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SOXF transcription factors in cardiovascular development
Andrew J. Lillya, Georges Lacaudb*, and Valerie Kouskoffa*
Cancer Research UK aStem Cell Hematopoiesis and bStem Cell Biology group
Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow
road, M20 4BX, UK.
*Corresponding Authors:
[email protected], phone: (44) 161 446 8381
[email protected], phone: (44) 161 446 8380
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Contents
Abstract
Keywords
Abbreviations
1. Introduction
2. Structure and function of SOXF factors
3. SOXF factors in cardiogenesis
4. Vascular and lymphatic development
4.1. SOXF factors in vasculogenesis and angiogenesis
4.2. SOXF factors in lymphangiogenesis
5. SOXF factors in the emergence of blood cells from hemogenic endothelium of
the vasculature
6. Conclusions
Conflicts of interest
Acknowledgements
References
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Abstract
Cardiovascular development during embryogenesis involves complex changes in
gene regulatory networks regulated by a variety of transcription factors. In this
review we discuss the various reported roles of the SOXF factors: SOX7, SOX17
and SOX18 in cardiac, vascular and lymphatic development. SOXF factors have
pleiotropic roles during these processes, and there is significant redundancy and
functional compensation between SOXF family members. Despite this, evidence
suggests that there is some specificity in the transcriptional programs they regulate
which is necessary to control the differentiation and behaviour of endothelial
subpopulations. Furthermore, SOXF factors appear to have an indirect role in
regulating cardiac mesoderm specification and differentiation. Understanding how
SOXF factors are regulated, as well as their downstream transcriptional target
genes, will be important for unravelling their roles in cardiovascular development and
related diseases.
Keywords: SOXF, cardiogenesis, vasculogenesis, angiogenesis,
lymphangiogenesis
Abbreviations: EPC, endothelial precursor cells; HMG, high mobility group; TAD,
transactivation domain; BRY, brachyury; ESC, embryonic stem cell; LECs, lymphatic
endothelial cells; Ra, ragged; Raop, ragged opossum; ISV, intersomitic vessels; EHT,
endothelial to hematopoietic transition; CDH, congenital diaphragmatic hernia.
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1. Introduction
The adult cardiovascular system is an extremely complex interconnected network
made up of the heart, vasculature and blood. These key components are derived
from a common mesodermal pool of progenitors that arises early in development:
from E6.5 in the mouse embryo. Differentiation along cardiac, vascular and
lymphatic endothelial lineages involve regulation of cell fate decisions as well as
intricate changes in cell type specific behaviour, which are controlled by dynamic
adjustments in transcriptional networks. A growing body of evidence indicates that
SOXF transcription factors are crucial regulators of endothelial cell differentiation and
behaviour in distinct subpopulations. These include: endothelial precursors (EPCs),
arterial and venous vascular endothelial cells, lymphatic endothelial cells as well as
endocardial cells. Furthermore, studies in xenopus and mouse embryos indicate an
indirect role for SOXF factors in cardiogenic mesoderm specification and
differentiation.
Despite the issue of redundancy and compensation between SOXF family members,
it is clear that each member has some specificity in terms of the transcriptional
program it regulates. In this review, we consider the various reported roles of the
SOXF family members in regulating cardiovascular development during mouse,
zebrafish and xenopus embryonic development.
2. Structure and function of SOXF factors
In most vertebrates, the SOXF subgroup of the SOX family transcription factors is
comprised of SOX7, SOX17 and SOX18 [1, 2]. Teleosts also possess a divergent
SOXF factor called Sox32, which is thought to have arisen through tandem gene
duplication of SOX17 [3]. Mammals have two isoforms of SOX17, with the short
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isoform missing the N-terminal region and half of the high mobility group (HMG)
domain [4]. However, the function of the short SOX17 isoform is currently unknown
[5]. Like other SOX factors, SOXF factors possess a highly conserved HMG DNA
binding domain which recognises the core DNA consensus sequence of AACAAT
(see Figure 1.) [6]. The HMG domain of SOX factors interacts with the minor groove
of the DNA helix causing bending of the DNA towards the major groove thus
resulting in localised changes in DNA structure [7, 8]. However, the role of the DNA
bending in relation to the biological function of SOX factors is poorly understood. All
of the murine, human and xenopus SOXF factors possess a conserved c-terminal -
catenin binding domain; whereas zebrafish Sox17 does not [3, 9-11]. Interactions
between SOX7, SOX17 or SOX18 with -catenin inhibits the activity of
-catenin/TCF transcriptional complexes, and therefore repress Wnt signalling [11].
SOXF factors also have a transactivation domain (TAD), required for mediating
transcriptional activation (see Figure 1.).
A hallmark of the SOX family of transcription factors is the ability to interact with
different DNA binding partner proteins to regulate specific transcriptional programs,
which is important due to the degenerate nature of the core SOX DNA consensus
sequence [12]. The HMG domain of SOX factors is often key in mediating the
interaction with other transcription factors, which is surprising given the similarity of
the HMG domain across the SOX family [13]. In terms of the SOXF subgroup,
SOX18 has been shown to physically interact with the transcription factor MEF2C in
endothelial cells via their respective HMG and MADS-box DNA binding domains, and
act synergistically to activate transcription [14]. It is well characterised that SOX17
physically interacts with OCT4 via their respective HMG and POU DNA binding
domains, which mediates cooperative DNA binding to specify endoderm
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development [15-17]. However, generally speaking, the interacting partners of
SOXF factors controlling various cardiovascular transcriptional programs have not
been well characterised.
3. SOXF factors in cardiogenesis
Cardiogenesis initiates when cardiogenic mesoderm cells, specified during
gastrulation, migrate through the primitive streak where they form the cardiac
crescent at around E7.5 in the mouse embryo [18, 19]. Cardiogenic mesoderm cells
subsequently differentiate along specific cardiac lineages including: myocardial,
endocardial, and smooth muscle cell lineages [20, 21]. During this period of cardiac
specification, endoderm derived growth factors such as BMPs are key in mediating
the formation and differentiation of myocardial and endocardial cells [22, 23]. At
around E8.0 the cardiac crescent fuses, forming a heart tube which grows and
undergoes looping forming the embryonic heart which starts to beat from E8.25 [24].
In the mouse embryo, Sox7 and Sox18 are expressed in vascular endothelial cells
located in the precardial region at E8.25, whereas Sox17 expression levels in this
region are low [25]. From E8.5 Sox7 and Sox18 are expressed in the heart tube and
from E12.5 in the endocardium and vascular endothelium of the heart [25, 26].
Sox17-/- mouse embryos display aberrant looping of the developing heart tube [25],
and SOX17 is essential for the specification of cardiac mesoderm in vitro [27]. Given
that SOX17 is highly expressed in developing endoderm [25, 28], together with the
importance of endoderm derived signals in regulating cardiac development, it is likely
that SOX17 regulates cardiac development in a non-autonomous fashion. In
agreement with this idea, Sox17 knockdown in differentiating embryonic stem cell
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(ESC) cultures decreased Hex expression: an endoderm gene known to be
important in regulating paracrine signals important for cardiac development [27, 29].
Studies in xenopus also indicate a non-autonomous role of SOXF factors in
regulating cardiogenesis. Sox7, Sox17and Sox18are important regulators of
endoderm development, and animal cap experiments have demonstrated that Sox7
and Sox18 are cardiogenic but Sox17 is not [30-32]. Inhibition of endodermal Wnt
signalling plays an important role in paracrine mediated regulation of cardiogenesis
[29]. However, whilst Sox7 and Sox18 inhibit Wnt signalling via interacting with -
catenin, mutational analyses demonstrated that the induction of cardiogenesis by
SOXF factors is reliant on the TAD rather than -catenin inhibition [30].
In vitro ESC differentiation has demonstrated that cardiac progenitors and
hemangioblasts are derived from mesodermal precursors expressing the
transcription factor Brachyury (BRY), which are specified in two distinct FLK1
expressing waves [21, 33]. The first BRY/FLK1+ hemangioblast population
differentiates along vascular endothelial and hematopoietic lineages. The second
BRY/FLK1 wave contains cardiac mesoderm population, which later express cardiac
markers such as CXCR4, MESP-1 and GATA4 [21, 33, 34]. Interestingly, it has
been demonstrated that SOX7 expression is significantly greater in the
hemangioblast (FLK1+/CXCR4-) compared with cardiac mesoderm populations
(FLK1+/CXCR4+), and therefore may drive vascular development over cardiac
differentiation [35].
4. Vascular and lymphatic development
The first blood vessels that form during embryogenesis develop in the extra-
embryonic yolk sac via vasculogenesis, which initiates following the formation of
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blood islands from mesodermal progenitors [36]. Cells on the inside of the blood
islands differentiate into blood cells, whereas cells on the outside differentiate in to
EPCs, which migrate and join with other blood islands. Subsequent migration and
differentiation of EPCs forms a primitive vascular plexus in the yolk sac [37, 38]. In
the embryo proper, EPCs migrate to form endothelial chords that differentiate into
the major arteries and veins [37, 39]. The primitive extra and intra-embryonic
vascular network subsequently undergoes angiogenesis involving the remodelling
and expansion of blood vessels [40].
In mice, lymphatic development initiates post arterial-venous blood vessel
differentiation, when a subset of endothelial precursors located at the cardinal vein
are specified to differentiate into lymphatic endothelial cells (LECs) [41]. The LECs
then migrate from the cardinal vein initially forming primary lymph sacs, which
develop by sprouting into the lymphatic vasculature [41]. In zebrafish, the early
stages of lymphangiogenesis are different from mice: lympho-venous sprouting
occurs in parallel before subsequent lymphatic and venous differentiation [42].
4.1. SOXF factors in vasculogenesis and angiogenesis
In the mouse embryo, SOX7 and SOX18 are expressed at the onset of endothelial
differentiation in the blood island region of the yolk sac from E7.5 [43, 44]. Single
cell PCR analysis of ETV2+/FLK1+/CD41- cells from E7.5 mouse embryos previously
shown to be highly enriched for EPCs [45], demonstrated that Sox7 is expressed in
97% of the EPC population compared with Sox17 (75%) and Sox18 (50%) (Figure
2). These data therefore indicate that SOX7 is likely to be an important regulator of
EPC behaviour and vasculogenesis at this early stage of development. By E8.25
Sox7 and Sox18 are expressed in the developing paired dorsal aortae, allantois and
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cardinal veins, whereas Sox17 is weakly expressed in the dorsal aortae but mainly
expressed in the endoderm at this stage [25, 46].
As vascular remodelling and angiogenesis proceeds from E8.5, Sox7 and Sox18
continue to be widely expressed throughout the developing vascular network: in the
dorsal aorta, cardinal vein and emerging intersomitic vessels (ISVs) [9, 25]. By E9.5
Sox17 expression levels increase in the dorsal aorta and to a lesser extent in the
intersomitic vessels [46]. More recently, wholemount staining demonstrated that
SOX17 is expressed in ephrin2+ arterial cells of the dorsal aorta at E10.5 as well as
the arteries of the yolk sac, but not in venous vasculature [47, 48]. In postnatal
mouse retinal vasculature SOX17 is expressed largely in arteries and arterioles,
whereas SOX7 and SOX18 are expressed in both arterial and venous vasculature
[49]. Collectively, these data indicate that SOX17 is more specific to endothelial
cells with arterial identity, whereas SOX7 and SOX18 are more broadly expressed
across the developing vascular network.
Mouse models have revealed critical roles for SOXF factors in vascular
development. Wat and colleagues investigated the role of SOX7 in congenital
diaphragmatic hernia (CDH): a genetic disorder characterised by recurrent
microdeletions at 8p23.1 that include the Sox7 gene [26]. Whilst SOX7
haploinsufficiency induced CDH in a fraction of adult mice, the complete knockout of
SOX7 was embryonic lethal from E10.5 onwards, with the mice displaying delayed
development, pericardial oedema and an absence of large blood vessels in the yolk
sac [26]. These data suggest that SOX7 deficiency induces cardiovascular failure
and blocks vascular remodelling in the yolk sac but the full characterization of
defects in Sox7-/- embryos remains to be performed.
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The importance of SOX18 in regulating vascular development became evident when
the underlying cause of the phenotype of ragged (Ra) and ragged opossum (Ra op)
mutant mice was found to be due to mutations in the Sox18 gene [44]. Ra
homozygote mice, rarely survive past weaning whereas Raop heterozygotes die
around E11.5, due to generalised oedema and hemorrhages indicative of
cardiovascular system defects [50, 51]. Interestingly, Ra mutations did not affect the
HMG domain but resulted in a truncated TAD [44]. Whilst Ra mutations could affect
the tertiary protein structure and therefore DNA binding capacity of SOX18, the data
indicates the importance of the TAD in providing the contacts necessary for
activating transcription.
Contrary to Ra mutant mice, initial observations of SOX18-/- mice were that they
displayed no gross abnormalities and no observable defects in the cardiovascular
system including in the formation of the dorsal aorta and ISVs [52]. However, more
recently a genetic interaction between SOX18 and VEGFD in regulating vascular
development has been demonstrated [53]. SOX18 and VEGFD double knockout
mice display dramatic vascular defects including haemorrhaging and dilation of the
dorsal aorta and cardinal vein from E11.5 [53]. Studies using a pure C57BL/6
background demonstrated that SOX18-/- mice develop extensive subcutaneous
oedema and die around E14.5 [54, 55]. It was also shown that SOX7 and SOX17
are upregulated in SOX18-/- mice of a mixed genetic background and can substitute
for SOX18 [55]. Despite this, the fact that SOX7 is indispensable for early vascular
development compared to SOX18 indicates that SOX7 regulates a transcriptional
program distinct from SOX18. SOX18 has been demonstrated to have a key role in
lymphangiogenesis and the pathogenesis of Hypotrichosis-Lymphedema-
Telangiectasia (HLT), which are considered in section 4.2.
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Zebrafish studies have confirmed a pivotal role for Sox7 and Sox18 in regulating
vascular development. sox7 and sox18 morphants individually display only mild
vascular defects, whereas sox7/sox18 double morphants exhibit severe defects in
arterial-venous morphogenesis, a lack of circulation in the trunk, and ISV branching
abnormalities [56-58]. More recently it has been established that a fraction of sox7-/-
zebrafish display a short circulatory loop and a lack of trunk circulation [59]. This
phenotype was found with 100% penetrance in double sox7-/-/sox18-/- zebrafish,
supporting the morpholino based studies that Sox7 and Sox18 play redundant roles
during vascular development in zebrafish [59]. sox7-/- as well as sox7/sox18 double
morphant zebrafish display increased expression of the venous marker flt4. [57, 59].
Zebrafish studies have demonstrated interplay between Sox7/Sox18 and notch
signalling in regulating vascular development. Sacilotto and colleagues
demonstrated that combinatorial knockdown of sox7, sox18 and rbpj resulted in a
loss of Dll4 expression in the vasculature [60]. Furthermore, the triple morphant
zebrafish displayed notable defects in arterio-venous segregation and retarded
development of ISVs [60]. A genetic interaction between sox7 and hey2 in vascular
development has also been demonstrated [59]. Furthermore, Sox7 acts upstream of
notch signalling as expression of the notch intracellular domain (NICD) rescues the
short circulatory loop phenotype of the sox7-/- zebrafish [59].
The first studies into the role of murine SOX17 in cardiovascular development were
hampered by gross morphological abnormalities developing in the trunk region at
E10.5 of Sox17-/- embryos caused by a depletion of definitive endoderm [28], making
investigation into cardiovascular development at these stages difficult. At E8.5,
before the definitive endoderm related defects, relatively mild cardiovascular defects
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were observed with Sox17-/- mice, which was more pronounced in Sox17-/-/Sox18-/-
mice again indicating redundancy between SOXF factors [25].
In order to circumvent these difficulties and to investigate the role of SOX17
specifically in endothelial cells, Corada and colleagues crossed Sox17flox/flox mice with
Tie2cre mice to create Sox17ECKO (endothelial conditional knockout) mice [47].
Sox17ECKO was embryonic lethal between E10.5 to E12.5, with embryos displaying a
block in vascular remodelling of the yolk sac, an absence of arteries and defective
patterning of the intersomitic blood vessels indicative of angiogenic defects [47].
Furthermore, at E10.5, it was observed that endothelial Sox17 deficiency resulted in
fusion and bypass circulation between the dorsal aorta and the cardinal vein: a
phenotype associated with loss of arterial and venous identity [47, 61]. Further
analysis demonstrated that inducing Sox17 deficiency in adult endothelial cells
resulted in an increase in venous genes such as CoupTFII and a decrease in arterial
genes such as Ephrin2, Notch4 and Dll4 [47]. ChIP analysis identified SOX17
binding to gene regulatory regions of Notch4, Notch1, Dll1 and Dll4 [47]. Although
none of these SOX17 target gene regulatory regions were confirmed by mutagenesis
the data suggests that SOX17 may directly regulate Notch signalling [47]. However,
the interaction between SOX17 and Notch is extremely complex as it has been
shown that Notch signalling can act upstream of SOX17 supressing its expression
[62]. Given the importance of Notch signalling in regulating arterial cell fate [63], it is
hypothesised that the interaction between SOX17 and Notch acts to control arterial-
venous specification of differentiating vascular endothelial cells during mouse
development.
Further studies have indicated that SOX17 may act together with FOXC2 and ETV2
to regulate arterial cell fate. FOXC2 and ETV2 are important regulators of EPC
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differentiation, and act together at FOX:ETS motifs in endothelial enhancers [64].
Robinson et al., demonstrated that an enhancer of endothelin converting enzyme 1
(ECE1) gene is initially active in EPCs, likely due to activity of FOXC2 and ETV2
[65]. As vascular development progresses, ECE1 becomes restricted to the arterial
endothelium [65]. Luciferase analysis demonstrated that SOX17 could synergise
with FOXC2 and ETV2 to drive activity of the ECE1 enhancer; however, a direct
interaction between these transcription factors was not investigated [65]. These data
suggest that SOX17 is required for maximal activation of arterial genes in cells that
are specified for arterial differentiation by interacting with other core endothelial
transcriptional machinery.
In contrast to Sox7 and Sox18, Sox17 has divergent roles in mice and zebrafish. In
zebrafish, sox17 is expressed in primitive red blood cells in the intermediate cell
mass but not in the developing vasculature [3]. The authors demonstrate that
knockdown of sox17 decreases the expression of primitive erythroid genes but not
definitive hematopoietic genes, indicating a specific role for Sox17 in regulating
primitive hematopoiesis [3]. Interestingly, zebrafish Sox17 does not possess a β-
catenin binding domain which may account for some differences with mice.
Furthermore, the teleost specific SoxF factor Sox32 is located adjacent to the sox17
locus and is thought to have arisen through tandem gene duplication. Sox32 has a
β-catenin binding domain and sox32 morphants display defects in vasculogenesis
and angiogenesis, whilst the loss of function sox32 mutant casanova shows defects
in endoderm formation [3, 66]. It is interesting to speculate that Sox32 in zebrafish
may perform some similar roles to SOX17 in mice.
4.2. SOXF factors in lymphangiogenesis
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An important role for SOX18 in regulating lymphangiogenesis was postulated after
the discovery of various mutations in the Sox18 gene in patients with HLT: a
developmental disorder characterised by swelling of the extremities caused by
defective lymphatic vasculature and dilation of the DA [67, 68]. Furthermore, the
phenotype of HLT patients mirrors that of Ra mutant SOX18 mice as well as SOX18
and VEGFD double knockout mice [44, 51, 67-69]. Since these studies, SOX18 has
been found to act at the onset of lymphangiogenesis. At the early stages of
lymphatic development, lymphatic endothelial precursor cells bud off from the
cardinal vein and express the transcription factor PROX1: an important regulator of
the lymphangiogenic transcriptional program [41, 70]. In the mouse embryo, SOX18
but not SOX17 or SOX7 is expressed in PROX1+ lymphatic endothelial precursor
cells surrounding the cardinal vein [54, 55].
Seminal studies by Francois and colleagues demonstrated that SOX18 mutant
mouse embryos have severely disrupted lymphatic development, and a complete
lack of migrating PROX1+ cells close to the cardinal vein [54]. Furthermore, Prox1
was shown to be a direct transcriptional target of SOX18 in differentiating ESC
cultures, whilst the expression of blood vascular endothelial genes such as Cdh5,
Tie2 and Vegfr2, was not affected by the overexpression of SOX18 [54]. Despite the
fact that SOX7 and SOX17 are not expressed in lymphatic endothelial precursor
cells, it has been shown that both can rescue the loss of SOX18 function by directly
upregulating Prox1 [55]. Collectively, these data indicate a pivotal role of SOX18 as
a master regulator working at the onset of lymphangiogenesis.
The role of SOX18 in zebrafish lymphangiogenesis is unclear due to conflicting
results. In zebrafish, sox18 is expressed around the posterior cardinal vein (PCV) in
the window of time crucial for the emergence of lymphatic progenitor cells [71, 72].
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Studies using morpholino and dominant negative mutant based approaches indicate
that Sox18 deficiency inhibits lymphatic sprouting and the emergence of lymphatic
progenitors resulting in lymphatic system defects [71]. The authors also show
cooperation between Sox18 and Vegfc in regulating lymphatic development,
indicating a genetic interaction [71]. A recent study demonstrated that the
transcription factor Mafba is important for controlling lymphatic endothelial precursor
migration and is downstream of Sox18 and Vegfc signalling [73]. However, sox18-/-
zebrafish have no detectable defects in the lymphatic system demonstrating that
Sox18 is dispensable for lymphatic development in contrast to mice [42]. Therefore,
the lymphangiogenic roles of SOX18 differ between mice and zebrafish; this is likely
to be partly due to mechanistic differences at the earliest stages of
lymphangiogenesis between the two organisms [42].
In contrast to angiogenesis, the role of neo-lymphangiogenesis as a platform for
tumour cell metastasis has only relatively recently been investigated in detail [74]. It
has been shown that tumour cells can release molecules such as VEGFA, C and D,
which signal to lymphatic vasculature inducing lymphatic vessel outgrowth and
subsequent tumour metastasis to the lymph nodes [75-77]. Whilst SOX18 is not
expressed in adult lymphatic vessels, SOX18 is re-expressed in LECs during tumour
growth in murine melanoma models [78]. Interestingly, SOX18 deficiency impairs
tumour induced neo-lymphangiogenesis resulting in a decrease in the rate of tumour
cell metastasis [78]. Given that SOX18 is not continually expressed in adult
lymphatic vasculature, it represents a promising molecular target for anti neo-
lymphangiogenesis based tumour therapy.
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5. SOXF factors in the emergence of blood cells from hemogenic endothelium
of the vasculature
Blood cells emerge from a specialised transient subpopulation of endothelial cells of
the vasculature called hemogenic endothelium, and a growing body of evidence
indicates an important role for SOXF factors in this process. In the yolk sac,
definitive blood progenitors emerge from E8.5, pre, during and post vascular
remodelling [48]. In the embryo proper blood progenitors emerge post vascular
remodelling from E10.5 in the dorsal aorta, vitelline and umbilical arteries [79].
SOX7 and SOX18 are broadly expressed across the developing vasculature at these
stages, whereas SOX17 is specifically expressed in the major arteries at E10.5, both
in the yolk sac and embryo proper.
During ESC cell differentiation SOX7 is highly expressed in hemogenic endothelium
and its expression is downregulated in emerging hematopoietic progenitor cells [80].
Maintenance of SOX7 expression beyond its normal time frame in these cultures
blocked the emergence of blood progenitors whilst maintaining the normally transient
hemogenic endothelium population [80]. Furthermore, SOX7 was found to bind and
activate the promoter of Cdh5, one of the key markers of hemogenic endothelium
[80].
In accordance with the role of SOX17 in regulating arterial specification, several
recent studies have highlighted a role for SOX17 in regulating the emergence of
blood progenitors from arterial based hemogenic endothelium in the embryo proper
at E10.5. Clarke and colleagues (2013), demonstrated that SOX17 is expressed in
hemogenic endothelium and critically required for the generation of long term
hematopoietic stem cells in vivo [81]. Further studies revealed that SOX17
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maintains arterial cell identity and represses the endothelial to hematopoietic cell
transition (EHT) by inhibiting the expression of RUNX1: a key hematopoietic cell
master regulator [82, 83]. Therefore, the downregulation of SOX17 in arterial
hemogenic endothelium is an important step in driving EHT. As SOX7 is more
broadly expressed than SOX17 early in vascular development, it is likely that SOX7
plays an important role in regulating the emergence of the first definitive blood
progenitors in the yolk sac. SOX17 however, probably in combination with SOX7,
regulates the emergence of HSPCs from the major arteries after vascular
remodelling.
6. Conclusions
SOX7, SOX17 and SOX18 have complex pleiotropic roles in regulating
cardiovascular development (Figure 3.). Furthermore, the redundancy and
compensation between the SOXF members has made research into their functions
challenging. The apparent different roles of SOX7, SOX17 and SOX18 during
development can at least be partially attributed to their different expression patterns.
In mouse embryogenesis, SOX17 is selectively expressed in arterial vasculature,
whereas the expression of SOX7 and SOX18 is more broadly detected and they are
expressed from earlier stages of vascular development. Furthermore, SOX18 is
expressed in LECs, whereas the other two are not. Therefore, understanding the
pathways and molecular mechanisms by which each SOXF factor is regulated will be
important for understanding their expression patterns and biological roles.
Despite the functional redundancy between SOXF factors, it is likely that there are
key distinctions in the transcriptional programs they regulate even when they are
expressed in the same cell type. For example, SOX7 and SOX18 have similar
17
expression patterns at early stages of mouse vascular development, but SOX7 -/-
mice die at E10.5 with severe cardiovascular defects, whereas SOX18 -/- mice die at
E14.5. In order to fully understand the different roles of SOXF factors, their global
DNA binding patterns need to be elucidated. This has been hampered by the large
cell numbers required for ChIP-seq, together with the rarity of different endothelial
cell populations during development. However, novel approaches such as DamID in
combination with ESC culture systems may provide alternative methods when ChIP-
seq is not feasible [84]. Furthermore, little is known about the DNA binding partners
of SOXF factors; therefore, unravelling the partner codes of SOX7, SOX17 and
SOX18 will be important in enhancing our understanding of SOXF factor regulation
of cardiovascular development.
The development of new vascular and lymphatic networks facilitates the proliferation
and metastases of tumour cells. Recent studies have shown that SOX17 and
SOX18 are important in controlling tumour neo-angiogenesis and neo-
lymphangiogenesis respectively [78, 85]; whilst the role of SOX7 has not yet been
investigated. These findings warrant further investigation into the role of SOXF
factors in promoting tumour vascular and lymphatic development, which may offer
novel therapeutic targets for the treatment of cancer.
Conflicts of interest
None.
Acknowledgements
This work was funded by BBSRC and CRUK grants.
Figure Legends
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Figure 1. The structure of murine SOXF factors. SOXF factors possess an N-
terminal HMG DNA binding domain, a transactivational domain (TAD) and a c-
terminal β-catenin binding domain. There are two isoforms of SOX17, the short
isoform is missing the N-terminal region and half the HMG domain, but its function is
unknown. The amino acids of the HMG domain show a high level of similarity
between the SOXF factors. The amino acids highlighted in black and grey are
common to three and two SOXF factors respectively.
Figure 2. SOX7 is broadly expressed across endothelial precursors. Single cell
qPCR analysis of ETV2+/FLK1+/CD41- EPCs cells from E7.5 mouse embryos. Data
are presented as the percentage of EPCs expressing Sox7, Sox17 and Sox18 above
the limit of detection (n=36 cells, chi-squared test).
Figure 3. SOXF factors have complex pleiotropic roles during murine
cardiovascular development. SOX7 is more highly expressed in hemangioblasts
compared to cardiac progenitors, and is broadly expressed across downstream
EPCs. SOX7 and SOX18 are expressed during vasculogenesis. After vascular
remodelling at E10.5, SOX17 is specifically expressed in arterial vasculature
whereas SOX7 and SOX18 are expressed in both arterial and venous vasculature.
SOX18 is a key regulator in the early stages on lymphangiogenesis from venous
vasculature. SOX7 and SOX17 play crucial roles in regulating the maintenance of
hemogenic endothelium and blood cell emergence. SOX17 in particular is also
expressed in the developing endoderm, where it inhibits Wnt signalling by inhibiting
β-catenin. This results in the release of endoderm paracrine signals that regulate
cardiac mesoderm specification and differentiation. Endocardial cells of the heart
express all three SOXF factors.
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