ECM-Dependent HIF Induction Directs Trophoblast Stem
Cell Fate via LIMK1-Mediated Cytoskeletal
Hwa J. Choi1, Timothy A. Sanders1, Kathryn V. Tormos1,4, Kurosh Ameri1, Justin D. Tsai1, Angela M. Park1,
Julissa Gonzalez2, Anthony M. Rajah1, Xiaowei Liu3,4, Diana M. Quinonez2, Paolo F. Rinaudo3,4,
1Department of Pediatrics, University of California San Francisco, San Francisco, California, United States of America, 2Department of Biology, San Francisco State
University, San Francisco, California, United States of America, 3Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco,
San Francisco, California, United States of America, 4Center for Reproductive Sciences, University of California San Francisco, San Francisco, California, United States of
America, 5Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, California, United States of
America, 6Developmental and Stem Cell Biology Program, University of California San Francisco, San Francisco, California, United States of America
The Hypoxia-inducible Factor (HIF) family of transcriptional regulators coordinates the expression of dozens of genes in
response to oxygen deprivation. Mammalian development occurs in a hypoxic environment and HIF-null mice therefore die
in utero due to multiple embryonic and placental defects. Mouse embryonic stem cells do not differentiate into placental
cells; therefore, trophoblast stem cells (TSCs) are used to study mouse placental development. Consistent with a
requirement for HIF activity during placental development in utero, TSCs derived from HIF-null mice exhibit severe
differentiation defects and fail to form trophoblast giant cells (TGCs) in vitro. Interestingly, differentiating TSCs induce HIF
activity independent of oxygen tension via unclear mechanisms. Here, we show that altering the extracellular matrix (ECM)
composition upon which TSCs are cultured changes their differentiation potential from TGCs to multinucleated
syncytiotropholasts (SynTs) and blocks oxygen-independent HIF induction. We further find that modulation of Mitogen
Activated Protein Kinase Kinase-1/2 (MAP2K1/2, MEK-1/2) signaling by ECM composition is responsible for this effect. In the
absence of ECM-dependent cues, hypoxia-signaling pathways activate this MAPK cascade to drive HIF induction and
redirect TSC fate along the TGC lineage. In addition, we show that integrity of the microtubule and actin cytoskeleton is
critical for TGC fate determination. HIF-2a ensures TSC cytoskeletal integrity and promotes invasive TGC formation by
interacting with c-MYC to induce non-canonical expression of Lim domain kinase 1–an enzyme that regulates microtubule
and actin stability, as well as cell invasion. Thus, we find that HIF can integrate positional and metabolic cues from within the
TSC niche to regulate placental development by modulating the cellular cytoskeleton via non-canonical gene expression.
Citation: Choi HJ, Sanders TA, Tormos KV, Ameri K, Tsai JD, et al. (2013) ECM-Dependent HIF Induction Directs Trophoblast Stem Cell Fate via LIMK1-Mediated
Cytoskeletal Rearrangement. PLoS ONE 8(2): e56949. doi:10.1371/journal.pone.0056949
Editor: Adam J. Engler, University of California, San Diego, United States of America
Received October 9, 2012; Accepted January 16, 2013; Published February 21, 2013
Copyright: ? 2013 Choi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The research was made possible by a grant from the California Institute for Regenerative Medicine (Grant Number TB1-01194) and the National
Institutes of Health (HL087754, HD072455). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Mammalian development occurs in a physiologically hypoxic
environment that drives the expression of dozens of genes via the
Hypoxia-inducible Factor (HIF) family of transcriptional regula-
tors . A heterodimeric transcription factor composed of alpha
and beta subunits, HIF can activate canonical target genes in
response to oxygen deprivation by directly binding to hypoxia
response elements (HRE) located within their regulatory regions
[2,3]. Due to the short half-life of alpha subunits, HIF activity can
be tightly regulated . Mitochondrial oxygen sensing mecha-
nisms produce highly labile reactive oxygen species to ensure that
HIF-a subunit stabilization occurs only under hypoxic conditions
. Oxygen gradients that are generated as a function of tissue
growth can thus activate HIF in a dynamic fashion to pattern the
developing embryo . Consistent with this, HIF activity is
required for embryonic development [7,8,9].
During mammalian gestation, the placenta forms a vital
transport interface between the maternal and fetal circulations
and its development is also dependent on HIF activity
[10,11,12,13]. In mice, two terminally differentiated cell types
are primarily responsible for placental function: 1. trophoblast
giant cells (TGC) and, 2. multinucleated syncytiotrophoblasts
(SynT) . TGCs anchor the placenta to the uterus and direct
maternal blood flow to the conceptus while SynTs perform the
transport functions of the placenta . The derivation of
trophoblast stem cells (TSCs) that differentiate into these placental
cells in vitro has enhanced our understanding of placental
development in vivo . Interestingly, however, TSC differenti-
ation using conventional techniques results primarily in the
PLOS ONE | www.plosone.org1February 2013 | Volume 8 | Issue 2 | e56949
production of TGCs that is associated with the stabilization of
HIF-a subunits independent of oxygen tension [10,17]. We
previously demonstrated that HIF induction is critical for TGC
differentiation as Hif-1/2a2/2(compound null) or Hif-1b (Arnt)2/
2TSCs (collectively referred to as HIF-null) fail to produce TGCs
in vitro, differentiating primarily into multinucleated SynTs,
indicating that HIF activity can suppress cell fusion and SynT
fate determination [10,17]. The mechanisms responsible for
differentiation-dependent HIF induction and how HIF ultimately
regulates cell fate in the placenta remain unknown, however.
Here, we show that TSC-extracellular matrix (ECM) interac-
tions provide positional cues during normoxia that trigger
differentiation-dependent HIF induction via signaling pathways
that intersect with metabolic responses to oxygen deprivation. We
find that altering the ECM substrate upon which TSCs are
cultured impacts differentiation-dependent HIF stabilization and
TSC fate. SynT formation is dependent on cell fusion – a process
that is associated with significant cytoskeletal reorganization
[18,19] [20,21]. Consistent with this, HIF-null TSCs exhibit
dramatic morphological changes upon syncytialization . We
now show that non-canonical HIF-2 activity, induced in response
to hypoxia or ECM composition, can prevent this process by
promoting Limk1 expression and subsequent cytoskeletal stabiliza-
ECM Composition Regulates TSC Fate and HIF Stability
Independent of O2Tension
TSC proliferation depends on Fibroblast Growth Factor 4
(FGF4) as well as the presence of fibroblast ‘‘feeder’’ cells or
fibroblast conditioned medium (Fib-CM) . In the absence of
either, TSCs default to a TGC differentiation program. While
screening for culture conditions that could maintain FGF4-
dependent TSC growth independent of fibroblasts or Fib-CM,
we identified the xeno-free defined ECM substrate, CELLstartTM
(Invitrogen) [22,23]. This ECM substrate is composed primarily of
Fibronectin, along with other ECM components , and thus
represents a physiologically relevant substrate for TSC culture
[25,26]. TSCs maintained on CELLstartTMin the presence of
FGF4, but without fibroblasts or Fib-CM, proliferated indefinitely
and expressed TSC-specific transcription factors such as CDX2
and EOMES (Fig. 1A–D), the levels of which dramatically
decreased following FGF4 withdrawal (not shown). Interestingly,
however, differentiation in 21% O2(room air) following FGF4
withdrawal of TSCs maintained on CELLstartTMpromoted cell
fusion and resulted primarily in the formation of multinucleated
SynTs (Fig. 1E, 1F), as opposed to the TGCs commonly observed
with TSCs maintained on fibroblasts or on TC plastic in Fib-CM
[16,17]. Importantly, differentiation under hypoxic (2% O2)
conditions could reverse this cell fate choice (Fig. 1G, 1H),
blocking SynT formation and generating TGCs expressing the
lineage specific transcription factor HOPX1 . Lineage-specific
gene expression analyses further confirmed that wild-type TSCs
differentiated following culture on CELLstartTMexpressed dra-
matically reduced levels of the TGC-specific markers Placental
lactogens I and 22, Proliferin and Cathepsin Q, and exhibited
increased levels of the SynT markers Tfeb and SynA, when
compared with genetically identical TSCs differentiated following
culture on TC plastic in Fib-CM (Fig. 1I). Importantly, this pattern
of gene expression was similar to, though more pronounced than,
that observed following differentiation of Arnt2/2or Hif-1/2a2/2
TSCs that form SynTs following culture on TC plastic in Fib-CM
(Fig. 1I)  . We therefore asked whether the alteration of
TSC fate following culture on CELLstartTMmight be due to
impaired HIF-a subunit stabilization that normally occurs during
differentiation using standard techniques . Indeed, TSCs
differentiated following culture on CELLstartTMin 21% O2failed
to stabilize HIF-2a and only slightly accumulated HIF-1a protein
levels, whereas differentiation in 2% O2induced both proteins
(Fig. 1J). Furthermore, TSCs derived from Vhlh2/2embryos,
which exhibit constitutively elevated HIF-1a and -2a due to lack
of VHL-dependent ubiquitination and proteasomal degradation
[2,3], still formed TGCs in 21% O2 following differentiation
despite maintenance on CELLstartTM(Fig. 1K). These results
suggest that TSC derivation on fibroblasts and maintenance on
TC plastic in Fib-CM provides a set of extracellular cues that
promote O2-independent HIF stabilization and subsequent TGC
formation during differentiation that are lost when TSCs are
maintained on the defined ECM substrate, CELLstartTM. To
understand the mechanisms responsible, we concentrated on cell
surface integrin expression, as these molecules play a central role
in cell-ECM interactions . TSCs maintained on CELLstartTM
were compared with TSCs maintained on TC plastic in Fib-CM.
Interestingly, culture on CELLstartTMcompletely blocked b3-
integrin expression in both undifferentiated and differentiated
TSCs (Fig. 2A–D), suggesting that ECM composition determines
cell surface integrin expression and thereby modulates down-
stream signaling and TSC fate. Importantly, while undifferentiat-
ed TSCs maintained on TC plastic in Fib-CM expressed this b3-
integrin, its cell surface expression was largely restricted to
differentiated TGCs, consistent with HIF induction being associ-
ated with TGC differentiation.
ECM- and Oxygen-dependent HIF Stabilization and TGC
Formation Occur via MAP2K1/2-dependent Signaling
We suspected that candidate pathways capable of integrating
ECM-dependent signals with oxygen-dependent inputs might
include members of the mitogen activated protein kinase (MAPK)
cascade. This is due to the fact each set of stimuli can
independently activate this signaling cascade  [30,31,32,33].
Consistent with this, pharmacological inhibition of MAP2K1/2
(MEK-1/2) activity prevented HIF-a subunit stabilization during
hypoxic (2% O2) differentiation of TSCs cultured on CELL-
startTM(Fig. 3A) and during normoxic (21% O2) differentiation
following culture on TC plastic in Fib-CM (Fig. 3B). Furthermore,
hypoxic TGC formation of TSCs following culture on CELL-
startTMcould be suppressed with the same MAP2K1/2 inhibitor
(Fig. 3C–E), while transient expression of constitutively active
MAP2K1 (Fig. 3F) promoted TGC formation under normoxic
conditions and dominant negative MAP2K1 allowed cell fusion
under hypoxic conditions (Fig. 3G). And finally, pharmacological
MAP2K1/2-inhibition could prevent TGC formation and pro-
moted SynT differentiation in TSCs that had been cultured on TC
plastic in Fib-CM (Fig. 3H, 3I). Northern blot analysis confirmed
that MAP2K1/2 inhibition in wild-type TSCs suppressed
expression of the TGC as well as spongiotrophoblast (SpT)
marker genes, Placental lactogen I and 4311, respectively, nearly to
levels observed in differentiated Arnt2/2TSCs (Fig. 3J). Collec-
tively, these results confirm that ECM- and oxygen-dependent
HIF-a subunit stabilization and subsequent TGC formation
occurs through a MAP2K1/2-dependent pathway.
Cytoskeletal Rearrangement is Central to MAP2K1/2-
mediated TGC Formation
Similar to mitochondrial responses to changing O2 levels,
dynamic integrin ligation in response to changes in ECM
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org2February 2013 | Volume 8 | Issue 2 | e56949
composition allow a cell to sense its environment by converting
positional information into downstream signals . These
frequently result in cytoskeletal reorganization  that can
promote cell migration or other alterations in cell behavior .
Additionally, trophoblast differentiation has been associated with
significant cytoskeletal changes [19,20,37]. We therefore examined
the cytoskeletal organization of differentiated control and Hif-1/
2a2/2TSCs that had been maintained on TC plastic in Fib-CM
and analyzed its association with MAP2K1/2 activation. TGCs
derived from control TSCs contained robust MTs extending the
length of the cell (Fig. 4A, 4D), while multinucleated SynTs
derived from Hif-1/2a2/2TSCs exhibited a disrupted microtu-
bule (MT) network consisting of ‘‘broken’’ appearing MT
fragments (Fig. 4B arrows, 4F). MT integrity associated strongly
with MAP2K1/2 activity, as control TGCs with robust MTs
stained strongly for the phosphorylated versions of the MAP2K1/
2 target MAPK3/1 (ERK-1/2) (Fig. 4A), while multinucleated
SynTs derived from Hif-1/2a2/2TSCs did not (Fig. 4B). Only
unfused SynT progenitors that did not contain ‘‘broken’’ MTs
continued to exhibit the active form of this kinase in Hif-1/2a2/2
TSCs (Fig. 4B, arrowheads). Additionally, we observed dramatic
differences in the actin cytoskeleton, with robust stress fibers noted
in TGCs (Fig. 4C) while SynTs exhibited a disorganized actin
cytoskeleton containing high amounts of diffusely distributed F-
actin (Fig. 4E). To formally test whether cytoskeletal integrity
could regulate TSC fate, we investigated whether pharmacological
MT or actin disrupting agents could promote SynT formation of
TSCs cultured using conventional techniques. Indeed, the MT
disrupting agent Taxol (Paclitaxel) (Fig. 4G) and the actin
disrupting agent cytochalasin B (Fig. 4H) inhibited TGC
Figure 1. HIF integrates ECM cues and Oxygen Levels to Direct TSC Fate. (A–D) Immunofluorescence microscopy of undifferentiated control
TSCs cultured on CELLstartTMwith anti-CDX2 and EOMES antibodies (blue=DAPI, red=CDX2, green=Eomes). (E, G) Phase contrast microscopy of
control TSCs maintained on CELLstartTMfollowing differentiation for 7 days under normoxic (21% O2) or hypoxic (2% O2) conditions. (F, H)
Immunofluoresce microscopy of control TSCs maintained on CELLstartTMfollowing differentiation for 7 days under normoxic (21% O2) or hypoxic (2%
O2) conditions with an anti-HOPX1 (red) antibody (blue=DAPI). (I) Quantitative RT-PCR analysis of Pl. I, Pl.II, Ctsq, Plf, Tfeb, SynA and SynB gene
expression in wild-type (+/+) TSCs differentiated for 7 days following culture on CELLstartTMor on TC plastic in Fib-CM, compared with Arnt2/2(2/2)
TSCs differentiated following culture on TC plastic in Fib-CM. p values ,0.05 versus wild-type Fib-CM indicated by an asterisk. (J) Immunoblot of HIF-
1a and -2a protein levels in whole cell lysates of wild-type TSCs differentiated for 7 days following culture on CELLstart at 21%O2or 2% O2. (K)
Quantitative RT-PCR analysis of Pl-1, Pl-2, Plf, Tfeb, SynA and SynB expression in Vhlh+/+and Vhlh2/2TSCs differentiated following culture on
CELLstartTM. p values ,0.05 versus wild-type indicated by an asterisk.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org3 February 2013 | Volume 8 | Issue 2 | e56949
formation and promoted the formation of multinucleated SynTs in
control TSCs that had been maintained on TC plastic in Fib-CM.
HIF-dependent Limk1 Expression Promotes TGC
We next focused our efforts on identifying cytoskeleton
regulatory molecules that may be misregulated in the absence of
HIF activity. Gene array studies indicated that expression of the
gene encoding Lim domain kinase 1 (LIMK1), an enzyme
responsible for regulating MT and Actin integrity , was
significantly downregulated in the absence of HIF activity (not
shown). Immunofluorescence microscopy confirmed that HIF-null
TS cells differentiated into SynTs expressed no detectable LIMK1
expression, while control TGCs expressed robust LIMK1 protein
levels in a perinuclear distribution (compare Fig. 5A and 5B).
Immunoblot analyses confirmed this (see below) and indicated that
the MAP2K1/2 inhibitor U0126 significantly decreased LIMK1
levels in wild-type TSCs differentiated following maintenance on
TC plastic in Fib-CM (Fig. 5H), consistent with its ability to block
HIF stabilization during differentiation. Furthermore, we found
that the LIMK1 target Cofilin was highly phosphorylated, also in a
perinuclear distribution, and therefore inactive in TGCs (Fig. 5D),
but not in SynTs (Fig. 5E), which frequently contained prominent
Cofilin rods that form when Cofilin is hyperactive  (Fig. 5E,
arrowheads). The residual phosphorylation of Cofilin rods in
SynTs may be due to their residual low-level LIMK2 levels (see
below) . Furthermore, transient LIMK1 expression in
differentiating Hif-1/2a2/2TSCs promoted the appearance of
large TGCs in the majority of transfected cells, as opposed to the
SynTs commonly observed following their differentiation (Fig. 5F,
5G). These results suggest that HIF-dependent LIMK1 expression
can regulate TSC fate downstream of ECM- or oxygen-dependent
MAP2K1/2 activation by modulating the cytoskeleton.
Non-Canonical HIF-2a Activity Drives TSC Fate via LIMK1-
mediated Cytoskeletal Stabilization
We next investigated how HIF regulates Limk1 gene expression
during TSC differentiation. We first determined whether we could
detect canonical HIF-DNA interactions in differentiated TSCs via
electrophoretic mobility shift assays (EMSA). As seen, control
TSCs maintained on TC plastic in Fib-CM and differentiated in
room air (21% O2) contained abundant HRE-bound HIF
complexes (Fig. 6A), consistent with our prior observations of
HIF activation during normoxic differentiation of TSCs main-
tained using conventional methods (13). Interestingly, however,
while two different HIF-1a-specific antibodies produced a ‘‘super-
shift, SS’’ in differentiated TSC nuclear extracts, a HIF-2a-specific
antibody did not (Fig. 6A), suggesting that HIF-1a was predom-
inantly responsible for canonical HRE-mediated gene expression
in differentiating TSCs. To interrogate the requirement for direct
HIF-DNA binding during TSC fate determination, we stably
reconstituted Hif-1/2a2/2TSCs  utilizing a PiggyBac trans-
poson system  either with HA-tagged wild-type HIF-1a or
HIF-2a individually, or with mutant forms of each lacking their
DNA binding basic domains (HIF-1aDb, HIF-2aDb) (Fig. 6B).
Deletion of the basic domain prevents HRE binding and canonical
target gene expression by HIF complexes without affecting their
stability . As expected, full-length versions of each were
capable of activating their respective canonical downstream target
genes in HIF-null TSCs, while versions lacking their basic domains
could not (Fig. 6C, 6D).
Figure 2. b3-Integrin (CD61) is downregulated in TSCs following culture on CELLstartTM. Immunofluoresce microscopy of wild-type TSCs
maintained on TC plastic in Fib-CM (A) or on CELLstartTM(B) in the presence of FGF4 and heparin (Undiff.) or following differentiation (Diff., C and D)
using an anti-CD61 antibody (green) (magnification 630X).
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org4February 2013 | Volume 8 | Issue 2 | e56949
dependence of LIMK1 expression. Interestingly, while neither
HIF-1a nor HIF-1aDb restored LIMK1 protein levels in Hif-1/
2a2/2TSCs to that observed in wild-type TSCs, both HIF-2a
and HIF-2aDb did (Fig. 7A, 7B), suggesting it to be a non-
canonical HIF-2-specific target gene. We suspected that the
HIF-2-specificity may be due to its known interaction with and
activation of c-MYC-dependent transcription [42,43]. To test
this, we immunoprecipitated HA-tagged HIF-2aDb in reconsti-
interaction (Fig. 7C). We next identified a 100% conserved c-
MYC binding E box (CACGTG) in the Limk1 promoter, but no
canonical HIF binding sites, and detected both c-MYC as well
as HIF-2a binding to this site in situ in differentiated TGCs by
chromatin immunopreciptation (Fig. 7D). We then tested
whether pharmacological c-MYC inhibition could block non-
thissystem,we investigated theHIF-a
canonical HIF-2a-dependent Limk1 gene expression, and found
that it could decrease LIMK1 protein levels in HIF-2aDb
expressing cells (Fig. 7E). This indicates that HIF-2a interacts
with c-MYC containing transcriptional complexes during TSC
differentiation and that this interaction contributes to Limk1
expression during TGC differentiation (Fig. 7F). Stable HIF-
2aDb expressing HIF-null TSCs, whether cultured on CELL-
start or on TC plastic in Fib-CM, failed to fuse into
multinucleated SynTs and differentiated along the TGC lineage
(Fig. 8A, 8B, 8C). Importantly, pharmacological cytoskeleton
disruption (Fig. 8D, 8F, 8G) could reverse this effect, promoting
SynT formation in these cells. Additionally, pharmacological
LIMK1 inhibition [44,45] promoted the formation of multinu-
cleated cells (Fig. 8E, 8H) in HIF-null TSCs reconstituted with
Figure 3. ECM- or oxygen-dependent HIF-a subunit stabilization and TGC formation are dependent on MAP2K1/2 activity. (A)
Immunoblot of whole cell lysates obtained from TSCs differentiated in 2% or 21% O2, with and without U0126, following culture on CELLstartTM, for
HIF-1a, -2a or a-Tubulin. (B) Immunoblot of whole cell lysates obtained from differentiated wild-type TSCs following culture on TC plastic in Fib-CM
with and without U0126 with a HIF-1a antibody. (C) (D) Immunofluorescence microscopy of TSCs maintained on CELLstartTMfollowing differentiation
for 7 days under hypoxic conditions without and with U0126 (10 uM) using anti b-Catenin antibodies (green) (blue=Dapi). (E) Quantitaive RT-PCR
analysis of Plf, Pl-I, Ctsq, 4311, Mash2, Tfeb and SynA expression following differentiation of wild-type TSCs cultured on CELLstartTMunder hypoxic
conditions without and with U0126. p values ,0.05 versus drug free control indicated by an asterisk. (F) Immunofluorescence microscopy using anti
HA (red) and b-Catenin (green) antibodies of control TSCs differentiated following culture on CELLstartTMunder 21% O2following transient
tranfection with constitutively active HA:MAP2K1 or under (G) 2% O2following transient transfection with dominant negative HA:MAP2K1. (H, I)
Immunofluorescence microscopy of wild-type TSCs differentiated following culture on TC plastic in Fib-CM in 21% O2with and without U0126 with
antibodies for HDAC2 (red) and E-Cadherin (green). (J) Northern blot analysis of lineage specific marker gene expression in wild-type TSCs maintained
on TC plastic in Fib-CM and differentiated with and without U0126, compared with differentiated Arnt2/2TSCs.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org5February 2013 | Volume 8 | Issue 2 | e56949
Collectively, our results indicate that oxygen- and canonical
target gene-independent HIF activity can drive TSC fate in
response to positional cues encoded by ECM components within
the TSC microenvironment (Fig. 9). Initially thought to function
as a mere scaffold, the ECM is now known to regulate many
aspects of cell behavior, including proliferation and growth,
survival, migration, and differentiation [28,46]. Primary compo-
nents of the ECM are structural proteins (e.g., collagens, laminins,
fibronectin, vitronectin and elastin) and specialized glycoproteins
that can interact with molecules having important biological
functions such as growth factors. The precise composition varies
by location. In the stem cell niche, the ECM can provide
instructive cues for cell fate decisions via the integrin family of
heterodimeric cell surface receptors [26,47,48]. In erythropoiesis,
for example, adhesion of primary erythroid progenitors to
fibronectin mediated by a4b1integrin is necessary for proper
proliferation in vitro . In this system, signals from the ECM
cooperate with signals from the soluble factor erythropoietin to
activate pathways necessary for terminal differentiation and
proliferation. In TSCs, altering their ECM in this way alters their
cell surface b3-integrin expression, and subsequent HIF induction
during differentiation. We have shown here that in TSCs, altering
their ECM in this way alters their cell surface b3-integrin
expression, and subsequent HIF induction during differentiation.
Supporting a link between HIF activity and ECM-dependent
integrin ligation, avb3 activation can trigger HIF accumulation in
some cancer cells  and HIF-deficiency negatively affects TSC
surface b3-integrin localization , suggesting that HIF-alpha
subunit stability can both be activated by, as well as further
promote, cell surface integrin activity. Importantly, trophoblast
adhesion to ECM is governed by integrins [52–53] and hypoxic
conditions promote trophoblast invasion in utero .
The mitogen activated protein kinase (MAPK) cascade is a
frequent point of convergence from multiple environmental inputs
[35,55]. We therefore reasoned that candidate signaling pathways
responsible for oxygen-dependent HIF induction that could
interface with ECM-dependent signaling would include members
of this family. The classical extracellular regulated kinase (ERK)/
MAPK signaling pathway is an obvious candidate because it: 1.
Regulates cell fate in a broad range of organisms [56,57,58,59]; 2.
Responds to hypoxia downstream of mitochondrial ROS 
and, 3. Is activated by integrin ligation . Importantly, in mice,
MEK1 (renamed MAP2K1), as well as its downstream target
ERK2 (renamed MAPK1), are critical regulators of placentation
[62,63,64,65]. Interestingly, MAP2K1 deficiency results in
placental malformation characterized by an excess of multinucle-
ated cells within affected placentas in vivo , similar to the
formation of multinucleated SynTs with HIF-deficient TSCs
in vitro, while genetic disruption of the MAP3K B-Raf results in
placental malformation associated with diminished HIF-1a
protein levels , consistent with our results.
Our results also shed novel insights into HIF-dependent cell fate
determination. We found that HIF-2acan interact with c-MYC to
enhance Limk1 gene expression and promote cytoskeletal integrity,
thereby enhancing TGC differentiation, via non-canonical means.
While HIF-2a has previously been shown to activate c-MYC-
dependent target genes [42,43], to our knowledge this is the first
demonstration of a role for this mechanism during normal
development. Importantly, c-MYC is required for normal
placentation in mice . Also, while HIF activity is generally
assumed to result from the reduced oxygen tension frequently
encountered during development, our results provide evidence
that ECM composition can be added to a growing list of O2-
independent factors that can also drive HIF-dependent develop-
mental programs such as Insulin-like Growth Factor 1 , or
Runx2-mediated HIF stabilization . Additionally, we show
that the dramatic cytoskeletal changes observed in SynTs are not a
simple by-product of cell fusion, but help regulate cell fate
decisions in the placenta. LIMK1 is likely activated downstream of
Rho kinase which is known to be induced during TGC formation
Figure 4. MAP2K1/2 inhibition and cytoskeletal rearrangement in differentiating HIF-null TSCs. Immunofluorescence microscopy of
terminally differentiated wild-type (+/+) TGCs (A) and Hif-1/2a2/2(2/2) SynTs (B) with an anti a-Tubulin (red), or p-MAPK3/1 (green) antibody
(arrows=microtubules, arrowheads=pMAPK3/1). (C) Confocal microscopy imaging of polymerized actin via FITC-phalloidin staining (green) or (D) a -
Tubulin (red) in terminally differentiated control TGCs (dashed line indicates approximate location of nucleus). (E) Confocal microscopy imaging of
polymerized actin via FITC-phalloidin staining (green) or (F) a-Tubulin (red) in terminally differentiated HIF-null SynTs. (G) Differentiation of control
TSCs in the presence of Taxol (G) or (H) Cytochalasin B (CB) promoted the formation of multinucleated cells (arrowheads) following culture on TC
plastic in Fib-CM. a-Tubulin (red) and E-Cadherin (green).
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org6February 2013 | Volume 8 | Issue 2 | e56949
. Interestingly, HIF stability can be influenced by MT integrity
[71,72], suggesting the possibility of a feed-forward mechanism
whereby MAPK-dependent HIF activity promotes MT integrity,
which further enhances HIF stability during TGC differentiation.
In addition to playing important roles during normal placental
development, the pathway outlined here is likely to be involved in
pregnancy complications such as preeclampsia wherein fetal
trophoblasts fail to properly invade maternal tissues. While
aberrant oxygenation and HIF activity have previously been
associated with preeclampsia [73,74], its ability to regulate LIMK1
has particular relevance, given that LIMK1 has recently been
shown to be important for tumor cell invasion in humans  as
well as collective cell migration in D. melanogaster . Further-
more, in addition to altered hypoxia signaling, ECM remodeling is
also frequently disrupted in PE , suggesting novel roles for HIF
in linking these disparate processes. Further investigation of the
intersection of these pathways by HIF activation should therefore
yield novel insights into the etiology of this intractable syndrome.
Mouse TSCs were derived on human placental fibroblasts as
previously described . Prior to differentiation experiments,
TSCs were subjected to a differential plating to remove fibroblasts
and subsequently cultured for two passages in 70% Fib-CM with
FGF4 and heparin . Differentiations were performed in
standard TSC medium without FGF4 or heparin, on tissue culture
Figure 5. HIF-dependent LIMK1 expression promotes TGC formation in TSCs. Immunofluorescence microscopy of terminally differentiated
control TGCs (A) and Hif-1/2a2/2SynTs (B) with a b-Catenin (red) and LIMK1 (green) antibody (arrows=perinunclear LIMK1 staining).
Immunofluorescence microscopy of terminally differentiated control TGCs (C) and Hif-1/2a2/2SynTs (D) with a b-Catenin (green) and p-Cofilin (red)
antibody (arrows=perinunclear p-Cof staining, arrowheads=cofilin rods). (E, F) Two representative images of TGC formation (arrows) following
transient myc-LIMK1 expression in Hif-1/22/2TSCs while untransfected cells primarily form SynTs (arrowheads) (red=myc-LIMK1, green=b-catenin).
(G) Quantification of the percentage of LIMK1 transfected HIF-null TSCs differentiated into TGCs vs. SynTs. (H) Immunoblot analysis of LIMK1 levels in
differentiated wild-type (+/+) TSCs without and with U0126 (U0). Integrated densitometry confirmed the decreased expression of LIMK1, relative to
total Cofilin, in control TSCs differentiated in the presence of U0126.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e56949
treated plates or 0.2% gelatin coated glass coverslips, for 7 days.
Vhlh+/2and2/2TSCs were generously provided by M. Celeste
Simon (U. Penn). Hypoxia (2% O2) was produced with the
Biospherix XVivo incubator. For differentiation in the presence of
inhibitors, TSCs were cultured in plain TSC medium containing
either 10 uM U0126 (Pierce Biotechnology, Rockford, IL), 5 uM
Paclitaxel (Taxol, Calbiochem), 10 ug/ml Cytochalasin B (Sigma),
60 uM c-MYC inhibitor (Sigma) or 10 uM LIMK inhibitor BMS-
Adaptation of TSCs on CELLstartTM
For the adaptation of TSCs to the xeno-free substrate
CELLstartTM(Invitrogen), cells were passaged from feeder culture
using mild trypsinization and plated on CELLstartTMcoated tissue
culture dishes according to manufacturer’s instructions. Cells were
passaged in TSC medium with FGF4 and heparin, but without
Fib-CM, upon reaching approximately 75% confluence. Follow-
ing 5–6 passages, TSC lines on CELLstartTMexhibited a distinct
morphology and maintained that morphology for greater than 20
generations in the absence of Fib-CM.
Northern blot hybridization was performed with the probes for
placental lactogen I and 4311 as described previously .
Immunoblotting and Immunofluorescence Staining
Whole cell lysates were prepared using a buffer consisting of
150 mM NaCl, 50 mM Tris-HCl (pH 7.4), containing 1 mM
EGTA, 1 mM EDTA, 1% Triton X-100, 1% SDS and 10%
glycerol. TSCs were incubated with PHEMT buffer (60 mM
Pipes, 25 mM Hepes, ph 6.9, 10 mM EGTA, 4 mM MgCl2and
0.5% Triton X-100) for 30 min (4uC) and centrifuged to
fractionate TSCs into soluble (supernatant) and insoluble fractions
(pellet). Immunoblotting was performed with ECL (Amersham) or
Odyssey Western blot methodologies and analyzed by Odyssey
infrared imaging system (LI-COR Biosciences, Lincoln, NE) using
the appropriate secondary antibodies. Briefly, 50–100 ug of whole
cell lysates were run on 7.5–12% SDS-PAGE gel, transferred on
PVDF membrane, applied with the primary antibody as indicated.
For immunoprecipitation, 500 ug of whole cell lysates were
immunoprecipitated with the indicated antibody and immuno-
Figure 6. Canonical vs non-canonical HIF target gene-expression in TS cells. (A) Electrophoretic mobility shift assay (EMSA) of differentiated
control TGC nuclear extracts with and without 2 different anti-HIF-1a antibodies (H1) or a HIF-2a antibody (H2) (‘‘supershift’’ SS, NS, non-specific
complexes.) (B) Schematic representation of full-length HIF-1a and HIF-2a, as well as versions lacking their DNA binding basic (b) domains (HLH, Helix-
loop-helix, PAS, Per-Arnt-Sim, ODDD, oxygen-dependent degradation domain). (C) Immunoblot detection of stable HA-epitope tagged HIF-1a, HIF-
1aDb, HIF-2a and HIF-2aDb protein, as well as respective target gene protein products in Hif-1/2a2/2TSCs. (D) Integrated densitometric
quantification of HIF target gene protein products relative to Actin expression in each respective cell line.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org8February 2013 | Volume 8 | Issue 2 | e56949
blotted. The following antibodies were used for immunoblotting,
EMSA, immunoprecipitaion and immunofluoresence staining:
CDX2 (Biogenex), EOMES (Orbigen), anti human/mouse HIF-
1a (R&D Systems, Minneapolis, MN), HIF-1a c-terminal (Cay-
man Chemical, Ann Arbor, MI), HIF-2a NB 100–122 (Novus
Biologicals), ARNT 2B10 (Abcam, Cambridge, MA), pMAPK3/1
(pERK; Cell Signaling, Danvers, MA), MAPK1(ERK2; Epi-
tomics, Burlingame, CA), a-Tubulin (NeoMarkers, Fremont, CA),
Ac- a-Tubulin (Sigma-Aldrich), LIMK1 (BD Biosciences), LIMK2
(Proteintech), Cofilin (BD Biosciences), p-Cofilin (CellSignal),
PDK1 (StressGen), BNIP3, VHL M-20, HOPX1 (Santa Cruz
Biotechnology, Santa Cruz, CA), b-catenin (Cell Signaling), c-
MYC (SCBT), HA (Zymed, South San Francisco, CA), GFP
(Aves), E-cadherin (BD Transduction Pharmingen), and HDAC2
(Zymed). Integrated densitometric analysis was performed using
Adherent cells were washed twice by addition of ice cold PBS to
the monolayer and disposal of the supernatant. 1 ml of freshly
made ice cold lysis/was buffer (50 mM Tris-HCl, 150 mM NaCl
pH 7.5, 1% Nonidet P40 0.5% sodium deoxycholate supplement-
ed with 1 complete tablet from Roche) was added to the washed
cell monolayers to achieve a concentration of 106–107cells/ml.
Cells were scraped into an eppendorf, and sonicated on ice with 5
pulses each for 8 seconds. Lysate was spun down at 13000 rpm for
5 minutes. Supernatant (except 200 ul) was put onto a new tube.
The un-lysed pellet was resuspended into the 200 ul remaining
lysate, and sonicated again, the tube centrifuged at 13000rpm for 5
minutes and the new lysate added to the original lysate. 50 ul of
this lysate was kept aside as input. To reduce background a pre-
clearing step was performed overnight. 50 ul of the homogeneous
protein G- agarose (Roche) suspension, equilibrated in the lysis
buffer, was added to the 1 ml lysate at 2–8uC on a rotating
platform overnight. Beads were then pelleted by centrifugation at
Figure 7. Canonical target gene-independent HIF-2 activity drives LIMK1 expression in TS cells via c-MYC interaction. (A)
Immunoblot analysis of LIMK1 protein levels in Hif-1/2a2/2TSCs stably reconstituted with full length HIF-1a or -2a, as well as versions lacking their
basic domains. (B) Immunoblot analysis of LIMK1 and LIMK2 expression in control (+), Hif-1/2a2/2(Hif2/2), and HIF-2a and HIF-2aDb reconstituted
Hif-1/2a2/2TSCs. Integrated densitometric analysis confirmed that both HIF-2a, as well as HIF-2aDb, restored LIMK1 expression to control levels in
Hif-1/2a2/2TSCs. (C) Immunoprecipitation with an anti-HA antibody of HA-tagged HIF-2aDb followed by immunoblot with anti-HA, c-MYC, b-
Catenin, a-Tubulin and GFP antibodies. (D) Schematic representation of E-box element identified within the Limk1 promoter. Chromatin
immunoprecipitation (ChIP) analysis indicated specific binding of c-MYC and HA-tagged HIF-2aDb to this element. (E) Immunoblot analysis of LIMK1
protein levels in HIF-2aDb expressing Hif-1/2a2/2TSCs without (-) or with (+) c-MYC inhibitor. Integrated densitometric analysis confirmed reduced
expression of LIMK1 relative to a-Tubulin in drug treated cells. (F) Schematic representation of HIF-2a interacting with MYC:MAX heterodimers at the
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org9 February 2013 | Volume 8 | Issue 2 | e56949
20006g for 2 minutes at 4uC. Supernatant was transferred to a
new tube. 50 ul of Agarose-coupled chicken anti-HA (Aves Labs,
Inc. Oregon) was equilibrated in the wash/lysis buffer, centrifuged
for 2 minutes at 20006g, and supernatant discarded. The cell
lysate was added to these beads and rotated (gentle end-over-end
mixing) overnight at 4uC. The lysate/bead complex was then
centrifuged for 2 minutes at 20006g. Pellet was washed 46 by
resuspending in lysis/wash buffer. A final wash was performed
once for 30 minutes. Beads were then resuspended in 90 ul of 26
SDS sample buffer, boiled for 10 minutes at 95uC. Beads were
collected by centrifugation at 27006g for 2 minutes at 4uC and
SDS-PAGE performed with the supernatant.
Cells were washed with D-PBS and cross-linked by 1%
formaldehyde (37 wt% from Sigma-Aldrich) for 10 min at 37uC.
Glycine (2.5M) was added and incubated in room temperature for
10 minutes. Cells were then washed 36in D-PBS for 5 minutes
and harvested with D-PBS in the presence of protease inhibitor
(EDTA-free Complete, Roche Applied Science). These cells were
then centrifuged at 3000 rpm for 5 minutes and lysis buffer (1%
SDS, 10 mM EDTA, 50 mM Tris-HCl, PH 8.1, with fresh
protease inhibitor) was added, sonicated and incubated overnight
at 65uC. Rnase A was added and incubated at 37uC, after which
2 ul 0.5 M EDTA, 4 ul 1 M Tris-HCL PH 8.1, 1 ul proteinase K
was added and incubated for 2 hours at 45uC to generate 200- to
500-bp DNA fragments, which were subsequently confirmed by
agarose gel electrophoresis. Pre-clearing was performed by using
50 ul protein G Sepharose (washed in dilution buffer, 0.01% SDS,
1.1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, PH 8.1,
150 mM MgCl2), 30 ul normal IgG, 20 ug salmon sperm and
rotated for 2 hours at 4uC. 20 ul was taken as input. Protein G
Figure 8. Non-canonical HIF-2-dependent LIMK1 expression promotes TGC formation. Immunofluorescence microscopy of HIF-2aDb
expressing Hif-1/2a2/2TSCs differentiated following culture on TC plastic in Fib-CM (A) or on CELLstartTM(B) (green=b-Cat, red=HA). (C) qRT-PCR-
based comparison of expression levels of the TGC (PLF, Pl-2, Ctsq), spongiotrophoblast (Mash2, 4311) and SynT (Tfeb, SynA) markers in Hif-1/2a2/2
and HIF-2aDb reconstituted HIF-null (Hif2/2:2aDb) TSCs differentiated for 7 days following culture on TC plastic in Fib-CM. p values ,0.05 versus HIF-
null indicated by an asterisk. (D) Immunofluorescence microscopy of HIF-2aDb expressing Hif-1/2a2/2TSCs differentiated following culture on TC
plastic in Fib-CM in the presence of Cytochalasin B or (E) the LIMK inhibitor (BMS-5 10 uM)(green=b-Cat). (F) qRT-PCR-based comparison of
expression levels of the TGC (PLF, Pl-1, Pl-2, Ctsq), spongiotrophoblast (Mash2, 4311) and SynT (Tfeb, SynA) markers in HIF-2aDb reconstituted HIF-
null (Hif2/2:2aDb) TSCs differentiated for 7 days following culture on TC plastic in Fib-CM without and with the actin cytoskeleton disrupting agent
cytochalasin B (Cyto B). p values ,0.05 versus drug free control indicated by an asterisk. (G) Immunofluorescence microscopy of HIF-2aDb expressing
Hif-1/2a2/2TSCs differentiated following culture on CellStartTMin the presence of Cytochalasin B or (H) the LIMK inhibitor (BMS-5 10 uM)(green=b-
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org10February 2013 | Volume 8 | Issue 2 | e56949
sepharose and antibody complexes were prepared by re-suspend-
ing protein G with dilution buffer, 1 ug antibody and incubated on
a rotator at 4uC overnight, and then washed twice with dilution
buffer and centrifuged at 3000 rpm for 1 minute. Precleared
samples were added to the antibody complex beads and rotated at
4uC overnight to collect antibody/antigen/DNA complex. Protein
G complex was centrifuged at 3000 rpm for 1 minute, and
supernatant removed. Protein G complex was washed sequentially
for 5 minutes twice with: low salt buffer (0.1% SDS, 1% Triton X-
100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, PH 8.1),
high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA,
500 mM NaCl, 20 mM Tris-HCl, PH 8.1), LiCl buffer (0.25 M
LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-
HCl, PH 8.1), with TE buffer 1 mM EDTA, 10 mM Tris-HCl,
PH 8.1). 200 ul of elution buffer was used containing 20 ul 10%
SDS, 20 ul 1 M NaHCO3, 160 ul H2O. 100 ul of elution buffer
was added to each tube containing the agarose/antibody complex
or the input and incubated in room temperature for 15 minuites.
Agarose complex was pelleted by centrifugation (50006g, 1
minute) and supernatant collected. This was repeated with another
100 ul elution buffer, and added to the first eluate (total volume
200 ul). Protein/DNA complexes were reversed to free DNA by
adding 8 ul 5 M NaCl, 1 ul 10 mg/ml RNase A, and incubated at
65uC overnight. DNA was purified by using spin columns from
Qiagen. Before purification, 4 ul 0.5 M EDTA, 8 ul 1 M Tris-
HCl, and 1 ul protein kinase k was added to each tube and
incubated at 45uC for 1 hour. Specifically bound purified DNA
fragments were visualized by PCR using specific primers: (forward)
59-tgcatgcaccctaaataaaaata-39, (reverse) 59- ccttgaggagcacacataac-
mRNA Expression Analysis by Real Time PCR
RNA was extracted using TRIzolH Reagent (Invitrogen), and
isolation was carried out according to manufacturer’s instructions.
2 mcg of RNA per sample was made into cDNA using MMLV
reverse transcriptase (Applied Biosystems). Prepared cDNA was
amplified using SYBRH Green PCR Master Mix (Life Technol-
ogies) and the Bio-Rad iCycler iQ multicolor real time PCR
detection system. Cycle threshold (Ct) values were normalized for
amplification using Hypoxanthine guanine phosphoribosyl transferase
(Hprt). Data analysis for real time quantitative PCR was done
using the deltaCt method. Primer sequences are as follows:
tctcatggggcttttgtctc. Placental Lactogen 1 (Pl-1) Sense – tggtgtcaagcc-
tactccttt, Antisense – caggggaagtgttctgtctgt. Placental Lactogen 2 (Pl-
2) Sense – ccaacgtgtgattgtggtgt, Antisense – tcttccgatgttgtctggtg.
Hypoxanthine guanine phosphoribosyl transferase (Hprt) Sense –aaacaatg-
caaactttgctttcc, Antisense – ggtccttttcaccagcaagct. Syncitin A (SynA)
Sense – tactcctgcccgatagatga, Antisense – ccgtttttcttaacagtgggt.
Syncytin B (SynB) Sense – ccaccacccatacgttcaaa, Antisense –
ggttatagcaggtgccgaag. Transcription factor EB (Tfeb) Sense –
aacaaaggcaccatcctcaa, Antisense – cagctcggccatattcacac. Tropho-
blast specific protein alpha (Tpbpa), Sense – cggaaggctccaacatagaa,
Antisense – tcaaattcagggtcatcaacaa. Mammalian achaete-scute homolog
2 (Mash2) Sense – TTTTCGAGGACGCAATAAGC, Antisense –
cactgctgcaggactcccta. Statistical analysis of real time PCR results
were performed as follows: All data points were performed in
triplicate. One-way analysis of variance of the results was
performed in Microsoft Excel 2007 to determine the presence of
significant differences within the data sets. When analysis of
variance indicated that a significant difference may be present, a
two-sample Student’s t-test was performed to compare experi-
mental data with appropriate controls . Statistical significance
was determined at a value of P,0.05 and is represented with an
Plasmid Constructs and Ectopic Expression
The full-length open reading frame of Hif-1a and Hif-2a were
PCR amplified from cDNA constructs. Deletional mutants were
generated removing the basic domain, Hif-1aDb (deletion of
amino acids 4–27) and Hif-2aDb (deletion of amino acids 6–24) by
high fidelity PCR. All expression constructs were modified to
include the 9 amino acid hemagglutinin epitope YPYDVPDYA
fused directly to the C-terminus and cloned into the ENTRD-
TOPO vector (Invitrogen). The integrity of the constructs was
confirmed by DNA sequencing. For expression in cell culture, a
derivative of the Piggybac transposon system was employed
allowing high efficiency expression. The parental plasmid EBXN
Figure 9. Model of HIF-dependent integration of positional and metabolic cues in the TSC niche. ECM composition regulates HIF
stabilization likely downstream of cell surface integrin ligation via MAP2K1/2 activation. Inside-out integrin signaling mechanisms may also be
operative. Oxygen sensing and signaling pathways intersect with this signaling cascade to stabilize HIF, when ECM-dependent cues are absent.
Stabilized HIF can act via canonical and non-canonical target genes. Non-canonical HIF-2, by interacting with MYC:MAX heterodimers, bind the Limk1
promoter to activate its expression. LIMK1 promotes microtubule and actin stability, critical for TGC formation, and thereby prevents SynT formation.
HIF, therefore, can integrate divergent environmental inputs from within the placenta to regulate cell fate via non-canonical gene expression.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org11February 2013 | Volume 8 | Issue 2 | e56949
containing the minimal Piggybac 59 and 39 inverted terminal
repeats as well as a CMV enhancer chicken Beta-actin promoter
expression cassette was modified to include the SV40 promoter
Blasticidin cassette allowing for eukaryotic selection in cell culture.
The plasmid was further modified to include the Invitrogen
Gateway Rfa cassette allowing for phiC31 mediated recombina-
tion. For monitoring transfection efficiency, EMCV IRES
upstream of palmitoylated EGFP was inserted, PBX2.2. A control
construct containing monomeric EGFP (Karel Svoboda, Addgene
Plasmid 18696) was inserted into the parental plasmid PBX2.1.
Transfection of TSC lines was performed with Lipofectamine
LTX and PLUS reagent (Invitrogen) in placental fibroblast-free
culture. A 2:1 molar ratio of Piggybac transposase helper plasmid,
PB, was combined with the transposon expression construct to
mediate integration and high level expression. Selection with
Blasticidin 5 mcg/ml was performed to identify stable integrants,
which were subsequently passaged on placental fibroblasts.
We thank Susan Fisher, Olga Genbacev, Anna Bakardjiev and Kevin
Shannon (UCSF) for reagents and thoughtful discussion of the manuscript
and M. Celeste Simon (Abramson Cancer Research Institute, UPenn) for
Vhlh2/2TSCs. We thank Torsten Wittmann (UCSF) for help with
confocal microscopy and thoughtful discussion, Natalie Ahn (HHMI, UC
Boulder) for MAP2K1 constructs, Michael Olson (Beatson institute for
Cancer Research, Glasgow) for LIMK1 constructs and MK Tee (WL
Miller Lab, UCSF) for assistance with genomics analyses. The contents of
this publication are solely the responsibility of the authors and do not
necessarily represent the official views of CIRM or any other agency of the
State of California.
Conceived and designed the experiments: HJC TAS EM PFR. Performed
the experiments: HJC TAS KVT KA JDT AMP JG AMR XL DMQ.
Analyzed the data: HJC KVT PFR EM. Wrote the paper: EM.
1. Simon MC, Keith B (2008) The role of oxygen availability in embryonic
development and stem cell function. Nat Rev Mol Cell Biol 9: 285–296.
2. Semenza GL (2009) Regulation of oxygen homeostasis by hypoxia-inducible
factor 1. Physiology (Bethesda) 24: 97–106.
3. Kaelin WG Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: the central role
of the HIF hydroxylase pathway. Mol Cell 30: 393–402.
4. Wang GL, Jiang BH, Rue EA, Semenza GL (1995) Hypoxia-inducible factor 1 is
a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc
Natl Acad Sci U S A 92: 5510–5514.
5. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, et al. (1998)
Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.
Proc Natl Acad Sci U S A 95: 11715–11720.
6. Dunwoodie SL (2009) The role of hypoxia in development of the Mammalian
embryo. Dev Cell 17: 755–773.
7. Ryan HE, Lo J, Johnson RS (1998) HIF-1 alpha is required for solid tumor
formation and embryonic vascularization. Embo J 17: 3005–3015.
8. Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, et al. (1998) Cellular and
developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha.
Genes Dev 12: 149–162.
9. Maltepe E, Schmidt JV, Baunoch D, Bradfield CA, Simon MC (1997) Abnormal
angiogenesis and responses to glucose and oxygen deprivation in mice lacking
the protein ARNT. Nature 386: 403–407.
10. Cowden Dahl KD, Fryer BH, Mack FA, Compernolle V, Maltepe E, et al.
(2005) Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast
differentiation. Mol Cell Biol 25: 10479–10491.
11. Kozak KR, Abbott B, Hankinson O (1997) ARNT-deficient mice and placental
differentiation. Dev Biol 191: 297–305.
12. Adelman DM, Gertsenstein M, Nagy A, Simon MC, Maltepe E (2000) Placental
cell fates are regulated in vivo by HIF-mediated hypoxia responses. Genes Dev
13. Maltepe E, Bakardjiev AI, Fisher SJ (2012) The placenta: transcriptional,
epigenetic, and physiological integration during development. J Clin Invest 120:
14. Rossant J, Cross JC (2001) Placental development: lessons from mouse mutants.
Nat Rev Genet 2: 538–548.
15. Watson ED, Cross JC (2005) Development of structures and transport functions
in the mouse placenta. Physiology (Bethesda) 20: 180–193.
16. Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J (1998) Promotion
of trophoblast stem cell proliferation by FGF4. Science 282: 2072–2075.
17. Maltepe E, Krampitz GW, Okazaki KM, Red-Horse K, Mak W, et al. (2005)
Hypoxia-inducible factor-dependent histone deacetylase activity determines
stem cell fate in the placenta. Development 132: 3393–3403.
18. Zheng QA, Chang DC (1991) Reorganization of cytoplasmic structures during
cell fusion. J Cell Sci 100 (Pt 3): 431–442.
19. Shibukawa Y, Yamazaki N, Kumasawa K, Daimon E, Tajiri M, et al. (2012)
Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell
Fusion. Mol Biol Cell.
20. Yoshie M, Kashima H, Bessho T, Takeichi M, Isaka K, et al. (2008) Expression
of stathmin, a microtubule regulatory protein, is associated with the migration
and differentiation of cultured early trophoblasts. Hum Reprod 23: 2766–2774.
21. Gauster M, Siwetz M, Orendi K, Moser G, Desoye G, et al. (2012) Caspases
rather than calpains mediate remodelling of the fodrin skeleton during human
placental trophoblast fusion. Cell Death Differ 17: 336–345.
22. Swistowski A, Peng J, Han Y, Swistowska AM, Rao MS, et al. (2009) Xeno-free
defined conditions for culture of human embryonic stem cells, neural stem cells
and dopaminergic neurons derived from them. PLoS One 4: e6233.
23. Swistowski A, Peng J, Liu Q, Mali P, Rao MS, et al. (2012) Efficient generation
of functional dopaminergic neurons from human induced pluripotent stem cells
under defined conditions. Stem Cells 28: 1893–1904.
24. Hughes CS, Radan L, Betts D, Postovit LM, Lajoie GA (2012) Proteomic
analysis of extracellular matrices used in stem cell culture. Proteomics 11: 3983–
25. Damsky CH, Fitzgerald ML, Fisher SJ (1992) Distribution patterns of
extracellular matrix components and adhesion receptors are intricately
modulated during first trimester cytotrophoblast differentiation along the
invasive pathway, in vivo. J Clin Invest 89: 210–222.
26. Armant DR (2005) Blastocysts don’t go it alone. Extrinsic signals fine-tune the
intrinsic developmental program of trophoblast cells. Dev Biol 280: 260–280.
27. Asanoma K, Kato H, Yamaguchi S, Shin CH, Liu ZP, et al. (2007) HOP/
NECC1, a novel regulator of mouse trophoblast differentiation. J Biol Chem
28. Hynes RO (2009) The extracellular matrix: not just pretty fibrils. Science 326:
29. Hamanaka RB, Chandel NS (2012) Mitochondrial reactive oxygen species
regulate cellular signaling and dictate biological outcomes. Trends Biochem Sci
30. Page EL, Robitaille GA, Pouyssegur J, Richard DE (2002) Induction of hypoxia-
inducible factor-1alpha by transcriptional and translational mechanisms. J Biol
Chem 277: 48403–48409.
31. Richard DE, Berra E, Gothie E, Roux D, Pouyssegur J (1999) p42/p44 mitogen-
activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-
1alpha) and enhance the transcriptional activity of HIF-1. J Biol Chem 274:
32. Emerling BM, Platanias LC, Black E, Nebreda AR, Davis RJ, et al. (2005)
Mitochondrial reactive oxygen species activation of p38 mitogen-activated
protein kinase is required for hypoxia signaling. Mol Cell Biol 25: 4853–4862.
33. Craig EA, Stevens MV, Vaillancourt RR, Camenisch TD (2008) MAP3Ks as
central regulators of cell fate during development. Dev Dyn 237: 3102–3114.
34. Campbell ID, Humphries MJ (2011) Integrin structure, activation, and
interactions. Cold Spring Harb Perspect Biol 3.
35. Butcher DT, Alliston T, Weaver VM (2009) A tense situation: forcing tumour
progression. Nat Rev Cancer 9: 108–122.
36. Scales TM, Parsons M (2011) Spatial and temporal regulation of integrin
signalling during cell migration. Curr Opin Cell Biol 23: 562–568.
37. Parast MM, Aeder S, Sutherland AE (2001) Trophoblast giant-cell differenti-
ation involves changes in cytoskeleton and cell motility. Dev Biol 230: 43–60.
38. Scott RW, Olson MF (2007) LIM kinases: function, regulation and association
with human disease. J Mol Med (Berl) 85: 555–568.
39. Minamide LS, Striegl AM, Boyle JA, Meberg PJ, Bamburg JR (2000)
Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt
distal neurite function. Nat Cell Biol 2: 628–636.
40. Yusa K, Rad R, Takeda J, Bradley A (2009) Generation of transgene-free
induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods
41. Jiang BH, Rue E, Wang GL, Roe R, Semenza GL (1996) Dimerization, DNA
binding, and transactivation properties of hypoxia-inducible factor 1. J Biol
Chem 271: 17771–17778.
42. Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, et al. (2004) HIF-
1alpha induces cell cycle arrest by functionally counteracting Myc. Embo J 23:
43. Gordan JD, Bertout JA, Hu CJ, Diehl JA, Simon MC (2007) HIF-2alpha
promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity.
Cancer Cell 11: 335–347.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org12 February 2013 | Volume 8 | Issue 2 | e56949
44. Scott RW, Hooper S, Crighton D, Li A, Konig I, et al. (2012) LIM kinases are
required for invasive path generation by tumor and tumor-associated stromal
cells. J Cell Biol 191: 169–185.
45. Ross-Macdonald P, de Silva H, Guo Q, Xiao H, Hung CY, et al. (2008)
Identification of a nonkinase target mediating cytotoxicity of novel kinase
inhibitors. Mol Cancer Ther 7: 3490–3498.
46. Guilak F, Cohen DM, Estes BT, Gimble JM, Liedtke W, et al. (2009) Control of
stem cell fate by physical interactions with the extracellular matrix. Cell Stem
Cell 5: 17–26.
47. Kim SH, Turnbull J, Guimond S (2012) Extracellular matrix and cell signalling:
the dynamic cooperation of integrin, proteoglycan and growth factor receptor.
J Endocrinol 209: 139–151.
48. Votteler M, Kluger PJ, Walles H, Schenke-Layland K (2012) Stem cell
microenvironments–unveiling the secret of how stem cell fate is defined.
Macromol Biosci 10: 1302–1315.
49. Eshghi S, Vogelezang MG, Hynes RO, Griffith LG, Lodish HF (2007)
Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in
erythropoiesis: integrins in red cell development. J Cell Biol 177: 871–880.
50. Skuli N, Monferran S, Delmas C, Favre G, Bonnet J, et al. (2009) Alphavbeta3/
alphavbeta5 integrins-FAK-RhoB: a novel pathway for hypoxia regulation in
glioblastoma. Cancer Res 69: 3308–3316.
51. Cowden Dahl KD, Robertson SE, Weaver VM, Simon MC (2005) Hypoxia-
inducible factor regulates alphavbeta3 integrin cell surface expression. Mol Biol
Cell 16: 1901–1912.
52. Wang J, Armant DR (2002) Integrin-mediated adhesion and signaling during
blastocyst implantation. Cells, tissues, organs 172: 190–201.
53. Sutherland A (2003) Mechanisms of implantation in the mouse: differentiation
and functional importance of trophoblast giant cell behavior. Dev Biol 258: 241–
54. Rosario GX, Konno T, Soares MJ (2008) Maternal hypoxia activates
endovascular trophoblast cell invasion. Dev Biol 314: 362–375.
55. Ramos JW (2008) The regulation of extracellular signal-regulated kinase (ERK)
in mammalian cells. Int J Biochem Cell Biol 40: 2707–2719.
56. Hsu JC, Perrimon N (1994) A temperature-sensitive MEK mutation demon-
strates the conservation of the signaling pathways activated by receptor tyrosine
kinases. Genes Dev 8: 2176–2187.
57. Wu Y, Han M, Guan KL (1995) MEK-2, a Caenorhabditis elegans MAP kinase
kinase, functions in Ras-mediated vulval induction and other developmental
events. Genes Dev 9: 742–755.
58. Umbhauer M, Marshall CJ, Mason CS, Old RW, Smith JC (1995) Mesoderm
induction in Xenopus caused by activation of MAP kinase. Nature 376: 58–62.
59. Kornfeld K, Guan KL, Horvitz HR (1995) The Caenorhabditis elegans gene
mek-2 is required for vulval induction and encodes a protein similar to the
protein kinase MEK. Genes Dev 9: 756–768.
60. Hamanaka RB, Chandel NS (2009) Mitochondrial reactive oxygen species
regulate hypoxic signaling. Curr Opin Cell Biol 21: 894–899.
61. Harburger DS, Calderwood DA (2009) Integrin signalling at a glance. J Cell Sci
62. Bissonauth V, Roy S, Gravel M, Guillemette S, Charron J (2006) Requirement
for Map2k1 (Mek1) in extra-embryonic ectoderm during placentogenesis.
Development 133: 3429–3440.
63. Saba-El-Leil MK, Vella FD, Vernay B, Voisin L, Chen L, et al. (2003) An
essential function of the mitogen-activated protein kinase Erk2 in mouse
trophoblast development. EMBO Rep 4: 964–968.
64. Hatano N, Mori Y, Oh-hora M, Kosugi A, Fujikawa T, et al. (2003) Essential
role for ERK2 mitogen-activated protein kinase in placental development.
Genes Cells 8: 847–856.
65. Giroux S, Tremblay M, Bernard D, Cardin-Girard JF, Aubry S, et al. (1999)
Embryonic death of Mek1-deficient mice reveals a role for this kinase in
angiogenesis in the labyrinthine region of the placenta. Curr Biol 9: 369–372.
66. Nadeau V, Guillemette S, Belanger LF, Jacob O, Roy S, et al. (2009) Map2k1
and Map2k2 genes contribute to the normal development of syncytiotropho-
blasts during placentation. Development 136: 1363–1374.
67. Galabova-Kovacs G, Matzen D, Piazzolla D, Meissl K, Plyushch T, et al. (2006)
Essential role of B-Raf in ERK activation during extraembryonic development.
Proc Natl Acad Sci U S A 103: 1325–1330.
68. Dubois NC, Adolphe C, Ehninger A, Wang RA, Robertson EJ, et al. (2008)
Placental rescue reveals a sole requirement for c-Myc in embryonic erythroblast
survival and hematopoietic stem cell function. Development 135: 2455–2465.
69. Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, et al. (2002) Insulin-like growth
factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial
growth factor expression, which is dependent on MAP kinase and phosphati-
dylinositol 3-kinase signaling in colon cancer cells. J Biol Chem 277: 38205–
70. Lee SH, Che X, Jeong JH, Choi JY, Lee YJ, et al. (2012) Runx2 protein
stabilizes hypoxia-inducible factor-1alpha through competition with von Hippel-
Lindau protein (pVHL) and stimulates angiogenesis in growth plate hypertro-
phic chondrocytes. J Biol Chem 287: 14760–14771.
71. Escuin D, Kline ER, Giannakakou P (2005) Both microtubule-stabilizing and
microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accu-
mulation and activity by disrupting microtubule function. Cancer Res 65: 9021–
72. Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, Swartz GM, et al. (2003)
2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and
dysregulating HIF. Cancer Cell 3: 363–375.
73. Burton GJ (2009) Oxygen, the Janus gas; its effects on human placental
development and function. J Anat 215: 27–35.
74. Pringle KG, Kind KL, Sferruzzi-Perri AN, Thompson JG, Roberts CT (2009)
Beyond oxygen: complex regulation and activity of hypoxia inducible factors in
pregnancy. Hum Reprod Update.
75. Zhang L, Luo J, Wan P, Wu J, Laski F, et al. (2012) Regulation of cofilin
phosphorylation and asymmetry in collective cell migration during morphogen-
esis. Development 138: 455–464.
76. Karthikeyan VJ, Lane DA, Beevers DG, Lip GY, Blann AD (2012) Matrix
metalloproteinases and their tissue inhibitors in hypertension-related pregnancy
complications. J Hum Hypertens.
77. Rieu I, Powers SJ (2009) Real-time quantitative RT-PCR: design, calculations,
and statistics. Plant Cell 21: 1031–1033.
Non-Canonical HIF Integrates Multiple Cues
PLOS ONE | www.plosone.org 13 February 2013 | Volume 8 | Issue 2 | e56949