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Brachyury cooperates with Wnt/β-Catenin signalling to elicit Primitive Streak like behaviour in differentiating mouse ES cells

The formation of the Primitive Streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic Stem Cells (ESCs) provide a good system to understand the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both Nodal/TGFβR and Wnt/β-Catenin signalling pathways specify cells to adopt a Primitive Streak like fate and direct them to undertake an epithelial to mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear as to what extent events in the embryo are able to be reproduced in culture. Here, we combine flow-cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs (mESCs) towards a Primitive Streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient Brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of Bra, whose expression is dependent on the EMT and ß-Catenin activity. As a consequence of our analysis, we propose that a major output of Brachyury expression is in controlling the velocity of the cells that are transiting out of the Primitive Streak.


Introduction
The development of an organism is the result of the proliferation, concomitant phenotypic diversification and spatial organisation of cells in the context of spatiotemporally controlled patterns of gene expression (Arias and Stewart, 2002;Gilbert, 2013;Wolpert and Tickle, 2010). The use of genetics to interrogate these processes has revealed that they are underpinned by the temporal iteration of coordinated interactions between signal transduction and transcription factor networks. Establishing the relationship between these molecular events and the emergence of cellular diversity is an essential step towards understanding the relationship between programs of gene activity and the process of morphogenesis that shapes cells into tissues and organs.
Gastrulation is one of the earliest events where it is possible to observe a convergence of fate specification and morphogenetic processes in embryos (Wolpert and Tickle, 2010). It occurs in all metazoan and encompasses a choreography of cell movements that transforms a group of seemingly identical epithelial cells, with species specific geometry, into the outline of an organism exhibiting an overt anterior-posterior organisation and three germ layers (ectoderm, mesoderm, endoderm) (Ramkumar and Anderson, 2011;Solnica-Krezel and Sepich, 2012;Tam and Gad, 2004). In chordates, gastrulation is led by a dynamic population of cells that gives rise to the mesoderm and the endoderm, defines and patterns the neuroectoderm and delineates the plane of bilateral symmetry (Nowotschin and Hadjantonakis, 2010;Tam and Gad, 2004). In mammalian embryos this population, called the Primitive Streak, is associated with the expression of the T-box transcription factor Brachyury (Bra) (Beddington et al., 1992;Fehling et al., 2003;Ramkumar and Anderson, 2011;Rivera-Pérez and Magnuson, 2005;Wilkinson et al., 1990). In the mouse, Bra is first expressed shortly after implantation in a group of cells at the boundary between the prospective embryonic and extraembryonic tissues. At stage E6.5, preceding the onset of gastrulation movements, Bra expression becomes restricted to the proximal posterior region of the embryo (Rivera-Pérez and Magnuson, 2005), at the position where under the influence of Nodal and Wnt signalling, the Primitive Streak is initiated as a dynamic structure that will progress towards the distal end of the epiblast, ploughing an anteroposterior axis (Arnold and Robertson, 2009). At the cellular level, gastrulation involves a sequence of highly organized Epithelial Mesenchymal Transition (EMT) movements that propagate through the tissue in a manner that resembles a travelling wave (Lim and Thiery, 2012;Thiery and Sleeman, 2006;Williams et al., 2011).
The first cells undergoing EMT invaginate and move towards the anterior contralateral side of the embryo. This movement accompanies the distal/anterior spread of the streak and thus, by the end of gastrulation, two thirds of the epiblast have been wrapped by the cells that have undergone gastrulation. This choreography of cell movements is characterized by the expression of a number of transcription factors at the leading edge of the EMT, in particular Brachyury (Wilkinson et al., 1990), Eomes (Russ et al., 2000) and Mixl1 (Robb et al., 2000).
Just anterior to the Primitive Streak there is a structure, the organizer, which does not undergo an EMT, shows homology with the Spemann organizer and will become the Node when the streak reaches the distalmost anterior region of the epiblast at about E7.5 (Beddington, 1994).
The organizer and the Node express Bra and, after E7.5 the Node regresses towards the posterior pole of the embryo, leaving the notochord in its wake, the Node and the notochord, continue to express Bra. After this time, Bra expression becomes restricted to a region in the tail that will undergo caudal extension to generate the caudal spinal cord and somatic mesoderm, (Beddington et al., 1992;Cambray and Wilson, 2007;J. C. Smith, 2004;Tam and Gad, 2004;Wilson et al., 1993). Genetic analysis has shown that in embryos, Bra is required for both movement of the cells through the Primitive Streak during axial extension and their specification into mesoderm and notochord posterior to somite 7 (Gluecksohn-Schoenheimer, 1938; B. L. Martin and Kimelman, 2008;Wilson et al., 1995).
The localization of Bra expression to the proximal posterior region of the epiblast is a useful reference for the onset of gastrulation and genetic analysis has identified a requirement for BMP, Wnt/β-Catenin, and Nodal for this event (Tam and Loebel, 2007). These studies have also shown that BMP sets up the expression of Nodal and Wnt3a, which, from the visceral endoderm, are likely to be the direct activators of Bra expression. How individual cells integrate Nodal and Wnt signalling with the expression of Bra to promote the directional EMT and how these events are coordinated across the cell population that is defined as the Primitive Streak is not known. While there are some reports of the gastrulation movements in mouse embryos with a cellular resolution (Ichikawa et al., 2013;Williams et al., 2011), these are descriptive and the visualization systems do not lend themselves to experimental perturbations.
In addition to gene expression analysis, ESCs also offer the opportunity to quantify proteins at the level of single-cells through live imaging. For these reasons ESCs could provide a useful model to understand the link between signals and morphogenesis in the context of the onset of gastrulation. However, in order to do this it is important to show that, in addition to patterns of gene expression, the differentiating ES cells share other features with the cells in the Primitive Streak, in particular EMT and its relationship to Bra expression.
Here we have used a combination of live cell imaging, immunocytochemistry and chemical genetics to analyse the onset and consequences of Bra expression in mESCs at the level of single cells. We observe that ES cells grown on gelatin and in the presence of Activin (Act) and Chiron (Chi, an agonist of Wnt/β-Catenin signalling), undergo an EMT associated with the expression of Bra and that the EMT itself contributes to Bra expression. We are able to separate the inputs of β -Catenin and Act in the onset of Bra expression and show that while β -Catenin is required for the up-regulation of Bra, Act is required for the velocity, and thereby the distance cells travel. We discuss our findings in the context of the emergence of the Primitive Streak during gastrulation and suggest that differentiation of ES cells into Bra expressing cells in culture provides a valuable system to study the mechanisms that specify the emergence of the Primitive Streak.

FACS analysis
GFP and RFP was assessed using a Fortessa flow cytometer (BD Biosciences). Analysis of data from single, live cells (DAPI-negative) was conducted using Flowjo software (Tree Star, Inc.). The GFP-positive populations from FACS data were analysed using a one-way ANOVA with Tukey's adjustment comparing the time-matched DMSO control and treatment; significance was set at p < 0.05.

Quantitative Image Analysis (QIA) and Confocal Microscopy
Immunofluorescence and image analysis carried out as described previously (Descalzo et al., 2012)  Data capture was carried out using Zen2010 v6 (Zeiss) and image analysis performed using Fiji (Schindelin et al., 2012).

Widefield, live-cell Microscopy and analysis
For live imaging, cells were imaged by widefield microscopy in a humidified CO 2 incubator (37°C, 5% CO 2 ) every 10 minutes for up to 96 hours using a 20x LD Plan-Neofluar 0.4 NA Ph2 objective with correction collar set to image through tissue-culture plastic dishes. An LED, white-light system (Laser2000) excited fluorescent proteins. Emitted light was recorded using an AxioCam MRm and recorded with Axiovision (2010) release 4.8.2. Analysis performed using Fiji (Schindelin et al., 2012) and associated plugins: MTrackJ (Meijering et al., 2012) or Circadian Gene Expression (CGE) (Dibner et al., 2009;Sage et al., 2010). When MTrackJ was used, the bright-field channel from the live cell imaging was used to manually identify cell nuclei, the centre of which was used as a seed point for the position of the cells.

The Onset of Brachyury Expression in culture
To probe the connection between the differentiation of mouse embryonic stem cells (mESCs) and the onset of Bra expression in the embryo, we used a mESC line bearing an insertion of GFP into the Bra locus (Bra::GFP) that has been shown to display, upon differentiation, characteristics associated with the Primitive Streak during development (Fehling et al., 2003;Gadue et al., 2006). We confirmed that this line faithfully reports the onset of Bra expression in culture by following, through FACS analysis, Bra::GFP expression in a variety of differentiation conditions (Fig. 1A) and comparing its expression profile to that of endogenous Bra protein and mRNA (Fig. 1B,C). When Bra::GFP-expressing cells are grown in neural differentiation conditions, there is no GFP expression ( Fig. S1) but growth in the presence of ActivinA (Act) and an inhibitor of GSK3, CHIR99021 (Chi), that mimics Wnt/ß-Catenin signalling) leads to a wave of Bra expression ( Fig. 1Ai) that parallels Bra mRNA and protein ( Fig. 1B, C). As this cell-line is heterozygous for Bra, it is likely to exhibit an haploinsuficient phenotype associated with the Bra locus (Dobrovolskaia-Zavadskaia, 1927; Rashbass et al., 1991;Wilson et al., 1993), but this should not interfere with the initiation of expression, which is the object of our study.
Individual treatment of the Bra::GFP mESCs with either Act (100 ng/ml) or Chi (3 μ M) resulted in a transient rise in the proportion of cells expressing GFP (Fig. 1Ai). In the case of Act alone we observe a peak of ~10% at 72 hours ( Fig. 1Ai), whereas treatment with Chi anticipates this peak with 25% of the total population expressing Bra::GFP at 48 hours ( Fig   1Ai). The combination of Act and Chi (Act/Chi) results in an increase in the proportion of cells expressing Bra::GFP (~33% GFP positive at 48-72h) which is sustained relative to Chi alone (Fig. 1Ai). These observations, extend previous ones (Gadue et al., 2006;Kubo et al., 2004) and suggest that both Act and Wnt/β-Catenin signalling can influence the progression of pluripotent cells towards a state characterized by the expression of Bra.
To understand the individual contributions of Act and Wnt/β-Catenin signalling to the expression of Bra::GFP, specific inhibitors targeting each pathway were added to the cell culture medium (Fig. 1Aii-v). Inhibition of β -Catenin signalling during Act treatment ( Fig.   1Aii,iii) resulted in complete ablation of Bra::GFP induction compared with the DMSO control (p-value < 0.05). A similar effect was observed upon inhibition of the Act signalling by SB43 (an inhibitor of Act/Nodal signalling) in the presence of Act or Chi (Fig. 1Aiv).
Taken together, these results are in agreement with previous observations (Gadue et al., 2006) that the onset of Bra::GFP by Act ligands not only requires active β -Catenin signalling, but that an active Act pathway is absolutely required for β -Catenin-mediated Bra induction.

An EMT associated with mesendodermal differentiation in ES cells
In the embryo, the onset of Bra expression is associated with spatiotemporally organized cellular movements that configure the process of gastrulation (Lim and Thiery, 2012; In order to assess whether this behaviour was also observed in ESCs in these conditions, we analysed the cellular activities associated with Bra expression in the adherent cultures by filming the behaviour of the cells in the presence of Act/Chi ( Fig. 2A), a condition that promotes the maximal Bra expression response (Fig. 1Ai). We observe that after 24 hours, the colonies characteristic of the pluripotent state loosen up and the differentiating cells form an epithelial monolayer from which they adopt a mesenchymal-like morphology and become motile (

Brachyury expression correlates with Nanog expression during ES cell differentiation
A hallmark of ES cell differentiation is the down-regulation in the expression of elements of the pluripotency network. To understand the relationship between this process and the onset of mesendodermal differentiation at the level of single cells, we used Quantitative Image Analysis (QIA) to monitor the expression of Bra in wild-type E14-Tg2A mESCs relative to that of Nanog, Oct4 and Sox2 following Act, Chi or Act/Chi treatment ( Fig. 3 & Fig. S3).
These results, together with those of the cell behaviours associated with the differentiation process, suggest that exposure of mESCs to Act/Chi triggers a developmental process that is very similar to that of the cells that undergo gastrulation in terms of gene expression (Gadue et al., 2006) as well as cell behaviour. Population-wise, cells in Act/Chi achieved higher instant velocities much earlier and sustained them for a larger period of time than in either Act or Chi alone (Fig 4B',D). Binning the velocities based on the time at which the cell analysis began (0-20h, 20-40h, 40-60h) revealed that cells did not have high velocities from the very beginning but these built up as time progressed (Fig 4B',E).

The onset of Brachyury expression is tightly linked to an EMT and cell motility in
Although the ability of Act or Chi to alter the instant velocities of individual cells was clear in our analysis, we resorted to statistical methods of random motion particle tracking to provide an objective measurement of the differences in cell motility between the different stimulation conditions ( Fig. 4D-G). In particular, we computed the mean-squared displacement (MSD) curves for each cell (Fig. 4F), which, despite being less intuitive than cell velocities, provide a robust analytical framework for rapidly moving cells. When considering all cells tracked over the entire course of the experiment, the evolution of the MSD in Act/Chi is much higher than in the case of individual Act or Chi stimulation (Fig 4E) i.e. cells treated with Act/Chi travel, on average, much larger distances in the same amount of time than cells in either Act or Chi.
In addition, in the three cases (Act, Chi or Act/Chi), the initial MSD increases approximately linearly with time, a sign of diffusive movement, and after the first three hours these plateau ( Fig. 4F). This levelling out of the MSD is due to a combination of both cell confinement as well as to the fact that we have only tracked cells that remain within the field of view (and thus we underestimate the MSD over long periods of time). Therefore, for each condition, we estimated an effective diffusivity (Supplemental Fig. S4). The evolution of the coefficient of diffusion over time indicates that cells in Act/Chi acquire a much larger degree of motility than cells in Act or Chi and that they do so both much earlier and for a longer period of time ( Fig. 4G) .
In addition, we studied the relationship between Bra expression and the dynamic behaviour of individual cells treated with Act/Chi. We found a correlation between Bra expression and cell movement and also that those cells with higher velocities had higher levels of the Bra::GFP We further analysed the trajectories of the cells to understand whether there was a particular bias to cell motion in terms of directionality and persistence (a continued direction of movement on a cell by cell basis; Fig. 4H & S4D). In all conditions tested (including both an N2B27 and Serum LIF control), the distribution of turning angles was far from isotropic ( Fig.   S4D, all conditions had a p-value < 0.00001 for the Kolmogorov-Smirnov test against the uniform distribution), a clear indication that regardless of the medium condition, when cells move, they do so with persistence and that the main difference between different conditions is the velocity and the ground covered by individual cells (Fig. S4D).
The importance of Wnt/β-Catenin signalling in the onset of Bra expression (Arnold et al., 2000;Yamaguchi et al., 1999)

Nodal/Activin and Wnt/β-Catenin signalling provide a link between the EMT and Bra expression
Our observations reveal a close relationship between the onset of Bra expression, the associated EMT and the activation of Wnt signalling. To establish a functional relationship between these three events, we first used CyclosporineA (CsA) to inhibit the EMT during the differentiation process and assessed the expression of Bra::GFP by FACS (Fig. 5A). CsA has been shown to inhibit Calcineurin thereby preventing both the phosphorylation and therefore the activation of nuclear factor of activated T-Cells (NFAT) (Clipstone and Crabtree, 1992;Li et al., 2011) and the transition from an epithelial towards a mesenchymal state (Mancini and Toker, 2009). In our experiments, CsA delayed the onset of Bra::GFP expression induced by Act alone and reduced the number of GFP-positive cells to a maximum of <10% at the end of the treatment (Fig. 5Aa). In the case of Chi, CsA resulted in an immediate reduction of Bra::GFP expression throughout the whole period of observation to levels similar to those observed with Act and CsA (Fig. 5Ab). Although simultaneous Act/Chi and CsA treatment showed an initial decrease in the proportion of GFP-positive cells after 24 hours, CsA treatment appeared only to delay the onset of Bra::GFP induction and shift the GFP-positive distribution by 48 hours (Fig. 5Ac).
In as confirmed with immunofluorescence), they remained within tight colonies and did not become motile (Fig. 5B,C). This is supported by the quantitative analysis of the cell , were unable to express Bra in the presence of Act and Chi (Fig. 6A,B). Time-lapse imaging revealed striking differences between the two β -Catenin mutant cell lines and the wild-type (Fig. 6C). Whereas by 24 hours E14-Tg2A mESCs began to show signs of the initiation of the EMT (dispersing cells with a mesenchymal appearance), cells mutant for β -Catenin remained tightly associated (Fig.   6C). As time progressed, the β -Catenin -/cells showed a much greater reduction in viability compared to the E14-Tg2A control and became motile (albeit much slower) however they remained within close proximity to the colony from which they were dispersing (Fig. 6B).
Manual tracking of these cells revealed that the speed at which cells move, and thereby indirectly the distance covered, over the time course was significantly shorter than the E14-Tg2A control, with a velocity much less than 36 μ m/hour for most of the time course (Fig.   6D,E, Fig. S6A). Unlike the E14-Tg2A control, the β -Catenin These results, in combination with the chemical genetics approach above, revealed that partial or complete loss of β -Catenin and disruption of its transcriptional activity impairs the ability of ESCs to both upregulate and undergo specification towards a Primitive Streak-like fate.

Nanog is required for the effect of Bra on the EMT
Our previous analysis on the correlations between Nanog and Bra expression (Fig. 3D,G) and a previous investigation in human ESCs detailing Nanog regulation of Bra (P. Yu et al., 2011) led us to follow the ability of Nanog -/-mESCs to express Bra and undergo EMT by both livecell imaging and QIA (Fig. S6). QIA analysis shows that following differentiation in Act/Chi, Nanog -/-mESCs express much lower levels of Bra and Sox2 (Fig. S6B). Live imaging of these cells by wide-field microscopy revealed that they undergo an EMT (Fig. S6C), although subtle differences can be observed in the dynamics of cell movements and in the morphology associated with their differentiation (Fig. S6C-E): analysis of the instant velocities revealed that Nanog -/-mESCs were less likely to engage in rapid velocities compared with the wildtype cells although not to the same extent as the ß-Catenin -/line (Fig. SD,E). These observations suggest that Nanog facilitates the up-regulation of Bra, though its presence is not a requirement for the initiation of the EMT program.

Brachyury expression is required for rapid cell movement following an EMT
In the embryo, the absence of Bra leads to truncations of the body axis and defects in axial extension, but does not have a major phenotype during gastrulation until the formation of the node (E7.5) (Beddington et al., 1992;Gluecksohn-Schoenheimer, 1938). This led us to test the behaviour of Bra mutant cells in the context of the ES cell differentiation; in particular, we asked whether the EMT program is activated as a consequence or is independent of the initiation of Bra transcription. To test this we analysed the properties of Bra -/-mESCs in culture (Fig. 7).
In all conditions, Bra -/cells were able to display the characteristics of an EMT, although the proportion of cells undergoing these changes appeared lower than the WT cells and the process was defective. In the presence of Act and Chi, although mutant lines were able to display movements associated with an EMT, immunofluorescent staining revealed that in most cells, the degradation of E-Cadherin and the subsequent nuclear localisation of β -Catenin were impaired (Fig. 7B). Nonetheless, by 96 hours there was a clear decrease of E-Cadherin in the membrane and some β -Catenin above background in the nucleus (Fig. 7B).
Manual tracking of the mutant cells over the time-course revealed their average instant velocity in Act and Act/Chi to be slower than the control Bra::GFP cells (Fig. 7C-D & Fig.   S7; Bra::GFP cells from Fig. 4) and therefore the distance travelled per time-step was less than the control cells (Fig. 7D). Interestingly, the effect of Chi on the Bra mutant cells was not as pronounced as that seen in Act or Act/Chi conditions: the probability of generating high velocities with Bra mutants was similar to the control cells with Chi treatment (Fig. 7D).
These observations indicate that Bra is not necessary for the EMT, but functions to promote high-velocity motion within Bra expressing cells, possibly facilitating their exit from the Primitive Streak. Together with the inhibitor studies and live-cell imaging of reporter cell lines, these data suggest that Bra, under the control of β -Catenin modulates the instant velocities of the cells as they enter the Primitive Streak fate and may control the expression of a factor, or factors that allow progression of the EMT and therefore specification of the Primitive Streak. It also shows that, for the most part, the effect of Chi on cell movement is not significant and is independent of Bra expression.

Discussion
It is thought that ESCs provide a unique model system for interrogating developmental processes in culture (Murry and Keller, 2008;Zhu and Huangfu, 2013). However, this possibility rests on the assumption that processes in tissue culture mimic events in the embryo and this has not been yet adequately tested. In the mouse embryo, the expression of Bra is tightly linked to the process of gastrulation and body extension, in particular, to a population of cells in the epiblast that experience a wave of EMTs associated with directional movement (Lim and Thiery, 2012;Thiery and Sleeman, 2006;Williams et al., 2011). This population sows the precursors for the definitive endoderm and the mesoderm and plays a central role in laying down the axial structure of mammalian embryos (Arnold and Robertson, 2009;J. C. Smith, 2004;Tam and Gad, 2004;Wilson and Beddington, 1997). Throughout gastrulation, Bra is expressed transiently within a moving population of cells, and a similar pulse of expression has been inferred from FACS analysis of cultured ES cells under the influence of Act and β -Catenin signalling (Fehling et al., 2003;Gadue et al., 2006;Hansson et al., 2009). We have confirmed this observation and extended it by showing that the onset of Bra expression is cell autonomous and that, like in the embryo, it is associated with an EMT (Ramkumar and Anderson, 2011;Tam and Gad, 2004;Williams et al., 2011)). Furthermore, we observe in culture that after the EMT, as cells become migratory, they lay down an ECM over which they move. This situation mimics the embryo where cells move over a bed of Fibronectin which has been shown to be important for fate specification (Cheng et al., 2013;Villegas et al., 2013). In the embryo, it is difficult to resolve the source of the Fibronectin, as it could be the epiblast or the ingressing cells. Our results would suggest that it is the ingressing cells that have expressed Bra that make an important contribution laying down the ECM.
In ESCs the expression of Bra is confined to a narrow temporal window, between days 3 and 4. If one assumes that ESCs are in a state similar to that of the preimplantation blastocyst (stage E4.0), the timing of Bra expression in differentiating ES cells, two days later, is similar to that of embryos, between E6.5-7.0, the time at which gastrulation commences (Snow, 1977;Tam and Gad, 2004). There are other features of the process in culture that parallel events in the embryo. In particular the association of Bra expression with β -Catenin signalling (Arnold et al., 2000;Ferrer-Vaquer et al., 2010;Tada and J. C. Smith, 2000), an EMT and the coexpression of both Nanog, and Oct4 with Bra during these processes (Hoffman et al., 2013;Thomson et al., 2011). There are reports that in human ESCs, Nanog is required for Bra expression (P. Yu et al., 2011) and here we have shown that the same is true in mouse ESC where the absence of Nanog dramatically reduces the expression of Bra and affects the behaviour of the cells.
Altogether, our observations support and extend the notion that in vitro differentiation of ESCs into a Bra expressing population, exhibits several parallels with the definition and behaviour of the Primitive Streak during mammalian gastrulation beyond gene expression profiles (Gadue et al., 2006;Izumi et al., 2007). This opens up the possibility of using ESCs to probe the molecular mechanisms linking cell fate and cell behaviour and, by comparing the evolution of the processes in cells and embryos, gain some insights into the emergence of collective behaviour from the activities of single cells.
Our results suggest an interplay between Act and Wnt/ß-Catenin signalling, the EMT and the activity of Bra in the specification and behaviour of cells in the Primitive Streak. Act initiates the EMT and the expression of Bra. The EMT triggers Wnt/ß-Catenin signalling that enhances the effect of Act on Bra which, in turn, promotes cell movement and cell fate (A. L. Evans et al., 2012;Gentsch et al., 2013). This module has the structure of a feed-forward loop. In agreement with these notions, Bra has been shown to control the expression of several components of the cytoskeleton and of canonical/non-canonical Wnt signalling (Hardy et al., 2008;Heisenberg et al., 2000;Shimoda et al., 2012;Tada and J. C. Smith, 2000) which are likely to promote movement and enhance the EMT. Downstream targets of Bra comprise members of the Wnt family which are likely to fuel movement. It is possible that the sluggish movement that we observe in the absence of Bra, is due to the activation of β -Catenin by Chi that might set in motion some of these mechanisms in a Bra-independent manner. In the absence of other elements, also controlled by Bra, the movement is greatly hampered.

A tissue culture model for Primitive Streak formation?
Differences between the events in the embryo and those in differentiating ESCs can be informative. An example is provided by the long range movement that we observe in differentiating ESCs which is not obvious in the embryo. During gastrulation, after their EMT, cells expressing Bra do not display long range movement as individuals but rather jostle as a group towards the proximal posterior pole and then ingress through the Primitive Streak (Williams et al., 2011). However, when they are explanted and placed onto ECM covered culture dishes the same cells can be observed to move individually, without a preferred direction but with some persistence/diffusivity (Hashimoto et al., 1987) in a manner that is very reminiscent to what we have described here for differentiating ESCs. These observations suggest that a main difference between Bra expressing ESCs and those in the embryo, is the confinement of the latter that restricts their movement and forces them to behave as a coherent collective, rather than becoming dispersed individual cells, as they do in the culture. It is interesting that the average velocity of the differentiating ESCs cells in Act/Chi (maximum average instant velocity of ~60µm/h; Fig. 4B') is within the same order or magnitude as that of the cells from Primitive Streak explants (average of 50µm/h on extracellular matrix-coated surfaces) (Hashimoto et al., 1987) and of migrating mesodermal cells within the embryo (46µm/h) (Nakatsuji et al., 1986). Although it is important to note that in our experiments, we were able to see a small proportion of cells that were able to travel at ~400 µm/s, albeit for short durations of time (Fig. 4B').
We observe a correlation between the levels of Bra and the velocity of the cell. Bra mutant cells are very delayed in migrating, only a few do migrate and when they do, they exhibit lower velocities relative to wild type. Similar observations have been made for cells from Bra mutant primitive streaks (Hashimoto et al., 1987) and indicate that an important function of Bra is to control the movement of the cells. On the other hand the combination of Act and Chiron promotes very high velocities which, in the confinement of the embryo, can result in strong directional forces.
These observations emphasize the importance of confinement in the behaviour of the cells in the Primitive Streak. At the onset of gastrulation the epiblast is a highly packed and dividing cell population. At this stage, movement towards and through the streak is likely to be due to large-scale, spatially constrained tectonic movements of the epiblast as a whole, with mechanical differences between the prospective anterior and posterior parts being responsible for the directional movement of the bulk population that has undergone EMT. Indeed imaging of the process of gastrulation in mouse embryos suggests that once cells have undergone EMT, Bra expressing cells are passively pushed towards the streak in a process that appears to be passive and not require convergence and extension, as it appears to be the case in frogs and fish (Williams et al., 2011). This notion of morphogenetic events underpinned by long range mechanical coordination of cells within tissues has been demonstrated during the convergence and extension movements of the neuroectoderm in the development of the nervous system in the zebra fish (Blanchard et al., 2009). Such long range effects might provide an explanation for the lack of a clear phenotype of Bra mutant cells during gastrulation . While this is likely to be the result of partial redundancy with Eomesodermin (Arnold et al., 2008;Slagle et al., 2011;Teo et al., 2011), it might be also be a reflection that at these early stages, the defects of Bra mutant cells are compensated by, mechanical large tissue coordination due to confinement. Bra mutant cells still undergo an EMT and therefore could be subject to these movements. As we have shown here, Bra mutant cells can initiate an EMT and therefore could be the subject of strong large scale forces that are likely to exist throughout the epiblast. After node formation, however, the strength of these forces might subside and regressing cells, particularly during axis extension, may come to rely more on the propulsion and navigational abilities driven by Bra. This is supported by the behaviour of mosaics of cells with different levels of Bra in WT embryos (Beddington et al., 1992;Wilson et al., 1995;Wilson and Beddington, 1997) and by our observation of a correlation between levels of Bra expression and cell velocity. Also, in agreement with this, wild type cells are outcompeted by cells expressing higher levels of Bra in the early gastrula (Wilson and Beddington, 1997). Therefore, cells with higher motility might be more prone to escape the tectonic movements of the tissue can 'overtake' WT cells.
Our results highlight the experimental possibilities provided by differentiating ESC as a first approximation to understand the mechanisms underlying events that, like gastrulation, are difficult to access in the embryo. Having established the similarities between the two systems, it will be important to exploit them to see how one could reproduce the Primitive Streak in culture through, for example, confining the movement of differentiating ES cells and attempting to create directionality by imposing spatially constrained forces.       immunostained for E-Cadherin, ß-Catenin, and Bra and imaged by confocal microscopy (Scale bar indicates 50μm, Hoechst marks the nucleus). In the absence of Bra, E-Cadherin and ß-Catenin are not effectively cleared from the membrane. (C, C') Instant velocities of Bra::GFP cells and Bra null cells in Act, Chi or Act/Chi. The thick black line in the individual graphs in indicates the average velocity for each condition, and is displayed in greater detail in (C'). (D) Histogram of instant cell velocities as measured from frame-to-frame displacements for both the Bra::GFP and Bra -/-mESCs. All the Bra::GFP live imaging data was displayed in Fig. 4.

Supplemental Movie Legends
Movie M1: Live imaging of Bra::GFP cells cultured in Act/Chi. Bra::GFP mESCs were plated in N2B27 supplemented with Act/Chi. As time progresses, cells begin to display EMTlike movements, become openly motile and begin to express Bra::GFP. Time in hours.
Movie M2: Live imaging of the ß-Catenin transcriptional reporter line (TLC2) following treatment with Act/Chi. The TCF/LEF::mCherry (TLC2) mESC reporter line was plated in N2B27 supplemented with Act/Chi. Cells initially expressed low and heterogeneous levels of the reporter which increased over time in the cells that would eventually undergo an EMT event and disperse from the colony. Following colony exit, cells began to down-regulate the reporter. Time in hours.

Supplementary Table and Legend
Table S1: Summary of the tracking data collected. For each cell line used in the live-cell imaging experiments within this investigation (left most column; 'cell type'), the information relating to the number of movies analysed, the temporal resolution (frames/h), spatial resolution (pixels/µm), the number of cells tracked, positions tracked and the average number of positions per cell for each medium condition (second column from the left; Medium conditions) to which the cells were exposed was tabulated. The total number of movies analysed and the total number of cells tracked throughout the investigation is recorded at the bottom of the