Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin
The events that drive the transition from pluripotency to lineage commitment remain poorly understood . It has been known for many years that BMP signalling protects pluripotency and suppresses neural specification ( Ying et al . , 2003a; Stern , 2006; Di-Gregorio et al . , 2007 ) , but the underlying mechanisms remain unclear . BMP signalling is also required for the formation of mesoderm in vivo ( Mishina et al . , 1995; Winnier et al . , 1995; Lawson et al . , 1999; Beppu et al . , 2000; Davis et al . , 2004 ) , and BMP is commonly used to induce mesoderm from embryonic stem ( ES ) cells ( Murry and Keller , 2008 ) . However , it is not clear how the effects of BMP on mesoderm differentiation relate to its pro-pluripotency and anti-neural effects: are these separable independent events or do they represent the outcomes of one common mechanism ? This question underlines our poor understanding of the mechanisms by which BMP influences neural and mesodermal specification . BMP acts through transcriptional upregulation of Inhibitor of Differentiation ( Id ) factors ( Ying et al . , 2003a; Zhang et al . , 2010 ) in order to prevent neural differentiation . Id factors generally act as dominant negative inhibitors of the bHLH family of transcription factors ( Norton , 2000 ) , but the mechanism by which Id proteins block neural induction is not known . Furthermore , it is not clear to what extent the pro-mesoderm effect of BMP within the epiblast is mediated by Id or by other BMP target genes: redundancy between the four Id family members may mask gastrulation phenotypes in Id mutants . We set out to examine more closely the effects of BMP and Id1 on neural and mesoderm differentiation by taking advantage of an ES cell differentiation system , which allows differentiation to be carefully monitored in a well-defined environment ( Ying and Smith , 2003 ) , and by using a reporter strategy to ask which cells usually express Id1 during early development . We find an unanticipated ability of BMP/Id to block differentiation by maintaining the expression of the cell adhesion molecule E-Cadherin ( Cdh1 ) . We find that loss of Cdh1 is tightly associated with neural as well as mesodermal differentiation and that this change in Cdh1 is a limiting requirement for neural differentiation . A number of recent reports ( Chou et al . , 2008; Soncin et al . , 2009; Li et al . , 2010; Redmer et al . , 2011; del Valle et al . , 2013; Faunes et al . , 2013 ) suggest that Cdh1 helps protect pluripotency . Despite this emerging appreciation of Cdh1 as a regulator of the pluripotent state , the upstream regulators of Cdh1 in pluripotent cells have not been reported . BMP favours mesenchymal to epithelial transitions in other contexts ( Kondo et al . , 2004; Samavarchi-Tehrani et al . , 2010 ) , but its ability to control Cdh1 activity during early fate specification has not previously been appreciated . We also find that BMP acts through Id1 to impose a proximal posterior identity on epiblast cells , priming them for mesodermal fates whilst transiently restraining them from overt mesoderm differentiation . Id1 may therefore play an early role in anterior-posterior ( AP ) patterning and mesoderm priming , independent from any effect on overt mesoderm differentiation . This helps to reconcile why BMP is required both for mesoderm differentiation and for the maintenance of pluripotency . Taken together , our data help to unify the distinct effects of BMP signalling during differentiation of pluripotent cells . BMP maintains high levels of Cdh1 , which help to protect the pluripotent state , whilst imposing a posterior identity that ultimately favours mesodermal over neural differentiation .
The BMP target gene Id1 has been reported to inhibit neural induction and instead favour either pluripotency or mesoderm differentiation from pluripotent cells ( Ying et al . , 2003a; Zhang et al . , 2010 ) , but the precise events controlled by Id1 , and the mechanism by which it acts , are not known . In order to address these questions , we first asked where Id1 is expressed in the early post-implantation embryo . It has been reported ( Jen et al . , 1997 ) that Id1 is expressed around the embryonic-anembryonic boundary and around the primitive streak of gastrulating mouse embryos , but it is not clear whether Id1 is expressed within pluripotent epiblast cells prior to their commitment to neural or mesodermal fate , or whether it is restricted to migrating mesodermal cells and to extraembryonic tissues during gastrulation . We decided to use a reporter strategy to examine the expression pattern of Id1 and its relationship to markers of pluripotency and differentiation during early development . We generated Id1-Venus ( Id1V ) reporter cells using a targeting construct designed to express a fusion between Id1 and Venus from the endogenous Id1 locus ( Nam and Benezra , 2009 ) ( Figure 1A , Figure 1—figure supplement 1A–E ) . This strategy has previously been demonstrated to faithfully report Id1 expression ( Nam and Benezra , 2009 ) . We used Id1V reporter cells to generate high-contribution chimeras in order to analyse and quantify Id1 expression specifically within the epiblast and its derivatives ( ES cells do not contribute to extraembryonic ectoderm in chimeras ) . 10 . 7554/eLife . 01197 . 003Figure 1 . The BMP target gene Id1 is expressed in the pluripotent epiblast . ( A ) Schematic illustrating the genotype of ES cells used for morula aggregation experiments . The coding sequence for an Id1-Venus fusion protein was targeted into one allele of the Id1 locus . ( B ) Proximal expression of Id1-Venus in E6 . 5 embryos generated using Id1-Venus ES cells marked with a constitutively expressed red mKate2-NLS lineage label . Scale bar = 30 μm . ( C ) Transverse section of mid-streak stage embryo ( E6 . 5 ) stained for DAPI , Id1-Venus , Nanog , T . Anterior ( ‘A’ ) and posterior ( ‘P’ ) regions of interest are defined based on expression of Nanog . Scale bar = 30 μm . ( D–I ) Quantification of antibody staining on sections of embryo shown in C . Staining of extraembryonic tissue was used as a negative control to define the gates in the dot plots . ( D ) Dot plots representing expression of Id1-Venus and Nanog in single cells in E6 . 5 epiblast and in the anterior and posterior regions of interest . ( E ) % Nanog-positive cells within the Id1-Venus-positive compartment in E6 . 5 epiblast . ( F ) Dot plots representing expression of Id1-Venus and T in single cells in E6 . 5 epiblast . ( G ) % T-positive cells within the Id1-Venus-positive compartment in E6 . 5 epiblast . ( H ) Dot plots representing expression of T and Nanog in single cells in E6 . 5 epiblast . ( I ) % Nanog-positive cells within the T-positive compartment in E6 . 5 epiblast . See Figure 1—figure supplement 1 for design and validation of Id1-Venus cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 00310 . 7554/eLife . 01197 . 004Figure 1—figure supplement 1 . Design and validation of Id1V reporter ES cells . ( A–D ) Southern blotting strategy and confirmation of successful targeting in clone 47 , marked with asterisk ( see ‘Materials and methods’ for details ) . ( E ) Validation by qRT-PCR that Id1 mRNA is enriched in Venus+ subpopulations of ES cells after separation by FACS . ( F ) E7 . 5 chimera containing Id1V ES cells labelled with red mKate2-NLS lineage label . Scale bar = 100 μm . ( G ) In situ hybridisation for Id1 in a wild type E7 . 5 embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 004 We first examined chimeras made with Id1V ES cells that were constitutively labelled with a fluorescent red mKate2-NLS protein , in order to examine the distribution of Id1V in the early post-implantation embryo . The expression pattern of Venus faithfully recapitulates the expression of Id1 mRNA in these chimeric embryos ( Figure 1—figure supplement 1F , G ) . Id1V is readily detectable at E6 . 5 throughout the proximal epiblast with expression declining more distally ( Figure 1B ) . The highest levels of Id1V therefore mark regions of the epiblast fated for mesoderm and surface ectoderm but not neural fates ( Cajal et al . , 2012; Li et al . , 2013 ) . We next generated chimeras using a line of Id1V ES cells that lack the red fluorescent lineage label in order to allow us to use antibody staining to quantify expression of both Nanog and T within individual Id1V+ cells at E6 . 5 ( Figure 1C–G ) . T was used to identify the posterior-most region of the epiblast and the primitive streak ( Rivera-Pérez and Magnuson , 2005 ) and Nanog was used to identify pluripotent cells within the posterior half of the epiblast ( Osorno et al . , 2012 ) . The majority ( >65% ) of Id1V+ cells in the proximal epiblast co-express Nanog , with this proportion increasing to >80% within the posterior regions of the proximal epiblast ( Figure 1D , E ) . In contrast , the majority ( >80% ) of Id1V+ cells do not express T , indicating that they are neither specified nor committed to a mesodermal fate at this stage of development ( Figure 1F , G ) . These data indicate that Id1V is expressed within pluripotent epiblast and is not restricted to the primitive streak or to differentiating mesoderm . We also confirmed that most ( >80% ) of the T+ cells at this stage co-express Nanog ( Figure 1H , I ) , confirming that T marks not only differentiating mesoderm but also pluripotent epiblast cells with a proximal posterior positional identity ( Osorno et al . , 2012 ) . We conclude that the direct BMP target gene Id1 is expressed in a subpopulation of Nanog+ epiblast cells prior to overt mesoderm differentiation , in addition to its previously described expression in migrating mesoderm ( Jen et al . , 1997 ) . We next asked what events are regulated by BMP/Id1 during differentiation of pluripotent cells ( Figure 2A ) . We examined the effect of BMP4 on cells cultured in serum-free conditions in the absence of any other exogenous growth factors . This system allows us to monitor the kinetics as well as the direction of differentiation . 10 . 7554/eLife . 01197 . 005Figure 2 . BMP signalling maintains Cdh1 expression during differentiation of pluripotent cells . ( A ) BMP blocks neural differentiation , maintains pluripotency and has both positive and negative effects on mesoderm differentiation , but the mechanisms by which BMP acts in each case are not well understood . ( B ) Sox1-GFP ES cells were placed under neural differentiation conditions for 4 days in the presence or absence of 5 ng/ml BMP and stained for markers as shown . Scale bar = 30 μm . ( C ) E14tg2α ES cells were placed under neural differentiation conditions for 4 days in the presence of 5 ng/ml BMP and stained for markers as shown . Scale bar = 30 μm . ( D ) qRT-PCR analysis of ES cells placed under neural differentiation conditions in the presence or absence of 5 ng/ml BMP , or under directed mesoderm differentiation conditions . ( E ) We propose that BMP imposes a posterior positional identity whilst delaying both EMT and overt mesoderm differentiation but permitting surface ectoderm differentiation . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to no-BMP4 control . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 005 We used Sox1-GFP reporter ES cells ( Ying et al . , 2003b ) to confirm that BMP4 blocks the upregulation of the early neural marker Sox1 ( Figure 2B ) and permits expression of the primitive streak/early mesoderm marker T ( Figure 2C , D ) , as expected ( Ying et al . , 2003a ) . However , despite expressing T , BMP-treated cells do not progress to overt mesoderm differentiation . Differentiation of epiblast to mesoderm is characterised by the loss of Oct4 and Cdh1 , whereas BMP4-treated cells remain uniformly Cdh1+ at least for the first 5 days of BMP treatment ( Figure 2B–D ) . Furthermore , in the presence of BMP4 , downregulation of Oct4 is delayed , and markers that characterise EMT and overt mesoderm differentiation are not upregulated within the 5-day time frame of this experiment ( Figure 2D ) . As a positive control , we checked that these markers were upregulated within 4 days in control cells that were put through a directed mesodermal differentiation protocol based on monolayer culture on collagen IV in the presence of serum ( Nishikawa et al . , 1998 ) ( Figure 2D ) . T is first expressed in the posterior proximal epiblast prior to overt mesoderm differentiation ( Rivera-Pérez and Magnuson , 2005 ) ( see also Figure 1H , I ) and marks a mesoderm-primed pluripotent state ( Kubo et al . , 2004; Bernemann et al . , 2011 ) . We therefore considered whether the ability of BMP4 to upregulate T may represent an effect on positional identity within the epiblast rather than a direct effect to drive mesoderm commitment . In support of this idea , we found that in the presence of BMP4 , T is coexpressed with Cdh1 ( Figure 2C ) . Not all cells persist in a T+ Cdh1+ state: a subpopulation of cells ( typically between 10 and 25% ) are able to differentiate into a Cdh1+ Tcfap2a ( AP2α ) + epithelial cell type similar to early surface ectoderm ( Mitchell et al . , 1991 ) ( Figure 2C ) . This is consistent with the recent finding that exogenous BMP4 promotes surface ectoderm differentiation when added to explants from the E7 . 0 mouse epiblast ( Li et al . , 2013 ) . We conclude that BMP4 , in the absence of other exogenous signals , imposes a T+ mesoderm-primed posterior positional identity but prevents cells from downregulating Cdh1 . BMP-treated cells are delayed in their differentiation and either persist in a Cdh1+ posterior-epiblast like state or ultimately differentiate into Cdh1+ surface-ectoderm-like cells ( Figure 2E ) . We next asked whether the BMP target gene Id1 mediates the effects of BMP to maintain Cdh1 expression during differentiation of pluripotent cells . We generated ES cells in which a Flag-tagged Id1 transgene is expressed in the presence of doxycyline ( dox ) . We first confirmed that dox-mediated induction of Id1 is able to block neural induction as expected ( Figure 3—figure supplement 1 ) . We next tested the effect of Id1 on EMT markers . Under serum-free monolayer conditions in the absence of exogenous growth factors , Id1 delays the loss of Cdh1 and suppresses the upregulation of EMT markers Cdh2 , Zeb1 and Zeb2 ( Thiery et al . , 2009 ) ( Figure 3A ) . 10 . 7554/eLife . 01197 . 006Figure 3 . Id1maintains Cdh1 expression and imposes a mesoderm-primed positional identity . ( A and B ) qRT-PCR analysis of dox-inducible Id1 ES cells placed under neural differentiation conditions ( N2B27 alone ) in the presence or absence of dox . ( C ) Dox-inducible Id1 ES cells placed under neural differentiation conditions ( N2B27 alone ) in the presence or absence of dox and stained for markers as shown . Scale bar = 50 μm . ( D ) Dox-inducible Id1 ES cells on day 4 under neural differentiation conditions ( N2B27 alone ) : T is coexpressed with Cdh1 and Oct4 . Scale bar = 50 μm . ( E ) Quantification of antibody staining of dox-inducible Id1 ES cells in the presence or absence of dox on day 0 and day 4 under neural differentiation conditions ( N2B27 only ) . A minimum of 750 nuclei were scored for d0 samples and a minimum of 7000 nuclei were scored for d4 samples . ( F ) We propose that the BMP target gene Id1 imposes a posterior positional identity whilst delaying both EMT and overt mesoderm differentiation . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to no-dox control . See Figure 3—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 00610 . 7554/eLife . 01197 . 007Figure 3—figure supplement 1 . Characterisation of Id1 overexpressing lines . ( A and B ) qRT-PCR analysis of dox-inducible Id1 ES cells placed under neural differentiation conditions ( N2B27 alone ) in the presence or absence of dox . ( C ) Dox-inducible Id1 ES cells placed under neural differentiation conditions for 5 days in the presence or absence of dox and stained for markers as shown . Scale bar = 100 μm . ( D ) Quantification of Flag staining of dox-inducible Id1 ES cells in the presence or absence of dox after 5 days of neural differentiation . A minimum of 2400 nuclei were scored for each condition . Parental E14tg2α ES cells were stained for Flag as a negative control . ( E and F ) qRT-PCR analysis of cell lines stably transfected with loxP flanked Id1 transgene ( Id1 ) or after Cre-mediated removal of the Id1 transgene ( Id1 reverted ) undergoing neural differentiation in N2B27 . ( G ) Antibody staining after 5 days of neural differentiation of cell lines stably transfected with loxP flanked Id1 transgene ( Id1 ) or after Cre-mediated removal of theId1 transgene ( Id1 reverted ) . Scale bar = 100 μm . ( H ) Schematic diagram of loxP flanked Id1 transgene ( 'Id1' ) . GFP becomes expressed after Cre-mediated removal of the Id1 transgene ( 'Id1 reverted' ) . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to no-dox or Id1-reverted controls . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 00710 . 7554/eLife . 01197 . 008Figure 3—figure supplement 2 . Id1 overexpressing cells differentiate preferentially into mesoderm rather than neuroectoderm or endoderm . qRT-PCR analysis of cell lines stably transfected with loxP flanked Id1 transgene ( Id1 ) or after Cre-mediated removal of the Id1 transgene ( Id1 reverted ) undergoing neural differentiation in N2B27 . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to Id1-reverted controls . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 008 We then examined the effect of Id1 on the rate and direction of differentiation . Id1 promotes the upregulation of T , a marker of posterior epiblast and primitive streak ( Blum et al . , 1992; Rivera-Pérez and Magnuson , 2005 ) , but delays the downregulation of the epiblast markers Oct4 , Fgf5 and Otx2 ( Figure 3B ) . These data are consistent with the idea that Id1 , like BMP , favours a posterior proximal epiblast identity whilst delaying overt differentiation . In keeping with this idea , Id1 favours the re-expression of Nanog , which marks posterior pluripotent epiblast , and Id1 also reduces expression of Sox2 , which marks anterior pluripotent epiblast ( Osorno et al . , 2012 ) ( Figure 3B ) . We confirmed that Nanog in this context is likely to be marking a posterior epiblast-like cell rather than an earlier naïve pluripotent state because other markers of naïve pluripotency , Klf4 , Rex1 and Esrrb , are rapidly lost in Id1 overexpressing cells , as they are in control cells ( Figure 3B , C ) . In further support of the idea that Id1 delays differentiation , we used quantitative immunofluorescence to demonstrate that in the presence of exogenous Id1 , T+ cells remain predominantly Cdh1+ and >90% of them coexpress Oct4 ( Figure 3D , E ) . These T+ cells are therefore unlikely to be differentiated mesoderm cells and are more likely to be equivalent to the T+ cells in the proximal epiblast of the early embryo that have not yet entered the primitive streak ( Rivera-Pérez and Magnuson , 2005 ) . Dox treatment does not entirely block differentiation ( Figure 3—figure supplement 1C ) . Neurons begin to emerge in the dox-treated cultures after 5 days . However , these neurons do not express the Flag-tagged Id1 transgenic protein . Indeed , around 50% of cells are negative for Flag on day 5 ( Figure 3—figure supplement 1D ) . Therefore , in order to address whether Id1 is sufficient to block differentiation , we turned to a constitutive expression system . We generated clonal lines of ES cells stably overexpressing Id1 under the control of the ubiquitously expressed ( Niwa et al . , 1991 ) CAGS promoter . In these cell lines , loxP sites flank the coding sequence for Id1 and upon Cre-mediated excision a downstream GFP cassette is expressed . We first confirmed that Id1 is able to block neural induction as expected , and that this phenotype can be reverted by Cre-mediated excision of the Id1 transgene ( Figure 3—figure supplement 1E–H ) . Unlike the dox-inducible system , this constitutive expression system is sufficient to completely block neurogenesis throughout the culture ( Figure 3—figure supplement 1G ) . Constitutive Id1 expression is sufficient to delay the downregulation of Oct4 ( Figure 3—figure supplement 2 ) and to block neurogenesis ( Figure 3—figure supplement 1G ) . However , it is not sufficient to completely block differentiation . Id1-overexpressing cells eventually differentiate predominantly into mesoderm rather than neurectoderm or endoderm , even under neural differentiation conditions ( Figure 3—figure supplement 2 ) . In this regard , exogenous Id1 does not recapitulate the effect of exogenous BMP4 described in Figure 2 . The ability of Id1-overexpressing cells to generate mesoderm at later time points under these conditions may reflect the progressive downregulation of the Id1 transgene ( Figure 3—figure supplement 1 ) , or it may reflect an Id1-independent effect of BMP4 on the later stages of differentiation . Taken together , these data suggest that the BMP target gene Id1 does not drive overt mesoderm differentiation but rather maintains Cdh1 and helps to maintain a primed pluripotent state similar to that of the post-implantation epiblast , whilst imposing a proximal posterior ( T+ Nanog+ ) epiblast identity that is primed for mesoderm differentiation . The data described above reveal an unexpected ability of BMP to maintain Cdh1 expression . We wondered how this may relate to the ability of BMP to block neural induction , given that Cdh1 is downregulated during the early stages of neural tube formation in vivo ( Hatta and Takeichi , 1986; Aaku-Saraste et al . , 1996; Karpowicz et al . , 2007 ) and during neural differentiation of ES cells ( Sterneckert et al . , 2010; Kamiya et al . , 2011 ) . We first asked whether the loss of Cdh1 occurs subsequently to neural differentiation or whether it is tightly associated with the differentiation process itself . Since the rate of neural differentiation varies considerably between individual cells , it was important to perform this analysis at the single cell level . We therefore performed single cell quantitative RT-PCR in order to monitor the transcriptional downregulation of Cdh1 , as cells proceed from pluripotency towards neural differentiation . We confirmed that Cdh1 is largely absent from cells that have upregulated early neural markers ( Figure 4A ) . Strikingly , we also observed a close correlation between the expression of Cdh1 and Oct4 at all stages of the differentiation process ( Figure 4B ) , consistent with the idea that Cdh1 is downregulated at precisely the time that cells transit from Oct4+ epiblast towards neurectodermal differentiation . We confirmed a similar correlation between loss of Oct4 and downregulation of Cdh1 in vivo: Cdh1 and Oct4 protein expression decline in synchrony within the developing neuroepithelium ( Figure 4C , D ) . 10 . 7554/eLife . 01197 . 009Figure 4 . Cdh1 is downregulated in synchrony with Oct4 during neural differentiation . ( A and B ) ES cells were placed under neural differentiation conditions . 40–80 single cells were analysed by qRT-PCR from the starting population ( Day 0 ) or at daily time points during differentiation . A shows expression levels of Cdh1 , Cdh2 , Oct4 and Sox1 in single cells analysed at day 3 of differentiation . The expression values for each factor are normalised to the maximum expression level found in this set of cells . The samples are sorted in ascending order of Cdh1 expression . B shows the Spearman’s rank correlation coefficients ( ρ ) between the named genes and Cdh1 at each time point . * indicates asymptotic p value <0 . 05 . ( C ) Oct4 and Cdh1 staining in 7 µm sections of embryos at E7 . 5 ( late streak stage ) or staged according to number of somite pairs ( s ) as indicated . Top row shows bright field images of embryos before sectioning with a white line indicating the location of the section shown in the bottom row . Scale bars = 100 μm . NT: Neural tube ( Yellow region in later stage embryos ) , SE: Surface ectoderm ( red region in later embryos ) . ( D ) Quantification of the average intensities of Oct4 and Cdh1 in different structures of the embryo at each stage . EPI: Epiblast ( yellow region and red region in LS embryo ) , Error bars represent the standard deviation of intensities in three different sections . Abbreviations are as described for C . ( E ) Sox1-GFP ES cells after 4 days in neural differentiation conditions stained for Cdh1 ( red ) and DAPI ( blue ) with Sox1-GFP in green . Scale bar = 30 μm . ( F ) E14tg2α ES cells after 4 days under neural differentiation conditions stained for Cdh1 ( red ) Keratin8 ( blue ) and Oct4 ( green ) . Scale bar = 30 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 009 We confirmed these results using Sox1-GFP neural reporter ES cells ( Ying et al . , 2003b ) to monitor early neural differentiation under uniform monolayer differentiation conditions . Under these conditions , around 20–40% of cells fail to become Sox1-GFP+ after 4 days; around half of these persist as undifferentiated ES cells whilst the other half differentiate into non-neural ( Sox1-negative ) cell types ( Ying and Smith , 2003; Lowell et al . , 2006 ) . The reasons for this heterogeneity in differentiation capacity are not fully understood . We observed that the subpopulation of cells that fail to respond appropriately to a neural environment by upregulating Sox1-GFP also fail to downregulate Cdh1 , either remaining as Oct4+ Cdh1+ undifferentiated cells or differentiating into Cdh1+ Krt8+ epithelial cells ( Figure 4E , F ) . We conclude that Cdh1 is downregulated in synchrony with Oct4 during neural differentiation , and that heterogeneity in neural differentiation capacity within populations of ES cells is associated with a failure to downregulate Cdh1 in a subpopulation of those cells . The data above confirm that the earliest stages of neural differentiation are accompanied by the downregulation of Cdh1 . We next asked whether this is simply a passive accompaniment to neural differentiation , or whether it is a limiting requirement , playing an active role in allowing cells to progress towards neural differentiation . Having confirmed that a subpopulation of cells fail to downregulate Cdh1 under neural differentiation conditions , and that these cells also fail to differentiate into neural cells ( Figure 4E , F ) , we next asked whether it is possible to rescue neural differentiation in this recalcitrant subpopulation by experimentally suppressing Cdh1 activity . We used function blocking antibodies ( Yoshida-Noro et al . , 1984 ) in order to abolish the adhesive abilities of Cdh1 . Blocking antibodies were added to Sox1-GFP reporter cells for the first 48 hr of neural differentiation and then removed , whereupon the cells rapidly regain normal cell-cell contacts . 1 day later , a minority of control cells are just starting to express Sox1-GFP , whereas the antibody-treated cells show strong Sox1-GFP expression throughout the culture . By the fourth day of differentiation , the antibody-treated cultures are almost uniformly Sox1-GFP+ ( Figure 5A , B ) . Flattened non-neural differentiated cells are readily apparent in control cultures , but these are absent from antibody-treated cultures ( Figure 5A ) . qRT-PCR analysis confirmed that early transient exposure to the blocking antibody accelerates the loss of the pluripotency marker Oct4 and the gain of the neural marker Sox1 ( Figure 5C ) . This indicates that transient loss of Cdh1 activity at the very start of the differentiation process is sufficient to drive ES cells efficiently towards neural differentiation . 10 . 7554/eLife . 01197 . 010Figure 5 . Loss of Cdh1 activity is a limiting requirement for neural differentiation . ( A–C ) FACS ( A and B ) and qRT-PCR ( C ) analysis of Sox1-GFP ES cells undergoing neural differentiation after exposure to Cdh1 blocking antibody ECCD1 for the first 48 hr of differentiation . Control indicates sister dishes not exposed to ECCD1 . ( D ) qRT-PCR analysis of Cdh1 null or parental D3 ES cells undergoing neural differentiation . ( E and F ) Dox-inducible TwistE cells during neural differentiation in the presence or absence of dox . Scale bar = 30 μm . ( F ) shows neurite frequency scored from the experiment illustrated in E . Neurites were scored as described in ‘Materials and methods’ . ( G ) qRT-PCR analysis of dox-inducible TwistE cells placed under neural differentiation conditions in the presence or absence of dox . ( H ) qRT-PCR analysis of dox-inducible TwistE cells on day 2 of neural differentiation in the absence of dox , the presence of dox throughout the experiment , or after removing dox 24 hr after initiating neural differentiation . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to no-antibody control , parental control , or no-dox controls . See also Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 01010 . 7554/eLife . 01197 . 011Figure 5—figure supplement 1 . Design and validation of TwE cell lines . ( A and B ) ES cells 5 days after episomal transduction ( CAGS promoter ) with empty vector , monomeric Twist1 , or Twist1-E47 tethered dimers . Cells are stained for DAPI and Vimentin ( A ) or analysed by qRT-PCR for EMT markers ( B ) . ( C ) Stable ES cell line TwistE ( 'TwE' ) expressing a dox-inducible Twist1-E47 tethered dimer or control parental cell line . Twist1 is upregulated approximately 25-fold when dox is added to ES cells for 48 hr under normal ES cell culture conditions . ( D ) The Flag-tagged Twist1-E47 fusion protein is induced within 12 hr of adding dox and is lost within 24 hr of removing dox from dox-inducible TwistE cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 011 As an alternative loss-of-function approach , we made use of Cdh1 null ES cells ( Larue et al . , 1996 ) . These cells do not form adherens junctions but do self-renew and retain multilineage differentiation potency . Cdh1 null ES cells differentiate into neural cells more rapidly and efficiently in comparison with parental controls ( Figure 5D ) . We sought to confirm these data using an alternative robust and regulatable system for suppressing Cdh1 activity . The transcription factor Twist1 is able to downregulate Cdh1 when bound to its heterodimerisation partner E47 ( Thiery et al . , 2009 ) . We engineered a construct designed to express Twist1 tethered by a flexible linker to E47: we refer to this heterodimeric fusion protein as TwistE . We confirmed that TwistE efficiently downregulates Cdh1 when expressed in ES cells by episomal transfection ( Figure 5—figure supplement 1A , B ) . We next generated ES cells lines that allowed dox-inducible expression of TwistE ( Figure 5—figure supplement 1C , D ) and made use of these dox inducible TwistE cell lines to suppress Cdh1 expression during neural differentiation . We reasoned that if downregulation of Cdh1 is a limiting requirement for neural induction , activation of TwistE should be able to enhance the efficiency of this process . This is indeed the case: activation of TwistE has a striking ability to accelerate neural differentiation . Cells that are treated with dox generate extensive networks of neurons after only 3 days of exposure to neural conditions , whilst untreated control sister dishes do not start to generate significant numbers of neurons until day 5 ( Figure 5E , F ) . Neural markers Sox1 , Zfp521 , Zeb2 , Blbp , Sox9 , Mash1 and Math1are upregulated prematurely and Oct4 is lost more quickly in dox-treated cultures ( Figure 5G ) . Activating TwistE only for the first day of differentiation is sufficient to accelerate the early neural programme ( Figure 5H ) , indicating that Cdh1 activity is normally a limiting factor at an early stage in the differentiation process . Taken together , these observations suggest that downregulation of Cdh1 is a limiting requirement for pluripotent cells to differentiate into neural cells . The data reported above indicate that BMP/Id can maintain Cdh1 , and that downregulation of Cdh1 is a limiting event during differentiation of ES cells . These findings raise the possibility that the mechanism by which BMP blocks differentiation could be through maintenance of Cdh1 . In order to test this directly , we asked whether suppression of Cdh1 is sufficient to rescue the differentiation block imposed by BMP4 . 1 ng/ml of BMP4 is sufficient to almost entirely inhibit neural induction and to maintain Cdh1 expression ( Figure 6A ) , as expected . Strikingly , suppression of Cdh1 via dox-induction of TwistE is sufficient to rescue neural differentiation in the presence of 1 ng/ml BMP . Tuj1+ neurons can be readily found in dox-treated cultures but are only rarely found in control sister dishes ( Figure 6A ) , and dox treatment rescues the expression of early neural markers Mash1 , Zfp521 , Sox9 and Pax3 to levels similar to those observed in the absence of exogenous BMP ( Figure 6B ) . Sox1 is downregulated before day 4 , in keeping with the ability of TwistE to accelerate the early stages of the differentiation process ( Figure 5G , H and data not shown ) . TwistE also abolishes the ability of BMP4 to sustain the expression of epiblast markers Oct4 , Cdh1 , Fgf5 and T ( Figure 6B ) . We conclude that under these conditions BMP acts primarily through Cdh1 to maintain the expression of epiblast markers and block the expression of neural markers . 10 . 7554/eLife . 01197 . 012Figure 6 . Suppression of Cdh1 rescues the BMP-imposed delay to differentiation . ( A ) Antibody staining for the indicated markers in dox-inducible TwistE cells after 4 days under neural differentiation conditions in the presence of 1 ng/ml BMP4 . Scale bar = 30 μm . ( B ) qRT-PCR analysis of dox-inducible TwistE cells after 4 days under neural differentiation conditions in the presence or absence of 1 ng/ml BMP4 . ( C ) Antibody staining for the indicated markers in dox-inducible TwistE cells after 4 days under neural differentiation conditions in the presence of 5 ng/ml BMP4 . ( D ) qRT-PCR analysis of dox-inducible TwistE cells under neural differentiation conditions in the presence or absence of 5 ng/ml BMP4 . ( E ) MODEL: we propose that BMP controls differentiation of pluripotent cells by suppressing the downregulation of Cdh1 . qRT-PCR data are represented as mean +/− standard deviation and * indicates p<0 . 05 relative to control samples treated with BMP but not treated with dox . DOI: http://dx . doi . org/10 . 7554/eLife . 01197 . 012 We noticed that the dox-treated cultures , in addition to containing abundant neurons ( Figure 6A ) , also contain many Tuj1-negative cells that do not have a neural morphology ( data not shown ) . This suggests that TwistE does not exclusively drive neural differentiation in this context but may be rescuing a more general delay to differentiation into multiple lineages . We speculated that BMP might have two independent effects: one to block multilineage differentiation by maintaining Cdh1 and another to influence the direction of differentiation independently of Cdh1 . In order to test this idea , we increased the dose of BMP to 5 ng/ml , with the aim of imposing a posterior positional identity that favours mesodermal and endodermal rather than neural fates ( Figure 2 ) . Under these conditions , activation of TwistE eliminates Cdh1 expression from the majority of cells and upregulates the mesenchymal and neural marker Vimentin ( Figure 6C ) , but we did not observe any Tuj1+ neurons either in the presence or the absence of dox treatment ( data not shown ) . We next confirmed that 5 ng/ml BMP4 upregulates posterior epiblast markers whilst delaying overt differentiation into mesoderm and endoderm ( Figure 6D , black bars ) , as expected . Under these conditions , suppression of Cdh1 via dox-induction of TwE efficiently rescues mesoderm and endoderm differentiation , but not neural differentiation ( Figure 6D ) . We conclude that part of the mechanism by which BMP helps to maintain pluripotency is through maintenance of Cdh1 , which interferes with efficient neural and mesodermal differentiation . Higher levels of BMP also impose a posterior identity that primes cells for mesoderm and is incompatible with a neural fate . Taken together , these findings help to explain why BMP can at the same time protect pluripotency and promote mesoderm differentiation .
Changes in the expression of the cell-cell adhesion molecule E-Cadherin ( Cdh1 ) are often associated with changes in cell identity throughout development and into adult life ( Thiery et al . , 2009 ) . For example , downregulation of Cdh1 is associated with the epithelial-mesenchymal transition ( EMT ) that accompanies the formation of mesoderm at the primitive streak and the emergence of neural crest cells from the dorsal neural tube ( Thiery et al . , 2009 ) . The formation of the early neural plate from the epiblast does not bear all the hallmarks of a classical EMT , but it is accompanied by loss of Cdh1 and gain of N-Cadherin ( Cdh2 ) and Vimentin ( Hatta and Takeichi , 1986; Aaku-Saraste et al . , 1996 ) . There is already evidence that EMT is required for overt mesoderm differentiation ( Martínez-Estrada et al . , 2010 ) , and we find here that downregulation of Cdh1 is similarly a limiting event for neural differentiation . Although it is well established that BMP signalling contributes to the maintenance of pluripotency ( Ying et al . , 2003a; Di-Gregorio et al . , 2007 ) , the underlying mechanisms remain unclear . We propose that BMP suppresses differentiation by maintaining expression of Cdh1 . We support this idea by showing that suppressing Cdh1 is sufficient to rescue the BMP-imposed block to multilineage differentiation . It is also unclear how BMP signalling favours mesodermal over neural fates . We find that the BMP target gene Id1 imposes a proximal posterior identity on undifferentiated cells , priming them towards mesoderm . The ability of Id1 to block neural differentiation is therefore unlikely to be explained entirely by inhibition of a neural-inducing transcription factor as is commonly assumed , nor by a direct activation of mesoderm differentiation , but rather to a shift in the positional identity of pluripotent cells that occurs in the absence of overt differentiation . This may explain why , to our knowledge , no Id-regulated pro-neural bHLH transcription factor has been found that explains the ability of Id1 to influence neural induction . Id1 null ES cells have a defect in the maintenance of pluripotency ( Romero-Lanman et al . , 2011 ) , consistent with our finding that Id1 delays exit from an epiblast-like state . We note , though , that the Id1 null phenotype is also likely to reflect an earlier pre-implantation role for Id1: to suppress the activity of Tcf15 ( Davies et al . , 2013 ) . The consequence of this would be to maintain expression of Nanog and suppress the transition to a T+ epiblast-like state ( Davies et al . , 2013 ) , in keeping with the Id1 null ES cell phenotype ( Romero-Lanman et al . , 2011 ) . The two effects of BMP , to maintain Cdh1 and to impose a posterior identity , can be uncoupled by varying the dose of BMP . Lower doses of BMP are sufficient to maintain Cdh1 and to suppress neural fate , but insufficient to impose a stable posterior identity . Strikingly , suppressing Cdh1 at these lower doses of BMP is sufficient to rescue neural differentiation , indicating that the primary mechanism by which BMP inhibits neural differentiation in this context is through its ability to control Cadherin activity . This is an unexpected finding that highlights the importance of cell adhesion and tissue organisation in controlling receptivity of cells to differentiation cues ( Stepniak et al . , 2009; Faunes et al . , 2013 ) . There is a growing body of evidence that Cdh1 influences growth factor receptiveness of ES cells , and that it can consequently stabilise particular pluripotent states ( Chou et al . , 2008; Soncin et al . , 2009; Li et al . , 2010; Redmer et al . , 2011; Hawkins et al . , 2012; del Valle et al . , 2013; Faunes et al . , 2013 ) , but it is not known what controls Cdh1 expression in this context . We fill this gap with our finding that BMP/Id maintains Cdh1 expression during differentiation of ES cells . Interestingly , Id1 is excluded from the anterior streak in vivo ( Jen et al . , 1997 ) , so it is possible that it plays a more general role in regulating EMT in the early embryo . In vivo , the BMP signals that are generated from the extraembryonic ectoderm not only block neural induction but are also required for formation of mesoderm ( Mishina et al . , 1995; Winnier et al . , 1995; Lawson et al . , 1999; Beppu et al . , 2000; Davis et al . , 2004 ) . However , BMP signalling is also necessary for maintenance of pluripotency ( Di-Gregorio et al . , 2007 ) , and mouse embryos in which BMP has been experimentally overactivated have an impairment in mesoderm differentiation that cannot easily be explained by models in which BMP drives mesoderm differentiation ( Aoyama et al . , 2012 ) . Our findings unite these observations by showing how BMP can prime cells for mesoderm fate by imposing a posterior identity , but restrain them from undergoing overt mesoderm formation by suppressing EMT . In frogs , antagonism of BMP by the organiser induces anterior-posterior axis formation . It has been proposed ( Constance Lane et al . , 2004 ) that BMP antagonism acts by stimulating mediolateral intercalation of caudal progenitors , releasing them to participate in the formation of the axis , and that the normal role of BMP is to preserve a reservoir of progenitors throughout axis elongation . We propose that BMP acts by a similar mechanism during differentiation of pluripotent mouse cells , preserving a population of undifferentiated cells by preventing the loss of Cdh1 . The mechanism by which BMP signalling controls EMT will be an interesting area for future study . This may be connected with BMP’s ability to antagonise Nodal signalling , given that Nodal is required for the initiation of EMT at the primitive streak ( Arnold and Robertson , 2009; Yan et al . , 2009 ) . Our work also has implications for controlling uniform differentiation of mouse ES cells . It has been known for some time that a subpopulation of ES cells tends to resist neural differentiation , but the reasons for this are not clear . We propose that this subpopulation of cells fail to respond appropriately to a neural-inducing environment because they are unable to downregulate Cdh1 . We demonstrate this directly by showing that experimental suppression of Cdh1 activity can rescue neural differentiation capacity in this recalcitrant subpopulation resulting in a more homogenous differentiation response . Importantly , this can be achieved by non-genetic means , such as function-blocking E-Cadherin antibodies , providing a convenient and readily applicable means to improve our control over pluripotent cells . In summary , we have identified the loss of Cdh1 activity as a limiting event for the earliest stages of neural differentiation and we propose that BMP controls differentiation in part by maintaining Cdh1 expression . The strategy of coupling changes in cell adhesion to transcriptional control of cell fate specification is used several times during development as a way of coordinating tissue morphogenesis ( Owens et al . , 2000; Martínez-Estrada et al . , 2010; Watt and Fujiwara , 2011 ) , and it will be interesting to investigate the implications of our findings for germ-layer formation in the early embryo .
ES cells were maintained in GMEM supplemented with 2-mercaptoethanol , non-essential amino acids , glutamine , pyruvate , 10% foetal calf serum ( FCS ) and 100 units/ml LIF on gelatinised tissue culture flasks ( Smith , 1991 ) . To generate dox-inducible Id1-overexpressing ES cells , E14tg2α ES cells were transfected with a plasmid containing a CAGS promoter driving expression of the reverse tetracycline-controlled transactivator ( rtTA ) , followed by an IRES and a Blasticidin S resistance gene . The cells were cultured in the presence of 20 µg/ml Blasticidin S for two passages . The DNA sequence corresponding to N-terminally 3 × Flag-tagged mouse Id1-001 was generated by overlapping extension PCR . This sequence was subcloned downstream of a tet-responsive promoter in a plasmid containing a Pgk promoter driving expression of a Hygromycin B resistance gene . This construct was transfected into the E14tg2α ES cells under Blasticidin S selection . The cells were subject to dual selection in 20 µg/ml Blasticidin S and in 200 µg/ml Hygromycin B prior to clone isolation and expansion . To generate Id1-overexpressing ES cells , E14tg2α ES cells were transfected with a previously described Id1-overexexpression plasmid ( Ying et al . , 2003a ) , containing a CAGS promoter driving the expression of Id1 cDNA , followed by an IRES , a Puromycin resistance gene ( Pac ) and a polyadenylation signal sequence ( pA ) . Id1-IRES-Pac-pAare flanked by loxP sites . Downstream of the second loxP site there is a GFP transgene , not expressed in Id1-overexpressing cells but activated after Cre-mediated excision of Id1-IRES-Pac-pA . Revertant cells were obtained by transfecting Id1-overexpressing cells with a circular plasmid encoding Cre recombinase and screening for expression of GFP . To generate Id1-Venus reporter lines , E14tg2α cells were electroporated with a targeting construct provided by H-S Song and R Benezra ( Sloan-Kettering Memorial Cancer Centre , NY , USA ) designed to express a fusion between Id1 and Venus from the endogenous Id1 locus . This construct is described in detail in Nam and Benezra ( 2009 ) . Correct targeting was determined by Southern blotting following the strategy described in Nam and Benezra ( 2009 ) . To generate a fluorescent lineage label , the sequence encoding the mKate2 red fluorescent protein was fused at its 3′ end prior to its stop codon with three repeats of the SV40 nuclear localisation signal sequence ( NLS ) . This sequence was subcloned downstream of a CAGS promoter and followed by an IRES and Pac . This construct was transfected into the Id1-Venus reporter cells and clones were isolated following Puromycin selection . Chimeric embryos were generated by morula aggregation . To generate dox-inducible Twist1-E47 ES cell lines: the DNA sequence encoding an N-terminally Flag-tagged mouse Twist1 tethered at its C-terminus to the N-terminus of mouse E47 through a 13 amino acid flexible linker of sequence TGSTGSKTGSTGS was generated by overlapping extension PCR . This sequence was placed under a tet-responsive promoter and transfected into the E14tg2A_AW2 cell line , which contains the coding sequence for the reverse tetracycline-controlled transactivator ( rtTA ) integrated into the Rosa26locus and expressed from the Rosa26 promoter . The E14tg2A_AW2 cell line is described in more detail elsewhere ( Zhou et al . , 2013 ) . Cells were plated at clonal density and clones expanded in ES cell culture conditions under Hygromycin B selection . The expression of Flag-Twist1-E47 was tested in a number of clones through qRT-PCR analysis ( using primers within Twist1 ) and western blot analysis ( probed by anti-Flag antibody ) over a 5-day time course of treatment in the presence or absence of doxycycline ( dox ) . A dox-inducible Twist1-E47 cell line was selected that had negligible leakiness of Twist1-E47 in the absence of dox , and moderate overexpression in the presence of dox . Cell lines for dox-inducible expression of monomeric N-terminally flag tagged Twist1 were generated in the same way . Monolayer neural differentiation is described in detail in Pollard et al . ( 2006 ) . Briefly , ES cells were washed to remove all traces of serum and then plated onto gelatin-coated tissue culture plastic in N2B27 serum-free medium . N2B27 consists of a 1:1 ratio of DMEM/F12 and Neurobasal media supplemented with 0 . 5% modified N2 ( made in house as described in Pollard et al . , 2006 ) , 0 . 5% B27 and 2- mercaptoethanol . Medium was changed every second day . Directed mesoderm differentiation on collagen IV was carried out as previously described ( Nishikawa et al . , 1998 ) . Primers used for qRT-PCR are described in Supplementary file 1 . All expression values are normalised to those of the housekeeping gene TBP . Experiments in Figure 6D were performed in four replicates , with paired replicates performed on different days . All other experiments were performed on three independent occasions . Statistical significance was calculated using a paired Student’s t test . RNA reverse transcription and cDNA pre-amplification from single cells were performed as previously described ( Dalerba et al . , 2011 ) with some modifications . Each well of a 96-well PCR plate was loaded with 5 µl 2X Reaction Mix , 0 . 2 µl Superscript III RT/Platinum Taq Mix ( with RNaseOUT Ribonuclease Inhibitor ) ( Invitrogen Cells Direct One-Step qRT-PCR kit , Life Technologies ) , 2 . 5 µl primer mix ( containing 200 nM of each gene-specific primer ) , 1 . 3 µl H2O . Single-cell suspensions were stained with 100 ng/ml DAPI . DAPI-negative cells were sorted into separate wells of the 96-well PCR plate . 32 cells were sorted into one well , to be used for serial dilution for generation of qRT-PCR standard curves . RNA reverse transcription and 22 cycles of cDNA pre-amplification were performed as previously described ( Dalerba et al . , 2011 ) . The cDNA was diluted 1:5 in H2O and 2 µl were loaded into each well of a Lightcycler 480 384 Multiwell plate , with 4 µl Lightcycler 480 Probes Master , 0 . 08 µl Universal ProbeLibrary probe ( Roche , Switzerland ) , 450 nM of each primer and H2O to 8 µl . qRT-PCR reactions were performed using a Lightcycler 480 II instrument with the following cycling conditions: 50°C for 2 min , 95°C for 10 min , then 40 cycles of 95°C for 15 s , 60°C for 1 minute . 1:2 serial dilutions of the 32 cells sorted into one well were used for the generation of standard curves . Samples with TBP expression lower than 10% of the population average were removed from further analyses . Spearman’s rank correlation coefficients for TBP-normalised gene expression values were calculated in R ( http://www . r-project . org/ ) using the rcorr ( ) function in the Hmisc package . For immunofluorescence analysis , cells were fixed in 4% paraformaldehyde ( PFA ) and incubated for 30 min in blocking buffer ( PBS , 3% Donkey serum and 0 . 1% Triton ) . Primary antibodies were diluted in blocking buffer and applied for 1 hr at room temperature or overnight at 4°C . After three washes in PBS , secondary antibodies conjugated to Alexa fluorophores ( Life Technologies ) were diluted at 1:1000 in blocking buffer and applied for 1 hr at room temperature . The cells were washed at least three times in PBS and visualised on an Olympus inverted fluorescence microscope . For nuclear counter staining , cells were incubated in 1 µg/ml DAPI ( Sigma ) for 10 min after immunostaining . Embryos were fixed in 4% PFA for 2 hr at room temperature . The PFA was quenched with NH4Cl for 30 min at room temperature . For wholemount staining , the embryos were permeabilised in PBS+0 . 5% Triton for 1 hr at room temperature , then transferred to blocking buffer overnight at 4°C . They were then incubated in primary antibodies for 48 hr at 4°C , subjected to five 10-min washes in PBS+0 . 1% Triton , incubated in secondary antibodies and 100 ng/μl DAPI for 48 hr at 4°C , then subjected to five 10-min washes in PBS+0 . 1% Triton . Staining of sectioned embryos ( 7 μm sections ) followed a similar protocol: the slides with gelatine-embedded sections were incubated at 55°C for 15 min to melt the gelatine , then permeabilised with PBS+0 . 5% Triton for 10 min at room temperature . The blocking and staining procedure is the same as that described for cells . After staining , the sections were mounted in Prolong Gold Antifade Reagent ( Life Technologies ) . Fluorescent mages were taken using a monochrome Retiga2000 camera using an Olympus IX51 inverted microscope or a Leica SPE confocal microscope . Images from the Olympus microscope were collected using Volocity software . 3D rendering was done using the 3DViewer plugin in Fiji ImageJ . Fluorochromes were Alexa 488 , 568 , 594 or 647 . Primary antibodies were obtained from the following sources: Neuronal beta-III tubulin ( TUJ1: Covance , Princeton , NJ ) , E-Cadherin ( ECCD1 for blocking experiments , ECCD2 for immunofluorescence: BD Biosciences , Franklin Lakes , NJ ) , N-Cadherin ( C32: BD Biosciences ) , Oct4 ( Santa Cruz , Dallas , TX ) , T ( R&D , Minneapolis , MN ) , Nanog ( MLC-51: eBioscience , San Diego , CA ) , Esrrb ( PP-H6705-00: Persaeus Proteomics , Japan ) , Klf4 ( R&D ) , Flag ( clone M2 HRP conjugated for Western Blot , or unconjugated for immunofluorescence: Sigma , St Louis , MO ) . Antibodies against Vimentin and Keratin8 were purchased from the Developmental Studies Hybridoma Bank , developed under the auspices of the NICHD and maintained by The University of Iowa , Department of Biological Sciences , Iowa City , IA 52242 . FACS analysis was performed using a Becton Dickinson FACS Calibur flow cytometer . FACS sorting was carried out on a FACS Aria . In situ hybridisation for Id1 was performed using a probe described in Gray et al . ( 2004 ) . The probe was generated using the following primers: F:AAGGTCGCGAGTGGCAGTGC R:GCCTGAAAAGTAAGGAAGGG . In order to quantify the fluorescence signal in each individual cell , we generated an automated pipeline for image analysis as described previously ( Davies et al . , 2013 ) or used tools provided through the Farsight project ( http://www . farsight-toolkit . org ) . In order to quantify neurite density after neural differentiation , five randomly selected fields ( for each condition: treatment and day of differentiation ) stained for Tuj1 were pictured . Each field was scored depending on the number of neurites present from 0 to 9 , where 0 means no neurites can be observed and 9 neurites cover almost the entire field; the scores for each condition were averaged .
Bone morphogenic protein ( BMP ) signalling contributes towards maintenance of pluripotency and favours mesodermal over neural fates upon differentiation , but the mechanisms by which BMP controls differentiation are not well understood . We report that BMP regulates differentiation by blocking downregulation of Cdh1 , an event that accompanies the earliest stages of neural and mesodermal differentiation . We find that loss of Cdh1 is a limiting requirement for differentiation of pluripotent cells , and that experimental suppression of Cdh1 activity rescues the BMP-imposed block to differentiation . We further show that BMP acts prior to and independently of Cdh1 to prime pluripotent cells for mesoderm differentiation , thus helping to reinforce the block to neural differentiation . We conclude that differentiation depends not only on exposure to appropriate extrinsic cues but also on morphogenetic events that control receptivity to those differentiation cues , and we explain how a key pluripotency signal , BMP , feeds into this control mechanism .
The human body is made up of about 200 different types of cell , all of which are descended from a single fertilised egg . As an embryo develops , its cells divide and specialise into distinct lineages . Cells in each lineage go on to form a restricted number of cell types that are required to make a specific tissue . As such , during early development , cells switch from being ‘pluripotent’ , with the potential to become the many different cell types , to committing to one particular cell lineage . Controlling this process involves a huge number of signalling proteins and pathways . One such protein is bone morphogenetic protein , or BMP for short , which has a number of different roles in embryo development: for example , it stops pluripotent cells turning into nerve tissue , and it also encourages embryonic stem cells to contribute to the ‘mesoderm’ of the early embryo ( which goes on to form the muscles , connective tissues and some blood cells ) . How these two actions are linked , and whether they depend on similar signalling pathways , was unknown . BMP is also known to trigger the production of proteins known as ‘Id factors’—which stands for ‘inhibitor of differentiation’ . Now , Malaguti et al . have investigated the roles of BMP and Id factors in controlling mouse embryo development and found , somewhat surprisingly , that these proteins needed help from a third protein to stop pluripotent cells turning into nerve tissue . This third protein , which is called E-Cadherin , normally helps cells to adhere to other cells . Malaguti et al . showed that losing this protein encourages cells to become either nerve or mesoderm tissues , and that a drop in E-Cadherin levels must occur before nerve tissue can form . Malaguti et al . also showed that encouraging cells to become part of the mesoderm requires BMP to activate another pathway , which does not require E-Cadherin . The two effects of BMP can be uncoupled by adjusting the levels of this protein . At low concentrations , BMP can keep cells pluripotent , but it cannot encourage cells to commit to a mesoderm fate . At higher doses , however , BMP ‘primes’ cells to respond to the signals that trigger their development into mesoderm tissue . The findings of Malaguti et al . suggest that manipulating both E-Cadherin and BMP signalling could improve our ability to generate useful cell types , such as neurons , from stem cells grown in laboratory cultures .