RESEARCH ARTICLE 3311
STEM CELLS AND REGENERATION
Development 140, 3311-3322 (2013) doi:10.1242/dev.091082
© 2013. Published by The Company of Biologists Ltd
MRAS GTPase is a novel stemness marker that impacts
mouse embryonic stem cell plasticity and Xenopus embryonic
cell fate
Marie-Emmanuelle Mathieu1,2, Corinne Faucheux1,2, Claire Saucourt1,2,*, Fabienne Soulet1,2,‡,
Xavier Gauthereau1,2, Sandrine Fédou1,2, Marina Trouillas1,§, Nadine Thézé1,2, Pierre Thiébaud1,2,¶ and
Hélène Bœuf1,2,¶
SUMMARY
Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide
a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have
led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of
the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we
used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein
sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that
MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate
the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows
gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved
and pleiotropic effects of MRAS in stem cells and early steps of development.
INTRODUCTION
Studies of stemness status in mouse embryonic stem cells (mESCs)
through transcriptomic, proteomic and drug screenings have
enabled the identification of many gene families potentially
involved in the maintenance of cell pluripotency, among which are
the regulators of Ras family (Chen et al., 2006; Ivanova et al., 2006;
Wray et al., 2010). Understanding the function of newly discovered
genes involved in the control of the pluripotency/differentiation
balance in stem cells requires the use of complementary models to
address cell biology and developmental questions in fast and
efficient ways. In this report, we have characterised the effects of a
novel regulated GTPase, muscle ras oncogene homologue (MRAS;
also known as muscle and microspikes RAS), in mESCs and in
Xenopus laevis. The convenient and efficient manipulation of gene
expression in Xenopus embryos, along with studies performed in
the mESC model, allow information concerning the basic conserved
function of genes during evolution to be examined.
mESCs are maintained in a pluripotent state in the presence of
the leukemia inhibitory factor (LIF) cytokine, which induces the
JAK1/STAT3/SOCS3 pathway that forms part of the pluripotency
circuitry along with Wnt and bone morphogenetic protein 4 (BMP4)
secreted factors, and the master genes such as Oct4 (Pou5f1), Nanog
and Sox2 (Boeuf et al., 1997; Duval et al., 2000; Niwa, 2007;
1
University of Bordeaux, CIRID, UMR 5164, F-33000 Bordeaux, France. 2CNRS,
CIRID, UMR 5164, F-33000 Bordeaux, France.
*Present address: IRHT, F-68100 Mulhouse, France
‡
Present address: INSERM U1029, F-33405 Talence, France
§
Present address: CTSA, F-92141 Clamart, France
¶
Authors for correspondence ([email protected];
[email protected])
Accepted 4 June 2013
Chambers and Tomlinson, 2009; Trouillas et al., 2009a). Extensive
microarray studies, performed in mESCs grown under pluripotent
conditions (with LIF) or induced to differentiate (24 hours or
48 hours without LIF) and restimulated for a short period of time by
LIF, have led to the identification of three LIF signatures (Pluri,
Spe-Lifind and Pleio-Lifind) (Mathieu et al., 2012; Trouillas et al.,
2009a; Trouillas et al., 2009b). An interesting feature of genes from
the Pluri cluster is their early downregulation upon LIF withdrawal
as compared with the master genes (Schulz et al., 2009; Trouillas et
al., 2009b).
Mras is a member of the R-ras subfamily of the large Ras family
and encodes a small GTPase (Díez et al., 2011). In mESCs, Mras is
in the Pluri gene cluster, with an expression profile similar to that
of Esrrb and Tcl1 (Ivanova et al., 2006; Trouillas et al., 2009b).
Mras expression is under the control of the KLF5 transcription
factor (Parisi and Russo, 2011; Parisi et al., 2010), with elevated
transcripts in specific brain areas, heart and the developing bones.
The protein is concentrated on plasma membrane-associated
structures (Kimmelman et al., 1997; Kimmelman et al., 2002;
Matsumoto et al., 1997; Watanabe-Takano et al., 2010). MRAS
expression and its GTPase activity are stimulated by nerve growth
factor (NGF) in pheochromocytoma PC12 cells, in which it triggers
ERK-dependent neuronal differentiation (Kimmelman et al., 2002;
Sun et al., 2006). MRAS expression is also stimulated by the BMP2
signalling pathway in myoblast C2C12 cells, in which it induces
osteoblast differentiation under the control of the p38 and JNK
kinases (Watanabe-Takano et al., 2010). Activated forms of MRAS,
such as the MRAS-G22V mutant which is constitutively in the
GTP-bound form, mimic inducers (NGF or BMP2) and trigger
neuronal or osteoblast differentiation in PC12 or C2C12 models,
respectively (Kimmelman et al., 2002; Sun et al., 2006; WatanabeTakano et al., 2010), induce formation of peripheral microspikes in
fibroblasts and participate in the reorganisation of the actin
DEVELOPMENT
KEY WORDS: LIF, Mras, mES cells, Neurogenesis, Pluripotency, Mouse, Xenopus
Development 140 (16)
3312 RESEARCH ARTICLE
Flow cytometry
Plated cells were washed once at room temperature with PBS, trypsinised
and counted. Cells were fixed for 20 minutes in 4% paraformaldehyde,
permeabilised with Perm/Wash Buffer (BD Biosciences) for 10 minutes and
labelled with 20 µl antibody for 1 hour with the Mouse Pluripotent Stem
Cell Transcription Factor Analysis Kit (BD Biosciences). After two washes
with the Wash Buffer for 10 minutes, cells were processed on a FACSCanto
II flow cytometer (BD Biosciences). The percentage of positive cells and the
mean fluorescence intensity (MFI) were obtained with DIVA software (BD
Biosciences).
RT-PCR and RT-qPCR
Total RNA from adherent ESCs was prepared with Trizol reagent as described
(Trouillas et al., 2009b). The reverse transcription reaction products were used
for qPCR with specific primer sets and relative expression was calculated
using Hprt (CGR8 cell line) or Erk2 (Mapk1) (E14 cell lines) as reference
genes. RNA from Xenopus embryo and explants was prepared and analysed
according to standard protocols (Sive et al., 2000; Naye et al., 2007). Primers
are listed in supplementary material Table S1.
Construction of expression vectors and establishment of stable
clones
Expression vector construction
RT-PCR-amplified fragments of Mus musculus Mras (NM_008624) were
inserted in fusion with EGFP cDNA in the NheI/XhoI site of the pCXN2
vector (Niwa et al., 1991). A 627 bp Xenopus laevis Mras cDNA was cloned
by RT-PCR from stage 20 embryo RNA into the EcoRI/XhoI sites of the
pCS2 vector (a gift from R. Rupp) using gene-specific primers: forward,
5⬘-cgcggaattcATGGCAACGAGTGCTGTTCTGAG-3⬘; reverse, 5⬘-cgcgctcgagTCACAATACGACACACTGCAGCCTG-3⬘.
Mouse Mras was cloned from pCAG-EGFP-MRAS into pCS2 by
digestion with NheI (end filled)/XhoI and ligation into the EcoRI (end
filled)/XhoI sites of pCS2. MRAS G22V and MRAS S27N encoding
sequences were cloned from the pEF-BOS-MycMras constructs (Sun et al.,
2006) into the BamHI and XbaI sites of pCS2. Plasmid constructs were
checked by DNA sequencing.
Derivation of mESC stable clones
This study was carried out in accordance with the European Community
Guide for Care and Use of Laboratory Animals and approved by the Comité
d’éthique en expérimentation de Bordeaux, No. 33011005-A.
To establish ESC clones expressing EGFP or EGFP-MRAS, CGR8 cells
maintained in LIF-containing medium were transfected using
Lipofectamine (Invitrogen) with 0.5-2 µg pCXN2-derived construct per 6cm Petri dish. After 2 weeks (for EGFP) and up to 4 weeks (for EGFPMRAS) of selection in the presence of 300 µg/ml G418, double-positive
clones (NeoR and EGFP+) were picked and grown in the presence of G418.
Images were captured on a Zeiss Axiovert 200 microscope with an
AxioCam HR camera and AxioVision 3.0 and Axioviewer 3.0 software.
Cell culture
Embryological methods
MATERIALS AND METHODS
Ethics statement
Heat map analysis was performed using the FunGenES website interface
(Schulz et al., 2009; Trouillas et al., 2009b).
Xenopus laevis embryos were obtained by standard procedures (Sive et al.,
2000) and staged according to Nieuwkoop and Faber (Nieuwkoop and
Faber, 1967). Animal cap explants were dissected from stage 8 or stage 11
embryos, treated with FGF2 or activin (R&D Systems) and cultured until
the control embryos reached the appropriate stage before RT-PCR analysis.
For microinjection experiments, capped mRNAs were synthesised using
the mMessenger mMachine SP6 Kit (Ambion). Injections were performed
into one blastomere at the 2-cell stage, with 250 pg lacZ mRNA as lineage
tracer. For knockdown experiments, 20-40 ng morpholinos (MOs) (Gene
Tools; supplementary material Table S2) were injected into 2-cell stage
embryos. In the animal cap assay, 500 pg Mras mRNA and 50 pg noggin
mRNA were injected into the animal pole of the 2-cell stage embryo in both
blastomeres.
Cell lysates and western blots
Whole-mount in situ hybridisation and immunohistochemistry
CGR8, E14Tg2aWT and E14Tg2a GeneTrap Mras (feeder-free) ESC lines
were grown as described (Trouillas et al., 2009b). The E14Tg2a GeneTrap
Mras cell line (E14Dmras) was purchased from the Mutant Mouse Regional
Resource Center (MMRRC) under the strain name BayGenomics ESC line
CSH312, stock number 014915-UCD. This cell line contains in exon 2 of
Mras an insertion of the β-geo fusion cDNA in one allele. A half dose of
Mras expression was validated with two sets of primers (sequences are
listed in supplementary material Table S1). The E14Tg2aWT (E14WT)
control cell line was purchased from ATCC.
Heat map analysis
Plated ESCs were rinsed twice at room temperature with PBS and lysed
directly in mild RIPA buffer (Trouillas et al., 2009b). Total protein (100 µg)
was loaded on 10% SDS-PAGE gels, transferred to nitrocellulose
membranes and incubated with anti-OCT4 (Abcam, 18976; 1:500), antiNANOG (Abcam, 21603; 1:1000) and anti-ERK2 (Santa Cruz Biotech, sc154; 1:5000) antibodies.
In situ hybridisation and immunohistochemistry were performed according
to standard protocols (Sive et al., 2000). For serial sections, embryos postfixed in MEMFA (0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4 and 3.7%
formaldehyde) were embedded in Paraplast (Sigma) and cut on a
microtome. The 12/101 antibody (Developmental Studies Hybridoma Bank)
was used at 1:2 dilution.
DEVELOPMENT
cytoskeleton (Matsumoto et al., 1997). In addition, these activated
forms are oncogenic in murine mammary epithelial cell lines (Ward
et al., 2004). MRAS function is potentially regulated by plexin B1,
a dual-function GTPase-activating protein (GAP) for RRAS and
MRAS, which remodels axon and dendrite morphology,
respectively (Saito et al., 2009).
There is a single Mras gene from nematodes to mammals and
in the ascidian embryo the orthologue has a role in neural
differentiation, suggesting an evolutionarily conserved function
(Keduka et al., 2009). The Xenopus embryo has contributed
substantially to the deciphering of the molecular mechanisms that
control embryo patterning, which relies on signalling pathways
that are highly conserved between mammals and amphibians
(Harland and Grainger, 2011; Heasman, 2006). LIF and its
receptors (gp130 and gp190), along with the Pluri genes (such as
Esrrb, Sod2, Cd9, Fzd5, Susd2 and Smarcd3), are conserved in
Xenopus laevis (our unpublished data). In addition, cells from the
animal part of the Xenopus blastula embryo (animal cap cells) are
similar to mammalian ESCs. They are pluripotent and able to
differentiate toward the lineage-specific cell types of the three
germ layers (Lan et al., 2009; Okabayashi and Asashima, 2003).
Moreover, the molecular mechanisms that govern self-renewal
mediated by Oct4 are conserved between mammals and Xenopus
(Morrison and Brickman, 2006; Abu-Remaileh et al., 2010). This
makes Xenopus a suitable model in which the basic conserved
functions of these genes can be studied during early
embryogenesis and organogenesis, two processes not easily
amenable in the mouse embryo. For instance, our recent work
using a combined approach of mESCs and Xenopus embryonic
cells has demonstrated a conserved function for a histone
methyltransferase adaptor protein in smooth muscle cell
differentiation (Gan et al., 2011).
In this study, we have carried out a functional analysis of Mras in
mESC and Xenopus models, leading to the characterisation of Mras
as a novel stemness marker that impacts on pluripotent cells and on
neurogenesis.
Mras in mESCs and Xenopus embryo
RESEARCH ARTICLE 3313
In vitro translation
In vitro transcribed mRNAs (500 ng) were translated in rabbit reticulocyte
lysate (Promega) and the reaction products were analysed by 12% SDSPAGE followed by autoradiography.
Sequence analysis
The Mus musculus MRAS protein sequence (NM_008624) was BLASTed
in the SWISS-PROT database and the sequences of various organisms were
retrieved and aligned using the MultiAlin tool (Corpet, 1988). Phylogenetic
analyses were performed with the following sequences: Xenopus laevis (Xl),
NP_001085506; Homo sapiens (Hs), NM_012219; Canis familiaris (Cf),
XP_534277; Mus musculus (Mm), NM_008624; Rattus norvegicus (Rn),
NM_012981; Gallus gallus (Gg), NM_204489; Danio rerio (Dr), 003201047.
Genes structure and intron size comparisons were retrieved from Ensembl.
Fig. 1. Mras has a unique expression profile among Ras and effectorof-Ras proteins. (A) Heat map analysis of expression profiles of Ras
members and regulators (Gef in red and Gap in blue) on a timescale of LIF
depletion from days 1 to 10 in mESCs. (B) RT-qPCR analysis of Ras family
members in cells grown with or without LIF for 24 hours or 48 hours and
reinduced for 25 minutes with foetal calf serum (FCS) or FCS + LIF. Mean
of expression with s.d. of triplicates is shown.
Persistent downregulation of Mras leads to a
decrease in the expression of core pluripotency
markers
To determine whether Mras controls the fate of pluripotent cells,
the expression of the core pluripotent genes (Oct4, Sox2 and Nanog)
and a selection of Pluri or differentiation markers was examined in
cells in which Mras expression was knocked down. We used a
clonal heterozygous GeneTrap Mras ESC line (E14Dmras), which
harbours an insertion of a β-geo cassette in exon 2 (see Materials
and methods). Mras expression was decreased by 50% in this cell
line when compared with the parental E14WT mESC line (Fig. 2A).
This mutated cell line, despite being alkaline phosphatase positive
in the presence of LIF, displays a reduced self-renewal efficiency
(7%, versus 35% for E14WT; supplementary material Fig. S1A).
To further characterise the deficit caused by decreased
expression of Mras, E14WT and E14Dmras were grown in the
presence or absence of LIF for 4 days. Whereas there was no
DEVELOPMENT
RESULTS
Mras, a new marker of stemness
Extensive microarray analyses performed with mESCs grown under
different culture conditions (pluripotency or differentiation
induction) have enabled the identification of novel markers of
pluripotent mESCs and the characterisation of a set of Pluri genes
that are highly expressed in undifferentiated mESCs (Schulz et al.,
2009; Trouillas et al., 2009a; Trouillas et al., 2009b). Here, we focus
on the expression profiles of Ras family members. Probe set
numbers of this gene family and related members (including the
Ras-Gap and Ras-Gef genes, 273 probe sets) were retrieved from
the Affymetrix 420.2 microarray and their expression profiles
analysed in pluripotent cells and after LIF withdrawal from 1 to 10
days. A heat map representation of a selection of genes (46 probe
sets) is presented (Fig. 1A).
This analysis showed heterogeneous expression profiles of Ras
members, and a particular profile for Mras. Indeed it is the only
member that is restricted in its expression to undifferentiated ESCs,
with a drastic reduction of expression as early as 1 day after LIF
withdrawal. We confirmed, using RT-qPCR analysis, that
expression of Mras, but not Hras, Kras, Rras or Eras, is regulated
by LIF (Fig. 1B). We also showed that Mras expression is under
LIF control only in undifferentiated cells. Indeed, Mras expression
is not induced by short LIF treatment in committed (24 hours minus
LIF) or differentiated (48 hours minus LIF) cells, in contrast to the
Lifind genes, which respond to LIF in ESC-derived committed and
differentiated cells and in other mature LIF-responsive cell types
(Trouillas et al., 2009a; Trouillas et al., 2009b; Mathieu et al., 2012).
These results revealed that Mras constitutes a new marker of
stemness.
3314 RESEARCH ARTICLE
Development 140 (16)
Fig. 2. Sustained knockdown of Mras
leads to decreased expression of Oct4
and Pluri genes. (A-C) RT-qPCR expression
analysis of (A) Mras and a selection of Ras
family members, (B) Pluri and master genes
and (C) differentiation markers in E14WT or
E14Dmras mESC lines grown with or without
LIF for 4 days. The average of three
independent experiments is shown with s.d.
*P<0.05, WT versus Dmras in the presence of
LIF; **P<0.01, WT or Dmras with LIF versus
without LIF. (D) Immunoblot of E14WT and
E14Dmras cell lysates cultured with or
without LIF for 4 days.
Overexpression of MRAS leads to different
cellular phenotypes
MRAS overexpression or its activation was previously shown to
induce differentiation in various cell models (Kimmelman et al.,
2002; Sun et al., 2006; Watanabe-Takano et al., 2010). We tested
the effect of overexpression of MRAS in mESCs grown with or
without LIF. MRAS protein expression was detected in 30-50% of
the transfected cells (data not shown) the day after transfection, with
an average of 10-fold overexpression of the transfected gene in
transient transfection versus endogenous Mras expression
(Fig. 3A,B, Fig. 4A).
Cells transfected with the EGFP-MRAS fusion protein display
specific morphological changes compared with EGFP control
cells. Formation of membrane microspikes was observed 3 days
after transfection and an increase in the proportion of
differentiated cells was observed after 8 days of selection
(Fig. 3A). Although differentiated cells could be maintained for
more than a month without apparent proliferation, they died after
the first passage following trypsin treatment. In addition, after 4
weeks in selective medium with LIF, stable EGFP-MRAS clones,
with a slow growth rate compared with the EGFP clones, were
recovered and amplified (Fig. 3B, Fig. 4A; supplementary
material Fig. S1B,C).
Our data suggest that Mras, like Oct4 and Sox2, behaves as a
rheostat gene that displays different morphological effects
depending upon its level of expression, which is heterogeneous
following transfection (Kopp et al., 2008; Niwa et al., 2000).
Stable clones overexpressing MRAS have
sustained expression of OCT4 and NANOG when
grown without LIF
To determine whether overexpression of MRAS impacts the core
pluripotent complex, clones expressing EGFP or EGFP-MRAS
were grown with or without LIF for 7 days. Sustained expression of
OCT4 was observed in Mras-overexpressing clones grown without
LIF (Fig. 4B; supplementary material Fig. S2). In addition, protein
expression was analysed by flow cytometry on the EGFP-positive
cell fractions. A representative analysis of four clones (EGFPMRAS-A to -D) displays sustained expression not only of OCT4
DEVELOPMENT
change in the expression of Ras family members (Fig. 2A),
significant downregulation in the expression of the Pluri genes
Ceacam1 and Esrrb and the master genes Oct4 and to a lesser
extent Nanog was observed in the mutated cell line, with a
concomitant decrease in OCT4 and to a lesser extent NANOG
protein (Fig. 2B,D). Under these conditions, no significant
differences in the expression of the differentiation markers tested
were detected between WT and Dmras cells (Fig. 2C). However,
we have reproducibly observed that the formation of embryoid
bodies (EBs) is slower and that they are smaller in Dmras versus
WT lines. Furthermore, analysis of differentiation markers at two
time points in differentiation (8-day EBs and 13 days after EB
plating) shows that there is a decrease in endodermal (Sox17) and
mesodermal [brachyury (T)] but not neuronal [Tuj1 (Tubb3) and
Map2] expression markers in Dmras versus E14WT only in plated
differentiated cells (data not shown).
These results suggest that the decrease in Mras expression, which
affects the proper balance in the expression of pluripotent genes,
leads to an unpoised state of the cells, which have lost, in part, their
potential to respond to differentiation cues.
Mras in mESCs and Xenopus embryo
RESEARCH ARTICLE 3315
Fig. 3. Overexpression of MRAS leads to
morphological changes in cells grown
with LIF. (A) mESCs transfected with EGFP
or EGFP-MRAS in selective medium with
LIF as indicated. (B) Selection of individual
clones grown for 4 weeks. Phase contrast
(left) and fluorescence (right) images are
shown. Scale bar: 100 μm.
Mras gene structure and protein sequence are
conserved among vertebrates
Xenopus MRAS protein shows 84% and 92% identity with the
zebrafish and human orthologues, respectively (Fig. 5A). This
identity is in agreement with the phylogenetic analysis of the Ras
family, which indicates that a single Mras orthologue is present in
the genome from tunicates to mammals (Keduka et al., 2009). Mras
gene structure is remarkably conserved among vertebrates and is
composed of five exons, interrupted by four introns with identical
splice site junctions, having a conserved coding capacity except for
the first exon of the zebrafish gene (Fig. 5B). The intronic regions
of mammalian Mras genes have a very long first intron compared
with those of the chicken, Xenopus and zebrafish orthologues
(Fig. 5C).
Xenopus Mras expression is maternal and zygotic
and restricted to the adult brain and ovary
Semi-quantitative RT-PCR analysis indicates that Mras is
expressed in unfertilised Xenopus eggs and at all embryonic stages
analysed, but that the maternal transcript level decreases after
blastula stage (stage 9) and zygotic expression starts at late neurula
stage (stage 17) (Fig. 6A). In the blastula embryo, Mras mRNA is
found throughout the embryo, with no difference between animal
and vegetal poles, whereas in the gastrula embryo (stage 10.5) it
is mainly found at the animal pole (Fig. 6B). In adult tissues, Mras
is mainly expressed in the brain (as in mammals) and ovary
(Fig. 6C).
Rohon-Beard cells and trigeminal neurons are the
major sites of expression of Mras in the Xenopus
embryo
We did not detect, through whole-mount in situ hybridisation, any
localised Mras expression in the early Xenopus embryo before stage
24, when a faint labelling was observed in a string of cells along
the nerve cord corresponding to Rohon-Beard (RB) cells, which are
primary sensory neurons (Roberts, 2000) (Fig. 7A,B). Later in
development, labelling also appears in the trigeminal neurons and
epiphysis and is clearly detected between somites on each side of the
neural tube (Fig. 7C-H).
Mras regulates neuronal differentiation
Mras overexpression induces the neuronal differentiation of PC12
mammalian cells (Kimmelman et al., 2002; Sun et al., 2006).
Moreover, microarray data analysis indicates that Mras is reexpressed in ESC-derived neuronal cells (Abranches et al., 2009;
DEVELOPMENT
but also of NANOG upon LIF withdrawal (Fig. 4C). This finding
could indicate that cell differentiation has been delayed upon MRAS
overexpression. The ERK and PI3K signalling pathways, which are
involved in cell differentiation and self-renewal in mESCs,
respectively, have been shown to be activated by MRAS in different
situations (Kimmelman et al., 2000; Kimmelman et al., 2002).
However, we did not observe a constitutive induction of these
pathways when Mras is overexpressed (supplementary material
Fig. S3).
Since MRAS-overexpressing clones grew slowly and were
difficult to maintain in culture, it was not possible to use them for a
more in-depth analysis. Therefore, to explore the functions of Mras
in cell fate decision we turned to the Xenopus model, which has
been fruitful for establishing the functional conservation of
pluripotent genes.
3316 RESEARCH ARTICLE
Development 140 (16)
Fig. 4. Overexpression of MRAS in the absence of
LIF leads to sustained expression of OCT4 and
NANOG. (A) Relative expression level of Mras as
determined by RT-qPCR. The endogenous level of
Mras in mESCs + LIF or without LIF for 24 hours or
transfected with 1 μg pCAG-EGFP (EGFP) (lanes 1-4)
is compared with the level of overexpressed Mras
after transfection with pCAG-EGFP-MRAS (lanes 5, 6)
the day after transfection. The expression of Mras in
stable clones selected for a month in G418 in
complete medium with LIF is shown for EGFP clones
(lanes 7-9) and EGFP-MRAS clones (lanes 10-12). The
mean fluorescence intensity (MFI) of transfected cells
is, on average, 15,000 the day after transfection.
(B) OCT4 and ERK2 western blot analysis of cell
lysates from stable clones expressing EGFP or EGFPMRAS with and without LIF. (C) FACS analysis of the
percentage of cells expressing OCT4, NANOG or
SOX2 among WT cells (the mean of three
experiments is shown with s.d.) or representative
individual clones expressing EGFP or EGFP-MRAS in
the presence or absence of LIF for 7 days. The mean
MFI is 2894 for clone A, 3000 for clone B, 3000 for
clone C and 3822 for clone D, with a background
autofluorescence of 100.
against Mras sequence (MO Mras) and a mismatch morpholino
(MO mismatch) were tested for their ability to block Xenopus
Mras translation. Only MO Mras reduced translation of Mras
mRNA (supplementary material Fig. S5). Morpholinos were
injected into one cell of 2-cell stage embryos and morphant
Fig. 5. Conservation of vertebrate Mras at both protein and genomic levels. (A) MRAS protein sequence comparison between dog (Cf, Canis
familiaris), human (Hs, Homo sapiens), mouse (Mm, Mus musculus), rat (Rn, Rattus norvegicus), chicken (Gg, Gallus gallus), Xenopus (Xl, Xenopus laevis) and
zebrafish (Dr, Danio rerio). Identical amino acids are black, highly conserved in blue and neutral in red. (B) Vertebrate Mras gene structure with exons
represented by black boxes, introns (I-IV) by a thin line and untranslated regions by white boxes. The length of the coding sequence (in nucleotides) is
indicated for each exon. The type of splice junction is indicated for each intron. (C) Intron size (in nucleotides) comparison between the different Mras
genes.
DEVELOPMENT
Schulz et al., 2009) (supplementary material Fig. S4). Because
Mras is expressed in RB cells of the Xenopus embryo, we
investigated the effects of its loss-of-function on neuronal
differentiation during development through the use of an antisense
morpholino oligonucleotide strategy. A morpholino directed
Mras in mESCs and Xenopus embryo
RESEARCH ARTICLE 3317
milder effect on interneurons and almost no effect on motor
neurons (Fig. 8).
The reduction in N-tubulin expression is specific as neither MO
mismatch nor MO standard control (GeneTools) impaired its
expression and, moreover, the injection of a Mras mRNA that is
insensitive to MO Mras can rescue N-tubulin expression (Fig. 8).
Additional markers of neuronal differentiation were also
investigated: Elrc, Runx1, Pak3 and Islet1 (Perron et al., 1999;
Souopgui et al., 2002; Carruthers et al., 2003; Brade et al., 2007).
The expression of Elrc (72%, n=77), Pak3 (71%, n=42) and Islet1
(81%, n=32) but not of Runx1 (3%, n=86) is reduced in MO Mras
morphants (Fig. 8). Together, these experiments indicate that Mras
might function upstream of Elrc, Pak3 or Islet1 in the regulation of
primary neuron formation but downstream or in parallel of Runx1
in the generation of RB sensory neurons.
embryos were analysed for the expression of the neuronal marker
N-tubulin, which marks zones of primary neurogenesis in the
embryo. In the stage 15 Xenopus embryo, N-tubulin is expressed
in three stripes of cells that correspond to primary motor, interand sensory neurons, respectively, the latter of which will become
RB sensory neurons (Chitnis et al., 1995). Mras knockdown by
injection of MO Mras resulted mostly in a reduction of the sensory
neuron stripe (75%, n=126) and of the trigeminal neurons, with a
MRAS maintains the competence of gastrula
embryo cells for FGF and activin induction and
stimulates neural differentiation
Embryonic cell patterning of the early Xenopus embryo relies on a
limited number of signalling pathways that influence cell fate
decisions that can be reproduced in pluripotent animal cap cells of
the blastula embryo (Heasman, 2006). We found that FGF2
stimulates Mras expression in animal cap cells, whereas activin or
BMP treatments have no significant effects (Fig. 10A). Knockdown
Fig. 7. Mras is expressed in Rohon-Beard cells and trigeminal neurons in the Xenopus embryo. (A-H) In situ hybridisation with Mras antisense
probe (A,B,E,F) or combined with immunostaining with the muscle-specific antibody 12/101 (C,D,G,H) on stage 24 (A-D) or stage 33 (E-H) embryos.
Lateral (A,C,E,G) and dorsal (B,F) views. The plane of the transverse sections shown in D and H are indicated in C and G, respectively. Arrowheads indicate
Mras expression in Rohon-Beard cells. Ep, epiphysis; No, notochord; Nt, neural tube; RB, Rohon-Beard cells; S, somites; Tn, trigeminal neurons.
DEVELOPMENT
Fig. 6. Mras expression is biphasic during Xenopus laevis
development. RT-PCR analysis of Mras expression. (A) Egg and
embryonic stages (st). (B) Stage 9 or 10.5 dissected embryos. AP, animal
part; VP, ventral part; DM, dorsal mesoderm; VM, ventral mesoderm; EN,
endoderm. An1, Vg1, Chordin (Chd), Wnt8 and Sox17 are used as controls.
Total embryo (E) was analysed in parallel. (C) Adult tissues. Sk.m, skeletal
muscle. Ornithine decarboxylase (Odc) and Ribosome protein like 8 (Rpl8)
were used as controls, and a reaction was performed in the absence of
reverse transcriptase (–RT).
Mras acts at a late step of neurogenesis and is
required for neuronal differentiation
The early pan-neural marker Sox2 is essential for neuroectoderm
formation, while Neurogenin is the earliest proneural gene
expressed within the neural plate (Kishi et al., 2000; Ma et al.,
1996). Mras depletion in the Xenopus embryo did not affect the
expression of either marker (0%, n=65 for Sox2 and n=54 for
Neurogenin), indicating a late role for Mras in neuronal
differentiation (Fig. 9).
A constitutively active mutant of Mras (G22V) has been shown
to induce neuronal differentiation of PC12 cells, whereas a
dominant-negative form of the protein (S27N) impaired
neuritogenesis (Sun et al., 2006). We tested whether these two forms
of Mras had an impact on neuronal differentiation. Whereas the
constitutively active form of MRAS induced increased expression
of N-tubulin (69%, n=86), the dominant-negative form decreased
N-tubulin expression (80%, n=25). Together, these data indicate that
Mras is acting at a late phase of neuronal differentiation and that its
activation is a prerequisite to its function during differentiation.
3318 RESEARCH ARTICLE
Development 140 (16)
Fig. 8. Inhibition of Mras blocks neuronal
differentiation. MO Mras, MO mismatch or MO
standard were injected into one blastomere of 2-cell
stage Xenopus embryos and morphants were analysed
by in situ hybridisation for N-tubulin, Elrc, Runx1, Pak3
and Islet1 expression. In rescue experiments, 500 pg
Mras mRNA was co-injected with MO Mras (MO Mras +
Mras). The injected side is on the right. Motor (m) inter(i) and sensory (s) primary neurons and the trigeminal
placode (tg) are shown (arrowhead). Arrows indicate
change in expression induced by MO Mras. Dorsal
views, anterior to bottom.
(Fig. 10D). Together, these results indicate that Mras can maintain
the mesodermal competence of pluripotent animal cap cells to FGF2
and activin. This property is not limited to mesoderm but can also
apply to endoderm, as shown by the expression of the endodermspecific gene Sox17 (Hudson et al., 1997) (Fig. 10D).
When considering the neurectoderm germ layer, the animal cap
test cannot be used because both early and late animal cap cells
respond to neurogenesis induction by the neural inducer noggin
(Lamb et al., 1993). When Mras is overexpressed in animal caps
with noggin, we observed a strong induction of the N-tubulin and
Ncam genes, indicating a positive effect of Mras on neuronal
differentiation (supplementary material Fig. S6). Together, these
results support a conserved role of Mras in neurogenesis and
describe a new function of this GTPase at the onset of gastrulation.
DISCUSSION
In this study, we have focused on Mras, which encodes a GTPase
that is preferentially expressed in undifferentiated mESCs and reexpressed in ESC-derived neuronal cells. The impact of Mras was
Fig. 9. Mras acts at a late step of neurogenesis and
is required for neuronal differentiation. MO Mras,
MO standard, or 500 pg mRNA encoding a
constitutively active form (G22V) or a dominantnegative form (S27N) of MRAS was injected into one
blastomere of 2-cell stage Xenopus embryos. Embryos
were analysed by in situ hybridisation for Sox2,
Neurogenin or N-tubulin expression. The injected side is
on the right. Arrows indicate change in expression.
Dorsal views, anterior to bottom.
DEVELOPMENT
with MO Mras did not affect mesoderm induction mediated by
FGF2 (Fig. 10B) or activin (data not shown), indicating that Mras
is not essential for mesoderm induction.
The Xenopus animal cap assay has been successfully used to
demonstrate the involvement of OCT4 and BRG1 in the
maintenance of pluripotency (Hansis et al., 2004; Snir et al., 2006).
Animal cap cells from the blastula embryo can be induced to form
mesoderm when treated with FGF2 or activin, but gastrula stage
embryos lose this property (Grimm and Gurdon, 2002; Snape et al.,
1987). Animal cap cells from the gastrula stage embryo injected
with Mras mRNA still express Xbra in response to FGF2
(Fig. 10C). Both Xenopus and mouse Mras showed this property
(data not shown). Other marker genes (Cdx4, Pnp and Esr5)
previously shown to be FGF targets (Branney et al., 2009) are also
re-expressed in late animal cap cells upon Mras expression
(Fig. 10C). Similarly, we tested the effect of Mras on the
responsiveness of animal caps cells to activin. When Mras mRNA
is overexpressed in the embryo, late animal cap cells can still
respond to activin treatment and express Xbra, Chordin and Xnr1
Fig. 10. Mras can maintain the competence of Xenopus gastrula
embryo cells for FGF and activin induction but is not required for
mesoderm induction. RT-PCR analysis of gene expression in Xenopus
animal caps cultured until the control embryo (lane E) reached stage 12.5.
(A) Activin, BMP or FGF2 treatment followed by Mras and Xbra expression
analysis. (B) Animal caps from Mras morphants (MO) or control embryos
treated with 50 or 100 ng/ml FGF2 (FGF50 or FGF100) and analysed for
Xbra expression. (C,D) Animal caps dissected at stage 8 (Early) or stage 11
(Late) from embryos injected with Mras or Brg1 mRNAs or uninjected
embryo (Control) were treated with FGF2 (F) or activin (A) or left
untreated (–) before expression analysis of Xbra, Cdx4, Pnp, Esr5, Xnr,
Chordin (Chd) or Sox17 (Sox). Odc was used as control, and a reaction was
performed in the absence of reverse transcriptase (–RT).
evaluated in two models of pluripotent cells – mESCs and Xenopus
blastula embryonic cells (animal cap) – and on the development of
the Xenopus embryo.
The involvement of Ras GTPase members in the maintenance of
cell pluripotency in mESCs was suggested by the fact that a
chemical inhibitor of RasGAP, which maintains Ras members in a
GTP-bound active conformation, allows the propagation of mESCs
in an undifferentiated pluripotent state in the absence of LIF (Chen
et al., 2006). In addition, a recent report has demonstrated the
importance of the nucleolar GTP-binding protein nucleostemin
(Gnl3) for the self-renewal of mESCs by direct action on the G1
phase of the cell cycle (Qu and Bishop, 2012).
Functional approaches have demonstrated various roles for Ras
members in mESCs: ERAS (ES cell-expressed ras) promotes ESC
proliferation and the formation of teratomas (Takahashi et al.,
2003); the activated KRAS mutant (G12V) alters the expression
of pluripotent markers, leading to growth maintenance even with
a low dose of LIF (Luo et al., 2007); and activated HRAS mutants
induce differentiation toward trophectoderm or extra-embryonic
endoderm lineages (Yoshida-Koide et al., 2004; Lu et al., 2008).
However, knockout mice failed to reveal any early embryonic
function as almost all individual Ras knockouts are viable and
RESEARCH ARTICLE 3319
fertile, except for Kras−/− mice, which display lethality at E12 (Ise
et al., 2000; Koera et al., 1997; Nuñez Rodriguez et al., 2006;
Umanoff et al., 1995). In this study, we have not observed a
compensatory effect in Ras member expression in ESC lines
expressing a half dose of Mras in the presence or absence of LIF.
Further studies will be required to determine which genes, if any,
might compensate for the absence of individual Ras members and
to reconcile the lack of an embryonic phenotype in knockout mice
with the crucial role played by some Ras members in the mESC
model.
Our results identify a novel role for MRAS, which was thus far
known for its involvement in differentiation processes (Kimmelman
et al., 2002; Sun et al., 2006; Watanabe-Takano et al., 2010). We
showed that downregulation of Mras expression in mESCs
deconstructs the stemness program by decreasing the expression of
Oct4 and of some Pluri genes, such as Ceacam1 and Esrrb, and
diminishes the self-renewal capacity of the cells. However, the
Dmras cell line and cells in which Mras has been repressed by
shRNA (Parisi et al., 2010) maintain their undifferentiated
morphology and are alkaline phosphatase positive, indicating that
compensatory effects allow cells to grow and to be amplified in LIFcontaining culture medium.
We also showed that overexpression of Mras leads to changes in
cell morphology, with more giant and spiky differentiated cells
observed within the first week of cell culture. However, we did not
detect any MRAS-dependent induction of the ERK pathway in these
early differentiated cells, which did not survive after the first
passage in culture (data not shown; supplementary material Fig.
S3). Stable clones overexpressing MRAS, which do eventually
grow in selective medium, exhibit, in contrast to the wild-type
clones, sustained expression of OCT4 and NANOG when cells are
deprived of LIF for 7 days, with no effect on SOX2 expression. This
indicates that MRAS activates a pathway that alters only the
OCT4/NANOG subcomplex and probably not the SOX2/OCT4/
NANOG complex. Oct4 is the paradigm of a rheostat effector gene,
which modulates the stemness/differentiation balance depending on
its level of expression (Niwa et al., 2000). In addition, an OCT4
dose-dependent switch from the Sox2 to Sox17 promoter triggers
mesodermal differentiation (Blin et al., 2010; Stefanovic et al.,
2009). Our data suggest that Mras behaves as a rheostat gene and
that there is a link between MRAS and OCT4 expression. In cells
deprived of LIF for a week, Oct4 mRNA expression diminishes
whether or not Mras is overexpressed (our unpublished data).
Therefore, we favour an MRAS-dependent post-translational
stabilisation of OCT4 that could lead to sustained expression of the
protein in Mras-overexpressing cells grown without LIF. Such posttranslational modifications (phosphorylation or ubiquitylation) alter
OCT4 stabilisation (Xu et al., 2009; Saxe et al., 2009). Interestingly,
Mras along with Tcl1, another member of the Pluri cluster (Trouillas
et al., 2009b), are direct targets of the KLF5 transcription factor,
which is involved in the maintenance of cell pluripotency (Parisi et
al., 2008; Parisi et al., 2010). MRAS could be a signalling relay of
KLF5, acting both on gene transcription and the protein stabilisation
machinery.
Because stable clones grew slowly, it was not possible to
determine whether they remain pluripotent in the absence of LIF
using teratoma formation. In addition, we observed a poor capacity
of the E14Dmras cell line to form EBs in comparison to WT cells,
and found that Brachyury (T) and Sox17 expression was decreased
in comparison to the WT situation in late differentiated cells,
indicating that a proper level of Mras is required for the cells to
maintain their pluripotency and to differentiate efficiently.
DEVELOPMENT
Mras in mESCs and Xenopus embryo
Therefore, we took advantage of the complementary Xenopus model
to address Mras functions in pluripotency and early development.
We used both the in vivo approach and the model of pluripotent
animal cap cells (Sasai et al., 2008). Mras is highly conserved
among vertebrates from zebrafish to human at both the gene and
protein levels, suggesting a selection pressure and conserved
function. As in mESCs, there are clearly two phases of Mras
expression in Xenopus, with a maternal expression that decreases
between the blastula and early gastrula stages followed by a zygotic
expression that increases in the early tadpole, a time when Mras
mRNA is found in RB cells and trigeminal neurons. RB neurons
are a transient population of primary sensory neurons that is
observed in non-mammalian vertebrates, such as zebrafish and
Xenopus, and is replaced in the late embryo by dorsal root ganglia
(Holland, 2009; Olesnicky et al., 2010; Roberts, 2000). Strikingly,
in the mouse embryo, Mras is also expressed in dorsal root ganglia
(Keduka et al., 2009). We conclude that the amphibian and murine
Mras genes share both strong sequence conservation and expression
pattern (Kimmelman et al., 2000).
Loss-of-function of Mras in morphant embryos results in a
consistent phenotype that is characterised by the suppression of
neuronal differentiation as assessed by N-tubulin, Pak3, Elrc or
Islet1 expression. This is supported by experiments in which a
dominant-negative form results in fewer neurons. By contrast, a
constitutively active form of MRAS stimulates neuronal
differentiation, expanding the N-tubulin expression domain (Fig. 9).
Although Mras has been shown to induce neuronal differentiation,
including neurite outgrowth in PC12 cells (Sun et al., 2006), our
data constitute the first report of the involvement of Mras during
vertebrate neurogenesis in vivo. We hypothesise that this property is
not restricted to amphibians but is shared by mammalian species
because the murine protein is able to rescue morphant embryos as
efficiently as the amphibian protein. Mras acts at a late phase of
neurogenesis, downstream of Sox2 and Neurogenin. Surprisingly,
Runx1 expression is unchanged in Mras morphant embryos.
Because Runx1 is expressed in RB progenitors and has been shown
to be required for their specification independently of Neurogenin
(Park et al., 2012), we hypothesise that Mras can act either
downstream of Runx1 in a common pathway or independently of
Runx1 in a parallel pathway.
In mESCs, a half dose of Mras leads to downregulation of
Ceacam1, Esrrb, Oct4 and, to a lesser extent, Nanog, whereas its
overexpression in LIF-deprived cells leads to sustained expression
of OCT4 and NANOG, suggesting a role in pluripotency
maintenance. We have demonstrated that gastrula cells that
overexpress Mras can be induced to form mesoderm when treated
with FGF2, suggesting that Mras can prolong their pluripotency, as
shown for Brg1 (Smarca4) (Hansis et al., 2004). Mras is also able
to maintain gastrula cell competence to form mesoderm, but also
endoderm, under the control of activin. An evaluation of whether
OCT4 expression is modulated in Mras morphant embryos was not
possible owing to the lack of a Xenopus OCT4 antibody. However,
we have not observed any variation in Oct4 expression at the
mRNA level in Mras morphants (data not shown).
In conclusion, we have observed in both models two phases of
Mras expression that could support two distinct roles. Mras
expression in LIF-treated ESCs declines after LIF removal and is reinduced during neural differentiation. In Xenopus, the elevated Mras
expression that is observed throughout the embryo until blastula
stage could be required for the maintenance of pluripotency,
whereas its localised expression in the presumptive neuroectoderm
of the gastrula embryo supports its function in neurogenesis. Indeed,
Development 140 (16)
in pluripotent cells neuralised by noggin, Mras expression
stimulates neuronal differentiation. Our work, along with the recent
report on the function of the GTP-binding protein nucleostemin in
the self-renewal of mESCs (Qu and Bishop, 2012), provide new
tools and insights for characterising and understanding the links
between GTP-binding proteins and stemness and the neurogenic
regulators in different models such as mESCs and the Xenopus
embryo. In addition, our study reinforces the use of complementary
models for a better understanding of gene functions in vertebrates
and demonstrates the involvement of Mras in cell pluripotency and
neurogenesis.
Acknowledgements
We thank members of our group for discussions; Sophie Daburon for
providing human LIF; Vincent Pitard and Santiago Gonzales for advice on flow
cytometry; Charline Maignan for experiments performed with the Dmras cell
line; Drs Sachinidis and Pradier for microarray data generated during the
FunGenES European program; Drs Kolde and Vilo for the FunGenES website;
Austin Smith for the CGR8 cells; Drs Endo, Bellefroid, Grainger, Pandur, Pieler,
Perron and Saint Jeannet for plasmids; Dr Goding for reading the manuscript;
and Lionel Parra Iglesias for Xenopus colony care. We acknowledge NIH
sponsorship of NHLBI-BayGenomics and the NCRR-MMRRC for providing the
ESC gene trap line.
Funding
This work was funded by the European consortium FunGenES [6th Framework,
project no. LSHG-CT-2003-503494]; CNRS; University Bordeaux Segalen; Ligue
National contre le Cancer; comité de Gironde; Association pour la Recherche
sur le Cancer [no. A08/2/1017]; Region Aquitaine; and SFR TransBiomed. M.E.M., C.S. and M.T. were funded by the Research Ministry, Region Aquitaine
and a FunGenES Fellowship, respectively.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
M.-E.M., C.F., C.S., F.S., X.G. and M.T. performed experiments and contributed
to data analysis. S.F. performed experiments. N.T. contributed to data analysis
and wrote the manuscript. P.T. and H.B. contributed to experimental design,
performed experiments, contributed to data analysis and wrote the
manuscript. All authors read and approved the final manuscript for
publication.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.091082/-/DC1
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