Download PDF

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Wnt/β-catenin and FGF signalling direct the specification and
maintenance of a neuromesodermal axial progenitor in ensembles
of mouse embryonic stem cells
ABSTRACT
The development of the central nervous system is known to result
from two sequential events. First, an inductive event of the
mesoderm on the overlying ectoderm that generates a neural plate
that, after rolling into a neural tube, acts as the main source of neural
progenitors. Second, the axial regionalization of the neural plate that
will result in the specification of neurons with different anteroposterior
identities. Although this description of the process applies with ease
to amphibians and fish, it is more difficult to confirm in amniote
embryos. Here, a specialized population of cells emerges at the end
of gastrulation that, under the influence of Wnt and FGF signalling,
expands and generates the spinal cord and the paraxial mesoderm.
This population is known as the long-term neuromesodermal
precursor (NMp). Here, we show that controlled increases of Wnt/
β-catenin and FGF signalling during adherent culture differentiation
of mouse embryonic stem cells (mESCs) generates a population
with many of the properties of the NMp. A single-cell analysis of
gene expression within this population reveals signatures that
are characteristic of stem cell populations. Furthermore, when this
activation is triggered in three-dimensional aggregates of mESCs,
the population self-organizes macroscopically and undergoes
growth and axial elongation that mimics some of the features of
the embryonic spinal cord and paraxial mesoderm. We use both
adherent and three-dimensional cultures of mESCs to probe the
establishment and maintenance of NMps and their differentiation.
KEY WORDS: Wnt signalling, Mesodermal, Morphogenesis, Neural,
Stem cells
INTRODUCTION
The nervous system comprises a cellular network that processes
sensory and motor information to generate patterns of activity and
behaviour in the organism. At the centre of this network there is a
large number of interconnected neurons that shape behaviours. In
mammals, the basic framework of the network is engineered during
development from a sheet of neuroepithelial progenitors that arise in
an anteroposterior sequence as the body plan unfolds. This process
can be followed through the expression of members of the Sox2b
Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 19 May 2014; Accepted 10 September 2014
family of transcription factors (Kamachi and Kondoh, 2013): Sox1,
Sox2 and Sox3 (Pevny et al., 1998; Graham et al., 2003).
Classical studies by Spemann and Mangold (Spemann and
Mangold, 1924; translated by Hamburger, 2001) showed that the
emergence of the nervous system requires an inductive event
mediated by a special group of cells, called the ‘organizer’, on a
layer of overlying neuroectodermal cells (Spemann and Mangold,
1924; De Robertis et al., 2000; De Robertis, 2009). Later,
experiments with Xenopus animal caps showed that the inductive
event is associated with an antagonism of BMP signalling and, by
extension, that BMP acts as a pan-neural repressor across the
ectoderm (Hemmati-Brivanlou and Melton, 1997a). Expression of
the BMP antagonists chordin, noggin and follistatin from the
organizer releases this repression locally and allows the dynamic
emergence of neural progenitors (reviewed by Hemmati-Brivanlou
and Melton, 1997b; De Robertis et al., 2001). However, the notion
that BMP antagonism is the main mechanism of neural induction
has been challenged; in particular, it has been suggested that other
signalling pathways, FGF/ERK and Wnt, are involved in the
acquisition of the neural fate independently of the inhibition of BMP
(Stern, 2005).
An important feature of the nervous system is its specialization
along the anteroposterior axis, which is most obvious in the structural
and functional differences between the fore-, mid- and hindbrain, and
the spinal cord. The emergence of these differences is thought to occur
in two steps. According to the ‘activation/transformation’ hypothesis
(Nieuwkoop et al., 1952; Nieuwkoop and Nigtevecht, 1954) the
neural plate is first specified, presumably by BMP antagonism, with
anterior characteristics; subsequently, posterior fates, including those
in the spinal cord, emerge by the action of a gradient of one or more
signalling molecules (Mangold, 1933; Nieuwkoop et al., 1952; Saxén
and Toivonen, 1961; reviewed by Stern et al., 2006). Experiments in
Xenopus have led to the suggestion that the transformative influence is
provided by a gradient of Wnt/β-catenin signalling (Kiecker and
Niehrs, 2001). In contrast to the situation in frogs, where fates are
assigned on an existing neural plate, there is evidence that in amniotes
the development of the cranial and hindbrain regions, and of the
spinal cord, are temporally and spatially separate (reviewed by Wilson
et al., 2009; Kondoh and Takemoto, 2012). While the anterior central
nervous system emerges from a neuroectodermal progenitor
population following neural induction (reviewed by Andoniadou
and Martinez-Barbera, 2013), work in chickens and mice has shown
that the spinal cord is derived from a specialized self-renewing
precursor population located within the growing caudal end of the
embryo (Brown and Storey, 2000; Mathis and Nicolas, 2000; Mathis
et al., 2001; Cambray and Wilson, 2002, 2007; Delfino-Machin et al.,
2005; Tzouanacou et al., 2009; Nowotschin et al., 2012). Cells within
this population exhibit some features of stem/progenitor cells (Mathis
4243
DEVELOPMENT
David A. Turner*, Penelope C. Hayward*, Peter Baillie-Johnson, Pau Rué , Rebecca Broome, Fernando Faunes
and Alfonso Martinez Arias‡
and Nicolas, 2000; Roszko et al., 2007; Tzouanacou et al., 2009) and
can give rise to mesodermal and neural progenitors of different
posterior axial levels (Brown and Storey, 2000; Cambray and Wilson,
2002; Tzouanacou et al., 2009; Tsakiridis et al., 2014). These cells,
named long-term axial neuromesodermal precursors (NMp), can be
identified because they co-express markers of the primitive streak
(Bra) and neuroectoderm (Sox2), as well as the homeobox gene Sax1/
Nkx1.2 (herein referred to as Nkx1.2), which is characteristic of the
caudal neural plate (Storey et al., 1998; Delfino-Machin et al., 2005;
Cambray and Wilson, 2007). Although it is possible to understand this
population in the context of the activation-transformation hypothesis
(Stern et al., 2006), it is also possible that it emerges de novo within the
primitive streak as a population that has never adopted a neural plate
identity. Analysis of the regulatory region of the Sox2 gene lends some
support for this possibility with the identification of an enhancer in the
Sox2 gene that drives its expression in the extending caudal neural
plate, also referred to as the stem cell zone or the caudal-lateral epiblast
(Wilson et al., 2009), within the progenitor population under the
control of Wnt and FGF signalling (Takemoto et al., 2006, 2011;
Kamachi et al., 2009).
Embryonic stem cells (ESCs) can be used as an experimental
system to probe the mechanisms that mediate the specification and
differentiation of cell populations in development. Recent analysis of
the behaviour of epiblast stem cells (EpiSCs) in response to Chiron,
an agonist of Wnt signalling, has revealed the emergence of a small
population of cells that co-expresses markers of the primitive streak
and Sox2, a blueprint of the NMp (Tsakiridis et al., 2014). Here, we
extend these observations to mouse ESCs (mESCs) and show that
modulation of Wnt signalling during controlled differentiation of
mESCs in adherent culture generates a transient NMp population in
an effective and reproducible manner. These progenitors require FGF
signalling and can be coaxed to differentiate into neural progenitors
and paraxial mesoderm with thoracic identities. Furthermore, using a
recently developed 3D culture system of mESCs (Baillie-Johnson
et al., 2014; van den Brink et al., 2014), we show that a similar
treatment of cell aggregates results in the emergence of a directional
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
extension that is neural in character and harbours, at its distal end, a
population that resembles the NMp in terms of gene expression and
phenotypic behaviour. We discuss the signalling requirements for the
emergence and differentiation of the NMp, and also the role that 3D
structures might play in its maintenance.
RESULTS
Wnt/β-catenin signalling promotes the emergence of
posterior axial fates in adherent mESC differentiation
In the postimplantation epiblast of the mouse embryo, cells become
allocated to one of two prospective fates: anterior neuroectoderm
(aNECT) that will give rise to the anterior nervous system; or,
posteriorly, to a rapidly expanding population that gives rise to the
mesoderm and the endoderm through the primitive streak (Arnold
and Robertson, 2009; Rossant and Tam, 2009). Aspects of this
decision-making event can be modelled in ESC cultures where fates
are controlled by extracellular signals. Thus, differentiation in the
presence of nodal, activin, BMP and Wnt/β-catenin signalling
promotes the emergence of a primitive streak-like population
(Gadue et al., 2005), whereas culture in N2B27 or serum and
retinoic acid (RA) generates neural precursors that can be monitored
in the expression of Sox2 and a Sox1::GFP reporter (Ying et al.,
2003; Abranches et al., 2009). After 2 days of differentiation in
N2B27, mESCs commit either to a neural or to a mesendodermal
fate in a signal-dependent manner (Thomson et al., 2011; Turner
et al., 2014b). This commitment is associated with a transient rise in
Wnt/β-catenin signalling (Faunes et al., 2013) (supplementary
material Fig. S1) that can be shown to promote both fates,
depending on the levels of nodal and activin: together with high
levels it promotes mesendodermal fates; in the context of low
levels, it is required for neural differentiation (Turner et al., 2014b),
as shown by the effects of inhibition of Wnt signalling (Fig. 1B).
This suggests that the activation of Wnt signalling observed during
the second day is part of a general priming of the differentiated state
and raises the question about its role in the development of the
nervous system.
Fig. 1. Wnt signalling is transiently active during neural differentiation and does not inhibit neural development. (A) The differentiation protocol.
(B) Population profiles of Sox1::GFP mESCs treated with continuous Serum+LIF (0-5 SL) or N2B27 (0-5 N) or for 2 days with N2B27 followed by either Chiron (2-5
Chi) or the tankyrase inhibitor XAV939 (2-5 XAV939). Black vertical bisecting line indicates the boundary between Sox1::GFP-negative and -positive, as
determined by the autofluorescence profile from wild-type (E14-Tg2A) cells. Population profiles normalized to the mode fluorescent value of the population.
(C) mRNA from Sox1::GFP mESCs treated with N2B27 for 5 days, or with N2B27 and a Chiron pulse on days 2-3 or 2-5 were analysed by qRT-PCR for
anterior (En1, Otx2, Six3), spinal cord (Hoxc5, Hoxc6, Hoxc9), neural (Sox1, Sox2) and mesoderm (Tbx6) identifiers. Significance tests were performed on this
set of experiments and are presented in supplementary material Fig. S2. Data are mean±s.d.
4244
DEVELOPMENT
RESEARCH ARTICLE
Wnt/β-catenin signalling has been shown to promote the
maintenance of neural progenitors and their maturation into
neurons (Zechner et al., 2003; Otero et al., 2004; Kim et al.,
2009; Slawny and O’Shea, 2011); this could account for the effects
of Wnt/β-catenin signalling during neural differentiation. However,
Wnt/β-catenin signalling also plays a role in the specification of
axial identities in developing embryos (Kiecker and Niehrs, 2001;
Nordström et al., 2002, 2006; Mazzoni et al., 2013) and we have
tested whether this is also the case during mESC differentiation.
mESCs differentiating in N2B27 adopt a neural fate and, after 5
days, express high levels of genes that are characteristic of mid/
hindbrain (En1, Otx2), but lack expression of spinal cord identifiers
(Hoxc5, Hoxc6, Hoxc9) (Fig. 1C). However, exposure to Chiron
after 2 days in N2B27 elicits the expression of thoracic Hox genes in
the absence of anterior ones (Fig. 1C), indicating that β-catenin
promotes the development of posterior fates in mESCs. To test
whether this effect required persistent signalling, cells were exposed
to Chiron for shorter periods of time. Exposure of mESCs to Chiron
between 48 and 72 h of differentiation elicits the emergence of
thoracic spinal cord identities at the expense of anterior fates
(Fig. 1C). A difference between the continuous and pulsed exposure
to Chiron can be observed in the cell types that emerge from this
population: in both cases, Chiron suppresses the differentiation of
anterior identities but, in the case of continuous exposure, in
addition to neural development Chiron promotes mesoderm
differentiation, as shown by the expression of Tbx6 (Fig. 1C).
These results are in agreement with the notion that high levels of
Wnt signalling lead to the emergence of a cell population with
posterior identities in the epiblast (Mazzoni et al., 2013; Tsakiridis
et al., 2014) and raises the issue of the mechanism through which
this is achieved.
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Wnt/β-catenin signalling promotes the emergence of an axial
progenitor population in adherent mESC differentiation
Exposure of mESCs to Chiron between 48 and 72 h elicits the
appearance of clusters of cells that co-express Sox2 and Bra
(Fig. 2A,B), a signature that is characteristic of a population of
cells that have been suggested to act as a precursor of the spinal cord
and the paraxial mesoderm in the embryo, the neuromesodermal
axial precursor (NMp) (reviewed by Wilson et al., 2009; Kondoh
and Takemoto, 2012). Addition of SB43, a TGFβ antagonist,
simultaneously with Chiron, abolishes the expression of Bra, and
thus of the Sox2+ Bra+ subpopulation. A similar effect is observed if
FGF signalling is inhibited by exposure to PD03, an inhibitor of
MEK. This suggests that the population is associated with the
specification of mesendodermal fates that have been shown to be the
source of this population.
Analysis of gene expression in cells 72 h after exposure to Chiron
between 48 and 72 h shows that they express Sox1 and Sox2, as well
as markers associated with the NMp: Bra, Nkx1.2 and Hoxb8
(Delfino-Machin et al., 2005) (Fig. 2C). These cells do not express
markers of anterior neural fate, e.g. En1 and Otx2, but exhibit low
levels of posterior ones, e.g. Hoxb1, Hoxc5, Hoxc6 and Hoxc9
(Fig. 2C). This signature requires ERK and nodal/activin signalling
(Fig. 2), and is dependent on Chiron, as cells differentiating in
N2B27 for the same period of time do not express Bra or posterior
markers, but do express anterior fates.
To study the emergence of the Sox2+ Bra+ Nkx1.2+ population in the
context of neural differentiation, we analysed gene expression
in subpopulations of cells expressing Sox1::GFP, a marker of mature
neural progenitors. After 3 days differentiating in N2B27, Sox1::GFPexpressing cells exhibit a complex expression profile with a prominent
negative population (Fig. 3A). Addition of Chiron during the third day
Fig. 2. Wnt/β-catenin signalling
promotes the emergence of an NMp
precursor in cell culture. (A) Confocal
images of Sox1::GFP mESCs exposed to
2 days N2B27 followed by 24 h Chiron alone
(Chi) or in combination with SB43 or PD03
(Chi+SB43 and Chi+PD03, respectively)
fixed and immunostained for Bra (green) and
Sox2 (red). Hoechst was used to identify the
nuclei. Scale bar: 50 µm. AFU, arbitrary
fluorescence units. (B) Quantitative image
analysis of the confocal images from the
conditions in A. Teal blue dots indicate cells
with no Bra (95% of cells in either Chi+PD03
or Chi+SB43); red dots correspond to cells
with significantly high levels of Bra protein
(percentage of Bra-positive cells indicated).
(C) Sox1::GFP mESCs were treated as
described in A prior to RNA extraction and
qRT-PCR analysis for genes associated with
neural (Sox1, Sox2), primitive streak (Bra),
anterior neural (En1, Otx2) and anterior/
posterior regional (Hoxb1, Hoxc5, Hoxc6,
Hoxc9) identities, and the NMp (Nkx1.2,
Hoxb8). Data were normalized to the housekeeping gene Ppia and are expressed as
sample concentrations±s.d. Significance
tests were performed on this set of
experiments and are presented in
supplementary material Fig. S2.
4245
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Fig. 3. Identification of the NMp amidst
differentiating neural cells on day 3 of
differentiation. (A) Population profiles of Sox1::
GFP mESCs analysed by FACS following
treatment with a 24 h Chiron pulse after 2 days
N2B27 (Chiron pulse, upper panel), 3 days N2B27
(middle panel) or 3 days serum+LIF (0-3 days SL,
lower panel). Grey vertical boxes highlight the
negative, low and high populations. (B) Cells sorted
from the negative, low and high Sox1::GFP
populations were analysed by qRT-PCR for the
presence of Nkx1.2 (red), Sox2 (green) and
Brachyury (Bra, purple). Data are mean±s.d.
Significance tests were performed on this set of
experiments and are presented in supplementary
material Fig. S3. (C) The negative, low and high
Sox1::GFP populations from the Chiron pulse
condition were fixed and stained for Brachyury and
Sox2. Quantitative image analysis of the fixed cells
shown below each condition. Teal blue dots
indicate cells with no Bra (95% of cells in
either Chi+PD03 or Chi+SB43), whereas red dots
correspond to cells with significantly high levels of
Bra protein ( percentage of Bra-positive cells
indicated). The NMp is found in cells negative or
low for Sox1::GFP.
Sox1::GFP negative population having the highest, with all Bra+ cells
expressing Sox2 (Fig. 3C). These results indicate that exposure of
mESCs to Chiron during day 3 of differentiation induces the appearance
of a population that has many of the hallmarks of the NMp that has been
postulated to emerge during the late stages of gastrulation.
A window of competence for the emergence of the axial
progenitor
To determine whether there is a period of competence for the
induction of the Sox2+ Bra+ population, we altered the timing of the
Fig. 4. A window of competence exists for the generation of the NMp. E14-Tg2A mESCs were exposed to either (A) a 24 h pulse of Chiron after 2, 3 or 4 days in
N2B27 (0-2N 2-3Chi, 0-3N 3-4Chi, 0-4N 4-5Chi, respectively) and fixed or (B) a 24 h pulse of Chiron after day 2 and returned to N2B27 for a further 24 or 48 h before
fixation and staining for Sox2 and Bra. (C) Schematic representation of the stimulation protocol. The highest proportion of cells that co-express Sox2 and Bra, and
therefore the highest proportion of NMp cells, occurs when cells are exposed to a 24 h pulse during days 2-3. Hoechst indicates the nuclei. Scale bars: 50 µm.
4246
DEVELOPMENT
reduces the Sox1::GFP high population in favour of the negative one
(Fig. 3A). To search for the NMp gene expression signature within this
profile, we used FACS to sort cells with no, low or high levels of Sox1::
GFP, and analysed them for the presence of the gene expression
signature of the NMp. The three subpopulations expressed Sox2, with
lowest levels in the negative population, but could be distinguished by
the levels of expression of Bra and Nkx1.2, which were highest in the
negative population, noticeable in the low and absent in the high
(Fig. 3B). Immunocytochemical analysis of the three populations
reveals differences in the proportion of Sox2+ Bra+ cells, with the
exposure to Chiron (Fig. 4A). Treatment of the differentiating cell
population between 72 and 96 h reduces the number of Sox2+ Bra+
cells and this number is further reduced if the exposure occurs
between 96 and 120 h (Fig. 4). When mESCs that had been exposed
to Chiron (Fig. 4B) between 48 and 72 h of differentiation are
returned to N2B27, the Sox2+ Bra+ population decays rapidly over
the next 2 days, and cannot be found by day 5 (Fig. 4). In both cases,
the cells maintain Sox2 expression. These results suggest that there
is a sensitive period on the third day of differentiation in which this
population can be induced and that, in the absence of signalling, the
population is not maintained and adopts a neural fate.
Single-cell analysis of ESC-derived NMps
To gather further information about the presence of NMp-like cells
in the negative and low Sox1::GFP populations of mESCs that had
been exposed to Chiron, we sorted 192 single cells from both
subpopulations (96 negative and 96 low). After a strict quality
control, we selected 89 cells (53+36, see supplementary material
Fig. S4 for details) and analysed them for expression of 85 genes,
including pluripotency markers, lineage markers, axial identifiers
and reporters of signalling pathway activity (see Materials and
Methods, Fig. 5 and supplementary material Fig. S4). We first tried
to order the cells using the conventional NMp signature: T/Bra,
Sox2, Nkx1.2 in a correlation analysis (Fig. 5, see also
supplementary material Figs S4 and S5). Most of the cells
analysed have detectable levels of Nkx1.2, with the expression of
Bra being heterogeneous and slightly correlated with the levels of
Nkx1.2 (correlation coefficient of ∼0.3, P<0.001). Surprisingly, the
expression of Sox2 mRNA is low in all cells except for a small group
and exhibits no significant correlation with the expression of
Nkx1.2; this is not a failure to amplify the mRNA as the pluripotent
population that can be observed within the Sox1::GFP-negative
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
population exhibits high levels of Sox2 expression (Fig. 5). A search
for genes whose expression is correlated with Nkx1.2 and Bra
identified Wnt3a, Fgf8, Foxb1, Lin28 and Mdb3 (Fig. 5 and
supplementary material Figs S4 and S5). In the embryo, Fgf8 and
Wnt3a are expressed in the area in which NMps reside (DelfinoMachin et al., 2005) and thus validate our analysis. Foxb1 is a
member of the Fkh family of transcription factors that has been
reported to be expressed in the NMp region during elongation (Ang
et al., 1993; Zhao et al., 2007), and thus represents a novel marker
for the NMp. However, Lin28, a microRNA-binding protein
(Viswanathan and Daley, 2010; Shyh-Chang and Daley, 2013),
and Mbd3, a core member of the NurD chromatin remodelling
complex (Kaji et al., 2006; Hu and Wade, 2012), have been
associated with pluripotency and reprogramming, and might
represent a signature of ‘stemness’. Next, we performed different
dimensionality reduction analyses of the data, including principle
component analysis (PCA) and linear discriminant analysis (LDA),
but these methods were unable to capture structural properties of the
cell population, most probably due to the heterogeneous nature of
the data. The method that performed best was multidimensional
scaling (Fig. 6), which was capable of separating the Sox1::GFPnegative and -low populations. From this analysis we can conclude
that the two populations are very similar but that the negative cells
contain a subpopulation that has not yet completed a transition from
the pluripotent epiblast, as evidenced by a prominent group of cells
with high levels of all pluripotency markers, including Sox2. The
analysis also allows visualization of differences in gene expression
between the cells from the two populations (Fig. 6).
The NMp population does not exhibit widespread expression of
lineage markers and lacks clear expression of axial markers, except
for Hoxb1, which is active in the NMp region in the embryo (van de
Ven et al., 2011). Interestingly, amidst the analysed population we
Fig. 5. Single-cell analysis of the NMp population derived from mESCs. mRNA was extracted from Sox1::GFP single cells expressing either low (L, 53 cells)
or negative (N, 36 cells) GFP levels and analysed for the indicated genes using Fluidigm technology. Gene expression levels for individual cells are shown
in colour-code and ordered according to the levels of Nkx1.2 expression (top). On the right, Pearson correlation coefficients of the expression of each given gene
to Nkx1.2, Bra or Sox2 are displayed and colour coded according to statistical significance level. The expression of pluripotency markers, as well as of Wnt
and FGF signalling pathway components in individual cells, is shown here (see supplementary material Figs S4 and S5 for other markers and extended
descriptions of data acquisition and processing).
4247
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
are likely to become mesoderm. During differentiation, continuous
exposure to both FGF and Chiron between 48 and 120 h amplifies the
emergence of Wnt/β-catenin-dependent mesodermal derivatives, as
reflected in Tbx6 expression (Fig. 7). To determine whether this is due
to an effect on the NMps, Sox1::GFP-negative and -low cells were
sorted from pulsed exposure to either Chiron or Chiron and FGF, and
each fraction was further cultured in either Chiron or Chiron and FGF.
After one and two passages, cells were tested for Sox1, Sox2 and Tbx6
expression. Only the Sox1::GFP originally negative population gave
rise to Tbx6-expressing cells and the number of Tbx6-positive cells
was enhanced in the presence of FGF (Fig. 7E). This analysis suggests
that, in culture, cells within the NMp-like population are in a state that
reflects the situation in the embryo in terms of signalling requirements,
particularly FGF and Wnt, and can differentiate into both neural and
paraxial mesodermal precursors in a signal-dependent manner.
Fig. 6. Dimensionality reduction analysis of single-cell expression data of
the NMp population derived from mESCs. Multidimensional scaling (MDS)
was applied to reduce the 85-dimensional single-cell profiles into a twodimensional dataset and facilitate visual inspection. In contrast to other
dimensionality reduction methods, MDS (top left panel) was able to
disaggregate, albeit only partially, low and negative Sox1:GFP cell populations
(blue and red dots, respectively). Expression levels of some of the assayed
genes are shown in colour and size code for each individual cell placed on the
corresponding MDS coordinates. The clusters of pluripotent cells within the
negative population are markedly highlighted with this data representation.
observed five cells that express Tbx6, Pdgfra and Dll1 and are likely
to be mesodermal, and 40 cells that express Sox1, N-cadherin and
Zfp521 and are likely to be neural (supplementary material Fig. S6).
These cells are negative for both Nkx1.2 and Bra, and represent
progenitors of the mesoderm and the nervous system. These results
are consistent with the notion that exposure of mESCs to Chiron
during the third day of differentiation elicits the emergence of cells
with the signature of the NMp.
FGF and Wnt signalling are active in NMps
The single-cell analysis reveals expression of targets of FGF (Spry2,
Spry4) and Wnt (e.g. Lef1, Dkk and Axin2) signalling in cells that
express Nkx1.2, an indication of FGF/ERK and Wnt/β-catenin
signalling in this population. This is consistent with results from
embryos (Storey et al., 1998; Mathis et al., 2001) and with the
requirements for these signalling pathways in the specification of
the NMp population (Fig. 2A,C). By contrast, there is no clear
indication of BMP or nodal signalling (supplementary material
Fig. S4). These observations led us to test the roles of FGF in the
specification and differentiation of the NMp population.
Exposure to both Chiron and FGF between 48 and 72 h leads to a
strong suppression of Sox1, a decrease in Sox2 levels and an increase in
Bra expression relative to Chiron alone (Fig. 7B,C). This results in an
increased number of NMps after exposure to these signals and
suggests that, in combination with Wnt/β-catenin signalling, FGF
promotes early progenitors at the expense of differentiating cells that
4248
Recently, we have developed a 3D mESC culture system that
mimics some of the morphogenetic events taking place during early
embryogenesis (Baillie-Johnson et al., 2014; van den Brink et al.,
2014). Using Sox1::GFP mESCs and exposing them to continuous
differentiation in N2B27, we observe the emergence of Sox1::GFPpositive cells throughout the aggregate (Fig. 8A) and a similar
pattern is observed if, on day 3, the aggregates are exposed to
DMH1, a BMP inhibitor (Fig. 8A; 2-5 DMH1), SB43, an activin/
nodal inhibitor (data not shown) or PD03 (Fig. 8A; 2-5 Chi+PD03).
The latter results are in agreement with previous observations that
differentiation of embryoid bodies in the absence of external signals
leads to the development of neural tissue (Watanabe et al., 2005;
Wataya et al., 2008).
When the aggregates are cultured in conditions that elicit the
NMp-like population in adherent culture, they exhibit a polarized
expression of Bra and undergo a polarized elongation that, after 2
further days in N2B27, has become 200-300 µm long and 20-40 µm
wide (Figs 8 and 9). The elongated region expresses Sox1::GFP and
Sox2, while the tip exhibits high levels of Bra and of Wnt signalling
(Fig. 8). Expression of Bra is confined to the population at the tip,
indicating that the elongation lacks a notochord (Fig. 8C). This
configuration is very reminiscent of the situation in the caudal lateral
epiblast of the developing embryo, where the NMp population is
thought to reside between stages 7.5 and 11.0 (Wilson et al., 2009).
To test for the presence of paraxial mesoderm in these aggregates,
we used mESC carrying a YFP reporter for Tbx6 (van den Brink
et al., 2014). As the aggregate begins to elongate, after it has
expressed Bra and Sox1 we observe mesenchymal-like cells that
express Tbx6::EYFP in the elongating region (Fig. 9A). Most of
these cells leave the aggregate and either float in the medium or
attach, preferentially, to the opposite end to the elongating tissue;
only cells that attach to the aggregate maintain expression of the
reporter (Fig. 9). These results suggest that, when elicited in three
dimensions, the NMps can organize themselves into structures that
resemble the initial steps of spinal cord development and can be
maintained autonomously for a number of days.
FGF regulates the extension of the aggregates
We also tested the role of FGF in the organization and development of
the elongates (Fig. 9). Suppression of MEK signalling from 72 to
120 h prevented the growth of the elongations (Fig. 9B′), indicating
that it is necessary to support the growth once it has started. Addition
of FGF during this period did not have a significant effect, indicating
that the endogenous levels produced by the aggregates are sufficient.
DEVELOPMENT
Emergence of an axial progenitor and polarized growth in 3D
aggregates of mESCs
RESEARCH ARTICLE
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
This effect was also seen for Wnt/FGF inhibition during the 48-72 h
period (Fig. 9D), consistent with our observations from adherent
culture that FGF signalling is necessary for the establishment of the
elongation. Blocking Wnt/β-catenin signalling also affects the
elongation, consistent with the effect of mutants in Wnt signalling
on axial development in the embryo (Aulehla et al., 2008; Dunty et al.,
2008; Aulehla and Pourquie, 2010).
DISCUSSION
We have shown that mESCs differentiated in adherent culture under
the influence of Wnt/β-catenin signalling give rise to cells with the
signature and potential of the axial NMps that have been described
in the embryo as giving rise to both paraxial mesoderm and neural
progenitors. The impact of Wnt/β-catenin signalling in the
emergence of this population is most effective on day 3 of
differentiation, when mESCs have been shown to transit through a
stage that is similar to that of the post-implantation epiblast
(Sterneckert et al., 2010; Turner et al., 2014b). This adds support to
a recent report that EpiSCs exposed to Wnt signalling develop
primitive streak fates, as well as a small subpopulation with the
characteristics of the NMp (Tsakiridis et al., 2014).
The NMp-like population that emerges in adherent culture
cannot be expanded efficiently under our culture conditions and
tends to differentiate into neural precursors and paraxial
mesoderm. Applying the same conditions to a 3D system of
mESC aggregates that we have developed to study the early stages
of embryogenesis, we observed the emergence of a phenotypically
similar population that now is maintained for at least 3 days, and
drives the polarized elongation of a structure containing,
principally, neural progenitors; the maintenance and elongation
of this population is driven by FGF signalling. These observations
support the suggestion that the spinal cord is derived from an axial
progenitor population and provide an experimental system, the
aggregates of mESCs, to identify and dissect the networks that
establish, maintain and differentiate this population (see also the
accompanying paper in this issue: van den Brink et al., 2014). This
system is complementary to those that already exist that can
generate forebrain and retina (Eiraku et al., 2008, 2011; Sasai
et al., 2012; Lancaster et al., 2013; Sasai, 2013), and it should
allow the study of the mechanisms that pattern axial derivatives, in
particular the nervous system (Sasai et al., 2012).
Origin of the NMp population
An important unresolved issue in developmental biology relates to
the embryological origin of the spinal cord in amniotes, whether its
progenitor population emerges from a posteriorization of an
anteriorly fated neural plate or as a separate population, induced
de novo in the primitive streak, which does not go through an
anterior fate (Stern et al., 2006; Wills et al., 2010). The first notion is
derived from classical experiments in frogs (Nieuwkoop et al.,
4249
DEVELOPMENT
Fig. 7. Role of FGF in NMp establishment
and differentiation. (A) Differentiation
protocols used throughout this figure.
(B) mRNA from E14-Tg2A mESCs treated
with a 24 h pulse of Chiron or Chiron and
FGF2 on days 2-3, or with Chiron or Chiron
and FGF2 from days 2-5 were analysed by
qRT-PCR for Bra and Tbx6 (top) or Sox1
and Sox2 (bottom). Data presented as
mean±s.d. Significance tests were
performed on this set of experiments and
are presented in supplementary material
Fig. S2. (C) Population profiles of Sox1::
GFP mESCs differentiated after 5 days
incubation with N2B27 with a 24 h pulse
between days 2 and 3 of either Chiron and
FGF or Chiron. Grey boxes indicate the
negative, low- and high-expressing
populations. mRNA from cells that were
sorted from the indicated populations and
conditions was analysed for the indicated
genes by qRT-PCR. Data presented as
mean±s.d. Significance tests were
performed on this set of experiments and
are presented in supplementary material
Fig. S3. (D) Sox1::GFP mESCs exposed to
Chi on days 2-3 were sorted based on the
level of expression (negative or low) and
stained for Sox2 and Bra. (E) Tbx6
expression from sorted Sox1::GFP mESCs
after a first passage under the indicated
conditions. N, N2B27; C, Chiron; F, FGF.
N-N-C means 2 days in N2B27 and 1 day in
Chiron, etc.
RESEARCH ARTICLE
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Fig. 8. Elongation process. (A) Examples of aggregates from Sox1::GFP
mESCs aggregated for 2 days in N2B27 before further treatment with N2B27,
which may include a 24 h pulse on day 3 with Chiron, 3 days of Chiron, or
Chiron and the MEK inhibitor PD03, or 3 days with the BMP inhibitor DMH1.
Aggregates were imaged on day 5 by wide-field epifluorescence microscopy.
The phase-contrast and fluorescence images shown are representative
examples. Aggregates exposed to a 24 h pulse of Chiron are able to show a
single large extension containing Sox1::GFP-expressing cells with a region at
the tip that is negative for the fluorescence reporter. (B) Wnt reporter TLC2
mESC aggregates differentiated in N2B27 with a 24 h pulse of Chiron on day 3
and imaged on day 5. (C) Aggregate as in B fixed and immunostained for
brachyury (white) and Sox2 (green). The fluorescent reporter is expressed
predominately in the aggregate extension corresponding to a Sox2+ Bra+
region. The white arrowhead indicates the region of highest Sox2 expression.
Hoechst was used to label the nuclei. Scale bars: 500 µm in A; 200 µm in B,C.
4250
Fig. 9. Signalling requirements in aggregates. (A) Stills from movies of
aggregates from Bra::GFP (top) or TBX6::EYFP mESCs, started at the time of
exposure to Chiron (after 2 days in N2B27). Expression of Tbx6 is delayed
relative to Bra. Notice the extrusion of cells from the Bra expression domain as
well as the elongation. (B,C) Summary of treatments applied to mESC
aggregates to test signalling requirements for the maintenance of the
elongation (B) and the establishment of the precursor population (C). In all
conditions, mESCs are formed as aggregates for 2 days in N2B27 (green)
before the medium is changed to include a 24 h pulse as indicated. Growth
conditions are abbreviated within the parentheses. (B′,C′) Proportion of
aggregates displaying elongated phenotypes from each growth condition. FGF
signalling is essential for the maintenance and establishment of the elongation,
whereas Wnt signalling is required for the establishment only. (D) Specific
comparisons demonstrate the requirements for Wnt and FGF signalling in
establishing the precursor population. Abbreviations as in B. Significance tests
were performed on this set of experiments and are presented in supplementary
material Fig. S7.
that are located laterally to the NSB. The elongations that we
observe in our aggregates support this possibility as they lack a
notochord.
In our experiments, we have observed that signalling on the third
day of differentiation is crucial for the commitment of cells to
particular lineages both in adherent and 3D culture. At this time,
differentiating mESCs transit through an epiblast-like state and
therefore our results parallel recent ones showing that transient
activation of Wnt signalling in EpiSCs can generate various
primitive streak-related populations (Tsakiridis et al., 2014).
Altogether, these observations favour the possibility that, at least
in culture, an NMp-like population can be generated from a
primitive streak-primed epiblast-like population without the need
for an anteriorly fated neural template. This is supported by the
observation that differentiation of mESCs continuously exposed to
DEVELOPMENT
1952) and finds support from modern work with Xenopus and
zebrafish. However, in amniotes, the origin of the spinal cord anlage
is spatially and temporally separate from that of the fore-, mid- and
hindbrain. For example, recent experiments can generate this
population directly from EpiSCs, which do not have a neural fate
(Tsakiridis et al., 2014), and experiments in chicken and mice
provide evidence for the existence of a distinct progenitor
population, adjacent to but distinct from the anterior neural plate,
that can give rise to neural tissue of different axial levels through
long term growth (Brown and Storey, 2000; Mathis and Nicolas,
2000). Although it cannot be ruled out that the NMp population
emerges from a group of cells that has an underlying anterior neural
fate, these results suggest that posterior fates might arise over time
and within cells that do not exist at the time of the initial
specification of the population, i.e. the mechanisms for the
specification of posterior neural fates might differ between
amniotes and anamniotes.
The NMp population emerges within the mouse node-streak
border (NSB) at the end of gastrulation and careful lineage-tracing
experiments have identified a subpopulation in the regressing node
that acts as a source of progenitors, at least for the ventral nervous
system (Cambray and Wilson, 2002, 2007). However, removal of
the node in chicken and mouse embryos does not affect axial
elongation. Genetic or mechanical ablation of the node in mouse
embryos before it regresses results in the loss of the notochord and
the floor plate, but it does not impair the emergence of a neural tube
with spinal cord characteristics, though it is smaller and lacks motor
neurons; the paraxial mesoderm is also affected in these embryos
(Ang and Rossant, 1994; Weinstein et al., 1994; Davidson et al.,
1999; Klingensmith et al., 1999). This observation suggests the
existence of alternative sources of progenitors for the spinal cord
RESEARCH ARTICLE
Signalling and the specification and differentiation of the
NMp population
In both adherent cultures and aggregates, the appearance of the
Sox2+ Bra+ population is associated with Wnt/β-catenin, as well as
with FGF/ERK signalling. Once the population has been
established, it appears to use cell-autonomous gene regulatory
networks fuelled, as in the embryo, by FGF/ERK signalling for its
maintenance and differentiation. Consistent with this, blockage of
FGF/ERK signalling after the establishment of the primordium,
inhibits its elongation, although it does not affect neural cells
generated prior to the inhibition. The details of the structure of this
population remain to be elucidated. Retrospective clonal analysis in
embryos has suggested the presence of a small, stem cell-like
population, with dual potential (Tzouanacou et al., 2009), but this
remains to be confirmed. Preliminary results of clonal analysis of
the NMp-like population generated in adherent culture have been
obtained (P.C.H. and D.A.T., unpublished).
Single-cell analysis of a population enriched for NMps reveals a
number of features that will require further examination. Most
strikingly, we observe a variation in the levels of Bra mRNA
expression in the population and a generally low level of Sox2
expression, which FGF makes very low. These heterogeneities
contrast with the more homogeneous and correlated expression of
the corresponding proteins. The discrepancy raises the possibility of
dynamic transcription time averaging at the level of the population
in some of the identifiers of the NMp. Such a situation has been
described before for some elements of the pluripotency network
(Munoz Descalzo et al., 2013) and might represent a general feature
of stem/progenitor cell populations (Martinez Arias and Brickman,
2011). The single-cell analysis also reveals the expression of one
new gene in the NMp: FoxB1. This gene had been associated with
the development of the diencephalon but lineage tracing and gene
expression place it within the NMp population (Zhao et al., 2007).
Finally, this analysis confirms that the population exhibits high FGF
and Wnt signalling, and identifies two genes associated with
pluripotency, Lin28 and Mbd3, expressed in these cells, suggesting
that they might share some features with mESCs.
In the future, it will be important to explore the properties and
long-term renewal and differentiation potential of the NMp-like
cells that we have generated in culture and in the 3D aggregates, and
compare them with those that have been recently obtained from
EpiSCs (Tsakiridis et al., 2014). In this regard, we notice that, under
the same conditions, the NMps in the aggregates are maintained
over a few days in a manner that they are not in adherent culture.
This is likely to be due to the ability of the cells in the aggregate to
generate their own niche. It will be interesting to investigate the
differences between the two systems to identify the factors that
create these differences.
Note added in proof
While this paper was being reviewed Gouti et al. (2014) have
reported a population of cells similar to the one described here
during the differentiation of both mouse and human ES cells.
MATERIALS AND METHODS
Routine monolayer cell culture, differentiation and aggregate
formation
E14-Tg2A, TCF/LEF::mCherry (TLC2), Sox1::GFP, TBX6::EYFP and
Bra::GFP mESCs were grown on tissue-culture plastic dishes as described
previously (Faunes et al., 2013; Turner et al., 2014b). For differentiation
experiments, cells were plated at a density of 4×103 cells/cm2 in a base
medium of N2B27 (NDiff 227, StemCells) supplemented with
combinations of activin A (100 ng/ml), CHIR99021 (Chiron; 3 μM),
XAV939 (1 μM), SB431542 (SB43; 10 μM), BMP4 (1 ng/ml), FGF2
(2.5 ng/ml), PD0325901 (PD03; 1 μM) and dorsomorphin-H1 (DM;
0.5 μM). Differentiation medium was replaced daily to reduce the
influence of increased concentrations of secreted factors.
To generate aggregates, 40 μl droplets of a 1×104 cells/ml solution (in
N2B27) were pipetted into each well of a sterile, non-tissue-culture treated,
bacterial grade, U-bottomed 96-well plate with a multichannel pipette. After
48 h, 150 μl of differentiation medium was added directly to the cells and
150 μl was replaced daily. It was essential that during medium replacements,
cells were agitated by the forceful ejection of medium to prevent their
adhering to the plate surface; medium was not changed during the initial
48 h of aggregate formation. For a full protocol and troubleshooting, see
Baillie-Johnson et al. (2014).
Flow cytometry and microscopy
Cells were analysed for GFP fluorescence using an LSR Fortessa (BD
Bioscience) with a 488 nm laser and emission was measured using a 530/30
filter. Forward- and side-scatter properties alongside DAPI exclusion
(405 nm laser and emission at 450/50) were used to select live, single cells
for analysis in FlowJo (TreeStar). Data were analysed using FlowJo
software. Sox1::GFP mESCs were sorted according to their GFP
fluorescence in a MoFlo sorter (Beckman Coulter) using the same laser
and filter sets described above. Immunofluorescence was performed as
described previously (Turner et al., 2014a); a list of primary antibodies and
staining conditions, as well as details of data capture are also fully described
previously (Turner et al., 2014a).
Epifluorescence, time-lapse imaging of adherent cells was performed
with an AxioObserver inverted microscope (Carl Zeiss) in a humidified CO2
incubator (37°C, 5% CO2). Images were captured every 10 min for the
required duration using a 20× LD Plan-Neofluar 0.4 NA Ph2 objective with
correction collar adjusted for imaging through plastic. All media were
changed daily. An LED white-light system (Laser2000, Kettering, UK)
provided illumination. The filter cubes GFP-1828A-ZHE (Semrock) and
Filter Set 45 (Carl Zeiss) were used for GFP and RFP, respectively. Emitted
light was recorded using an AxioCam MRm and recorded with Axiovision
release 4.8.2. For live imaging of cell aggregates, aggregates within a 35 mm
non-tissue-culture grade bacterial dish were imaged every 10 min within a
humidified (37°C, 5% CO2) Biostation IM (Nikon). Analysis performed
using Fiji (Schindelin et al., 2012).
Quantitative RT-PCR
Protocols used are as described previously (Faunes et al., 2013) (see
supplementary material Figs S2 and S3). The primers used are available in
supplementary material Table S2. The population qRT-PCR experiments
are data from one experiment (each sample analysed in triplicate) and are
reflective of two independent experiments, whereas, due to constraints of
sample availability, the sorted subpopulation qRT-PCR data are from one
experiment with each sample analysed in duplicate or triplicate where
possible. The Sox1::GFP population profiles gained in each condition have
been analysed on multiple occasions and are both reproducible and stable.
Fluidigm
A list of primers and details of the protocols followed for the Fluidigm analysis
have been described previously (Turner et al., 2014b) and are available in
supplementary material Table S3 and supplementary materials and methods.
Acknowledgements
We thank James Briscoe and Val Wilson for sharing unpublished information and,
together with Ben Steventon and Kate Storey, for constructive and helpful
discussions throughout this work; Bertie Gottgens and Cristina Pina for help with the
Fluidigm single-cell analysis; Nigel Miller for help with the flow cytometry; Sonja
S. Nowotschin and Anna Katerina Hadjantonakis for the Tbx6 reporter; Christoph
Budjan for help with the immunostaining; and Tina Balayo for assistance with the
tissue culture of the aggregates.
4251
DEVELOPMENT
activin and Chiron, which suppresses the neural fate, results in the
appearance of some Sox2+ Bra+ cells (Fig. 1A).
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Competing interests
The authors declare no competing financial interests.
Author contributions
A.M.A., D.A.T. and P.C.H. conceived the project; D.A.T., P.H., P.B.-J., R.B. and F.F.
performed the experiments; P.R. and P.C.H. analysed the data; A.M.A. and D.A.T.
wrote the manuscript.
Funding
This work is funded by a European Research Council Advanced Investigator Award
(to D.A.T., P.R. and P.C.H.) and by a Project Grant from the Wellcome Trust (R.B.) to
A.M.A.; by an Engineering and Physical Sciences Research Council studentship
(to P.B.-J.); and by a Postdoctoral fellowship [Beca Chile 74100037 to F.F.].
Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.112979/-/DC1
References
Abranches, E., Silva, M., Pradier, L., Schulz, H., Hummel, O., Henrique, D. and
Bekman, E. (2009). Neural differentiation of embryonic stem cells in vitro: a road
map to neurogenesis in the embryo. PLoS ONE 4, e6286.
Andoniadou, C. L. and Martinez-Barbera, J. P. (2013). Developmental
mechanisms directing early anterior forebrain specification in vertebrates. Cell.
Mol. Life Sci. 70, 3739-3752.
Ang, S.-L. and Rossant, J. (1994). HNF-3 beta is essential for node and notochord
formation in mouse development. Cell 78, 561-574.
Ang, S. L., Wierda, A., Wong, D., Stevens, K. A., Cascio, S., Rossant, J. and
Zaret, K. S. (1993). The formation and maintenance of the definitive endoderm
lineage in the mouse: involvement of HNF3/forkhead proteins. Development 119,
1301-1315.
Arnold, S. J. and Robertson, E. J. (2009). Making a commitment: cell lineage
allocation and axis patterning in the early mouse embryo. Nat. Rev. Mol. Cell Biol.
10, 91-103.
Aulehla, A. and Pourquie, O. (2010). Signaling gradients during paraxial
mesoderm development. Cold Spring Harb. Perspect. Biol. 2, a000869.
Aulehla, A., Wiegraebe, W., Baubet, V., Wahl, M. B., Deng, C., Taketo, M.,
Lewandoski, M. and Pourquié , O. (2008). A beta-catenin gradient links the clock
and wavefront systems in mouse embryo segmentation. Nat. Cell Biol. 10,
186-193.
Baillie-Johnson, P., van den Brink, S. C., Balayo, T., Turner, D. A. and Martinez
Arias, A. (2014). Generation of aggregates of mouse ES cells that show symmetry
breaking, polarisation and emergent collective behaviour in vitro. Biorxiv.
Brown, J. M. and Storey, K. G. (2000). A region of the vertebrate neural plate in
which neighbouring cells can adopt neural or epidermal fates. Curr. Biol. 10,
869-872.
Cambray, N. and Wilson, V. (2002). Axial progenitors with extensive potency are
localised to the mouse chordoneural hinge. Development 129, 4855-4866.
Cambray, N. and Wilson, V. (2007). Two distinct sources for a population of
maturing axial progenitors. Development 134, 2829-2840.
Davidson, B. P., Kinder, S. J., Steiner, K., Schoenwolf, G. C. and Tam, P. P. L.
(1999). Impact of node ablation on the morphogenesis of the body axis and the
lateral asymmetry of the mouse embryo during early organogenesis. Dev. Biol.
211, 11-26.
De Robertis, E. M. (2009). Spemann’s organizer and the self-regulation of
embryonic fields. Mech. Dev. 126, 925-941.
De Robertis, E. M., Larraı́n, J., Oelgeschlä ger, M. and Wessely, O. (2000). The
establishment of Spemann’s organizer and patterning of the vertebrate embryo.
Nat. Rev. Genet. 1, 171-181.
De Robertis, E. M., Wessely, O., Oelgeschlager, M., Brizuela, B., Pera, E.,
Larrain, J., Abreu, J. and Bachiller, D. (2001). Molecular mechanisms of cell-cell
signaling by the Spemann-Mangold organizer. Int. J. Dev. Biol. 45, 189-197.
Delfino-Machin, M., Lunn, J. S., Breitkreuz, D. N., Akai, J. and Storey, K. G.
(2005). Specification and maintenance of the spinal cord stem zone.
Development 132, 4273-4283.
Dunty, W. C., Jr., Biris, K. K., Chalamalasetty, R. B., Taketo, M. M., Lewandoski,
M. and Yamaguchi, T. P. (2008). Wnt3a/beta-catenin signaling controls posterior
body development by coordinating mesoderm formation and segmentation.
Development 135, 85-94.
Eiraku, M., Watanabe, K., Matsuo-Takasaki, M., Kawada, M., Yonemura, S.,
Matsumura, M., Wataya, T., Nishiyama, A., Muguruma, K. and Sasai, Y.
(2008). Self-organized formation of polarized cortical tissues from ESCs and its
active manipulation by extrinsic signals. Cell Stem Cell 3, 519-532.
Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S.,
Sekiguchi, K., Adachi, T. and Sasai, Y. (2011). Self-organizing optic-cup
morphogenesis in three-dimensional culture. Nature 472, 51-56.
4252
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Faunes, F., Hayward, P., Munoz-Descalzo,
S., Chatterjee, S. S., Balayo, T., Trott,
̃
J., Christophorou, A., Ferrer-Vaquer, A., Hadjantonakis, A.-K., DasGupta, R.
et al. (2013). A membrane-associated β-catenin/Oct4 complex correlates with
ground-state pluripotency in mouse embryonic stem cells. Development 140,
1171-1183.
Gadue, P., Huber, T. L., Nostro, M. C., Kattman, S. and Keller, G. M. (2005). Germ
layer induction from embryonic stem cells. Exp. Hematol. 33, 955-964.
Gouti, M., Tsakiridis, A., Wymeersch, F. J., Huang, Y., Kleinjung, J. et al. (2014).
In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for
Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity.
PLoS Biol. 12(8), e1001937.
Graham, V., Khudyakov, J., Ellis, P. and Pevny, L. (2003). SOX2 functions to
maintain neural progenitor identity. Neuron 39, 749-765.
Hamburger, V. (2001). Induction of embryonic primordia by implantation of
organizers from a different species. Int. J. Dev. Biol. 45, 13-38.
Hemmati-Brivanlou, A. and Melton, D. (1997a). Vertebrate embryonic cells will
become nerve cells unless told otherwise. Cell 88, 13-17.
Hemmati-Brivanlou, A. and Melton, D. (1997b). Vertebrate neural induction. Annu.
Rev. Neurosci. 20, 43-60.
Hu, G. and Wade, P. A. (2012). NuRD and pluripotency: a complex balancing act.
Cell Stem Cell 10, 497-503.
Kaji, K., Caballero, I. M., MacLeod, R., Nichols, J., Wilson, V. A. and Hendrich,
B. (2006). The NuRD component Mbd3 is required for pluripotency of embryonic
stem cells. Nat. Cell Biol. 8, 285-292.
Kamachi, Y. and Kondoh, H. (2013). Sox proteins: regulators of cell fate
specification and differentiation. Development 140, 4129-4144.
Kamachi, Y., Iwafuchi, M., Okuda, Y., Takemoto, T., Uchikawa, M. and Kondoh,
H. (2009). Evolution of non-coding regulatory sequences involved in the
developmental process: reflection of differential employment of paralogous
genes as highlighted by Sox2 and group B1 Sox genes. Proc. Jpn. Acad. B
Phys. Biol. Sci. 85, 55-68.
Kiecker, C. and Niehrs, C. (2001). A morphogen gradient of Wnt/beta-catenin
signalling regulates anteroposterior neural patterning in Xenopus. Development
128, 4189-4201.
Kim, W.-Y., Wang, X., Wu, Y., Doble, B. W., Patel, S., Woodgett, J. R. and Snider,
W. D. (2009). GSK-3 is a master regulator of neural progenitor homeostasis. Nat.
Neurosci. 12, 1390-1397.
Klingensmith, J., Ang, S.-L., Bachiller, D. and Rossant, J. (1999). Neural
induction and patterning in the mouse in the absence of the node and its
derivatives. Dev. Biol. 216, 535-549.
Kondoh, H. and Takemoto, T. (2012). Axial stem cells deriving both posterior
neural and mesodermal tissues during gastrulation. Curr. Opin. Genet. Dev. 22,
374-380.
Lancaster, M. A., Renner, M., Martin, C.-A., Wenzel, D., Bicknell, L. S., Hurles,
M. E., Homfray, T., Penninger, J. M., Jackson, A. P. and Knoblich, J. A. (2013).
Cerebral organoids model human brain development and microcephaly. Nature
501, 373-379.
Mangold, O. (1933). Über die Induktionsfä higkeit der verschiedenen Bezirke der
Neurula von Urodelen. Naturwissenschaften 21, 761-766.
Martinez Arias, A. and Brickman, J. M. (2011). Gene expression heterogeneities
in embryonic stem cell populations: origin and function. Curr. Opin. Cell Biol. 23,
650-656.
Mathis, L. and Nicolas, J. F. (2000). Different clonal dispersion in the rostral and
caudal mouse central nervous system. Development 127, 1277-1290.
Mathis, L., Kulesa, P. M. and Fraser, S. E. (2001). FGF receptor signalling is
required to maintain neural progenitors during Hensen’s node progression. Nat.
Cell Biol. 3, 559-566.
Mazzoni, E. O., Mahony, S., Peljto, M., Patel, T., Thornton, S. R., McCuine, S.,
Reeder, C., Boyer, L. A., Young, R. A., Gifford, D. K. et al. (2013). Saltatory
remodeling of Hox chromatin in response to rostrocaudal patterning signals. Nat.
Neurosci. 16, 1191-1198.
Munoz Descalzo, S., Rue, P., Faunes, F., Hayward, P., Jakt, L. M., Balayo, T.,
Garcia-Ojalvo, J. and Martinez Arias, A. (2013). A competitive protein
interaction network buffers Oct4-mediated differentiation to promote
pluripotency in embryonic stem cells. Mol. Syst. Biol. 9, 694.
Nieuwkoop, P. and Nigtevecht, G. V. (1954). Neural activation and transformation
in explants of competent ectoderm under the influence of fragments of anterior
notochord in Urodeles. J. Embryol. Exp. Morphol. 2, 175-193.
Nieuwkoop, P. D., Boterenbrood, E. C., Kremer, A., Bloesma, F. F. S. N.,
Hoessels, E. L. M. J., Meyer, G. and Verheyen, F. J. (1952). Activation and
organisation of the central nervous system in amphibians. J. Exp. Zool. 120,
1-108.
Nordströ m, U., Jessell, T. M. and Edlund, T. (2002). Progressive induction of
caudal neural character by graded Wnt signaling. Nat. Neurosci. 5, 525-532.
Nordströ m, U., Maier, E., Jessell, T. M. and Edlund, T. (2006). An early role for
WNT signaling in specifying neural patterns of Cdx and Hox gene expression and
motor neuron subtype identity. PLoS Biol. 4, e252.
Nowotschin, S., Ferrer-Vaquer, A., Concepcion, D., Papaioannou, V. E. and
Hadjantonakis, A.-K. (2012). Interaction of Wnt3a, Msgn1 and Tbx6 in neural
DEVELOPMENT
RESEARCH ARTICLE
versus paraxial mesoderm lineage commitment and paraxial mesoderm
differentiation in the mouse embryo. Dev. Biol. 367, 1-14.
Otero, J. J., Fu, W., Kan, L., Cuadra, A. E. and Kessler, J. A. (2004). Beta-catenin
signaling is required for neural differentiation of embryonic stem cells.
Development 131, 3545-3557.
Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge, R. (1998). A role
for SOX1 in neural determination. Development 125, 1967-1978.
Rossant, J. and Tam, P. P. L. (2009). Blastocyst lineage formation, early embryonic
asymmetries and axis patterning in the mouse. Development 136, 701-713.
Roszko, I., Faure, P. and Mathis, L. (2007). Stem cell growth becomes predominant
while neural plate progenitor pool decreases during spinal cord elongation. Dev.
Biol. 304, 232-245.
Sasai, Y. (2013). Next-generation regenerative medicine: organogenesis from stem
cells in 3D culture. Cell Stem Cell 12, 520-530.
Sasai, Y., Eiraku, M. and Suga, H. (2012). In vitro organogenesis in three
dimensions: self-organising stem cells. Development 139, 4111-4121.
Saxé n, L. and Toivonen, S. (1961). The two-gradient hypothesis in primary
induction: the combined effect of two types of inducers mixed in different ratios.
J. Embryol. Exp. Morph. 9, 514-533.
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch,
T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B. et al. (2012). Fiji: an
open-source platform for biological-image analysis. Nat. Methods 9, 676-682.
Shyh-Chang, N. and Daley, G. Q. (2013). Lin28: primal regulator of growth and
metabolism in stem cells. Cell Stem Cell 12, 395-406.
Slawny, N. A. and O’Shea, K. S. (2011). Dynamic changes in Wnt signaling are
required for neuronal differentiation of mouse embryonic stem cells. Mol. Cell.
Neurosci. 48, 205-216.
Spemann, H. and Mangold, H. (1924). Über Induktion von Embryonalanlagen
durch Implantation artfremder Organisatoren. Arch. Mikrosk. Anat.
Entwicklungsmechanick 100, 599-638.
Stern, C. D. (2005). Neural induction: old problem, new findings, yet more questions.
Development 132, 2007-2021.
Stern, C. D., Charite, J., Deschamps, J., Duboule, D., Durston, A. J., Kmita, M.,
Nicolas, J.-F., Palmeirim, I., Smith, J. C. and Wolpert, L. (2006). Head-tail
patterning of the vertebrate embryo: one, two or many unresolved problems?
Int. J. Dev. Biol. 50, 3-15.
Sterneckert, J., Stehling, M., Bernemann, C., Araú zo-Bravo, M. J., Greber, B.,
Gentile, L., Ortmeier, C., Sinn, M., Wu, G., Ruau, D. et al. (2010). Neural
induction intermediates exhibit distinct roles of Fgf signaling. Stem Cells 28,
1772-1781.
Storey, K. G., Goriely, A., Sargent, C. M., Brown, J. M., Burns, H. D., Abud, H. M.
and Heath, J. K. (1998). Early posterior neural tissue is induced by FGF in the
chick embryo. Development 125, 473-484.
Takemoto, T., Uchikawa, M., Kamachi, Y. and Kondoh, H. (2006). Convergence
of Wnt and FGF signals in the genesis of posterior neural plate through activation
of the Sox2 enhancer N-1. Development 133, 297-306.
Takemoto, T., Uchikawa, M., Yoshida, M., Bell, D. M., Lovell-Badge, R.,
Papaioannou, V. E. and Kondoh, H. (2011). Tbx6-dependent Sox2 regulation
determines neural or mesodermal fate in axial stem cells. Nature 470, 394-398.
Thomson, M., Liu, S. J., Zou, L.-N., Smith, Z., Meissner, A. and Ramanathan, S.
(2011). Pluripotency factors in embryonic stem cells regulate differentiation into
germ layers. Cell 145, 875-889.
Development (2014) 141, 4243-4253 doi:10.1242/dev.112979
Tsakiridis, A., Huang, Y., Blin, G., Skylaki, S., Wymeersch, F., Osorno, R.,
Economou, C., Karagianni, E., Zhao, S., Lowell, S. et al. (2014). Distinct Wntdriven primitive streak-like populations reflect in vivo lineage precursors.
Development 141, 1209-1221.
Turner, D. A., Rué , P., Mackenzie, J. P., Davies, E. and Martinez Arias, A.
(2014a). Brachyury cooperates with Wnt/β-Catenin signalling to elicit primitivestreak-like behaviour in differentiating mouse ES cells. BMC Biol. 12, 63.
Turner, D. A., Trott, J., Hayward, P., Rue, P. and Martinez Arias, A. (2014b). An
interplay between extracellular signalling and the dynamics of the exit from
pluripotency drives cell fate decisions in mouse ES cells. Biol. Open 3, 614-626.
Tzouanacou, E., Wegener, A., Wymeersch, F. J., Wilson, V. and Nicolas, J.-F.
(2009). Redefining the progression of lineage segregations during mammalian
embryogenesis by clonal analysis. Dev. Cell 17, 365-376.
van den Brink, S. C., Baillie-Johnson, P., Balayo, T., Hadjantonakis, A.-K.,
Nowotschin, S., Turner, D. A. and Martinez Arias, A. (2014). Symmetry
breaking, germ layer specification and axial organisation in aggregates of mouse
embryonic stem cells. Development 141, 4231-4242.
van de Ven, C., Bialecka, M., Neijts, R., Young, T., Rowland, J. E., Stringer, E. J.,
Van Rooijen, C., Meijlink, F., Novoa, A., Freund, J.-N. et al. (2011). Concerted
involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal
neural tube and cloacal derivatives from the posterior growth zone. Development
138, 3451-3462.
Viswanathan, S. R. and Daley, G. Q. (2010). Lin28: A microRNA regulator with a
macro role. Cell 140, 445-449.
Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki,
H., Watanabe, Y., Mizuseki, K. and Sasai, Y. (2005). Directed differentiation of
telencephalic precursors from embryonic stem cells. Nat. Neurosci. 8, 288-296.
Wataya, T., Ando, S., Muguruma, K., Ikeda, H., Watanabe, K., Eiraku, M.,
Kawada, M., Takahashi, J., Hashimoto, N. and Sasai, Y. (2008). Minimization of
exogenous signals in ES cell culture induces rostral hypothalamic differentiation.
Proc. Natl. Acad. Sci. USA 105, 11796-11801.
Weinstein, D. C., Ruiz i Altaba, A., Chen, W. S., Hoodless, P., Prezioso, V. R.,
Jessell, T. M. and Darnell, J. E., Jr. (1994). The winged-helix transcription factor
HNF-3 beta is required for notochord development in the mouse embryo. Cell 78,
575-588.
Wills, A. E., Choi, V. M., Bennett, M. J., Khokha, M. K. and Harland, R. M. (2010).
BMP antagonists and FGF signaling contribute to different domains of the neural
plate in Xenopus. Dev. Biol. 337, 335-350.
Wilson, V., Olivera-Martinez, I. and Storey, K. G. (2009). Stem cells, signals and
vertebrate body axis extension. Development 136, 1591-1604.
Ying, Q.-L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003). Conversion of
embryonic stem cells into neuroectodermal precursors in adherent monoculture.
Nat. Biotechnol. 21, 183-186.
Zechner, D., Fujita, Y., Hü lsken, J., Mü ller, T., Walther, I., Taketo, M. M.,
Crenshaw, E. B., III, Birchmeier, W. and Birchmeier, C. (2003). beta-Catenin
signals regulate cell growth and the balance between progenitor cell expansion
and differentiation in the nervous system. Dev. Biol. 258, 406-418.
Zhao, T., Zhou, X., Szabó , N., Leitges, M. and Alvarez-Bolado, G. (2007). Foxb1driven Cre expression in somites and the neuroepithelium of diencephalon,
brainstem, and spinal cord. Genesis 45, 781-787.
DEVELOPMENT
RESEARCH ARTICLE
4253