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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3754-3763
SCL Expression in the Mouse Embryo Detected With a Targeted
lacZ Reporter Gene Demonstrates Its Localization to
Hematopoietic, Vascular, and Neural Tissues
By
Andrew G. Elefanty,
C. Glenn Begley,
Lynne Hartley,
Bette Papaevangeliou, and
Lorraine Robb
From The Walter and Eliza Hall Institute of Medical Research, the
Cooperative Research Centre for Cellular Growth Factors and the Rotary
Bone Marrow Research Laboratories, Victoria, Australia.
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ABSTRACT |
The helix-loop-helix transcription factor SCL (TAL1)
is indispensable for blood cell formation in the mouse embryo. We have explored the localization and developmental potential of cells fated to
express SCL during murine development using SCL-lacZ mutant mice in which the Escherichia coli lacZ
reporter gene was `knocked in' to the SCL locus. In addition
to the hematopoietic defect associated with SCL deficiency, the
yolk sac blood vessels in SCLlacZ/lacZ embryos
formed an abnormal primary vascular plexus, which failed to undergo
subsequent remodeling and formation of large branching vessels.
Intraembryonic vasculogenesis in precirculation
SCLlacZ/lacZ embryos appeared normal but, in
embryos older than embryonic day (E) 8.5 to E9, absolute anemia leading
to severe hypoxia precluded an accurate assessment of further vascular
development. In heterozygous SCLlacZ/w embryos,
lacZ was expressed in the central nervous system, vascular endothelia,
and primitive and definitive hematopoietic cells in the blood, aortic
wall, and fetal liver. Culture of fetal liver cells sorted for high and
low levels of  galactosidase activity from
SCLlacZ/w heterozygous embryos indicated that there
was a correlation between the level of SCL expression and the
frequency of hematopoietic progenitor cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE HELIX-LOOP-HELIX transcription factor
SCL (TAL1) was initially cloned as a partner in the
t(1;14) translocation, which characterized certain human T-cell
leukemias.1 It was subsequently shown to be indispensable
for blood cell formation in the mouse because SCL-deficient
embryos displayed an absolute anemia, which resulted in retarded growth
by 9 days of gestation (E9) and death by E9.5 to 10.5.2,3
SCL was also critically required for the development of
hematopoiesis in the adult mouse4,5 and for the induction
of hematopoietic genes during embryonic stem cell
differentiation.6
Complementary studies examining the expression pattern of SCL
in hematopoietic populations in mice and humans demonstrated selective
expression in primitive CD34+ cells and in erythroid,
megakaryocytic, and mast cell lineages.7-11 SCL
expression decreased as CD34+ cells differentiated into
myeloid cells and analyses of SCL expression in erythroid
colonies showed more abundant expression in colonies at early
timepoints during their development.9,11-14 Consistent with
these observations, retroviral transfer of SCL into human hematopoietic precursors preferentially enhanced erythroid and megakaryocytic colony formation.15,16
These data have spawned a model in which SCL is portrayed as a
`master regulator of hematopoiesis'17 and lies at the
apex of a pyramid of transcription factors controlling hematopoietic lineage commitment.18 Of course, this view of the role of
SCL may be oversimplified, as the complete molecular program of
hematopoietic commitment is likely to require a complex, interdependent
network of regulatory genes. Indeed, it has been shown that SCL can
form a multiprotein complex in hematopoietic cells, which includes the
transcription factors E2A and GATA-1, linked by the transcription factor binding proteins LMO2 and Ldb-1.19 It is instructive that the extreme phenotype displayed by SCL knockout embryos is recapitulated in LMO2 knockout mice,20,21 but not
in animals targeted at the other loci. This implies that SCL is
critical for the expression of genes needed to initiate hematopoiesis, that a multiprotein complex is required (hence the need for the bridging protein LMO2), but that the other known components of this complex are at least partly redundant at this stage.
Although the gene ablation and gene expression studies provided strong
circumstantial evidence for the role of SCL as a hematopoietic stem cell regulator, a functional correlation between endogenous SCL expression and progenitor cell activity in adult
hematopoietic cells was first provided by the analysis of mice in which
the Escherichia coli lacZ reporter gene had
been "knocked in" to the SCL locus (SCL-lacZ
mice).22 This analysis indicated that SCL was
expressed in all of the progenitors examined, including day 12 spleen
colony-forming cells (CFCs) and progenitors for erythroid, myeloid, and
lymphoid lineages. Thus, hematopoietic cells could be stratified into
functional groups based on their expression of a developmentally
relevant transcription factor.
To further define the role of SCL in embryogenesis, we have
examined the expression pattern of galactosidase during development in both heterozygous (SCLlacZ/w) and
SCL-null (SCLlacZ/lacZ) SCL-lacZ
mutant mice. These studies showed abnormalities in vascular
development, which were more severe in the yolk sac than in the embryo
in early SCLlacZ/lacZ embryos. Examination of mid-
and later-gestation SCLlacZ/w heterozygous embryos
demonstrated SCL expression in hematopoietic, vascular, and
neural tissues. Culture of fetal liver cells sorted for high and low
levels of galactosidase activity from SCLlacZ/w
heterozygous embryos indicated that there was a correlation between the
level of SCL expression and progenitor cell activity in the embryo, as well as in the adult mouse.
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MATERIALS AND METHODS |
SCL-lacZ mice.
The generation of SCL-lacZ mice has been described
previously.22 As a consequence of this "knock-in"
strategy, all SCL coding sequences were deleted and expression
of the lacZ gene is controlled by SCL regulatory elements.
Histochemical staining for galactosidase activity.
Embryos were fixed in 0.2% glutaraldehyde (E7.5 to E11.5) or 0.2%
glutaraldehyde/1% formaldehyde (>E12.5) in wash buffer
(phosphate-buffered saline [PBS])/2 mmol/L MgCl2/5 mmol/L
EGTA/ 0.02% NP-40/0.01% sodium deoxycholate) for 60 minutes on ice.
The yolk sac and amnion were opened and the roof of the fourth
ventricle and the abdomen were pierced in older embryos to improve
access of solutions. Embryos were washed 3 times for 30 minutes at room
temperature in wash buffer and incubated overnight at 37°C in
staining solution (100 mmol/L NaPO4, pH 7.3/5 mmol/L
K4 Fe(CN)6 .3H2O/5 mmol/L
K3 Fe(CN)6/2 mmol/L MgCl2/5 mmol/L
EGTA/0.02% NP-40/0.01% sodium deoxycholate/0.6 mg/mL
5-bromo-4-chloro-3-indolyl--D-galactopyranoside [X-gal]). Stained embryos were washed twice for 5 minutes in PBS/3% dimethyl sulfoxide (DMSO) and postfixed in 4%
paraformaldehyde in PBS overnight at 4°C. Fetal livers and brains
were dissected from embryos for whole organ staining, fixed in 0.2%
glutaraldehyde in wash buffer for 40 minutes on ice, washed, and
stained as above. Histological sections of paraffin-embedded lacZ
stained embryos and tissues were counterstained with nuclear fast red.
In some cases, embryos were fixed in 0.2% glutaraldehyde/PBS for 30 minutes, infiltrated with 30% sucrose/PBS, embedded in Tissue-Tek
O.C.T. compound (Sakura Finetek, Torrance, CA) and
frozen. Frozen sections were washed and stained for lacZ as above.
Immunohistochemistry.
Whole-mount staining of embryos and yolk sacs with
anti-PECAM-1 (platelet endothelial cell adhesion
molecule-1) monoclonal antibody (MEC13.3; Pharmingen, San Diego, CA)
was performed as described.23 Stained embryos were embedded
in paraffin, sectioned, and counterstained with nuclear fast red.
Fluorescence-activated cell sorting (FACS)-Gal analysis.
The FACS-Gal assay was performed essentially as
described.24 Fetal livers from E11.5 to E18.5
SCLlacZ/w mice or their wild-type littermates were
dissociated by triturating into 300 µL of PBS supplemented with 5%
fetal calf serum (FCS). The cells were washed once with the same
solution and resuspended in 20 to 300 µL of PBS/5%FCS and warmed to
37°C. Hypotonic loading was accomplished by diluting the cells 1:1
with warmed 2 mmol/L fluorescein di--D-galactopyranoside (FDG)
(Sigma Chemical Co, St Louis, MO) and incubating at 37°C for 2 minutes. Loading was stopped by adding 10 vol ice-cold PBS/5%FCS and
intracellular hydrolysis of FDG to fluorescein by galactosidase was
allowed to proceed on ice for 3 hours. Propidium iodide was added to a final concentration of 1 µg/mL before analysis. Cells were analyzed and sorted on a FACStar Plus (Becton Dickinson, San Jose, CA).
Clonogenic assays for hematopoietic progenitors.
Fractionated and unfractionated E12.5 fetal liver cells from
SCLlacZ/w mice or their wild-type
SCLw/w littermates were cultured in 0. 9%
methylcellulose for myeloid and erythroid colony formation as
described.25 The number of cells cultured for each fraction
was titrated to produce approximately 100 colonies per 1 mL culture at
day 7. Day 7 myeloid and day 7 blast-forming unit-erythroid (BFU-E)
colonies were stimulated by the combination of 1,000 U/mL interleukin-3
(IL-3) and 4 U/mL human erythropoietin (Epo) and day 2 colony-forming
unit-erythroid (CFU-E) colonies by 4 U/mL human Epo. CFU-E were scored
using an inverted microscope and day 7 colonies were scored using a dissection microscope.26
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RESULTS |
Abnormal intraembryonic and yolk sac vessel formation in
SCLlacZ/lacZ embryos.
In SCL-lacZ embryos, expression of lacZ was detected at
embryonic day (E)7.5 in the extraembryonic mesoderm
(Fig 1A and B) in a speckled pattern reminiscent of FLK-1 expression27 and consistent with the previously demonstrated expression of SCL in the mesodermal precursors of yolk sac blood
islands.28,29 At this stage of development, homozygous
SCLlacZ/lacZ embryos, which were destined to die
due to absolute anemia by E9.5, were indistinguishable from
developmentally normal SCLlacZ/w heterozygotes.
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| Fig 1.
LacZ staining of whole mount E7.5 to 8.5 days
postcoitum (dpc) SCLlacZ/lacZ
embryos. (A) E7.5 embryo showing speckled staining (arrow) in the yolk
sac. (B) Sagittal section of E7.5 embryo indicating that lacZ staining
is confined to the extraembryonic mesoderm (m); endoderm (e); amnion
(am). (C) Lateral and (D) anterior views of early somite (E8) embryos
demonstrating specific lacZ staining of vascular structures in
SCLlacZ/lacZ embryos (left). No staining is seen in
SCLw/w fetuses (right); cvp, cerebral vascular
plexus; avp, allantoic vascular plexus; en, endocardium; da, dorsal
aorta. (E) Ventral view of early somite (E8.0)
SCLlacZ/lacZ embryo, demonstrating migration and
alignment of angioblasts to form the dorsal aorta (da), allantoic
vascular plexus (avp), and the vitelline vein (vv), which receives
blood from the yolk sac. (F) E8.5 embryo showing the somitic arteries
(sa) sprouting from the dorsal aorta.
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| Fig 2.
Histological sections of lacZ-stained
SCLlacZ/lacZ embryos. (A) Section through headfold
and trunk of E8 embryo showing lacZ staining in the angioblasts forming
the cerebral vascular plexus (cvp) and the dorsal aorta (da). Staining
in (B) endocardium (en) and (C) somitic arteries (sa) in an E8.5
embryo. Note the large number of apoptotic cells (ap) in the neural
tube in (C). Original magnification: (A), ×200; (B and C), ×400.
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In early somite stage ( E8) homozygous
SCLlacZ/lacZ embryos, galactosidase was
expressed in the primitive heart and the cells lining the dorsal aorta
and the cerebral and allantoic vascular plexuses (Fig 1C through E).
Microscopic examination of sections of stained embryos confirmed that
lacZ expression was confined to the endocardium and endothelia (Fig 2A
and B). Consistent with previous observations,2,3 these
data confirmed that SCL was dispensable for embryonic
vasculogenesis, the specification of angioblasts from ventral mesoderm,
their appropriate migration within the embryo, and alignment to form major vascular structures. Development in the majority of
SCLlacZ/lacZ embryos continued until E9, by which
time the embryos had a beating heart and had turned. As shown in Fig
1F, lacZ expression now also marked the somitic arteries sprouting from
the dorsal aorta, indicating that there was no intrinsic failure of
angiogenesis. These vessels are shown histologically in Fig 2C. Marked
regions of neuronal cell death were already evident by this time,
presumably secondary to hypoxia.
Although lacZ expression was readily detectable in homozygous
SCLlacZ/lacZ embryos, staining for lacZ expression
in SCLlacZ/w heterozygous embryos during early
development was weak, probably because the SCL promoter
directed a low level of transcription and only 1 copy of the
lacZ gene was present. Therefore, the appearance of the
developing embryonic vasculature in wild-type
(SCLw/w and SCLlacZ/w) and
SCLlacZ/lacZ embryos was directly compared using an
antibody to the highly expressed endothelial surface marker PECAM-1
(CD31).30 In early somite embryos, the pattern of PECAM-1
staining of the nascent intraembryonic vasculature was very similar in
wild-type and homozygous SCLlacZ/lacZ embryos
(Fig 3A). This was identical to the pattern
of lacZ expression observed in
SCLlacZ/lacZ embryos of a similar
developmental stage (compare Fig 3A with Fig 1E). In contrast, vascular
development in the yolk sac was markedly abnormal in
SCLlacZ/lacZ embryos by E8, earlier than had been
reported previously.2,31 While wild-type embryo yolk sacs
harbored a well-developed, clearly demarcated primary vascular plexus,
SCLlacZ/lacZ yolk sacs contained only a
disorganized network of fine vascular channels (compare Fig 3C with
3B). By the 10 somite stage, PECAM-1 expression was reduced in
the dorsal aorta and the cerebral vascular plexus of
SCLlacZ/lacZ embryos (Fig 3D). In wild-type
embryos, cardiac looping had commenced, but in
SCLlacZ/lacZ embryos, the heart was still a
straight tube and there was considerable pericardial edema (Fig 3D and
E). Consistent with the lacZ staining shown in Fig 1F, PECAM-1
expression was evident in the forming somitic arteries at E9 in
SCLlacZ/lacZ embryos (Fig 3F). Even though
intraembryonic cell death was already widespread histologically by E9
(Fig 2C), marked growth retardation and vascular degeneration in
SCLlacZ/lacZ embryos did not become grossly
apparent until E9.5 (Fig 3G). The yolk sac vessels in these embryos had
failed to develop significantly from their disorganized state at E8,
while large PECAM-1-positive vascular trunks were prominent in
wild-type yolk sacs (compare Fig 3B and C with Fig 3H and I). In
histological sections of these embryos, PECAM-1 staining in the yolk
sac was apparent in the endothelium and a subset of the hematopoietic
cells in the blood islands in wild-type E8.5 (10 somite stage)
embryos32
(Fig 4A), while expression was limited to the endothelium in the bloodless channels of the SCLlacZ/lacZ yolk sacs (Fig 4C).
The complex cardiac looping and clearly defined vasculature of the
normal embryo contrasted with the linear heart and dilated dorsal
aorta, cardinal, and perineural veins displayed by the
SCLlacZ/lacZ embryos (Fig 4B and D).
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| Fig 3.
Anti-PECAM-1 monoclonal antibody staining of wholemount
E8 to 9.5 dpc wild-type (wt) and SCLlacZ/lacZ
( /) embryos and yolk sacs. (A) through (C) E8 embryos showing a
similar pattern of anti-PECAM-1 staining in wt and / embryos
(A), but a poorly formed primary vascular plexus in the / yolk
sac (C) compared with the wt yolk sac (B). (D) and (E) At E8.5, somitic
arteries are beginning to develop from the dorsal aorta. A dilated
pericardial sac (arrowed), delayed cardiac looping, and diminished
cerebral vasculature are evident in the / embryo. The pericardial
sac was dissected away in (E) to display anti-PECAM-1 staining in the
endocardium. (F) E9 embryos showing incomplete somitic vessels in the
/ embryo, which is undergoing apoptosis. (G) Preterminal,
severely growth-retarded E9.5 mutant embryo (/) displaying loss
of vascular architecture. (H) Large branching vitelline vessels in wt
yolk sac at E9.5 contrast with the / yolk sac (I), which retains
a poorly formed capillary plexus.
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| Fig 4.
Histological sections of anti-PECAM-1 monoclonal
antibody-stained E8.5 dpc wt and SCLlacZ/lacZ
(/) embryos and yolk sacs. Anti-PECAM-1 stains both the
endothelium and a subset of the hematopoietic cells in a wt yolk sac
(A), but is restricted to the endothelium in the / yolk sac (C).
(B) Section through the trunk of a wt embryo showing anti-PECAM-1
staining of the endocardium and endothelia. (D) In the / embryo,
the anti-PECAM-1 antibody recognizes the endocardium of a more linear
heart tube and dilated intraembryonic vessels. Original magnification:
(A and C), ×400; (B and D), ×200.
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| Fig 5.
LacZ staining in the central nervous system of
heterozygous (SCLlacZ/w) embryos. (A and B) At
E11.5, lacZ staining is apparent in the presumptive motor neurons in
the ventral neural tube (arrows). (C) Lateral and (D) dorsal views at
E12.5 and (E) lateral and (F) dorsal views at E13.5, showing expression
of lacZ in the developing midbrain. At E18.5 (G) and (H) and in the
adult mouse (I and J), lacZ expression is strongest in the superior
colliculi, but lower levels persist in the inferior colliculi. Weak
lacZ expression is evident in tracts on the ventral surface of the
hindbrain (H) and (J).
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| Fig 6.
Histological sections of lacZ-stained hematopoietic and
vascular tissues in SCLlacZ/w embryos. (A) Sagittal
section through the dorsal aorta of an E9.5 embryo demonstrating lacZ
staining of variable intensity in circulating hematopoietic cells. (B)
Cytocentrifuge preparation of E13.5 embryonic blood showing lacZ
staining in a definitive erythrocyte. (C) Endothelial lacZ expression
in a cerebral artery of an E13.5 embryo. (D) The dorsal aorta of an
E11.5 embryo demonstrating lacZ staining in the endothelium and in a
ventral aortic wall cluster of hematopoietic cells (arrow). All the
cells in the cluster were positive, but this cannot be appreciated in a
single focal plane. (E) and (F) LacZ staining in E11.5 fetal liver of a
wt (E) and heterozygous SCLlacZ/w embryo (F)
showing specific staining in large megakaryocytes and smaller
erythroblasts. Original magnification: (A, C through F), ×400; (B),
×1,000.
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Galactosidase expression in
SCLlacZ/w embryos shows the hematopoietic,
vascular, and neural expression patterns of SCL.
The major sites of lacZ expression in mid- and later-gestation
SCLlacZ/w embryos were the hematopoietic
compartment and the developing nervous system, consistent with the
sites of SCL mRNA and protein expression detected in previous
studies.9,28,33 In the central nervous system, weak galactosidase expression was first observed at E10.5 ventrally in the
presumptive spinal cord (Fig 5A and B), in a similar distribution to
that described for SCL in the frog34 and the
zebrafish.35 At E12.5, expression in the midbrain was
evident laterally in the tegmentum and in the fiber tracts of the
posterior commissure and proceeded to encompass most of the tectum and
the tegmentum by E13.5 (Fig 5C through F). Later in embryogenesis,
expres-sion of galactosidase was strongest in the superior tectum,
in the region that would ultimately become the superior colliculi and
weaker expression persisted in the inferior tectum, the site of the
definitive inferior colliculi (compare E18.5 and adult panels in Fig 5G
and I). LacZ expression was also apparent in fiber tracts on the
ventral surface of the brainstem in embryonic and adult brains and in
cortical radiations and descending tracts from the tectum in adult
brains (Fig 5H and J and data not shown).
Most primitive, yolk sac-derived erythoid cells in E9.5
SCLlacZ/w embryos expressed galactosidase,
although the level of expression varied from cell to cell (Fig 6A).
However, by E12.5, lacZ expression was no longer apparent in the
nucleated, primitive erythrocytes, although some of the earliest
circulating definitive erythocytes transiently retained galactosidase activity (Fig 6B).
Between E10.5 and E11.5, there is a switch from primitive hematopoiesis
in the yolk sac to definitive hematopoiesis in the fetal liver. This
transition is associated with a brief period of definitive
hematopoietic activity in the aorta-gonad-mesonephros region of the
embryo, a derivative of the paraaortic splanchnopleura.36 It is hypothesized that these hematopoietic progenitor cells form intravascular clusters comprising 4 to 15 cells attached to the wall of
the aorta and some of its branches.37 As anticipated, endothelial expression of galactosidase was seen in
SCLlacZ/w embryos, although expression was quite
weak (Fig 6C). In addition, however, there were small aggregates of
lacZ-positive cells along the ventrolateral aortic wall of E10.5 to
E11.5 embryos (Fig 6D). Isolation of these SCL-expressing cells
will be required to determine whether they represent the definitive
hematopoietic precursors in the aorta-gonad-mesonephros region. Strong
galactosidase activity in SCLlacZ/w embryos was
apparent in the fetal liver, the dominant hematopoietic organ from
E11.5 until birth. On histological section, there was intense lacZ
staining of megakaryocytes and weaker expression in erythoid cells (Fig
6E and F).
From E10.5, embryos were large enough to allow a more accurate
quantification of lacZ expression in circulating blood cells through
FACS-Gal analysis. As shown in Fig 7A, yolk
sac-derived blood cells from E10.5 SCLlacZ/w
embryos showed elevated galactosidase activity compared with SCLw/w embryonic blood. As the circulating
primitive erythroid populations became morphologically more mature, the
level of galactosidase activity fell (compare the profiles from
E12.5 with E10.5 to E11.5 in Fig 7A). The subpopulations of
lacZ-positive cells seen in E12.5 and E13.5 blood were shown by
histochemical staining to be enucleated erythrocytes (eg, Fig 6B).
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| Fig 7.
Representative FACS profiles showing galactosidase
activity in (A) E10.5 to E13.5 embryonic blood cells and (B) E11.5 to
E18.5 fetal liver cells from SCLlacZ/w (solid line)
and SCLw/w (dashed line) embryos. (A) galactosidase activity in SCLlacZ/w yolk
sac-derived erythroid cells decreased after E11.5. The major peaks
represent fluorescence derived from primitive erythroid cells and the
minor peaks of high galactosidase activity seen in the E12.5 and
E13.5 samples were in the initial wave of definitive, enucleated
erythrocytes (see Fig 6). Results are representative of analyses
performed on 2 litters of embryos for each time point. (B) In the
SCLlacZ/w fetal livers, the percentage (mean ± standard deviation [SD]) of cells expressing high levels of galactosidase activity at each developmental time point are shown.
Results are representative of analyses performed on 1 to 3 litters of
embryos for each time point.
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The level of galactosidase activity in
SCLlacZ/w fetal liver cells from E11.5 to E18.5 was
determined using the FACS-Gal technique. At E11.5, subsets of fetal
liver hematopoietic cells expressing high and low levels of galactosidase were discernible (Fig 7B). Over time, there was a
progressive decrease in the percentage of lacZ-positive cells. This was
most readily appreciated as a decrease in the percentage of
lacZhigh cells, from approximately 55% of the fetal liver
at E11.5 to approximately 10% at E18.5.
We have previously demonstrated that SCL was expressed in
hematopoietic progenitor cells in the adult mouse.22
Therefore, we determined whether the level of SCL expression in
the fetal liver, inferred by the measurement of galactosidase
activity, could also be used as a criterion to enrich for hematopoietic progenitor cells. The distribution of erythroid and myeloid CFCs in the
lacZhigh and lacZlow fractions from E12.5
SCLlacZ/w fetal livers was determined. This stage
of development represented the earliest time point at which sufficient
numbers of fetal liver cells could be harvested for analysis from
individual embryos. As seen in Fig 8A,
45% of SCLlacZ/w and 0.5% of
SCLw/w in E12.5 fetal liver cells were
lacZhigh. Culture of lacZhigh and
lacZlow fetal liver cells demonstrated significant
enrichment for erythroid and myeloid CFCs in the lacZhigh
compared with the lacZlow fraction (Fig 8B). The enrichment
for erythroid CFCs (10-fold for CFU-E and 20-fold for BFU-E) was
greater than for myeloid CFCs (5-fold) suggesting higher levels of
SCL expression in erythroid rather than myeloid progenitors.
Most CFCs were in the lacZhigh fetal liver fraction,
consistent with our previous findings in the adult bone marrow
(Table 1).
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| Fig 8.
Enrichment for myeloid and erythroid CFCs in the
lacZhigh fraction of fetal livers from
SCLlacZ/w mice. (A) Representative FACS profiles of
forward scatter plotted against galactosidase activity (measured as
fluorescence) for FACS-Gal-labeled E12.5 fetal liver cells from
SCLlacZ/w and SCLw/w embryos.
Sort windows and the percentage of cells in each window are shown for
lacZlow and lacZhigh fractions for each
genotype. (B) Frequency of d2 CFU-E and d7 BFU-E and myeloid (GM) CFC
in cultures of unsorted and sorted fetal liver fractions from
SCLlacZ/w mice cultured in Epo (for d2 CFU-E) or
IL-3/Epo (for d7 BFU-E and GM-CFC). Values represent the mean ± SD
from replicate cultures of 10 fetal livers from 3 litters of embryos.
The percentage of fetal liver cells, which were sorted into each
fraction, is indicated.
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DISCUSSION |
In this study, we have used the SCL-lacZ "knock-in" mice
to accurately delineate SCL-expressing regions in the brain,
hematopoietic tissues, and vasculature throughout embryogenesis.
Linking galactosidase expression to transcription from the
SCL promoter allowed us to use lacZ staining to map the
distribution of cells fated to express SCL in both heterozygous
(SCLlacZ/w) and SCL-null
(SCLlacZ/lacZ) embryos. This has enabled us to
explore in detail the previously identified vascular abnormalities seen
in SCL-null embryos.2,31 A comparison of lacZ
staining in SCLlacZ/w mice with the SCL
expression patterns determined in prior studies validated galactosidase expression as a facsimile for SCL expression and
extended the previous findings.9,28,29,33,38 The expression analysis of SCL homologues in Xenopus laevis and
Danio rerio has shown that SCL is expressed in the
mesodermal precursors of blood and endothelial cells in these species
also,34,35,39,40 suggestive of a conserved role for
SCL in blood formation throughout vertebrate evolution.
LacZ expression in embryonic blood and fetal liver provided additional
insights into the pattern of SCL expression in developing hematopoietic cells. Because SCL expression decreases as
erythroblasts mature,11,12,14 the synchronous fall in galactosidase activity in SCLlacZ/w circulating
primitive erythroblasts after E11.5 was consistent with the hypothesis
that primitive erythropoiesis occurs as a single
"wave."41,42 Similarly, the progressive decline in
galactosidase activity in the developing fetal liver may have reflected its increasing complement of more mature hematopoietic cells
in addition to its decreasing progenitor content.26
Consistent with this, there was a strong correlation between progenitor
cell frequency and lacZ expression in the fetal liver, as previously reported for experiments using adult bone marrow cells.22
A number of studies have documented the presence of definitive
hematopoietic progenitors in the paraaortic splanchnopleura or its
derivative, the aorta-gonad-mesonephros, in the embryo before the
development of fetal liver hematopoiesis.36 It is likely
that the hematopoietic precursor activity resides in clusters of cells
associated with the ventral wall of the aorta and the umbilical artery.
Cells forming these clusters, which have been observed in the
mouse,37,43 human,44-46 and
chick,47 express a coterie of genes associated with
hematopoietic progenitors.36,43,46,48-50 Our data indicates
that murine aortic clusters express SCL. This result
complements a recent in situ hybridization study in human embryos in
which these aorta-associated cells were shown to express SCL.46
The pattern of galactosidase expression in the ventral spinal cord
and the developing midbrain of SCLlacZ/w mice
mirrored the results of earlier studies in mouse
embryos9,28,33 and bore great similarity to the pattern and
kinetics of SCL expression in the central nervous system of
amphibians and teleost fish. 34,35,40 We extended these
studies to late embryonic and postnatal life, demonstrating that the
lacZ-positive sites in the mouse embryonic midbrain developed into the
superior and inferior colliculi, laminated cortical structures
concerned with reflex movements of the eyes and head in response to
visual and auditory stimuli.51,52 It is interesting that
members of several protein classes known to interact with SCL,
notably the GATA proteins,53-56 have overlapping patterns
of expression in the central nervous system. However, delineation of
the precise role played by SCL in neural development awaits the
generation of mutant animals in which SCL is specifically deleted in these cells.
At a gross morphological level, SCL-null embryos developed
normally until the early somite stage,2,3 when circulation between the yolk sac and the embryo is established. However, in the
absence of hematopoietic cells, SCL-null embryos died at
E9.5.2,3 Therefore, there existed only a narrow window
in which the SCLlacZ/lacZ embryos could be
evaluated for developmental defects free from any confounding effects
of hypoxic cell death. The pattern of lacZ staining at E7.5 indicated
that SCL was not required for the migration of mesoderm into
the yolk sac. The yolk sac endothelial cells in precirculation
SCLlacZ/lacZ embryos may have represented the
limited differentiation potential of SCL-null yolk sac
hemangioblasts. The failure of these endothelia to form more than
rudimentary capillary networks provided evidence for a cell-autonomous
requirement for SCL in yolk sac vascular development as
recently suggested by Visvader et al.31
In contrast to the minimal development present in the yolk sac,
vascular development in precirculation SCLlacZ/lacZ
embryos was more extensive. The pattern of lacZ staining in endocardium and in the dorsal aorta, allantoic, and cerebral vascular plexuses matched the vascular expression of PECAM-1 seen in both wild-type and
SCLlacZ/lacZ embryos at this stage. These data
implied that the specification of embryonic angioblasts and their
appropriate differentiation and migration within the embryo to form
vessels were not dependent on the SCL protein.
The vascular abnormalities we have described in this study are similar
to those seen in a number of mouse strains in which genes have been
disrupted by gene targeting. Embryos null for transforming growth
factor (TGF)-1 or its receptor, TGF- RII, formed yolk sacs with
an abnormal primary vascular plexus and reduced hematopoiesis, which
closely mimicked the phenotype of SCL-null
embryos.57,58 As was the case in SCL-knockout
embryos, early embryonic vasculature developed normally. Yolk sac
endothelium was completely absent from embryos lacking the
transcription factor myocyte enhancer factor (MEF)-2C and, in this
instance, embryonic vasculogenesis was also severely perturbed,
although hematopoietic cells were present in the yolk
sac.59 It was interesting that SCLlacZ/lacZ embryos displayed a milder vascular
defect, but more severe hematopoietic abnormality than
MEF2C / embryos. In embryos lacking
FLK-1 (VEGF-R2) or its major ligand, VEGF,
specification of endothelial precursors occurred, but organized blood
vessels were absent and hematopoiesis was markedly
impaired.49,50,60-62 Abnormalities in yolk sac vascular
maturation or remodeling were also prominent in embryos deficient in
the heterodimeric bHLH-PAS transcription factor, hypoxia inducible
factor (HIF)-1,63-65 the endothelial TIE2
receptor,66,67 and its ligand, Ang-1,68 the ETS-family transcription factor, TEL,69 and the major
VEGF receptor expressed on lymphatics, VEGF-R3.70
Similarly, embryos deficient in ephrinB2 or in both
EphB2 and EphB3 receptors displayed lethal defects in
remodeling of yolk sac and cerebral primary vascular
plexuses.71,72 Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on SCL-null embryos showed intact
expression of ephrinB2, and its ligand EphB4 (data not shown).
A striking feature of the SCLlacZ/lacZ embryos (and
many of the other knock-out mice described above) was the greater
severity of the vascular defect in the yolk sac than in the embryo.
This suggests that endothelial precursors in different sites in the
mouse embryo may differ in their requirements for certain growth
factors or transcription factors, including SCL. This may be
analogous to the situation in the chick, in which two populations of
angioblasts, one with and one without hematopoietic potential, have
been described.73,74 However, the precise molecular
pathways, which regulate vascular and hematopoietic specification and
the way in which SCL participates in this process, remains to
be elucidated.
| |
ACKNOWLEDGMENT |
We thank Dr Frank Battye for assistance with cell sorting and Dr Thomas
Sato for providing the anti-PECAM-1 staining method. Koula
Kosmopoulos, Rachel Mansfield, Viki Lapatis, Dora Kaminaris, and Jenny
Parker provided expert technical assistance. We thank the staff of the
Walter and Eliza Hall Institute animal facility for the care of our
mice and Simon Olding for the production of figures.
| |
FOOTNOTES |
Submitted May 17, 1999; accepted July 26, 1999.
Supported by the Lions Special Fellowship (A.G.E.) and the Fraser
Fellowship (C.G.B.) of the Anti-Cancer Council of Victoria, the Sylvia
and Charles Viertel Charitable Foundation (L.R.), the National Health
and Medical Research Council (Canberra), the Australian Federal
Government Cooperative Research Centres Program, and the Bone Marrow
Donor Institute.
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.
Address reprint requests to Andrew G. Elefanty, FRACP PhD, The
Walter and Eliza Hall Institute of Medical Research, PO Royal
Melbourne Hospital, Victoria 3050, Australia; e-mail:
elefanty{at}wehi.edu.au.
| |
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In vivo fate-tracing studies using the Scl stem cell enhancer: embryonic hematopoietic stem cells significantly contribute to adult hematopoiesis
Blood,
April 1, 2005;
105(7):
2724 - 2732.
[Abstract]
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J. R. Gothert, S. E. Gustin, J. A. M. van Eekelen, U. Schmidt, M. A. Hall, S. M. Jane, A. R. Green, B. Gottgens, D. J. Izon, and C. G. Begley
Genetically tagging endothelial cells in vivo: bone marrow-derived cells do not contribute to tumor endothelium
Blood,
September 15, 2004;
104(6):
1769 - 1777.
[Abstract]
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Y. Chen, M. M.W. Chong, R. Darwiche, H. E. Thomas, and T. W.H. Kay
Severe Pancreatitis with Exocrine Destruction and Increased Islet Neogenesis in Mice with Suppressor of Cytokine Signaling-1 Deficiency
Am. J. Pathol.,
September 1, 2004;
165(3):
913 - 921.
[Abstract]
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E. Ravet, D. Reynaud, M. Titeux, B. Izac, S. Fichelson, P.-H. Romeo, A. Dubart-Kupperschmitt, and F. Pflumio
Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis
Blood,
May 1, 2004;
103(9):
3326 - 3335.
[Abstract]
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B. Gottgens, C. Broccardo, M.-J. Sanchez, S. Deveaux, G. Murphy, J. R. Gothert, E. Kotsopoulou, S. Kinston, L. Delaney, S. Piltz, et al.
The scl +18/19 Stem Cell Enhancer Is Not Required for Hematopoiesis: Identification of a 5' Bifunctional Hematopoietic-Endothelial Enhancer Bound by Fli-1 and Elf-1
Mol. Cell. Biol.,
March 1, 2004;
24(5):
1870 - 1883.
[Abstract]
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R. Lahlil, E. Lecuyer, S. Herblot, and T. Hoang
SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression
Mol. Cell. Biol.,
February 15, 2004;
24(4):
1439 - 1452.
[Abstract]
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R. Martin, R. Lahlil, A. Damert, L. Miquerol, A. Nagy, G. Keller, and T. Hoang
SCL interacts with VEGF to suppress apoptosis at the onset of hematopoiesis
Development,
February 1, 2004;
131(3):
693 - 702.
[Abstract]
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M. Gering, Y. Yamada, T. H. Rabbitts, and R. K. Patient
Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1
Development,
December 22, 2003;
130(25):
6187 - 6199.
[Abstract]
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N. Minegishi, N. Suzuki, T. Yokomizo, X. Pan, T. Fujimoto, S. Takahashi, T. Hara, A. Miyajima, S.-i. Nishikawa, and M. Yamamoto
Expression and domain-specific function of GATA-2 during differentiation of the hematopoietic precursor cells in midgestation mouse embryos
Blood,
August 1, 2003;
102(3):
896 - 905.
[Abstract]
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N. Byrd, S. Becker, P. Maye, R. Narasimhaiah, B. St-Jacques, X. Zhang, J. McMahon, A. McMahon, and L. Grabel
Hedgehog is required for murine yolk sac angiogenesis
Development,
March 3, 2003;
129(2):
361 - 372.
[Abstract]
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M. Ema, P. Faloon, W. J. Zhang, M. Hirashima, T. Reid, W. L. Stanford, S. Orkin, K. Choi, and J. Rossant
Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse
Genes & Dev.,
February 1, 2003;
17(3):
380 - 393.
[Abstract]
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A. H. Hart, L. Hartley, K. Sourris, E. S. Stadler, R. Li, E. G. Stanley, P. P. L. Tam, A. G. Elefanty, and L. Robb
Mixl1 is required for axial mesendoderm morphogenesis and patterning in the murine embryo
Development,
August 1, 2002;
129(15):
3597 - 3608.
[Abstract]
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A. M. Sinclair, A. J. Bench, A. J. C. Bloor, J. Li, B. Gottgens, M. L. Stanley, J. Miller, S. Piltz, S. Hunter, E. P. Nacheva, et al.
Rescue of the lethal scl-/- phenotype by the human SCL locus
Blood,
May 13, 2002;
99(11):
3931 - 3938.
[Abstract]
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B. Gottgens, L. M. Barton, M. A. Chapman, A. M. Sinclair, B. Knudsen, D. Grafham, J. G.R. Gilbert, J. Rogers, D. R. Bentley, and A. R. Green
Transcriptional Regulation of the Stem Cell Leukemia Gene (SCL) --- Comparative Analysis of Five Vertebrate SCL Loci
Genome Res.,
May 1, 2002;
12(5):
749 - 759.
[Abstract]
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Y. S. Chung, W. J. Zhang, E. Arentson, P. D. Kingsley, J. Palis, and K. Choi
Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression
Development,
January 12, 2002;
129(23):
5511 - 5520.
[Abstract]
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M.-J. Sanchez, E.-O. Bockamp, J. Miller, L. Gambardella, and A. R. Green
Selective rescue of early haematopoietic progenitors in Scl-/- mice by expressing Scl under the control of a stem cell enhancer
Development,
December 1, 2001;
128(23):
4815 - 4827.
[Abstract]
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A. W. Roberts, L. Robb, S. Rakar, L. Hartley, L. Cluse, N. A. Nicola, D. Metcalf, D. J. Hilton, and W. S. Alexander
Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3
PNAS,
July 31, 2001;
98(16):
9324 - 9329.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION