In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm Identity

Timed pulses of WNT and FGF signaling convert human and mouse pluripotent stem cells into neuromesodermal progenitors that can be directed to differentiate into spinal cord and paraxial mesoderm cells


Introduction
The differentiation of embryonic stem cells (ESCs) to specific cell types offers insight into developmental mechanisms and has potential therapeutic applications. For example the differentiation of neural progenitors (NPCs) from monolayers of ESCs seeded in serum free conditions is a model of neural induction and regional patterning [1]. In the absence of additional signals, NPCs differentiated from ESCs adopt an anterior-dorsal neural (telencephalon) identity [1,2]. The addition of Sonic Hedgehog (Shh) ventralises these neural progenitors, mimicking the in vivo role of Shh [3,4]. Exposing NPCs to retinoic acid (RA) results in the repression of anterior identity and the induction of genes that typify hindbrain and anterior spinal cord (cervical) identity [5]. This has been taken as support for the idea that newly generated NPCs are by default anterior and are then posteriorised by exposure to specific extrinsic signals [6,7]. It is notable, however, that RA is actively excluded in the progenitors of the posterior spinal cord after gastrulation [8] and that commonly used ESC differentiation protocols do not efficiently generate neural cells of the more posterior spinal cord such as thoracic and lumbar spinal cord cells marked by posterior Hox gene expression, including Hoxc8-10 expression [9].
The anterior and posterior nervous system has distinct origins [10][11][12]. Anterior epiblast expresses Otx2 and contributes cells to the anterior nervous system [2,13] whereas spinal cord progenitors are located posteriorly [14][15][16]. Clonal analysis indicates that the spinal cord shares a common lineage, at least in part, with the trunk paraxial mesoderm that forms the somites [15]. The dualfated neuromesodermal precursors (NMPs) of these tissues are located in the node-streak border (NSB), caudal lateral epiblast (CLE) cell layer adjacent to the regressing node and the chordoneural hinge of the tail bud [13,14,17,18]. Cells in these regions coexpress the neural marker Sox2 and nascent mesoderm marker Brachyury [8,19,20]. Genetic lineage tracing experiments confirm that many spinal cord cells previously expressed Brachyury [21] indicating that as cells from regions harbouring NMPs move into the neural tube they downregulate Brachyury but maintain Sox2 expression and consolidate neural identity. By contrast, NMPs that enter the primitive streak delaminate basally, downregulate Sox2 and acquire expression of the paraxial mesoderm marker Tbx6 [22] en route to somite formation. Strikingly, in embryos lacking Tbx6, paraxial mesoderm cells express Sox2 and transdifferentiate into neural cells, providing additional support for the inter-relationship between spinal cord and somitic mesoderm [22][23][24]. As yet, however, the existence of NMPs has only been revealed in vivo and the inaccessibility of this population makes them difficult to study.
The region occupied by NMPs is exposed to Wnt and Fgf ligands [16]. These signals are required for body axis elongation [16] and both Wnt and Fgf signalling have been implicated in mesoderm and neural induction [22,[25][26][27][28][29][30][31]. In vivo and in vitro evidence has suggested that Wnt signalling is responsible for posteriorising tissue by inducing posterior Hox genes [29,32,33]. Together, the data suggest that the generation of posterior neural tissue and paraxial mesoderm proceeds by Wnt and Fgf signalling inducing a neuromesodermal bipotential intermediate. To test this idea, we developed an efficient in vitro differentiation method for spinal cord and paraxial mesoderm from mouse and human pluripotent stem cells. We show that carefully timed and calibrated pulses of Wnt and Fgf signalling generate a population of cells that transiently coexpress Sox2 and Brachyury in which the expression of posterior Hox genes are induced. Transcriptome analysis is consistent with the equivalence of these cells to the NMPs found in vivo. In vivo grafting and directed in vitro differentiation confirm the ability of NMPs to assume spinal cord or paraxial mesoderm cell fates. We further show that Brachyury is not required for the production of posterior neural cells or for the induction of posterior Hox genes, hence separating the posteriorising and mesoderm inducing functions of Wnt signalling. Taken together the data define a means to generate posterior neural and paraxial mesodermal tissues in vitro and illustrate how the directed differentiation of stem cells provides novel insight into developmental mechanism.

Generation of Neural Progenitor Cells with Spinal Cord Identity
To identify conditions for the generation of posterior neural cells from monolayers of mouse ES cells (mESCs), we cultured mESCs in serum free media containing bFgf for 3 days (D1-D3) and then transferred these to media lacking bFgf for an additional 2 days [1] ( Figure 1A). This resulted in the induction of a postimplantation epiblast-like intermediate by D2, indicated by the downregulation of the ''naïve'' pluripotency marker Zfp42 (Rex1) and the upregulation of the epiblast marker Fgf5 ( Figure 1F) [34]. At this stage, Pou5f1, which is expressed in both mESCs and epiblast-like cells, is maintained ( Figure 1F) [34]. In all experiments a Shh agonist, SAG, was added at D3 in order to generate a predictable ventralised identity for subsequent comparisons. The transcriptome of cells was then analysed at D5 by mRNA-seq. Consistent with previous studies [35][36][37][38][39], cells in these conditions had acquired an anterior neural identity (N A ), exemplified by the expression of Otx1 and Otx2 [2]. The presence of SAG induced the expression of ventral neural markers ( Figure S1A). Addition of retinoic acid (RA) and SAG to differentiating mESCs at D3 downregulated anterior neural markers (e.g. Otx2, Six3, Lhx5) and instead genes typical of hindbrain identity, including Hoxa2, Hoxb2, Mafb, Epha4 and Ephb2 were expressed ( Figure 1B) [40]. However markers of spinal regions of the neural tube, such as the 59 Hox genes Hoxc6, Hoxc8 and Hoxc9 were not detected ( Figure 1B) [5,9]. Changing the timing or concentration of RA used in these experiments did not result in the efficient induction of more posterior spinal cord identity [29].
To recapitulate the sequence of signalling events that generate the spinal cord, we seeded mESCs into serum free media containing bFgf. At D2, Wnt signalling was induced by the addition of the Wnt agonist CHIR99021 (CHIR). bFgf and Wnt agonist were removed at D3 and cells exposed to media containing RA and SAG until D5. Examination of gene expression profiles indicated that cells subjected to the FGF/CHIR/RA regime expressed genes characteristic of the spinal cord including high levels of 59 Hox genes Hoxb6, Hoxb8, Hoxc6, Hoxc8, Hoxc9 and low levels of the anterior neural and brainstem markers Otx2 and Mafb ( Figure 1B). Together, the data suggested that a brief pulse of Wnt signalling between D2-D3 was sufficient to posteriorise differentiating mESCs. We termed the neural cells generated in this regime N P cells and cells that display anterior and brainstem identity N A and N H , respectively ( Figure 1A).
We confirmed the posteriorisation and neural identity of N p cells using qRT-PCR and immunostaining ( Figure S1C-D). Analysis of the time course of Hox gene expression in N H and N P cells indicated that their temporal sequence of induction matched the in vivo time course [40]: Hoxb1 was induced within 12 h of exposure to Wnt signalling, whereas more 59 Hox genes were induced later ( Figure 1C). Notably the more posterior Hox genes, e.g, Hoxc6 and Hoxc9 were not induced in N H cells. In N p cells Hoxc6, Hoxc8 and Hoxc9 were strongly induced at D4 ( Figure 1C). Delaying the addition of CHIR to differentiating mESCs until D3 resulted in a concomitant shift in the timing of Hox gene induction ( Figure S2A-C). Furthermore, in agreement with studies indicating that RA represses the most posterior Hox genes [41], exposure of cells to FGF/CHIR without subsequent

Author Summary
Stem cells are providing insight into embryo development and offering new approaches to clinical and therapeutic research. In part this progress arises from ''directed differentiation'' -artificially controlling the types of cells produced from stem cells. Here we describe the directed differentiation of mouse and human pluripotent stem cells into cells of the spinal cord and paraxial mesoderm (the tissue that generates muscle and bone that is normally found adjacent to the spinal cord). During embryo development, spinal cord and paraxial mesoderm arise from a shared group of precursors known as neuromesodermal progenitors (NMPs). We show that signals to which NMPs are exposed in embryos can be used to generate NMPs from pluripotent stem cells in a dish. We define conditions for the conversion of these NMPs into either spinal cord or mesoderm cells. Using these conditions, we provide evidence that the decision between spinal cord and mesoderm involves a gene, Brachyury, that promotes mesoderm production by inhibiting spinal cord generation. Together the data illustrate how mimicking normal embryonic development allows the generation of specific cell types from stem cells and that this can be used to analyse cells that are otherwise difficult to study.  [74]. In N H cells Hoxb1 and Hoxb2 are induced prior to Hoxc4. However, the more posterior Hox genes are not induced. By contrast, in N P conditions the 59 Hox genes Hoxc6, Hoxc8 and Hoxc9 are induced at D4 and their expression is maintained at day 5. (Note log 2 scale). (D) Immunohistochemistry indicates that N H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the addition of RA induced Hoxc10 characteristic of the lumbar spinal cord ( Figure S2E). Finally we passaged N H and N P cells at D5 and allowed them to differentiate until D8, at which point we assayed the expression of genes expressed in motor neurons (MNs). Both N H and N P cells adopted a neuronal morphology and expressed the neuronal marker class III b-tubulin (Tuj1). The N H cells acquired a posterior hindbrain MN identity evident by the coexpression of Hoxb4 and the cranial motor neuron marker Phox2b [42] ( Figure 1D). In the case of N P cells however, only a few Hoxb4 expressing cells were detected ( Figure S1D) and most of the b-tubulin expressing neurons acquired a Hoxc6 and Hoxc9 identity characteristic of neurons of the brachial and thoracic spinal cord, respectively [43] (Figure 1E,G). Moreover N P cells expressed Olig2, a marker of somatic motor neuron progenitors, as well as the differentiated MN markers Hlxb9 and Islet1/2 [3] ( Figure 1E). Taken together these data indicate that similar to the situation in vivo [44] and in embryoid bodies [29] exposure of monolayers of differentiating ESCs to a combination of Wnt, Fgf and RA signalling generates spinal cord cells.

Generation of N P Cells Proceeds via Neuromesodermal Progenitors
To address how the combination of Wnt and Fgf signalling induces spinal cord identity we examined gene expression in differentiating ESCs at D2.5 and D3 (Figure 2A). ESCs that had been exposed to Fgf/Wnt signalling for 12 h (D2.5) and 24 h (D3) induced the expression of Cdx2 and the mesoderm transcription factors Brachyury and Tbx6 ( Figure 2B). Recombinant Wnt3a protein had a similar activity to CHIR in these assays ( Figure S2F). By contrast, ESCs cultured in the absence of Wnt agonist, expressed significantly lower levels of these genes ( Figure 2B). These data suggest that Wnt signalling, in combination with Fgf, is initiating a mesodermal transcriptional program. This is consistent with the loss of mesoderm in mouse embryos lacking Wnt3a [45] and the induction of Brachyury by b-catenin [28].
It was also noticeable that the levels of Sox2 mRNA were transiently reduced in D2.5 and D3 N P cells treated with FGF/ CHIR ( Figure 2B). We therefore assayed Sox2 and Brachyury proteins by immunostaining in D3 N P and N A cells. Strikingly, the level of Sox2 protein was similar in N P and N A cells, consistent with the long half-life of Sox2 protein [46]. Moreover, ,80% of N P cells coexpressed Brachyury and Sox2 ( Figure 2C) whereas only a small number of N A cells expressed Brachyury. These data suggest that the exposure to bFgf and Wnt signalling induces a cell identity reminiscent of the dual-fated neuromesodermal progenitors present during axial elongation in the CLE [15,44] ( Figure  S4E). If D2-D3 N P cells represent NMPs, they should form mesoderm. To test this, we transferred cells at D3 into media containing Wnt agonist but lacking bFgf. In these conditions (termed Meso) the expression of Sox1, Sox2 and Brachyury were downregulated and several genes characteristic of paraxial mesoderm, including Tbx6 and Msgn1 [47], were significantly upregulated ( Figure 2D). Immunostaining revealed that more than 90% of cells in this condition expressed Tbx6 protein at D5 ( Figure 2E). By D8 Desmin, the intermediate filament protein of muscle sarcomeres [48] and the muscle transcription factor MyoD were highly expressed ( Figure 2E). Thus the continued exposure of cells to Wnt signalling induces a paraxial mesodermal identity that differentiates to a muscle-like identity. This provides evidence that ESCs exposed to Wnt and bFgf at D2-D3 represent bipotential neuromesodermal cells that can differentiate into either mesoderm or neural tissue.
We next tested the in vivo potential of NMP cells. For these experiments we took advantage of the chick. Cells with NMP-like behaviour have been identified in chick [16] and chick embryos provide an accessible and experimentally tractable vertebrate host for grafts of mouse ESCs [49]. We grafted small groups of DiI labelled D3 N A cells, not exposed to Wnt signalling, or D3 NMPs, exposed to Fgf/Wnt signalling for 24 h, into the caudal lateral epiblast of Hamburger-Hamilton (HH) stage 8-9 chick embryos ( Figure 2F). Analysis of embryos 24 h later revealed efficient incorporation and migration of the NMP cells to both the neural tube and the somites ( Figure 2G). Transplanted cells from a single graft contributed to multiple anterior-posterior levels and most embryos showed contribution to both spinal cord and somites ( Figure 2G-J). In several embryos grafted cells were also observed in the tail bud of the embryo as well as the neural tube and somites ( Figure 2J). Contribution to endoderm was not observed. By contrast, transplanted N A cells showed somewhat lower rates of engraftment and contributed only to the neural tube and not to somites ( Figure 2K-L). These data confirm the bipotency of the in vitro derived NMP cells and demonstrate that similar to in vivo NMPs [15] [44] they contribute to both neural and paraxial mesoderm lineage. Single cell and clonal analysis, in vivo and in vitro, will be necessary to test the potency of individual cells and to understand the molecular mechanism by which neural and/or mesodermal progeny are generated from NMP.

A Distinct Transcriptional Programme Identifies Neuromesodermal Progenitors
We took advantage of the in vitro differentiation to analyse the transcriptional programmes that generate each of the neural and mesodermal lineages ( Figure 3A). Principal component analysis of the transcriptomes indicated that each differentiation pathway could be clearly distinguished ( Figure 3B). Strikingly, the first principal component (PC) appeared to represent developmental time and the second PC the tissue identity of the differentiated cells. The data revealed a set of genes that distinguished N P , N A , N H cells and Meso cells ( Figure S3 and Tables S2, S3). These included the upregulation of Mafb and Phox2b in N H samples and the upregulation of posterior Hox genes, notably Hoxc6, Hoxc8 and Hoxc9 in N P samples. By contrast, the induction of genes such as Tbx6, Hes7 and Hoxc8 and Hoxc9 in D5 cells subjected to mesodermal conditions confirmed the posterior paraxial identity of these cells. Moreover, the analysis indicated a bifurcation in the transcriptional programmes that generate anterior neural and brainstem cells from those that produce posterior neural and paraxial mesodermal cells. It was notable that gene expression typical of paraxial mesoderm was evident at D4 of N P differentiation suggesting a gradual separation of neural and mesodermal identity. Together these data provide a molecular correlate to the distinct cellular origins of anterior and posterior neural tissue [15] and identifies the NMP state as the branch point in the developmental trajectories.
We identified genes upregulated in NMPs compared to mESCs at D1 and N A cells at D3. Comparing these to genes induced in D5 neural and mesodermal cells revealed a large intersection. standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N P conditions. All data used to generate the plots of Figure 1 can be found in Data S1. doi:10.1371/journal.pbio.1001937.g001 Thus, in part, NMPs have a transcriptional programme that is a combination of neural and mesodermal gene expression. In addition however, a set of ,240 genes appeared uniquely upregulated in NMP cells (Table S1). These included the transcription factors Brachyury, Nkx1.2 (also known as Sax1), which is expressed in the stem zone of midgestation embryos [50,51] Mixl1 [52], Wnt3a and Cdx2 which are expressed in the primitive streak and nascent mesoderm [32,33]. In addition Follistatin, which plays a key role in neural induction by blocking TGFb signalling [53] and components of the Fgf signalling pathway, which is implicated in mesoderm induction [16], are upregulated in NMPs ( Figure 3C). Together these data support the idea that exposure of differentiating ESCs to Fgf/Wnt signalling between D2 and D3 induces a bipotential neuromesodermal population equivalent to that found in vivo in the CLE [15,16,18,22] and that the balance and timing of these two signals influences the further differentiation of these cells into neural or mesodermal tissues.

Generation of NMPs from Mouse Epiblast Stem Cells
The activation of Wnt signalling in differentiating mouse epiblast stem cells (EpiSCs) leads to a modest induction of   Brachyury/Sox2 coexpressing cells, suggestive of NMP identity [19]. To improve the efficiency of this induction we adapted our mESC protocol to take account of the more advanced developmental state of EpiSC compared to mESCs ( Figure 1F). Accordingly, we exposed EpiSCs to a range of CHIR (Wnt) and bFgf concentrations and assayed the expression of Sox2 and Brachyury ( Figure S4A). Maximal proportions of Sox2/Brachyury coexpressing cells resulted from 3 mM CHIR and 20 ng/ml bFgf (hereafter referred to as FGF/CHIR) ( Figure 4A, Figure S4A).
Assaying a broader panel of genes supported the idea that FGF/ CHIR was inducing NMP identity. The expression of the pluripotency factor Nanog was undetectable and the majority of the Sox2 expressing cells expressed minimal levels of Oct4, suggesting that they had exited pluripotency ( Figure 4B, Figure  S4D). Moreover, the acquisition of Brachyury/Sox2 coexpression coincided with an upregulation of Wnt3a, Cdx2 and Nkx1.2 as well as trunk Hox genes ( Figure 4B), characteristic of embryo and mESC derived NMPs. Consistent with this, the paraxial/somitic mesoderm markers Tbx6 and Meox1 and the neural factor Sox1 were expressed in these conditions ( Figure S5A). Immunostaining indicated that by D3 of differentiation Tbx6 and Sox2 expression were mutually exclusive ( Figure S5B). By contrast the expression of genes characteristic of anterior neural plate (e.g. Otx2 and Six3) and endoderm (Foxa2) [54] were largely absent in FGF/CHIR conditions ( Figure S5A). Collectively, these data indicate that, similar to mESCs, stimulation of Wnt and Fgf signalling in mouse EpiSCs leads to the induction of an NMP state.
The developmental potential of differentiated mouse EpiSCs has previously been tested by transplantation into mouse embryos [19,55]. We therefore grafted EpiSC-derived NMPs constitutively expressing GFP into the NSB of E8.5 embryos. After 48 h in culture, we observed extensive incorporation of GFP expressing cells (15/15 embryos) ( Figure 4C-D). Sections from these embryos revealed integration of transplanted cells into the somites and presomitic mesoderm of host embryos (10/10) and neural tube (4/ 10) ( Figure 4D). We did not observe contributions to endoderm or other tissues. Antibody staining for paraxial mesoderm (Tbx6), somite/dermomyotome (Pax3), neural (Sox2) and floor plate (Foxa2) markers confirmed that the engrafted cells had acquired the marker expression of their host environment ( Figure 4E). Moreover, examination of the rostral limit of labelling using the somite level as a reference revealed that grafted EpiSC derived NMPs behaved similarly to homotopic grafts of microdissected E8.5 NSB cells [56]. Strikingly, few cells grafted into the node of E7.5 embryos showed any incorporation (2 out of 8 embryos had 8-10 incorporated cells/embryo), suggesting that these conditions produce a population incompatible with gastrulation-stage development. Similarly, cells differentiated for 24 h in FGF/CHIR did not incorporate into the NSB of E8.5 embryos (n = 5) ( Figure 4D). Collectively, these results suggest that 48 h treatment of EpiSCs with FGF/CHIR results in coexpression of Brachyury/Sox2 (up to 90%, Figure S4B) and generates NMPs that functionally resemble their in vivo counterparts.

Directed Differentiation of Human ES Cells to NMPs
The resemblance of mouse EpiSCs to human embryonic stem cells (hESCs) prompted us to ask whether an analogous FGF/ CHIR treatment regimen was sufficient to generate human NMPs. Treatment of three independent hESC lines with CHIR and bFgf from D0-D3 downregulated NANOG and OCT4 and upregulated the suite of NMP expressed genes-BRACHYURY, NKX1.2 and CDX2-similar to mouse ESCs and EpiSCs ( Figure 5C, Figure S4D). SOX2 expression was maintained in this population and up to ,80% of cells co-expressed SOX2 and BRACHYURY ( Figure 5B, Figure S4B). We also observed the spontaneous upregulation of paraxial mesoderm/somite markers (TBX6, MSGN1, MEOX1). ( Figure S5C). By contrast, the expression of a lateral plate (KDR) and an endoderm (FOXA2) marker were minimal ( Figure S5C). Thus FGF/CHIR treated hESCs appear to adopt an NMP identity and are likely to represent the in vitro correlates of the SOX2 and BRACHYURY co-expressing cells found in the caudal epiblast of human embryos [8]. Consequently we dubbed these cells hNMPs.
To test the potency of hNMPs, we treated hESCs with FGF/ CHIR for 72 h to drive the generation of BRACHYURY + / SOX2 + cells and then re-plated them for a further 48 h in serum free media without additional factors to promote the induction of spinal cord identity ( Figure 5A). We termed these cells N P and compared them to neural cells derived from hESCs using a dual SMAD inhibition protocol involving Nodal and BMP inhibitors (SB/LDN) [57]. Both conditions induced neural identity, exemplified by increased levels of SOX2, TUBB3 and PAX6 ( Figure 5D). As expected, neural cells generated using dual SMAD inhibition expressed the anterior marker OTX2 but lacked expression of HOX genes ( Figure 5D). By contrast, neural cells derived from NMPs expressed SOX1 and the posterior HOX genes HOXC6, HOXC8 and HOXC9 but not OTX2 ( Figure 5D). A similar expression profile was obtained after treatment with RA and dual Shh agonists SAG and purmorphamine (Pur). This also induced expression of the motor neuron progenitor marker OLIG2 ( Figure S5D). Antibody staining verified HOXC8 expression in N P conditions and revealed that the majority of HOXC8 + cells co-expressed SOX2, confirming their neural identity ( Figure 5E, F). Treatment of neural cells for 48 h with FGF/CHIR following 72 h dual SMAD inhibition did not result in HOXC8 induction suggesting that posteriorisation is necessary before or concomitant with neural induction ( Figure S5E). Together these data suggest that neural differentiation of hNMPs generates spinal cord progenitors similar to mNMPs.
We next tested whether hNMPs differentiate into mesoderm by culturing them in the presence of CHIR alone. This resulted in the expression of paraxial/somitic mesoderm markers TBX6, MSGN1 and MEOX1 ( Figure S5D), but little if any expression of KDR, a lateral plate mesoderm marker ( Figure S5D). Taken together these findings provide evidence of a human NMP population that gives rise to spinal cord and paraxial mesoderm derivatives but not anterior neurectoderm or lateral plate mesoderm. Moreover, a similar set of developmental cues induces and directs NMPs in human and mouse, consistent with a similar ontogeny of trunk tissues in these species.

Brachyury Is Required for the Induction of Mesoderm but Not Posterior Neural Identity
The ability to generate NMPs in vitro allows experimental investigations of trunk development that are challenging or impossible in vivo. For example, although the requirement for Brachyury in mesoderm formation is well-established [58][59][60], the truncation of embryos lacking Brachyury has complicated analysis of its role in the elaboration of spinal cord identity. In zebrafish, a non-autonomous role for Brachyury orthologues has been identified [60]. It is unclear whether in mammals Brachyury is required directly to maintain NMPs and therefore generate spinal tissue or indirectly via Wnt induction to establish a mesodermal niche that signals to generate or maintain posterior neural tissue. To address this we took advantage of Brachyury null mESCs (BTBR10) derived from embryos lacking Brachyury [61].
Assaying Brachyury null cells at D3 of differentiation indicated that, in contrast to wild-type ESCs, Tbx6 expression was not upregulated by exposure to FGF/CHIR signalling, whereas Cdx2 and Hoxb1 expression were induced ( Figure 6B). This is consistent with the lack of posterior mesoderm induction in Brachyury mutant embryos and prompted us to address the fate of Brachyury mutant cells that would normally form mesoderm. In wild type cells exposed to Meso conditions, Tbx6 was highly expressed at D5 (Figure 6D), as were Desmin and MyoD at D8 ( Figure 6E). By contrast Brachyury null cells subjected to the same conditions  and Brachyury null cells (Bra 2/2 ) with and without CHIR. In wild-type cells activation of Wnt signalling induces the expression of these three genes. In the absence of Brachyury while Cdx2 and Hoxb1 continue to be induced by Wnt signalling, Tbx6 induction is lost. (C) qRT-PCR analysis of the expression of mesodermal, neural and posterior marker genes at D5 of differentiation in wt and Bra 2/2 ESCs exposed to CHIR from D2-D5 (Meso failed to differentiate into paraxial mesoderm as indicated by the absence of Tbx6 ( Figure 6D). Instead these cells expressed Sox1, Sox2 and posterior Hox genes (Hoxc6 and Hoxc9) at D5 ( Figure 6C) and differentiated into b-Tubulin expressing neurons ( Figure 6E). These data indicate that within mouse NMPs, Brachyury not only specifies mesodermal identity via mechanism(s) in addition to its direct stimulation of Wnt signalling, but also represses neural identity. In the absence of Brachyury, NMPs adopt a neural differentiation route. Thus the induction of posterior neural tissue is not dependent on Brachyury. Moreover the data separate the mesoderm inducing and posteriorising activity of Wnt signalling and provide evidence that posteriorisation of the CNS is not dependent on mesoderm derived signals.
What could be responsible for the induction of posterior Hox genes? Analysis of the transcriptome data revealed the induction in NMPs of the Cdx genes Cdx1, 2 and 4, which have been implicated in the regulation of Hox gene expression ( Figure 6F) [29,62]. Induction of both Cdx1 and Cdx2 were detectable within 12 h of FGF/CHIR exposure and the levels of all three genes increased further at D3 and D4 of N P differentiation and at D5 of Meso differentiation. Moreover, the induction of Cdx2 by Fgf/ Wnt signalling was maintained in Brachyury null ESCs ( Figure 6B). Thus the induction of Cdx proteins by Fgf/Wnt signalling represents a good candidate for the posteriorisation of NMPs. Moreover the temporal accumulation of Cdx levels following Wnt exposure might provide a timing mechanism for the progressive induction of increasingly more posterior Hox genes.

Discussion
We describe the in vitro generation of bipotential neuromesodermal progenitors from both mouse and human pluripotent stem cells that are capable of producing posterior neural tissue and paraxial mesodermal tissue. This recapitulates the behaviour of NMPs residing in the CLE and NSB [15,22] (Figure 6G). Moreover, we provide evidence that Wnt signalling has two distinct functions in NMPs, initiating a mesodermal differentiation programme by regulating Brachyury expression and independently posteriorising these cells. It is also likely that Brachyury maintains NMPs during axis elongation by forming a positive feedback loop with Wnt gene expression as has been previously shown [60]. Strikingly, a neuromesodermal precursor is also present in ascidian embryos [63]. Similar to vertebrates, the induction of these cells depends on the timing of Wnt and Fgf signalling [64,65]. Moreover the mesoderm and posterior nervous system of many arthropods, including short germband insects, arises from a shared progenitor population that is exposed to Wingless signalling and expresses Cdx [66]. Thus molecular and cellular features of the development of the neural and mesodermal components of the trunk appear to be evolutionarily conserved across bilaterian embryos. This emphasizes the distinct developmental origins of cells that form anterior and posterior regions of bilaterian embryos, suggesting an explanation as to why it has proved difficult to generate spinal cells and skeletal muscle from ESCs. More generally, the ability to produce and manipulate NMPs in vitro has the potential to increase the efficiency with which cell types derived from posterior neural and paraxial mesodermal tissue can be generated and analysed.

Cell Culture and Differentiation
The mouse ES cell lines, HM1 [67] and BTBR10 [68] were maintained in ES cell medium [69] with 1000 U/ml LIF (Chemicon) on mitotically inactive primary mouse embryo fibroblasts. To initiate differentiation, ES cells were removed from feeders by dissociation using 0.05% trypsin and then plated onto tissue culture plates for two short successive periods (20-30 mins) to remove feeder layers. To induce differentiation, the cells were plated on CellBINDSurface dishes (Corning) precoated with 0.1% gelatin (Sigma) at a density of 5610 3 cells cm 22 in 'N2B27' medium. This medium comprised Advanced Dulbecco's Modified Eagle Medium F12 (Gibco) and Neurobasal medium (Gibco) (1:1), supplemented with 16N2 (Gibco), 16B27 (Gibco), 2 mM L-glutamine (Gibco), 40 mg/ml BSA (Sigma), 0.1 mM 2-mercaptoethanol. Cells were grown in N2B27 supplemented with 10 ng/ml bFgf (R&D) for 3 days (D1-D3) and then were transferred into serum free media without bFgf (D3-D5). To induce ventral hindbrain identity NPCs (N H ) 100 nM RA (Sigma) and 500 nM SAG (Calbiochem) was added from D3-D5. Spinal cord identity (N P ) was induced by the addition of 5 mM CHIR99021 (Axon) or 100 ng/ml Wnt3a (R&D) from D2 to D3 followed by 100 nM RA, 500 nM SAG from D3-D5. To induce mesodermal differentiation the cells were treated with CHIR99021 from D2-D5. To induce terminal differentiation, cells were trypsinised and plated as single-cell suspension on plates coated with Matrigel (BD Biosciences) at a density of 1610 5 cells cm 22 in N2B27 medium supplemented with bFgf (10 ng/ml). The next day bFgf was removed and cells were left to differentiate for an additional 3 days. conditions). Posterior Hox genes Hoxc8 and Hoxc9 are induced in both wt and Brachyury null cells. However, in contrast to wild-type cells neural markers Sox1 and Sox2 are expressed only in Bra 2/2 cells exposed to Meso conditions. (D) Immunostaining of Tbx6 and Sox2 at D5 of Meso differentiation in Bra 2/2 and wild-type ESCs. Wild-type cells efficiently differentiate to paraxial mesoderm and expresses Tbx6 but not Sox2. By contrast Bra 2/2 cells differentiate to a neural identity exemplified by Sox2 expression in the absence of Tbx6. (E) At D8 wt cells cultured in CHIR express Desmin/MyoD but not b-Tubulin (Tuj1) whereas Bra 2/2 cells fail to produce Desmin/MyoD and differentiate into neurons expressing b-Tubulin (Tuj1). (F) The time course of Cdx gene expression in posterior neural (N P ) and mesodermal inducing conditions (Meso). Cdx genes are transiently induced in posterior neural cells but continuously upregulated in mesodermal cells. (Note, log 2+ scale). All data used for the plots can be found in Data S6. (G) Model for the generation of spinal cord and paraxial mesodermal tissue from ESCs. ESCs cultured in N2B27 with FGF generate anterior but not posterior neural tissue. The activation of Wnt signalling in differentiating ESCs results in the generation of a bipotential neuromesodermal progenitor, equivalent to those found in the CLE of the embryo, which generate spinal cord or paraxial mesodermal tissue. Wnt signalling activates homeodomain proteins of the Cdx family in these progenitors that could account for the posteriorisation. In addition, Wnt signalling activates the mesodermal specifier Brachyury (Bra) that is required for Tbx6 induction and the repression of Sox2. The induction of Brachyury induces the Brachyury-Wnt autoregulatory loop that is necessary for mesoderm induction. In the absence of this gene ESCs differentiate into posterior neural tissue even in the presence of continued Wnt signalling. doi:10.1371/journal.pbio.1001937.g006 The mouse EpiSC line R04-GFP [55] was routinely maintained in N2B27 supplemented with Activin A (20 ng/ml; R&D Systems) and bFgf (10 ng/ml; Peprotech) as previously described [70]. For differentiation of EpiSCs into NM progenitors approximately 1500-2000 cells/cm 2 were plated on fibronectin (Sigma)-coated wells in N2B27 medium supplemented with CHIR99021 (3 mM; Signal Transduction Division, Dundee) and bFgf (20 ng/ml). For grafting experiments the initial plating density was 2500 cells/cm 2 and cells were plated on either fibronectin or gelatin.
Human ESC lines MasterShef 5 and 7 (a gift of Prof. Harry Moore, University of Sheffield) and a Sox2GFP reporter line (a gift of Dr Andrew Smith, University of Edinburgh) were cultured in Essential 8 TM medium on Geltrex TM -coated plates. For hNMP differentiation cells were pre-treated for 1 h with ROCK inhibitor Y-27632 (10 mM; Calbiochem), dissociated with accutase and plated at approximately 10,000 cells/cm 2 (Sox2-GFP hESCs) or 80,000 cells/cm 2 (MasterShef5 and 7 hESC lines) on fibronectincoated wells in N2B27 medium supplemented with 3 mM CHIR99021/20 ng/ml bFgf and Y-27632 (10 mM). The medium was replaced the following day with fresh N2B27 containing the same components minus the ROCK inhibitor. For directed differentiation of hESCs, cultures were differentiated in the presence of CHIR99021/bFgf for 72 h as described above. For neural/spinal cord differentiation 72 h CHIR99021/bFgf-differentiated cells were treated with Accutase (Sigma) and transferred onto Geltrex (Life Technologies)-coated plates either in N2B27 alone or N2B27 supplemented with RA (0.1 mM; Sigma), SAG (0.5 mM; Calbiochem) and purmorphamine (1 mM; Calbiochem) for 48 h. For mesodermal differentiation 72 h CHIR99021/bFgf differentiated cells were cultured in N2B27 supplemented with CHIR99021 (3 mM) for a further 48 h. For dual SMAD inhibition Sox2-GFP hES cells were plated at 10,000 cells/cm 2 on Geltrex TM -coated wells in N2B27 supplemented with LDN193189 (100 nM; Stemgent) and SB431542 (10 mM; Sigma). This was followed either by re-plating and culture in N2B27 or in N2B27/CHIR99021 (3 mM)/bFgf (20 ng/ml) for a further 48-72 h. All experiments involving hES cells have been approved by the UK Stem Cell Bank steering committee.

Grafting of Mouse NMP Cells in Chick and Mouse Embryos
To graft NMP and N A cells into the CLE of stage HH8-9 chick embryos, plates of appropriately prepared cells were labelled for 10 mins at 37uC with DiI and washed 3 times with PBS. N2B27 medium was added and cells were incubated for 30 mins. Small clumps of cells were mechanically detached from the plate and transplanted using a manually pulled glass needle. Groups of 100-200 cells were grafted into the caudal lateral epiblast and the eggs were incubated for a further 24 h. Embryos were then fixed in 4% paraformaldehyde (PFA) for 60 mins at 4uC. Fixed embryos were cryoprotected by equilibration in 15% sucrose and then cryosectioned (14 mm). Images were taken using an Apotome2 (Zeiss) and Leica confocal microscope TCS-SP5.
For mouse embryo grafting, r04-GFP EpiSC were flow sorted for GFP expression using a BD FACSAria II sorter and plated overnight in EpiSC conditions followed by Fgf/Wnt for 24 h or 48 h. Mouse embryo grafting (,10 cells/embryo), culture and imaging were performed as described previously (Huang et al., 2012).

Immunofluorescence
Cells were fixed for 10 minutes at 4uC in 4% paraformaldehyde in phosphate buffer saline (PBS), then washed in PBST (PBS with 0.1% Triton X-100). Blocking was for 1 h in PBST with 3% donkey serum at room temperature. Primary and secondary antibodies were diluted in PBST containing 1% donkey serum. Cells were incubated with primary antibodies overnight at 4uC, with secondary antibodies at room temperature for 2 h, mounted with DAPI containing Prolong Antifade (Molecular Probes), and fluorescent images were taken using an inverted Leica SP5 confocal microscope or an Apotome 2 microscope or an Olympus IX51 inverted microscope (Olympus). Embryo processing and immunohistochemistry on tissue sections was performed as described previously [55]. Whole embryos were imaged using a Nikon NZ100 dissecting microscope, and sections were imaged in an Olympus BX61 fluorescence compound microscope. Nuclear segmentation followed by single cell fluorescence quantification was performed as described previously [70]. The following primary antibodies were used: mouse anti-Hoxc6 . anti-Nanog (2.5 mg/ ml) (14-5761-80, eBioscience); anti-Oct4 (1 mg/ml) (N-19, Santa Cruz), HoxC8 (5 mg/ml) (Abcam). Secondary antibodies were anti-mouse, anti-rabbit, anti-goat and anti-rat, Alexa's (488, 568, 647) from Molecular Probes.

Reverse Transcription-Quantitative PCR analysis
Total RNA was isolated from cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions and digested with DNase I (Qiagen) to remove genomic DNA. First strand cDNA synthesis was performed with Superscript III system (Invitrogen) using random primers and amplified using Platinum SYBR-Green (Invitrogen). For QPCR the Applied Biosystems 7900HT Fast Real time PCR or the Light Cycler 480 SYBR Green I Master Mix (Roche) systems were used. PCR primers were designed using Primer3 software. All experiments were performed in biological duplicates or triplicates for each time point analysed. Expression values were normalized against the b-actin or the TATA-binding protein (TBP) and standard deviations were calculated and plotted using Prism 6 software (GraphPad). Primer sequences are available upon request.

RNA-Sequencing and Data Analysis
Total RNA was processed according to the TruSeq protocol (Illumina). Three separate RNA libraries (biological replicates) were barcoded and prepared for each time point. Library size, purity and concentration were determined using Agilent Technologies 2100 Bioanalyzer with a DNA specific chip (Agilent DNA-1000). For sequencing, four samples were loaded per lane of an Illumina Genome Analyzer Hiseq2500. The sequence files generated each contained approximately 30million reads per sample. Reads were aligned to the Ensembl transcriptome mm10 using Bowtie2 and TopHat2 [71]. Per gene counts were collated using HTseq-count [72] and normalized using the DESeq R package [73]. Data analysis, PCA and Biplots were performed using custom scripts in R and MATLAB (MathWorks). RNA-seq data are available in the Array express database (http://www.ebi. ac.uk/arrayexpress) under accession number E-MTAB-2268. Figure S1 mESC derived neural progenitor cells respond to ventralising and posteriorising signals. (A) Changes in the expression of the indicated ventral progenitors markers over time in N A , N H , and N P conditions (RNA-seq data) exposed to the Shh agonist SAG. Nkx2-1 is induced only in N A cells as expected, whereas Nkx2-2 is induced in all conditions. (B) Expression profile of Sox genes from D1 to D5 of differentiation in N A , N H , and N P conditions. Sox1, specific for neural identity, is induced in all three conditions at D5 (RNA-seq data). (Note, log 2+ scale). (C) Expression of posterior Hoxb and Hoxc gene clusters analysed by qRT-PCR in N A , N H and N P conditions at D5. (Note, log 2 scale). This validates the mRNA-seq data shown in Figure 1B. (D) Otx2 is strongly expressed only in N A cells as shown by immunostaining at D5 of differentiation. By contrast Hoxb4 is strongly expressed in N H but in not N A or N P cells (representative images shown). All data used to generate the plots in Figure S1 can be found in Data S7. (TIFF) Figure S2 Wnt controls the timing of Hox gene induction. (A) Schematic illustrating the two differentiation conditions used in this experiment. In condition I, CHIR is added from D2 to D3, whereas in condition II CHIR is added from D3 to D4. (B) qRT-PCR shows the rapid induction of Hoxb1 after CHIR addition. (C-D) qRT-PCR analysis shows that the timing of induction of Hoxb and Hoxc genes depends on the timing of Wnt treatment. (Note, log 2 scale). (E) Cells exposed to a short pulse of FGF/CHIR, but not RA, express Hoxc10 at D8 of differentiation. (F) Immunostaining for Brachyury/Sox2 at day 3 of differentiation after a short pulse with Wnt3a/Fgf instead of CHIR/Fgf. Recombinant Wnt3a substituted for CHIR and NMP cells coexpressing Brachyury + and Sox2 + were generated to a similar extent. All data used to generate the plots in Figure S2  . In all cases qPCR results are represented as log 10 ratio of expression versus untreated EpiSCs (mouse) or hES cells (human). Anterior, anterior neural plate; PXM/SOM, paraxial/somitic mesoderm; LPM, lateral plate mesoderm; END, endoderm; PNP, posterior neural plate; SP, spinal cord; RA, retinoic acid; Pur, purmorphamine n/d, not determined. All data used to generate the plots in Figure S5 can be found in Data S10. (TIFF)

Supporting Information
Table S1 List of genes induced only in NMP cells. NMP specific genes were identified by collating genes significantly upregulated in NMP cells compared to D1 ES cells and D3 N A cells that were not upregulated in D5 neural or mesoderm cells. Genes are shown with their Ensembl gene id number, short gene name and in each of the comparisons the fold change and p adjusted value is calculated using DESeq.