© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
FOXF1 inhibits hematopoietic lineage commitment during early
mesoderm specification
ABSTRACT
The molecular mechanisms orchestrating early mesoderm
specification are still poorly understood. In particular, how alternate
cell fate decisions are regulated in nascent mesoderm remains mostly
unknown. In the present study, we investigated both in vitro in
differentiating embryonic stem cells, and in vivo in gastrulating
embryos, the lineage specification of early mesodermal precursors
expressing or not the Forkhead transcription factor FOXF1. Our data
revealed that FOXF1-expressing mesoderm is derived from FLK1+
progenitors and that in vitro this transcription factor is expressed in
smooth muscle and transiently in endothelial lineages, but not in
hematopoietic cells. In gastrulating embryos, FOXF1 marks most
extra-embryonic mesoderm derivatives including the chorion, the
allantois, the amnion and a subset of endothelial cells. Similarly to the
in vitro situation, FOXF1 expression is excluded from the blood
islands and blood cells. Further analysis revealed an inverse
correlation between hematopoietic potential and FOXF1 expression
in vivo with increased commitment toward primitive erythropoiesis in
Foxf1-deficient embryos, whereas FOXF1-enforced expression
in vitro was shown to repress hematopoiesis. Altogether, our data
establish that during gastrulation, FOXF1 marks all posterior primitive
streak extra-embryonic mesoderm derivatives with the remarkable
exception of the blood lineage. Our study further suggests that this
transcription factor is implicated in actively restraining the
specification of mesodermal progenitors to hematopoiesis.
KEY WORDS: Gastrulation, Hemangioblast, Mesoderm
INTRODUCTION
At the early stages of embryonic development, the multipotent
epiblast gives rise to the three germ layers which are subsequently
specified to form all tissues of the developing organism (Tam and
Loebel, 2007). The initiation of germ layer formation occurs
through a process known as gastrulation in which cells from the
epiblast undergo an epithelial to mesenchymal transition and
ingress through the primitive streak to generate mesoderm and
endoderm (Tam and Behringer, 1997). Cells exiting the posterior
part of the primitive streak give rise to the extra-embryonic and
lateral plate mesoderm. In gastrulating embryos, the extraembryonic mesoderm contributes to the formation of the yolk sac
blood islands (Palis et al., 1995), the allantois, the amnion, the
1
Cancer Research UK Stem Cell Hematopoiesis Group, Cancer Research UK
Manchester Institute, The University of Manchester, Wilmslow Road, Manchester
2
M20 4BX, UK. Department of Chemistry and Molecular Biology, University of
3
Gothenburg, Gothenburg 40530, Sweden. Cancer Research UK Stem Cell Biology
Group, Cancer Research UK Manchester Institute, The University of Manchester,
Wilmslow Road, Manchester M20 4BX, UK.
*Authors for correspondence ([email protected];
[email protected])
Received 24 March 2015; Accepted 11 August 2015
chorion and the mesothelium lining of the exocoelomic cavity (Tam
et al., 1997). Fusion of the allantois and chorion later establishes the
connection between the placenta and the embryo, forming the
umbilical cord (Downs, 1998). Endothelial and smooth muscle
lineages forming the vasculature and both the endocardium and
myocardium of the heart are also derived from the mesoderm
emerging from the posterior primitive streak (Kinder et al., 2001,
1999). Some of these early events of embryonic development can be
recapitulated in vitro upon the differentiation of pluripotent
embryonic stem cells (ESCs), providing an alternative model
system to the in vivo settings for the study of cell fate specification
during development (Keller, 2005; Lacaud et al., 2004). The
molecular mechanisms controlling the specification of mesoderm
are starting to be unraveled but the overall transcriptional network
orchestrating this process is still poorly understood. In particular,
how alternate cell fates are regulated in nascent mesoderm is mostly
unknown.
The specification of blood and endothelial lineages is tightly
associated, depending on similar signaling pathways and
transcription factors for their emergence (Costa et al., 2012a;
Moignard et al., 2013). Soon after gastrulation, the hemangioblast,
found within the primitive streak, gives rise to blood precursors
through a specific population of endothelial cells with hemogenic
properties, i.e. the hemogenic endothelium (Eilken et al., 2009;
Huber et al., 2004; Lancrin et al., 2009b). Whereas it is clear that not
all endothelial cells are derived from hemangioblast precursors, it is
still not understood how a specific subset of mesoderm becomes
committed to the hematopoietic fate. The FLK1-VEGF signaling
axis and the ETS transcription factor ETV2 have been shown to be
essential very early in mesoderm for the generation of both
endothelial and hematopoietic lineages (Kataoka et al., 2011; Lee
et al., 2008; Shalaby et al., 1995; Wareing et al., 2012b). However,
both FLK1 (KDR – Mouse Genome Informatics) and ETV2 are
widely expressed in nascent mesoderm and are unlikely to be
responsible for restricting mesoderm specification to blood and/or
endothelium fate. Directly regulated by ETV2, the bHLH
transcription factor SCL (TAL1 – Mouse Genome Informatics) is
required for both primitive and definitive hematopoiesis through its
control of hemogenic endothelium formation (Lancrin et al., 2009b;
Porcher et al., 1996; Wareing et al., 2012b). Specification to the
cardiac tissues was shown to depend on a distinct set of
transcriptional regulators. The heart is composed of several
lineages, including cardiomyocytes, endothelial and smooth
muscle cells and is formed by two sources of progenitors derived
from the first heart field formed by the cardiac crescent and the
second heart fields derived from pharyngeal mesoderm (Olson,
2006). The transcription factor MESP1, a master regulator of
cardiac development, is expressed in the primitive streak, and is the
earliest marker of cardiogenesis (Saga et al., 1996, 2000). Upon
enforced expression, MESP1 promotes the generation of all cardiac
lineages, controlling the downstream cascade of transcriptional
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Maud Fleury1, Alexia Eliades1, Peter Carlsson2, Georges Lacaud3, * and Valerie Kouskoff1, *
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
regulators including NKX2.5, GATA4, HAND1/2 and MEF2C
(Bondue et al., 2008; Lindsley et al., 2008). In contrast, little is
known about the specification of the mesoderm to the mesothelium
that forms an epithelial monolayer lining the exocoelomic cavity in
early embryos and all body cavities and organs later in life
(Mutsaers, 2004). Throughout development and in the adult
organism, the mesothelium is implicated in the control of fluid
and solute transport, and in the regulation of inflammation and
wound healing. Apart from its mesodermal origin, nothing is known
about the molecular control underlying the specification of this
lineage that shares a common mesodermal origin with the
cardiovascular system. Overall, the molecular mechanism that
regulates the allocation and patterning of extra-embryonic
mesoderm to the various lineages of the cardiovascular system
and mesothelium is still unknown. How MESP1, ETV2, SCL and
other transcriptional regulators are integrated into a network
activating or repressing cell fate decisions remains to be defined.
Forkhead box (FOX) transcription factors belong to an
evolutionary conserved family of proteins named after the
Drosophila forkhead gene whose mutation results in a spiked
head phenotype. There are 44 FOX proteins in the mouse genome,
all characterized by a forkhead DNA-binding motif, and assembled
into 19 subgroups based on sequence homologies inside and outside
the forkhead domain (Jackson et al., 2010). FOX proteins are
implicated in multiple key aspects of embryonic development and
adult homeostasis ranging from proliferation and differentiation to
metabolism and senescence. FOXF1, one of the two members of the
FOXF subgroup, is widely expressed in extra-embryonic and lateral
plate mesoderm (Kalinichenko et al., 2003; Mahlapuu et al., 2001b)
and its deletion leads to early embryonic lethality with a complete
absence of vasculogenesis and mis-expression of hematopoietic and
vascular markers (Mahlapuu et al., 2001b). The vasculogenesis
defect can be restored to some level in explant cultures by BMP4
addition, indicating that the BMP signaling axis acts downstream
of FOXF1 (Astorga and Carlsson, 2007). At the late stage of
embryonic development, heterozygosity or mutation in FOXF1 is
linked to lung malformation and alveolar capillary dysplasia
involving the abnormal development of the vascular capillary
system in lungs (Kalinichenko et al., 2001; Mahlapuu et al., 2001a;
Stankiewicz et al., 2009). Given its expression pattern and null
phenotype, we became interested in the potential role of FOXF1
during mesoderm specification to the cardiovascular system. In
order to precisely characterize its expression, to track and isolate
FOXF1-expressing mesoderm, we generated ESCs and transgenic
mouse lines carrying a Foxf1-venus knock-in allele. In the present
study, using both in vitro and in vivo systems, we investigated when
and where FOXF1 is expressed at the early stages of mesoderm
development and how FOXF1 expression might contribute to
mesoderm specification during gastrulation.
analysis of Foxf1::venus ESCs and differentiated embryoid bodies
(EBs) cells showed no significant alteration in hematopoietic
output and expression of a panel of selected genes when compared
to wild-type ESCs (supplementary material Figs S1C and S2). EB
cells were next analyzed by flow cytometry for the presence of
FLK1 and FOXF1::VENUS expression. Upon differentiation,
FOXF1::VENUS was first detected within the mesodermal
FLK1+ population (Fig. 1B), and within 12 h of culture a FLK1−
FOXF1::VENUS+ population appeared and was maintained upon
further differentiation (supplementary material Fig. S3, upper
panels). To delineate how FOXF1::VENUS+ cells were generated,
three subpopulations were sorted according to their relative
expression of FLK1 and FOXF1::VENUS then reaggregated and
further cultured for up to 48 h (Fig. 1C). The reaggregated cells
were then analyzed by flow cytometry at different times during
the culture. The sorted FLK1+ FOXF1::VENUS+ cells almost
exclusively retained their expression of FOXF1::VENUS during the
culture, while progressively downregulating FLK1 expression
(Fig. 1C). A large fraction of FLK1+ FOXF1::VENUS− cells
quickly gave rise to FOXF1::VENUS+ cells which then downregulated
FLK1 expression, hence resulting in a phenotypic subpopulation
similar to that obtained from the FLK1+ FOXF1::VENUS+. Finally,
the cell population negative for both markers first upregulated FLK1
expression followed by the upregulation of FOXF1::VENUS within
the FLK1-expressing cells. Together these data suggest an in vitro
progression in which mesoderm cells first express FLK1 and then
upregulate FOXF1 expression.
We next assessed the expression of FOXF1::VENUS upon
further commitment of the FLK1+ population toward the
hematopoietic and endothelial lineages. To this end, sorted
FLK1+ mesodermal cells were further differentiated for 4 days in
VEGF-containing hemangioblast culture conditions which promote
the differentiation to endothelium and hematopoiesis (Fig. 1D).
Although FOXF1::VENUS+ cells were detected from day 1 of the
culture and remained present throughout the culture period, the vast
majority of these cells did not express the hematopoietic marker
CD41 (integrin α2b – Mouse Genome Informatics) (Ferkowicz
et al., 2003; Mikkola et al., 2003) or the endothelial marker CD144
(cadherin 5 – Mouse Genome Informatics) (Fig. 1D; gating controls
supplementary material Fig. S4A). Similar results were observed for
CD41 expression when FOXF1::VENUS expression was followed
in differentiating EBs for up to day 7 (supplementary material
Fig. S3, lower panels). Altogether, these data reveal that, upon
in vitro differentiation FOXF1::VENUS+ cells are generated from
the FLK1+ mesodermal population and that this transcription factor
is not expressed in hematopoietic cells. Surprisingly, FOXF1::
VENUS-expressing cells represent a large fraction of the
differentiated cells within the hemangioblast culture or whole EB
culture.
RESULTS
FOXF1 expression is initiated within the early FLK1+
mesoderm population during in vitro differentiation
FOXF1 expression defines a non-hemogenic vascular
progenitor population within the FLK1+ mesoderm
population in vitro
To track the expression of FOXF1 at the earliest stage of mesoderm
commitment, we generated an ESC line carrying the H2B-VENUS
reporter protein under the control of Foxf1 regulatory sequences
(hereafter referred to as FOXF1::VENUS), through knocking in a
reporter cassette in the first exon of the Foxf1 allele (Fig. 1A;
supplementary material Fig. S1A,B). This ESC line allowed
monitoring the emergence of Foxf1 expression in conjunction
with cell surface markers upon in vitro differentiation to
hematopoietic, endothelial and smooth muscle lineages. The
To explore the biological characteristics of FOXF1-expressing
cells, FLK1+ FOXF1::VENUS+ and FLK1+ FOXF1::VENUS−
populations were sorted from day 2.5 EBs and analyzed for the
expression of genes known to be involved in lineage decisions
during mesoderm differentiation. As shown in Fig. 2A, whilst as
expected the expression of Foxf1 was enriched in the FOXF1::
VENUS+ population, the expression of genes involved in
hemogenic endothelial specification (Scl, Gata2, Sox7) (Costa
et al., 2012b; Pimanda et al., 2007) were enriched in the FOXF1::
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Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
VENUS− population compared to FOXF1::VENUS+. Similarly,
genes involved in cardiac lineage cell fate decision (Nkx2-5 and
Mesp1) (Chen and Fishman, 1996; Saga et al., 1999) were more
predominantly expressed in the cell population negative for FOXF1
expression. We also assessed the hematopoietic output of these
populations through differentiation in hemangioblast culture
followed by flow cytometry and clonogenic analysis. Upon
differentiation, FLK1+ FOXF1::VENUS− cells produced
hematopoietic cells as demonstrated by the generation of a high
frequency of CD41+ cells (Fig. 2B, upper panels; gating controls
supplementary material Fig. S4B). This population also produced
FOXF1::VENUS+ cells, but those FOXF1-expressing cells were
negative for CD41 expression. In contrast, the sorted FLK1+
FOXF1::VENUS+ cells produced very few CD41+ cells and mostly
maintained their expression of FOXF1::VENUS upon further
differentiation (Fig. 2B, lower panels). To define the clonogenic
potential of the cells generated during this culture period, cells were
harvested after 2 days of culture and assessed for progenitor
potential in semi-solid hematopoietic condition. Whereas cells
derived from the FLK1+ FOXF1::VENUS− culture generated
hematopoietic colonies, cells derived from the FLK1+ FOXF1::
VENUS+ culture produced very few colonies (Fig. 2C). During the
hemangioblast culture, both populations gave rise to CD144+
endothelial cells and αSMA+ smooth muscle cells (Fig. 2D),
although the FLK1+ FOXF1::VENUS+ cells produced a higher
frequency of αSMA+ cells and a lower frequency of CD144+ cells
than the FLK1+ FOXF1::VENUS− population. Taken together,
these results reveal that FOXF1 expression marks a non-hemogenic
population within the FLK1+ mesodermal population generated
during ESC differentiation in vitro. These FOXF1-expressing
mesoderm cells display a vascular-restricted differentiation
potential, generating both endothelial and smooth muscle cells
but no or very few hematopoietic cells upon culture.
To also characterize the population of FOXF1::VENUS+ cells
observed at later stage of EB differentiation, co-staining with
various cell surface markers was performed at EB day 6 (Fig. 3A).
This analysis revealed that most FOXF1::VENUS+ cells had
downregulated FLK1 expression by day 6 of EB differentiation
whereas CD140a (PDGRFα) and TIE2 were highly expressed by
FOXF1::VENUS+ cells. Gene expression analysis further revealed
high levels of both smooth muscle markers Sma (Acta2) and Sm22
[transgelin (Tagln)] in the FOXF1::VENUS+ population whereas
hematopoietic genes (Gata1, Gata2) were not expressed in this
fraction (Fig. 3B). Finally, upon further culture FOXF1::VENUS+
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DEVELOPMENT
Fig. 1. FOXF1 expression is initiated within
the early FLK1+ mesoderm population
during in vitro differentiation. (A) Graphical
representation of Foxf1 WT allele and Foxf1::
venus targeted allele. H2b-venus was
knocked in at the first exon of the Foxf1 gene.
(B) Foxf1::venus ESCs were differentiated as
embryoid bodies (EBs) and analyzed by flow
cytometry for the expression of FOXF1::
VENUS and FLK1 at different stages of
differentiation. (C) At day 2 (d2.0) of
differentiation, EBs were dissociated and
sorted based on their expression of FLK1 and
FOXF1::VENUS, then cultured as
reaggregates. Aggregates were analyzed by
flow cytometry for FLK1 and FOXF1::VENUS
expression at 12 h, 24 h or 48 h after
reaggregation. (D) FLK1+ cells from day 2.5
EBs were sorted and differentiated on
gelatinized plates in hemangioblast medium.
Cells were harvested every day and analyzed
by flow cytometry for the expression of
FOXF1::VENUS and the hematopoietic
marker CD41 (top row) or the endothelial
marker CD144 (bottom row). All data are
representative of at least 3 independent
experiments.
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Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
cells isolated from day 6 EBs rapidly gave rise to smooth muscle
cells as demonstrated by extensive αSMA staining (Fig. 3C).
Together, these data suggest that in late differentiating EBs Foxf1
expression marks cells fated to the smooth muscle lineage.
FOXF1 is expressed in all extra-embryonic tissues in
gastrulating embryos except in the blood islands
To define the expression pattern of FOXF1 during gastrulation and
to compare in vitro and in vivo data, the Foxf1::venus ESC line was
used to generate a Foxf1::venus transgenic mouse line. At the early
stage of embryonic development ( primitive streak to headfold
stage), FOXF1::VENUS was detected in the extra-embryonic
mesoderm and the lateral plate mesoderm of the embryo proper
(Fig. 4A) as previously documented using in situ hybridization
(Mahlapuu et al., 2001b; Peterson et al., 1997). In particular,
FOXF1::VENUS was homogenously expressed in the mesothelium
lining the exocoelomic cavity and the amnion as well as in the
allantois but FOXF1::VENUS was not detected in the nascent
mesoderm of the primitive streak. To define the progressive
changes in FOXF1 expression during gastrulation and to correlate
this pattern with the in vitro data presented above, sequential stages
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of gastrulation, as previously defined (Downs and Davies, 1993),
were analyzed by flow cytometry for FLK1 and FOXF1::VENUS
expression (Fig. 4B and supplementary material Fig. S5A). At the
early streak (ES) stage, the first FOXF1::VENUS-expressing cells
were detected within the FLK1+ population, similar to the onset of
FOXF1 expression during in vitro differentiation. From the midstreak (MS) stage, FLK1− FOXF1::VENUS+ cells were detected
and both FLK1+ FOXF1::VENUS+ and FLK1− FOXF1::VENUS+
populations increased progressively as gastrulation proceeded to
late streak (LS) and neural plate (NP) stages. At the NP stage (OB:
no allantoic bud, EB: early allantoic bud) and early headfold (EHF)
stage, most of the FLK1+ cells co-expressed FOXF1::VENUS,
whereas at the later headfold stage (LHF) a clear FLK1+ FOXF1::
VENUS− population was observed. To define the anatomical
localization of these distinct populations, neural plate Foxf1::venus
embryo sections were stained for FLK1 expression (Fig. 4C).
The few double positive cells were found spread throughout the
extra-embryonic mesoderm, allantois and embryonic mesoderm
whereas most of the FOXF1::VENUS+ negative for FLK1
expression were detected in the mesothelium and allantois
(Fig. 4Ci-iii).
DEVELOPMENT
Fig. 2. FOXF1 expression defines a non-hemogenic vascular progenitor population within the FLK1+ mesoderm population in vitro. (A) Gene expression
analysis (quantitative RT-PCR) of day 2.5 EB cells sorted based on FLK1 and FOXF1::VENUS expression (n=3, mean±s.e.m., P-values were obtained using a
paired t-test.). (B,D) Day 2.5 EB cells were sorted based on FLK1 and FOXF1::VENUS expression and differentiated on gelatinized plates in hemangioblast
medium. Cells were harvested every day and analyzed by flow cytometry for the expression of FOXF1::VENUS and the hematopoietic marker CD41 (B), the
endothelial marker CD144 (D, left panel) or the smooth muscle marker αSMA (D, right panel). (C) At day 2 of differentiation, cells were harvested and seeded in
semi-solid hematopoietic medium. Hematopoietic colonies were scored after 4 days (n=4, each point represents an independent experiment, mean±s.d.).
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Of interest, no cells within the developing blood islands
expressed detectable levels of FOXF1::VENUS (Fig. 4Ciii,iv),
whereas FLK1+ FOXF1::VENUS+ endothelial cells were detected
at the endoderm interface of the blood islands in older embryos
(Fig. 4Civ, arrowheads). Flow cytometry analysis showed that
a very small frequency of CD41+ cells co-expressed FOXF1::
VENUS+ (supplementary material Fig. S5B) raising the possibility
that FOXF1 might be expressed in some of the mesoderm
precursors giving rise to hematopoiesis, although the bulk of
CD41+ cells did not express FOXF1::VENUS. To determine
whether Foxf1-expressing mesoderm was under the same molecular
control which specifies the mesodermal progenitors giving rise to
the hematopoietic and vascular system, we next analyzed FOXF1::
VENUS expression in the context of Etv2 deficiency. We and others
have previously shown that the ETS transcription factor ETV2 is
expressed in mesodermal progenitors which give rise to both
hematopoietic and vascular system, and that Etv2−/− embryos lack
both lineages (Kataoka et al., 2011; Lee et al., 2008; Wareing et al.,
2012b). Whilst FLK1+ cells are still detected at the expected
frequency in Etv2−/− embryos, the TIE2+ population is drastically
reduced (Wareing et al., 2012a); we therefore analyzed the
co-expression of FOXF1::VENUS and TIE2 in wild-type,
heterozygous or homozygous null Etv2 embryos (Fig. 4D). In
ETV2-expressing embryos, both TIE2+ FOXF1::VENUS+ and
TIE2− FOXF1::VENUS+ populations were detected. In contrast, in
Etv2−/− embryos the TIE2+ FOXF1::VENUS+ population was
strongly reduced whereas the TIE2− FOXF1::VENUS+ population
was present at the expected frequency. Together these data reveal
that most of the FOXF1-expressing mesoderm is specified
independently of ETV2 and the hematopoietic and endothelial
lineages. However, the small fraction of FOXF1::VENUS+ cells that
co-express TIE2 is dependent on ETV2, suggesting that some
mesoderm progenitors giving rise to the hematopoietic and
endothelial lineages do express FOXF1 during their ontogeny. As
we observed in vitro that a large fraction of the FOXF1::VENUS+
population co-expressed the smooth muscle marker αSMA
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Fig. 3. FOXF1::VENUS+ cells in day 6 EBs are committed to the smooth muscle lineage. (A) Representative flow cytometry analysis of day 6 embryoid body
(EB) cells from wt and Foxf1::venus ESCs for the indicated cell surface markers. (B) qPCR analysis of sorted FOXF1::VENUS– and FOXF1::VENUS+
day 6 EB cells for the indicated genes. Each point represents an independent sorted cell population (n=4). PCR data were normalized to β-actin levels.
(C) Immunofluorescence staining for αSMA (red) and DAPI (blue) of FOXF1::VENUS+ cells sorted from day 6 EBs and cultured for 3 days in the presence of
VEGF and FGF. Scale bar: 100 µm. All data are representative of 4 independent experiments.
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(Fig. 2D), we assessed in vivo the co-expression of FOXF1 and
αSMA during gastrulation. At the early headfold stage, αSMA
expression was mainly detected in the chorion where it overlapped
with FOXF1::VENUS expression (Fig. 5A). At the late headfold
and early somite stage, αSMA expression was now also observed in
the mesothelium lining the exocoelomic cavity and the prospective
heart field (Fig. 5B-D). At these stages, FOXF1::VENUS and
αSMA were clearly coexpressed in the mesothelium; a few αSMA+
FOXF1::VENUSlow were also observed in the presumptive cardiac
mesoderm (Fig. 5C-D).
Overall, we observed a similar pattern of FOXF1 emergence
in vivo and in vitro with FOXF1::VENUS expression first detected in
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emerging FLK1+ mesodermal cells followed by a progressive
increase in the frequency of FOXF1::VENUS+ cells that do not
express FLK1. Most cells of the allantois and all the mesothelium
were FLK1− FOXF1::VENUS+. Interestingly, although FOXF1 was
not detected within the blood islands giving rise to hematopoiesis, a
few CD41+ cells expressed FOXF1::VENUS.
Increasing Foxf1 expression levels correlate with
decreasing hematopoietic potential in vivo
To further characterize the FOXF1::VENUS expressing and nonexpressing populations in vivo, cells from pooled E7.5 NP stage
embryos were sorted according to their relative expression
DEVELOPMENT
Fig. 4. Analysis of FOXF1::VENUS expression in gastrulating Foxf1::venus+ embryos. (A) Whole mount staining of VENUS (green) in embryos at the
primitive streak (PS), neural plate (NP) and headfold (HF) stages showed that FOXF1::VENUS is expressed in the lateral plate and extra-embryonic tissues. Blue
is DAPI counterstaining. Scale bars: 100 µm. (B) At each stage of gastrulation, embryos were dissociated and analyzed by flow cytometry for the expression of
FLK1 and FOXF1::VENUS. ES, early streak; MS, mid-streak; LS, late streak; NP/OB, neural plate/no bud; NP/EB, neural plate/early bud; EHF, early headfold;
LHF, late headfold. (C) Immunostaining of VENUS (green) and FLK1 (red) in NP embryos. (i) Sagittal section of an early NP embryo and higher magnification of
the anterior (ii) and posterior (iii) region. (iv) Sagittal section of the posterior part of a late NP embryo. Blue is DAPI counterstaining. Scale bars: 100 µm. (D) Flow
cytometry analysis of FOXF1::VENUS and TIE2 expression in double transgenic Foxf1::venus + Etv2 +/+, Foxf1::venus + Etv2 +/− and Foxf1::venus + Etv2 −/− E7.5
NP embryos. (A,C) Abbreviations: al, allantois; am, amnion; BI, blood island; e, embryo proper; x, extra-embryonic region; xc, exocoelomic cavity.
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of FLK1 and FOXF1::VENUS (Fig. 6A) and processed for
transcriptomic analysis. Interestingly, despite VENUS detection
by flow cytometry, Foxf1 transcripts in the FLK1+ FOXF1::
VENUS+ population were detected by Affymetrix array at the same
background level as in the FLK1+ FOXF1::VENUS− population,
whereas the Foxf1 transcripts were detected at a high level in the
FLK1− FOXF1::VENUS+ population (Fig. 6B). To investigate this
discrepancy in Foxf1 transcript levels, qPCR analysis was
performed on single cells sorted from primitive streak (PS), neural
plate (NP) and headfold (HF) embryos. Although FLK1+ FOXF1::
VENUS− cells expressed very low or no detectable Foxf1 transcripts
(Fig. 6C), the expression of Foxf1 in FLK1+ FOXF1::VENUS+ cells
was highly heterogeneous across the gastrulating stages analyzed.
At the PS stage, Foxf1 transcripts were detected at similar levels in
FOXF1::VENUS cells expressing or not FLK1 but at the later NP
and HF stages, an increasing frequency of FLK1+ FOXF1::
VENUS+ cells expressed low or no detectable levels of Foxf1
transcripts (Fig. 6C). In contrast, Foxf1 transcripts remained overall
at a higher level in FLK1− FOXF1::VENUS+ cells when compared
to FLK1+ FOXF1::VENUS+ cells in both NP and HF stages.
Together these data demonstrate that FLK1+ FOXF1::VENUS+
cells are only transiently expressing Foxf1. As expected, single cell
analysis is more sensitive than global transcriptomic arrays in
detecting small differences and heterogeneity in cell populations.
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Fig. 5. Co-expression of FOXF1::VENUS and αSMA in gastrulating Foxf1::venus embryos. Whole mount staining of VENUS (green) and αSMA (red) in
embryos ranging from early headfold stage (A), late headfold stage (B,C), to early somite stage (D). Left panel shows overlay of 5 consecutive images. z-stack is
5 µm in total. Scale bars: 100 µm. The right panels show higher magnification of the boxed areas in the left panels. Scale bars: 25 µm. Blue is DAPI
counterstaining. Abbreviations: al, allantois; am, amnion; BI, blood island; ch, chorion; h, heart; xc, exocoelomic cavity.
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
Fig. 6. Characterization of FOXF1/FLK1-expressing subpopulations in gastrulating embryos. (A) Representative dot plot of pooled E7.5 Foxf1::venus
(NP stage) embryos sorted according to their expression of FOXF1::VENUS and FLK1. (B) Affymetrix array-derived log2 expression values of Foxf1 transcript
in the sorted populations. (C) qPCR analysis of Foxf1 transcript levels in single cells sorted from gastrulating embryos at the indicated stage. Each point represents
an individual cell, PCR data were normalized relative to β-actin levels (error bars represent mean±s.d.). (D) E7.5 embryos were sorted based on the expression of
FLK1 and FOXF1::VENUS and cells were processed for transcriptome analysis (n=2 biological samples). Heatmap represents hierarchical clustering of
differentially expressed genes (fold change >2 between at least 2 populations, average value of biological samples). The most informative function annotations
terms for each cluster are indicated on the right. (E) Heatmaps of selected genes from the corresponding clusters shown in (D). (F-H) E7.5 embryos (NP stage)
were dissociated and sorted based on the expression of FLK1 and FOXF1::VENUS and cultured on OP9 stroma. After 2 days of differentiation, cells were
harvested and analyzed by flow cytometry for the expression of the hematopoietic markers CD41 and CD45 (upper panel), or the endothelial marker CD144
(lower panel). In F, OP9 cells are gated out. After 3 days of differentiation, cells were harvested and seeded in semi-solid hematopoietic medium. Hematopoietic
colonies were scored after 4 days. G,H: Mac, macrophage colonies; Ery/Mac, primitive erythroid and macrophage colonies; Ery, primitive erythroid colonies; e.e.,
equivalent embryo. n=5. Mean±s.e.m. is shown in C and G, P-values were obtained using a paired t-test.
In the transcriptomic analysis, differentially expressed genes with
2-fold or more changes in expression levels between at least two of
the sorted populations were clustered and submitted to Gene
3314
Ontology annotation to highlight enriched biological functions
within each population (Fig. 6D; supplementary material Table S1).
This analysis identified three main clusters of differentially
DEVELOPMENT
RESEARCH ARTICLE
expressed genes: cluster i containing genes with increased
expression in FLK1+ FOXF1::VENUS− cells, cluster ii containing
genes with increased expression in FLK1+ FOXF1::VENUS− and
FLK1+ FOXF1::VENUS+ cells and cluster iii containing genes with
increased expression in FLK1− FOXF1::VENUS+ cells. However,
the Gene Ontology analysis revealed important overlap within the
biological functions attributed to each of these clusters (Fig. 6D).
Most notably, genes found in clusters i and ii were enriched for
‘hematopoiesis’ function whereas genes found in all three clusters
were enriched for ‘development of blood vessel’ function. Results
of this analysis highlighted the close relationship and common
mesodermal origin between the populations analyzed. Nevertheless,
a close inspection of specific genes differentially expressed within
each cluster revealed important insights into the molecular identity
of these three embryonic populations. Cluster i, which contained
genes with the highest expression in the FLK1+ FOXF1::VENUS−
population, included many genes directly implicated in
erythropoiesis (Fig. 6Ei) such as Hbb-bh1, Hbb-y, Hbb-b2, Klf1,
Gata1, Nfe2, all expressed in primitive erythroid cells, the first
blood cells to be specified from the mesoderm (Kingsley et al.,
2013; Palis, 2008). In contrast, cluster ii genes, enriched in both
FLK1+ populations, comprised many genes encoding known
regulators of endothelial and hematopoietic development
(Fig. 6Eii) such as Egfl7, Etv2, Fli1, Foxc2, Gata2, Hhex or
Sox17 (De Val et al., 2008; Kubo et al., 2005; Pimanda et al., 2007;
Wareing et al., 2012a), suggesting a common hematopoietic and
endothelial potential in these two populations. Finally, genes found
in cluster iii, for which expression was highest in the FLK1−
FOXF1::VENUS+ population, included many Hox genes
(Fig. 6Eiii), suggesting that this population contained mesoderm
fated to other developmental pathways as suggested by the Gene
Ontology analysis. This cluster was also enriched for genes related
to muscle and contractile function (Acta1, Sma, Cnn2, Myl9, Tagln,
Tnnc1, Tnni2), most likely linked to the αSMA-expressing
mesothelial subset of the cells within this population.
To evaluate the hematopoietic and endothelial potential of these
three populations, sorted cells from E7.5 embryos (NP stage) were
cultured on OP9 stroma to allow differentiation toward these two
lineages. Flow cytometry analysis revealed that to some extent all
sorted populations could generate CD41+ cells during the culture,
although only the FLK1+ FOXF1::VENUS− cells displayed robust
hematopoiesis producing high frequencies of CD41+/CD45+ cells.
The FLK1+ FOXF1::VENUS+ cells could produce some CD41+
CD45+ cells and FLK1− FOXF1::VENUS+ cells produced very
limited frequencies of CD41+ cells only (Fig. 6F, upper panel). This
differential hematopoietic potential was further confirmed using
clonogenic assay which showed that hematopoietic progenitors
were enriched in the FLK1+ FOXF1::VENUS−-derived cells
(mean±s.e.m.=23.4±9.2 colonies per cultured equivalent embryo)
compared with the FLK1+ FOXF1::VENUS+ cells (7.1±3.5),
whereas the FLK1− FOXF1::VENUS+ population was devoid of
hematopoietic progenitors (0.2±0.1) (Fig. 6G). No significant
differences in lineage output were observed between the two FLK1+
populations (Fig. 6H). Similarly, all populations displayed
endothelium potential as shown by CD144 staining (Fig. 6F,
lower panel), although the frequency of CD144+ cells generated was
inversely correlated with the expression of FOXF1::VENUS.
Altogether, these data revealed that at this early stage of
embryonic development the relative expression of FLK1 and
FOXF1 delineates specific subsets of mesoderm cells. The absence
of FOXF1 expression in FLK1+ cells marks a population of cells
highly enriched for both endothelium and hematopoietic potential
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
and also contains the population of mesodermal progenitors already
committed to primitive erythropoiesis. The transient expression of
FOXF1 marks a population of FLK1+ cells that contain both
endothelium and hematopoietic potential but it is not clear if and
how they differ from the FLK1+ FOXF1:VENUS− population.
Finally, the FOXF1+ population that does not express FLK1 is
enriched for mesothelial cells and mesodermal progenitors fated to
other developmental pathways.
FOXF1 expression impairs the hematopoietic potential of
mesodermal precursors
To establish whether FOXF1 might participate in lineage choices
during mesoderm differentiation, we generated an ESC line in
which FOXF1 linked to the GFP reporter protein via a 2A peptide
can be expressed upon doxycycline addition using a Tet-on
inducible system (thereafter referred to as iFoxf1), as previously
described (Gandillet et al., 2009; Kyba et al., 2002). Upon EB
differentiation, FLK1+ mesodermal cells were sorted and cultured in
hemangioblast condition to allow differentiation to hematopoietic,
endothelial and smooth muscle lineages (Stefanska et al., 2014).
The doxycycline-mediated induction of FOXF1 expression from
day 0 or day 1 of the culture dramatically decreased the
hematopoietic output as shown by flow cytometry analysis of
CD41 and CD45 expression (Fig. 7A, upper panel), clonogenic
replating assay (Fig. 7B) and the absence of hemogenic endothelial
clusters giving rise to hematopoietic cells (supplementary material
Fig. S6A) as previously shown in this culture system (Lancrin et al.,
2009b). Interestingly, this block in hematopoietic differentiation
was irreversible as removal of doxycycline even after only 12 h of
induction was unable to restore the generation of hematopoietic cells
(Fig. 7A, lower panel). Similar results were also obtained when the
induction of FOXF1 expression was performed during EB culture
with doxycycline added at day 3, 4 or 5 (supplementary material
Fig. S6B). Induction of FOXF1 expression from day 0 of the culture
also impaired endothelial differentiation as shown by a decrease in
the frequency of CD144+ cells produced in the culture (Fig. 7C,
upper panel, Dox d0-3). In contrast, an increase in the frequency of
αSMA+ cells was observed (Fig. 7C lower panel). When FOXF1
expression was induced at day 1 of the culture (Dox, d1-3), the
endothelial potential was similar when compared to the noninduced culture condition, but the frequency of αSMA+ cells
remained high. These results suggest that the induction of FOXF1
expression in mesodermal progenitors impairs hematopoietic
differentiation potential, whereas vascular differentiation potential
toward endothelial and smooth muscle lineages is maintained or
enhanced.
Given the profound and irreversible block in hematopoiesis
induced by FOXF1, we next investigated whether FOXF1 acts in a
cell-autonomous manner to shut down hematopoiesis or through the
production of soluble factors, as FOXF1 has been shown in vivo to
control BMP4 transcription (Astorga and Carlsson, 2007). For this
purpose, FLK1+ Tomato-labeled cells (iTomato cells) were
differentiated together with FLK1+ iFoxf1 cells in hemangioblast
culture condition (Fig. 7D). Upon addition of doxycycline,
iTOMATO+ cells were able to differentiate toward hematopoiesis
at similar frequencies in the single or co-culture (Fig. 7E,F)
whereas GFP+ (iFoxf1) cells were not able to generate CD41+ nor
CD45+ cells, supporting a cell-autonomous block of hematopoietic
potential upon the induction of FOXF1 expression. Because FOXF1
appeared to inhibit hematopoietic specification, embryos deficient
for FOXF1 expression might harbor increased frequency of
mesoderm progenitors committed to hematopoiesis. To test this
3315
DEVELOPMENT
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RESEARCH ARTICLE
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
hypothesis, embryos from Foxf1::venus mice intercrosses were
replated in clonogenic assays. Neural plate stage was chosen for this
analysis as this stage marks the onset of primitive hematopoiesis and
developmental defects are not yet observed in Foxf1 null embryos.
Although wild-type and heterozygous Foxf1::venus embryos had
similar frequencies of primitive erythroid progenitors, Foxf1::venus
homozygous null embryos had on average 6-fold more primitive
erythroid progenitors (Fig. 7G). Altogether, these data suggest that
during gastrulation, mesoderm specification toward hematopoiesis
is negatively regulated by FOXF1.
DISCUSSION
The use of a knock-in VENUS reporter system allowed us for the
first time to track, isolate and analyze mesodermal progenitors and
3316
their derivatives expressing the transcription factor FOXF1. Both in
differentiating ESCs and in gastrulating embryos, we observed that
FLK1+ mesodermal cells emerge prior to FOXF1-expressing cells
whilst later a population of FOXF1+ FLK1− was observed and
became prominent over time. Additionally, in vitro sorted FLK1+
mesoderm gave rise to FOXF1-expressing cells that progressively
lost FLK1 expression. Consistent with these findings, cell tracing
experiments using a Flk1-LacZ reporter allele have shown that all
extra-embryonic mesoderm was derived from FLK1-expressing
cells (Ema et al., 2006). Altogether, these findings allow us to
further dissect and understand the sequential commitment steps
taking place during early mesoderm specification (Fig. 8).
One of the most striking and surprising observations of our study is
that of all the extra-embryonic mesoderm derivatives, hematopoiesis
DEVELOPMENT
Fig. 7. FOXF1 blocks the hematopoietic potential of mesodermal precursor. (A,C) FLK1+ cells from i-Foxf1-2a-gfp EB day 2.5 were sorted and cultured with
or without doxycycline (Dox) for the specified period of time (d0-1 and d1-2 denote a 12 h pulse-chase during the night from d0 to d1 or d1 to d2, respectively).
After 3 days of differentiation, cells were analyzed by flow cytometry for the expression of the hematopoietic markers CD41 and CD45 (A), the endothelial marker
CD144 (C, upper panel) or the smooth muscle marker αSMA (C, lower panel). (B) At day 2 of differentiation, cells were harvested and seeded in semi-solid
hematopoietic medium. Hematopoietic colonies were scored after 4 days. Mean±s.e.m. is shown, n=6, P-values obtained by t-test. (D) Schematic representation
of the experimental design for determining cell autonomous activity of FOXF1-induced block of hematopoiesis. FLK1+ cells were sorted from day 2.5 EBs derived
from i-Tomato or i-Foxf1-2a-gfp ESCs, then cultured on gelatinized plates in hemangioblast medium in the presence of doxycycline either alone or in co-culture
(ratio 1/1). (E) After 3 days of differentiation, cells were harvested and analyzed by flow cytometry for the expression of the hematopoietic markers CD41 and
CD45 (Tomato+ or GFP+ cells are gated as indicated). (F) Summary of CD41+ frequencies obtained in all experiments. (G) Primitive erythroid colony count
obtained from clonogenic replating of wild-type (WT), heterozygous (HET) or homozygous null Foxf1::venus (KO) embryos at the neural plate stage. Each point
represents an independent embryo.
RESEARCH ARTICLE
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
is the only one that does not express FOXF1. During the in vitro
differentiation of ESCs, we observed a clear dichotomy between the
expression of FOXF1 and CD41; this mutually exclusive pattern of
expression was observed in FLK1+ sorted cells differentiated in
hemangioblast culture as well as in differentiating EB culture over
time. Furthermore, when FLK1+ FOXF1::VENUS+ cells were sorted
and further cultured, they gave rise to very few CD41+ hematopoietic
cells that did not express FOXF1::VENUS. In gastrulating
embryos, we observed a similar trend, blood islands were devoid of
FOXF1::VENUS expression and by flow cytometry the vast majority
of CD41+ cells did not express FOXF1::VENUS. However, we
did observe in vivo a few CD41+ cells expressing FOXF1::VENUS
and the FLK1+ FOXF1::VENUS+ population gave rise to low
hematopoietic output when cultured ex vivo. In line with this
observation, analysis of FOXF1::VENUS expression in Etv2deficient embryos revealed that a subset of FOXF1::VENUS+
mesodermal progenitors co-expressing TIE2 was under the control
of ETV2 expression, a master regulator of blood and endothelium
specification (Kataoka et al., 2011; Lee et al., 2008; Wareing et al.,
2012b). Finally, global transcriptomic analysis revealed the
expression of many transcriptional regulators of hematopoietic and/
or endothelial fate including Etv2 in the FLK1+ FOXF1::VENUS+
population. Given that the allantois is the main mesodermal tissue
where both FOXF1 and ETV2 are expressed at the E7.5 stage of
development (Kataoka et al., 2011; Wareing et al., 2012a), these
FLK1+ FOXF1::VENUS+ ETV2+ cells are most likely localized to
this mesoderm derivative which has been shown previously to give
rise to hematopoiesis autonomously (Zeigler et al., 2006). Altogether,
these findings reveal that in gastrulating embryos a subset of FOXF1expressing cells can give rise to hematopoietic cells. Interestingly, this
was not observed in vitro in differentiating Foxf1::venus ESCs and
could be due to the culture conditions not being permissive for the
generation of all mesodermal subsets.
As in vitro FOXF1 expression and hematopoiesis appeared
mutually exclusive, we investigated the outcome of FOXF1enforced expression during ESC differentiation and observed that
the generation of hematopoietic cells was inhibited by FOXF1
expression. Furthermore, short-term FOXF1 exposure was sufficient
to block blood cell production and even previously specified CD41+
cells were lost upon FOXF1-enforced expression. These findings
seem in opposition with the detection of a FLK1+ FOXF1::VENUS+
population generating hematopoiesis in gastrulating embryos.
However, at the time of isolation, these FLK1+ FOXF1::VENUS+
cells expressed highly heterogeneous levels of Foxf1 transcripts,
with many cells no longer expressing Foxf1 transcript. This suggests
that this cell population did express Foxf1 earlier during ontogeny
but that Foxf1 transcription had already been shut down at the time of
isolation. Most likely, VENUS was still detected in these cells due to
differential stability or half-life of this protein. One most likely
interpretation is that FLK1+ FOXF1::VENUS+ progenitors are able
to generate blood cells because FOXF1 is no longer expressed. The
fact that FOXF1 blocks hematopoietic specification could explain
the previously published observation of ectopic hematopoiesis in the
amnion of Foxf1-deficient embryos in which lack of FOXF1
expression resulted in the inappropriate specification of mesoderm
progenitors to blood derivatives (Mahlapuu et al., 2001b). This is
in line with our observation that Foxf1-deficient embryos have a
higher frequency of primitive erythroid progenitors than wild-type
embryos.
In vitro, we observe that FOXF1::VENUS is expressed by a high
frequency of cells in hemangioblast culture and differentiating EBs;
by day 6 of differentiation EB cells either express CD41 and are
hence committed to hematopoiesis or they express FOXF1 and
might represent mesenchyme or mesothelium progenitors which
rapidly give rise to smooth muscle upon further culture. It is
interesting to note that in the adult organism, the mesothelium
retains remarkable progenitor characteristics with the ability to
differentiate into myofibroblast, smooth muscle and endothelium
upon injury or specific signals in organs such as lung (Que et al.,
2008), heart (Zhou et al., 2008) or liver (Li et al., 2013; Rinkevich
et al., 2012). In line with these observations, FOXF1 has been
shown to be implicated later in life in the regulation of visceral
smooth muscle cells (Hoggatt et al., 2013) and the formation of
embryonic vasculature (Ren et al., 2014). The Foxf1::venus ESC
line will be helpful in future studies to dissect how Foxf1 expression
is regulated during mesoderm specification. It was shown in vivo
that Hedgehog signaling from the extra-embryonic endoderm is
responsible for Foxf1 activation in the yolk sac mesoderm but not in
the allantois (Astorga and Carlsson, 2007); it will be interesting to
define in vitro whether hedgehog inactivation prevents Foxf1
transcription in all or only a subset of mesodermal cells, possibly
defining different types of mesoderm derivatives in vitro. During
embryonic development, cell fate specification is orchestrated by
tightly regulated molecular and cellular processes. Understanding
how these processes occur in vivo is important from a fundamental
perspective but it is also crucial in helping us devise optimal
protocols for the in vitro derivation of specific lineages from ESC to
be used in the clinic for therapeutic applications.
3317
DEVELOPMENT
Fig. 8. Schematic depiction of early
mesoderm specification. During
gastrulation, FOXF1-expressing
mesoderm is generated from FLK1+
mesoderm cells. FOXF1 expression
marks most extra-embryonic derivatives
with the remarkable exception of the
blood islands and blood cells. Most of the
FOXF1-expressing mesoderm is
specified independently of ETV2 and is
localized in the amnion and mesothelium.
However, a small subset of FOXF1+ cells
are ETV2-dependent, are likely localized
in the allantois and might give rise to a
subset of hematopoietic and endothelial
lineages. The sequential expression of
critical genes is depicted here with genes
expressed shown in green and genes not
expressed shown in red.
RESEARCH ARTICLE
Development (2015) 142, 3307-3320 doi:10.1242/dev.124685
MATERIALS AND METHODS
Gene expression analysis
ESC and mouse lines
Total RNA was extracted with RNeasy Plus Mini or Micro kit (Qiagen), and
was reverse-transcribed into cDNA with random hexamer oligonucleotides
using Omniscript RT kit (Qiagen). Real-time PCR was performed on an
ABI7900 (Applied Biosystems) using Universal ProbeLibrary System
(Roche) with TaqMan Universal Master Mix (Applied Biosystems).
Expression data were calculated relative to β-actin as 2−ΔCt.
An inducible iFoxf1-2aGFP ESC line was established as previously described
(Gandillet et al., 2009; Kyba et al., 2002). Briefly, a plasmid containing Foxf1
cDNA fused to 2a-GFP was inserted into the modified HPRT locus of Ainv18
ESC by Cre-mediated recombination. ESC clones with a restored functional
Neomycin gene were selected using G418. The same protocol was used to
establish an iTomato ESC line, with introduction of Tomato cDNA in the
modified HPRT locus. For generation of the Foxf1::venus ESC line,
homology arms were amplified with Phusion DNA Polymerase (NEB) and
cloned into a vector containing a H2B-venus reporter gene and an FRTflanked PGK/EM7-NeoR cassette. Ainv18 mouse ES cells were
electroporated with linearized targeting vector and selected using G418.
Correctly targeted clones were identified by PCR, confirmed by southern blot
analysis and the FRT-PGK-NeoR-FRT cassette was removed by transiently
expressed flipase recombinase. To generate a corresponding mouse line,
Foxf1::venus ESCs were injected into blastocysts and animals in subsequent
generations were backcrossed with C57BL/6 mice. Double transgenic Foxf1::
venus + Etv2 +/− mice were obtained by mating Foxf1::venus and Etv2 +/−
animals (Wareing et al., 2012b). Timed matings were set up between Foxf1::
venus mice, Foxf1::venus and ICR mice, or double transgenic Foxf1::venus +
Etv2 +/− mice. The morning of vaginal plug detection was defined as day 0.5 of
embryonic development. Gastrulating embryos were staged using
morphological landmarks (Downs and Davies, 1993). All animal work was
performed under regulation governed by the Home Office Legislation under
the Animal Scientific Procedures Act (ASPA) 1986.
Cell culture and differentiation
ESC lines were maintained, differentiated and cultured in hemangioblast
media as described (Lancrin et al., 2009a; Sroczynska et al., 2009). Where
indicated, 1 µg/ml of doxycycline was added to the culture medium. For
reaggregation assay, sorted cells were seeded at 5×105 cells/ml in ultra-low
attachment culture plates in embryoid bodies (EB) differentiation media.
Populations sorted from E7.5 embryos were seeded onto irradiated OP9
stromal cells in hemogenic endothelium media (Mukouyama et al., 2000)
supplemented with 1 µg/ml osteopontin. For hematopoietic progenitor
assays, cells were plated in 1% methylcellulose as previously described
(Sroczynska et al., 2009).
Single cell expression analysis
Cell lysis, cDNA synthesis and specific gene target amplification was
performed using the CellsDirect One-Step qRT-PCR kit (Invitrogen) as
previously described (Moignard et al., 2015). The assay mix contained a
mix of primers for genes including Foxf1 and β-actin from Sigma. Real-time
PCR was performed as described above in the Gene expression analysis
section. Expression data were normalized to β-actin and plotted as 2−ΔCt.
Microarray analysis
Total RNA was extracted and amplified using SuperAmp kit (Miltenyi).
Gene expression analysis was performed using Affymetrix GeneChip
Mouse Exon 1.0 ST Array, and data were processed using Affymetrix Power
Tools software package (Affymetrix) for RMA normalization and DABG
detection. The expression level of a gene was calculated based on the
median expression of its exonic probesets called present. Annotations and
cross-mappings between probesets, exons and genes were obtained from
Ensembl mouse genome build 70 using the R/BioConductor package
annmap. Differential expression analysis was performed on gene-level data
using the R/BioConductor package limma. Hierarchical clustering (Pearson
correlation) was performed using GENE-E platform (www.broadinstitute.
org/cancer/software/GENE-E/) and Function Annotation terms were
obtained using Ingenuity Pathway Analysis (IPA) software (Ingenuity
Systems). Datasets are deposited in the GEO repository under accession
number GSE66574.
Acknowledgements
We thank the staff at the Flow Cytometry, Histology, Advanced Imaging and
Molecular Biology Core facilities of CRUK Manchester Institute for technical support.
Competing interests
The authors declare no competing or financial interests.
EBs or embryos were trypsinized (TryplE, Gibco) for 3 min, and the singlecell suspension was stained at 4°C with a combination of antibodies. For
intracellular staining, cells were fixed in PBS 2% paraformaldehyde and
permeabilized in 0.5% saponin. Antibodies were purchased from eBioscience
(FLK1-bio #13-5821, FLK1-APC #17-5821, CD41-PE #12-0411, CD41PE-Cy7 #25-0411, CD41-eFluor450 #48-0411, CD140a-APC #17-1401,
CD144-AlexaFluor647 #51-1441, Tie2-PE #12-5987, SA-peCy7 #25-4317),
Biolegend (CD45-PerCP-Cy5.5 #103132) or Sigma Aldrich (αSMA-Cy3
#C6198). Dead cells were excluded using Hoechst 33258 staining (Life
Technologies). Cells were sorted using AriaII or Influx and analyzed on
FACSCalibur, LSRII or LSRFortessa cytometer (BD Biosciences).
Immunohistochemistry
For tissue sections, embryos were fixed in PBS 4% paraformaldehyde,
embedded in gelatin-sucrose and snap-frozen. Cryosections (7 μm) were
blocked with 10% goat serum (DAKO), incubated with primary antibodies
followed by fluorochrome-labeled species-specific secondary antibodies.
Cultured cells were fixed in PBS 4% paraformaldehyde and blocked with
serum prior to immunostaining. Whole-mount stainings were performed as
previously described (Yokomizo et al., 2012). Primary antibodies used were
rat anti-mouse Flk1 (BD Pharmingen #550549), anti-αSMA-Cy3 (Sigma
Aldrich #C6198) and rabbit anti-GFP (MBL #598). Sections and cultured
cells samples were mounted with ProLong Gold antifade reagent with DAPI
(Molecular Probes) and images were acquired using a Low-light Zeiss
Axiovert 200 M system and an Andor iXon DU888+ camera. Whole mount
samples were counterstained with DAPI and images were acquired using a
Leica (SP8 MP CSU) Two Photon Confocal Microscope.
3318
Author contributions
M.F. designed and performed experiments, analyzed the data and wrote the
manuscript. A.E. designed and performed experiments and analyzed the data. P.C.
provided reagents. G.L. and V.K. designed and supervised the research project,
analyzed the data, and wrote the manuscript.
Funding
This work is supported by Cancer Research UK. A.E. was supported by an EMBO
long term post-doctoral fellowship.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.124685/-/DC1
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