~Mutations
~.'~
in S1 or W loci lead to variable degrees
of anemia and sterility in
Both CFU-S and, probably, PGC express
and can be maintained in culture
with SCF in combination with other factors?"" In addition,
SCF can act as a chemotactic agent6' and therefore be involved in the spatial organization of CFU-S and PGC. Although it has been shown that in vitro, soluble SCF does
not exert a chemotropic effect on PGC,65 it cannot be excluded that a membrane-associated form of SCF may control
this process. Leukemia-inhibitory factor (LIF), which is expressed on day 10 of development in the mouse
was shown as survival and proliferative factor both for primitive hematopoietic progenitors" and PGC.64-66
Experimental
evidence has been obtained that TGF-P 1 is required for yolk
sac hematopoiesi~~~
and is partially responsible for migration
of PGC into genital ridges6' As a chemotropic
TGF-/31 could play a similar role in CFU-S distribution
within the embryo at the pre-liver stage of hematopoietic
development. Fine dissection of the AGM region shows that
CFU-S are more widely distributed in the AGM region than
PGC. PGC are located in genital ridges, whereas CFU-S are
located in the vicinity of the dorsal aorta as well. This suggests that, in addition to some presumptive common growth
factors for CFU-S and PGC, there could exist some factors
specifying differential behavior of these progenitors. Further
experiments are necessary to find out what growth factors
are really responsible for the expansion and spatial distribution of the HSC pool during the transition from the YSI
AGM region to FL stage of hematopoiesis.
ACKNOWLEDGMENT
Wethank E. Dzierzak(London, UK), I.L. Chertkov (Moscow,
A. Elefanty (MelRussia), J. Dick (Toronto,Ontario,Canada),
bourne, Australia), and D. Abraham (London, UK) for their helpful
Office of
comments on the manuscript. We also thank the MOSCOW
Kodak Corp forproviding us withKodacolorGold 1 0 0 filmsand
making prints.
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CFU”S8 DEVELOPMENT IN THE MOUSE EMBRYO
NOTE ADDED IN PROOF
When this article was submitted, a manuscript suggesting
the hematopoietic potential of PGCs in vitro was published
(Rich, Blood 86~463,1995). This work is consistent with
our observation of the colocalization of Cm-Ss and PGCs
within the embryo body. However, to show that this is not
just a result of reprogramming of PGCs into ES cells during
culture (Matsui et al, Cell 70:841, 1992), further experiments
need to be performed. Weare currently developing an experimental system that should enable us to identify cells giving
rise to early hematopoietic activity within the AGM region
(Medvinsky and Dzierzak, manuscript in preparation).
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1996 87: 557-566
Development of day-8 colony-forming unit-spleen hematopoietic
progenitors during early murine embryogenesis: spatial and temporal
mapping
AL Medvinsky, OI Gan, ML Semenova and NL Samoylina
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