GATA6 Mediates Endoderm and Mesoderm Progenitor Fate Driven by WNT and NODAL Signaling

GATA6 Mediates Endoderm and Mesoderm Progenitor Fate Driven by WNT and NODAL Signaling | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results GATA6 Mediates Endoderm and Mesoderm Progenitor Fate Driven by WNT and NODAL Signaling Miriam Gordillo , Rebecca Wu , Kelly M. Banks , Neranjan de Silva , View ORCID Profile Todd Evans doi: https://doi.org/10.1101/2025.06.20.660704 Miriam Gordillo 1 Department of Surgery, Weill Cornell Medicine , New York, NY, 10065, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rebecca Wu 1 Department of Surgery, Weill Cornell Medicine , New York, NY, 10065, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kelly M. Banks 1 Department of Surgery, Weill Cornell Medicine , New York, NY, 10065, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Neranjan de Silva 1 Department of Surgery, Weill Cornell Medicine , New York, NY, 10065, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Todd Evans 1 Department of Surgery, Weill Cornell Medicine , New York, NY, 10065, USA 2 Hartman Institute for Therapeutic Organ Regeneration, Weill Cornell Medicine , New York, NY 10065, USA 3 Center for Genomic Health, Weill Cornell Medicine , New York, NY 10065, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Todd Evans For correspondence: tre2003{at}med.cornell.edu Abstract Full Text Info/History Metrics Preview PDF SUMMARY During gastrulation, NODAL and WNT signaling drives distinct gene regulatory networks (GRNs) to promote pluripotency exit, specify progenitor fate, and direct lineage differentiation. Here, we employ murine embryonic stem cell directed differentiation toward endoderm and mesoderm lineages to reveal a crucial role of GATA6 during anterior primitive streak-derived progenitor segregation. GATA6 is known to direct by default extra-embryonic endoderm. To promote definitive endoderm fate, GATA6 reinforces the endoderm GRN driven by NODAL while repressing the mesoderm GRN through down-regulation of WNT activity. During progression of mesoderm differentiation, GATA6 induces a switch in the transcriptional output regulated by WNT, promoting lateral mesoderm specification and repression of paraxial mesoderm fate. Thus, this single transcription factor mediates formation of four distinct tissue types, depending on integration of NODAL/WNT activities. Regulation of Eomes and T/Bra expression during anterior primitive streak progression is an essential function of GATA6 during the specification of endoderm or mesoderm fate. INTRODUCTION The interaction between signaling pathways and transcriptional networks controls the specification of cell lineages during gastrulation. Definitive endoderm (DE) and mesoderm (ME) development requires the concerted activity of the NODAL and WNT pathways within signaling gradients formed in the primitive streak ( Robertson, 2014 ). In this context, NODAL (a member of the TGFβ superfamily) and WNT cooperate to induce a progenitor population known as mesendoderm ( Estarás et al., 2015 ; Funa et al., 2015 ; Kubo et al., 2004 ; Reid et al., 2012 ; Rodaway and Patient, 2001 ; Zorn and Wells, 2007 ). After mesendoderm formation, NODAL and WNT control the segregation of DE and ME progenitors through unknown molecular mechanisms. In vivo, study of the segregation of these lineages is complicated by the transient and rapid mesoderm/endoderm bifurcation in the primitive streak ( Mittnenzweig et al., 2021 ). Work in multiple species indicates that high levels of NODAL promote DE fate, and lower activity levels promote ME ( Robertson, 2014 ). Furthermore, after an initial requirement for WNT signaling, early DE progenitors committed to a SOX17+ DE fate become WNT independent by an unknown mechanism ( Pour et al., 2022 ). Recent evidence demonstrates the dynamic nature of the interaction of NODAL and WNT to induce different fates. In mice, constitutively active WNT signaling pushes epiblast cells to posterior and middle streak ME progenitors at the expense of anterior ME progenitors, and it was proposed that WNT cooperates with NODAL to promote the differentiation of anterior ME derivatives ( Hernández-Martínez et al., 2024 ). Using geometrically constrained models of human pluripotent cell differentiation, WNT3a was shown to induce NODAL expression, which then drives DE differentiation. In this context, NODAL activity also restricts posterior ME specification, and if NODAL is inhibited, cell fate is diverted to paraxial ME, indicating the ability of NODAL to modulate WNT signaling ( Ortiz-Salazar et al., 2024 ). A similar in vitro approach using CHIR99021 (CHIR), a GSK3β inhibitor that stabilizes β-catenin and is commonly used as a WNT agonist ( Bennett et al., 2002 ), found that small variations in CHIR concentration induced distinct Nodal activity levels and dynamics which in turn determined the choice of DE or ME fate ( Robles-Garcia et al., 2024 ). Interpretation of extracellular signals by transcription factors (TFs) involves cell-type specific protein complexes that drive tissue-specific gene regulatory networks (GRNs). During exit of pluripotency, NODAL and WNT regulate the expression of genes encoding TFs including T/Bra , Eomes , Gata4 , and Gata6 , that dismantle the pluripotency network and establish the GRNs that will drive ME and DE fate ( Brown et al., 2011 ; Dunn et al., 2004 ; Faial et al., 2015 ; Funa et al., 2015 ; Liu et al., 2011 ; Morgani and Hadjantonakis, 2020 ; Senft et al., 2018 ; Zorn and Wells, 2007 ). BRACHYURY and EOMES are essential regulators of pluripotency exit, neuroectoderm fate repression, and DE and ME differentiation and play pivotal roles during entry and exit from the mesendoderm stage ( Arnold et al., 2008 ; Faial et al., 2015 ; Gentsch et al., 2017 ; Herrmann et al., 1990 ; Tosic et al., 2019 ). T/Bra is a WNT target and direct activator of the ME regulatory network driven by Tbx6 that controls paraxial ME formation and patterning ( Hofmann et al., 2004 ; Mariani et al., 2021 ; Yamaguchi et al., 1999 ). Eomes is a critical regulator of the early DE and anterior ME GRNs ( Probst and Arnold, 2017 ). GATA6 is quite remarkable, as it is indispensable for the development of extraembryonic endoderm, while also playing an essential role in DE and ME development and organogenesis ( Carrasco et al., 2012 ; Hayashi et al., 2016 ; Jun et al., 2023 ; Keijzer et al., 2001 ; Kozhemyakina et al., 2014 ; Morrisey et al., 1998a ; Sam et al., 2020 , 2024 ; Xuan et al., 2012 ; Zhao et al., 2008 ). Mutations in GATA6 are associated with human pancreatic and gall bladder agenesis, congenital heart defects, and hepatobiliary malformations ( Allen et al., 2012 ; Chao et al., 2015 ; De Franco et al., 2013 ). GATA6 function in DE and ME has recently been shown to involve interactions with EOMES and SMAD2/3 ( Bisson et al., 2024 ; Chia et al., 2019 ; Heslop et al., 2021 , 2022 ). Despite advances in understanding that NODAL, WNT, and the above TFs regulate the exit of pluripotency and entry into mesendoderm fate, mechanisms at the exit from mesendoderm towards specification of DE or ME remain unclear. Here, we investigate the role of GATA factors in the transcriptional networks driven by NODAL and WNT during lineage segregation from mesendoderm. Previous reports on the function of Gata4 or Gata6 during mouse embryonic stem cell (ESC) differentiation demonstrated their capacity to direct visceral endoderm but not DE development ( Capo-chichi et al., 2010 ; Fujikura et al., 2002 ; Holtzinger et al., 2010 ; Morrisey et al., 1998b ; Soudais et al., 1995 ). Using serum-free conditions to avoid the known inhibitory effect of FBS on DE differentiation ( D’Amour et al., 2005 ; Kubo et al., 2004 ), we confirmed a critical role for Gata6 in murine DE differentiation, consistent with results using human ESCs ( Chia et al., 2019 ; Fisher et al., 2017 ; Shi et al., 2017 ; Tiyaboonchai et al., 2017 ). We find that Gata6 expression in mesendoderm progenitors drives DE fate by reinforcing and propagating the DE GRN controlled by NODAL while repressing the ME GRN controlled by WNT. Yet, unexpectedly, during ME differentiation induced by sustained WNT activation, GATA6 promotes lateral plate ME fate at the expense of paraxial ME by modulating the transcriptional output of WNT. Eomes and T/ Bra are among the first targets of GATA6-mediated transcriptional regulation, essential for the transition of mesendoderm progenitors to DE or ME. In this manner, a single TF, GATA6, directs early steps of organogenesis from distinct germ layers for specification of four distinct early progenitor fates. RESULTS Expression of GATA Factors Drives Definitive Endoderm Specification We used a defined serum-free medium and Activin A to efficiently induce DE progenitors from mouse ESCs ( Figure S1A ). To track DE progenitors, flow cytometry was used to analyze the c-KIT and CXCR4 double-positive population ( Gouon-Evans et al., 2006 ). However, in no-Activin or low-Activin conditions, these markers were unreliable compared to the combination of EpCAM/CXCR4 to discriminate DE from other populations ( Figure S1B, S1C ). Thus, although both combinations were analyzed as validation, we quantified the EpCAM/CXCR4 population as DE throughout this study. High concentrations of Activin A promote specification of DE, while concentrations below 25 ng/ml do not induce DE progenitors or the expression of DE markers ( Figure S1C and S1D ). Transcriptome analysis shows enrichment of DE signatures derived from single-cell RNA-seq of mouse embryos with 75 ng/ml Activin A ( Figure S1E ). In a time-course analysis for the expression of DE TFs induced by Activin A at 75 ng/ml, we observed three waves of expression of transcription factors of the DE Gene Regulatory Network (GRN, Figure S1F ). Eomes , FoxA2 , and Mixl1 are the earliest expressed followed by Gata6 , Sox17 , FoxH1 and Lhx1 , and finally Gata4 , Hesx1 , Hhex , and FoxA3 (blue, green, and red lines, respectively, Figure S1F ). To investigate the role of Gata4 and Gata6 in DE development, we first tested the ability of each to drive DE development using previously established mESC lines that allow conditional expression of Gata4 or Gata6 upon the addition of doxycycline (+Dox; Figure 1A ) ( Holtzinger et al., 2010 ; Turbendian et al., 2013 ). Initial induction of Dox under conditions of 75 ng/ml Activin showed a significant increase in DE progenitors, suggesting that forced expression of GATA4 or GATA6 can enhance the effect of Activin A during DE specification ( Figure 1B , GATA6; Figure S1G , GATA4). Next, the GATA factors were induced using just 10 ng/ml Activin A, a concentration that on its own does not specify DE ( Figure S1C ). Indeed, DOX-induction of either GATA factor generates a significant DE population comparable in numbers to that induced by 75 ng/ml Activin A ( Figure 1B , Gata6 ; Figure S1G , Gata4 ). qPCR analysis confirmed the increased expression of multiple DE markers ( Figure 1C , Gata6 ; Figure S1H , Gata4 ). Gene list analysis of the Gata6 -induced transcriptome using Enrichr ( Chen et al., 2013 ) shows a high enrichment of genes associated with DE development ( Figure S1I ). Gene Set Enrichment Analysis (GSEA) using sets extracted from embryonic mouse single cell RNA-seq data in the literature shows enrichment of early DE signatures ( Figure 1D ). Download figure Open in new tab Figure 1. Expression of GATA4 or GATA6 can drive murine definitive endoderm specification, and GATA6 is required. (A) Schematic diagram of the protocol for directed differentiation and induced expression of GATA factors. Embryoid body (EB) formation was initiated on day 0 and cells reaggregated on day 2 in the presence of Activin A. Doxycycline (+Dox, 30 ng/ml) was added (or not, -Dox) to induce GATA factor expression starting on day 3. (B) Quantification by flow cytometry of CXCR4+/EpCAM+ DE progenitors on day 4.5 of differentiation after induction of Gata6 expression. See Figure S1B for gating strategy. (C) Gene expression analysis by RT-qPCR for key DE markers expressed on day 4 of differentiation using 10 ng/ml Activin A. (D) GSEA analysis of RNA-seq gene expression data at day 4 of differentiation (+Dox vs –Dox; 10ng/ml Activin A) using mouse embryo endoderm enriched gene sets obtained from single-cell RNA-seq data previously published ( Ibarra-Soria et al., 2018 ; Peng et al., 2019 ; Pijuan-Sala et al., 2019 ). NES=normalized enrichment score; FDR=false discovery rate. (E) Gene expression of DE (purple) and VE (green) classifier gene sets previously identified through single-cell RNA-seq of mouse embryo cells (Nowotschin et al., 2019). Expression as normalized counts was obtained from RNA-seq data at day 4 of differentiation of cells grown in 10 ng/ml Activin A, +Dox, compared to cells grown in 75 ng/ml Activin A. (F and G) RT-qPCR analysis of tissue markers in DE progenitors that underwent further hepatic differentiation (F) or mid-hindgut differentiation (G). (H) DE differentiation of WT, Gata4 -/- , Gata6 -/- , and Gata4 -/- /Gata6 -/- mESCs. The left panel shows quantification of DE progenitors by flow cytometry; right panel shows viability analysis by flow cytometry. Cells were differentiated as depicted in Figure S1A in the presence of 75 ng/ml Activin A. (I) Gene expression analysis by RT-qPCR of key DE markers in WT, Gata4 -/- , Gata6 -/- , and Gata4 -/- /Gata6 -/- mESCs. Data in B, C, F, G, and I are presented as mean ± SEM. n ≥ 3 biologically independent experiments. Statistical significance in C, F, and G was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Statistical significance in B was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. Statistical significance in I was evaluated with one-way ANOVA followed by Dunnett’s test for multiple comparisons relative to WT. The early DE lining of the primitive gut tube comprises a mixture of DE and VE progenitors with highly similar overall gene expression profiles (Nowotschin et al., 2019). However, DE and VE cells can be distinguished using specific classifier gene sets identified through single-cell RNA-seq of embryo cells. Using these classifier sets, we confirmed that the DE progenitors generated by induction of GATA factors correspond to DE progenitors that express low levels of genes predictive of the VE class and high levels of genes predictive of the DE class ( Figure 1E ). In addition, these sets of genes are expressed at a similar level as the cells differentiated with a high concentration of Activin, indicating that GATA factors direct the same type of DE generated by high activity of the NODAL/Activin signaling pathway. Replating Day 5 EBs in hepatoblast or mid-hindgut differentiation conditions showed that DE progenitors generated by expression of GATA factors differentiate efficiently into these cell types derived from anterior or posterior DE ( Figure 1F-G , Gata6 ; Figure S1J-K , Gata4 ). Using mouse Gata6 -/- ; Gata4 -/- ; and Gata4 -/- , Gata6 -/- ESCs generated by gene targeting ( Molkentin et al., 1997 ; Zhao et al., 2008 , 2005 ), Gata6 (and not Gata4 ) function was found to be essential for DE formation ( Figure 1H-I ). An essential role for GATA6 in DE development was also reported using human pluripotent cells ( Fisher et al., 2017 ; Shi et al., 2017 ; Tiyaboonchai et al., 2017 ), and our data indicate it is a conserved program. However, in contrast to human pluripotent cells, the deficiency in DE development from murine Gata6 null cells is not the result of cell death ( Figure 1H ) ( Fisher et al., 2017 ; Tiyaboonchai et al., 2017 ). Taken together, our results demonstrate the capacity of either GATA4 or GATA6 to induce functional DE progenitors and show a conserved requirement for Gata6 in DE formation. Gata6 Reinforces the NODAL Pathway to Drive Endoderm Specification After induction of Gata6 in the context of a low dose of Activin A, expression levels of Nodal and its direct downstream target, Lefty1 , are increased ( Figure 2A ). In addition, enrichment analysis of the GATA6-induced transcriptome highlights gene sets regulated by the TGFβ/NODAL signaling pathway and SMAD2, SMAD3, and SMAD4, the main effectors of the NODAL pathway ( Figure S2A-S2B ). GSEA using sets of genes regulated by SMADs gathered from previous studies ( Chia et al., 2019 ; Chiu et al., 2014 ; Heslop et al., 2022 ; Kim et al., 2011 ; Senft et al., 2018 ) also reveals the up-regulation of multiple sets of direct SMAD2/3 targets after Dox-induction of Gata6 ( Figure 2B ). qPCR analysis of SMAD2/3/4 direct targets Lhx1 , Cer1 , and Eomes ( Chiu et al., 2014 ), shows that transcript levels are Activin A dose-dependent ( Figure S2C ) and that Gata6 ( Figure 2D ) or Gata4 ( Figure S2D ) upregulate the expression of these genes. RNAseq analysis shows the extensive transcriptional regulation of other multiple SMAD2/3/4 direct targets by GATA6 ( Figure 2C ). Transcript levels of FoxH1 , a TF essential for NODAL signaling and DE specification ( Chiu et al., 2014 ; Hoodless et al., 2001 ; Slagle et al., 2011 ), are also Activin A dose-dependent ( Figure S2C ) and enhanced by GATA factors ( Figure 2D , Gata6 ; Figure S2D , Gata4 ). Furthermore, direct targets of FOXH1 are also upregulated by expression of Gata6 , as indicated by GSEA analysis ( Figure 2B ). Expression analysis of SMAD2/3 direct targets Eomes , Lhx , and Cer1 in Gata6 -/- cells shows that Gata6 is necessary to maintain their expression ( Figure 2E ). However, there is no significantly impaired expression level of FoxH1 or Nodal or decreased pathway activity as reflected by the normal expression level of Lefty1 in the absence of GATA6 ( Figure S2E ). To test the ability of GATA6 to specify DE independently of NODAL signaling, Gata6 expression was induced with Dox in the absence of Activin A, and this failed to generate DE ( Figure 2F ). In addition, inhibition of Activin type I receptors ALK5, ALK4, and ALK7 with the A83-01 inhibitor at low or high doses of Activin A blocked DE specification despite the forced expression of Gata6 ( Figure 2G and S2F ), indicating that Gata6 requires a NODAL signal to induce DE. Together, these results indicate that Gata6 is an essential component of the NODAL regulated network that reinforces and propagates activity of the pathway to drive the DE regulatory network. Download figure Open in new tab Figure 2. GATA6 reinforces the NODAL pathway. (A) Gene expression analysis by RT-qPCR for NODAL pathway markers Nodal and Lefty1 on day 4 of differentiation using 10 ng/ml Activin A). (B) GSEA using the Gata6 -induced transcriptome comparing sets of genes regulated by TGFβ/NODAL pathway effectors, as described from previous studies, including direct SMAD2/3 targets. (C) The heat map from Gata6 -induced RNA-seq analysis shows the extensive transcriptional regulation of multiple SMAD2/3/4 direct targets as defined previously ( Kim et al., 2011 ). (D) Gata6 -induced gene expression analysis by RT-qPCR for known SMAD2/3/4 direct targets on day 4 of differentiation using 10 ng/ml Activin A. (E) Gene expression analysis by RT-qPCR for Eomes , Lhx1 , and Cer1 during endoderm differentiation using 75 ng/ml Activin A shows that Gata6 -/- cells cannot maintain expression of these genes. (F) Based on flow cytometry, specification of DE is dose dependent, and without induction of Gata6 expression requires a high dose of Activin A, but with induction of Gata6 a low dose is sufficient. Nevertheless, with no-dose (0 ng/ml), Gata6 fails to induce DE. (G) As quantified by flow cytometry, specification of DE by induction of Gata6 (in 10 ng/ml Activin A) requires active Nodal/Activin A signaling, as it is blocked by addition of the A83-01 ALK4/5/7 type I receptor inhibitor. For all panels, n≥3 independent differentiation experiments. Data in A, D-G are presented as mean ± SEM. Statistical significance in A, D-F was evaluated by unpaired, two-tailed Student’s t test for each two-group comparison. Statistical significance in G was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. GATA Factors Promote the Transition of Mesendoderm Toward Definitive Endoderm Fate We hypothesized that GATA factors drive mesendoderm exit towards DE fate at the expense of ME fate. GSEA analysis of the transcriptome of cells at low concentration of Activin A with or without Dox revealed the capacity of GATA6 ( Figure 3A ) or GATA4 ( Figure S3A ) to promote the DE regulatory network and that DE specification is coupled with their ability to suppress the ME regulatory network. The qPCR analysis for transcript levels of key ME TFs and markers at this early stage of differentiation confirms that expression of Gata6 ( Figure 3B ) or Gata4 ( Figure S3B ) causes down-regulation of genes essential for ME development. In mice, mesendoderm progenitors are identified by the co-expression of FoxA2 and T/Bra ( Kubo et al., 2004 ). After segregation from mesendoderm, DE progenitors upregulate FoxA2 and downregulate T/Bra , while ME progenitors down-regulate the expression of FoxA2 and upregulate T/Bra ( Figure S3E ). The qPCR analysis 24 hr after Dox-induction of Gata6 ( Figure 3C ) or Gata4 ( Figure S3C ) shows upregulation of FoxA2 and down-regulation of T/ Bra . Flow cytometry analysis confirms the down-regulation of T/Bra expression ( Figure 3D , Gata6 ; Figure S3D , Gata4 ). Flow cytometry analysis for FOXA2 was not possible using currently available antibodies. Therefore, to monitor co-expression of FoxA2 and T/Bra , we generated a Gata4 inducible cell line in the background of a dual reporter line containing GFP cDNA targeted to the T/Bra locus and a truncated version of the human CD4 cDNA targeted to the Foxa2 locus ( Cheng et al., 2008 ). This cell line allowed evaluation of the progression of double-positive (GFP-BRA + /CD4-FOXA2 + ) mesendoderm progenitors after the induction of Gata4 . Kinetic analysis of this population shows that Dox-induction of Gata4 results in a steady increase of the CD4-FOXA2 + /GFP-BRA - population ( Figure S3F ), while the mesendoderm GFP-BRA + /CD4-FOXA2 + population decreases ( Figure S3G ). Thus, the results indicate that GATA factors promote the exit of mesendoderm progenitor towards DE fate at the expense of ME fate. Download figure Open in new tab Figure 3. GATA6 drives mesendoderm cells to definitive endoderm fate. (A) GSEA using RNA-seq data of cells at day 4 with or without Dox-induced expression of Gata6 at low concentration of Activin A shows enrichment for gene sets representing the definitive endoderm regulatory network and suppression for those of the mesendoderm regulatory network. (B) Gene expression analysis by RT-qPCR for key mesoderm drivers on day 4 of differentiation following Dox-induction of Gata6 and culture in 10 ng/ml Activin A. (C) Gene expression analysis by RT-qPCR for FoxA2 and T/Bra on day 4 of differentiation using 10 ng/ml Activin A. (D) Flow cytometry quantification for BRA+ cells at defined stages of directed differentiation with or without induction of Gata6 using 10 ng/ml Activin A. (E and F) Gene expression analysis by RT-qPCR with or without Dox-induced expression of Gata6 for key early drivers of DE specification (E) or ME specification (F) from day 3 to day 4 of differentiation using 10 ng/ml Activin A. (G) GSEA of RNA-seq data using gene sets regulated by EOMES or BRACHYURY, as reported in previous studies. (H) Representative western blotting analysis using lysates from WT, Gata4 -/- or Gata6 -/- lines for expression of SOX17 and EOMES shows that both proteins fail to accumulate in cells lacking GATA6 at day 5 of differentiation using 75 ng/ml Activin A. (I) Gene expression analysis by RT-qPCR shows a failure to down-regulate T/Bra at day 5 in cells lacking GATA6 during differentiation using 75 ng/ml Activin A. (J) Western blotting analysis using lysates from WT, Gata4 -/- or Gata6 -/- lines for expression of OCT4, GATA4, or BRA confirms accumulation of BRA in cells lacking GATA6 at day 5 of differentiation using 75 ng/ml Activin A. Data in B-F and I are presented as mean ± SEM. Statistical significance was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. To assess how GATA factors promote the transition to DE while repressing ME fate, the temporal expression levels of key DE and ME transcription factors were analyzed following the Dox-induced expression of Gata6 . Eomes is the earliest DE transcription factor upregulated by 6 hr following Gata6 induction ( Figure 3E ). In contrast, T/Bra is significantly downregulated as early as six hours after Gata6 induction ( Figure 3F ). GSEA analysis of RNAseq data using gene sets regulated by EOMES and BRACHYURY, as reported in previous studies ( Faial et al., 2015 ; Harland et al., 2021 ; Schüle et al., 2023 ; Tsaytler et al., 2023 ) revealed that gene signatures positively regulated by EOMES are generally upregulated upon Gata6 induction, whereas those positively regulated by BRACHYURY are down-regulated ( Figure 3G ). Moreover, genes negatively regulated by EOMES showed decreased expression levels following Gata6 induction, while those negatively regulated by BRACHYURY were upregulated. The expression levels of Eomes and T/ Bra were next measured during directed specification of DE in cells lacking Gata6 . The qPCR analysis revealed that Eomes transcript levels decrease at day 4-5 of Activin-induced DE differentiation in Gata6 -/- cells, and western blotting assays confirmed that EOMES protein fails to accumulate at the same time ( Figure 2E and 3H ). In contrast, qPCR and western blotting analyses demonstrated that T/Bra expression levels are increased and maintained at higher levels in Gata6 -/- cells during differentiation toward DE ( Figure 3I and 3J ), indicating that Gata6 is necessary for the downregulation of T/Bra during DE specification. Collectively, these results strongly indicate that GATA6 promotes specification into DE fate predominantly through activation of the TGFβ/NODAL endodermal regulatory network via activating expression of Eomes . Concurrently, GATA6 inhibits the mesodermal gene regulatory network by suppressing T/Bra expression. GATA Factors Negatively Regulate WNT Signaling and Grant WNT Independence to Endoderm Progenitors During Endoderm Specification GSEA analysis and GO term and pathway enrichment analysis of differentially down-regulated genes following Dox-induced Gata4/6 expression revealed WNT/β-catenin signaling, an essential regulator of ME formation ( Arnold and Robertson, 2009 ), as one of the main pathways down-regulated by the expression of Gata6 ( Figure 4A ) or Gata4 ( Figure S4A ). As T/Bra is a WNT target and WNT signaling in concert with T box factors (including T/Bra and Tbx6 ) regulate ME development ( Arnold et al., 2000 ; Hofmann et al., 2004 ; Mariani et al., 2021 ), we hypothesized that GATA factors modulate WNT signaling during ME and DE lineage choice. Axin2 is widely used as a readout for the activity of the canonical WNT pathway ( Jho et al., 2002 ), and we confirmed that Axin2 transcript levels are reduced following Dox-induced expression of either Gata6 ( Figure 4B ) or Gata4 ( Figure S4B ). In contrast, transcript levels of Axin2 were maintained abnormally high in Gata6 -/- cells during the transition to DE and beyond, indicating that Gata6 function is necessary to regulate WNT signaling activity during DE specification ( Figure 4C and S4C ). To better understand the activity of the WNT pathway in this in vitro system, the expression level of Axin 2 at different concentrations of Activin A was evaluated from Day 2, when Activin A was added, to Day 5, when DE specification was complete. Activity of the pathway (based on Axin2 transcript levels) increases sharply in the first 24 hr but is only maintained at Activin concentrations below 25 ng/ml ( Figure 4D ). Interestingly, with Activin A at concentrations that can induce DE progenitors (25 ng/ml and above), the peak of activity at day 3 is followed by a sharp decrease in the next 24 hr when transcript levels of Axin2 are significantly lower compared to lower concentrations of Activin A conditions ( Figure 4D and S4D ). This suggests that WNT signaling activity is quickly down-regulated during the transition of mesendoderm to DE, which is in agreement with recent results with human ESCs showing that WNT signaling is required initially for DE induction, yet cells already specified to DE fate become independent of external WNT activation ( Pour et al., 2022 ). Download figure Open in new tab Figure 4. GATA6 negatively regulates WNT signaling and grants WNT independence during DE specification. (A) GO term and pathway enrichment analysis of differentially down-regulated genes at day 4 following Dox-induced Gata6 expression shows WNT/β-catenin signaling as one of the main down-regulated pathways. (B) Gene expression analysis by RT-qPCR shows down-regulation of the canonical WNT direct target gene Axin2 at day 4 in cells induced to express Gata6 during differentiation using 10 ng/ml Activin A. (C) Gene expression analysis by RT-qPCR to measure relative Axin2 transcript levels at indicated time points in cells lacking GATA6 compared to WT cells during differentiation using 75 ng/ml Activin A. Statistical analysis is reported in Figure S4C . (D) Gene expression analysis by RT-qPCR at indicated time points to measure relative Axin2 transcript levels in WT cells induced by different concentrations of Activin A. Statistical analysis is reported in Figure S4D . (E) WT cells were induced at day 2 to promote DE specification with 75 ng/ml Activin A and then inhibited for WNT signaling with XAV939 (XAV) at day 3 or day 4. Flow cytometry was used to quantify DE specification compared to control vehicle-treated (DMSO) samples. (F) Analysis for DE specification at day 5 was carried out as in (E) except with (+Dox) or without (-Dox) induction at day 3 of Gata6 expression in either 10 ng/ml (top) or 75 ng/ml Activin A (bottom). (G) Analysis for DE specification at day 5 was carried out as in (F) except with addition at day 3 of 0, 3, 6, or 9 uM of the WNT pathway activator CHIR99021 (CHIR). (H) GSEA of RNA-seq data at day 4 comparing Dox-induced cells versus DMSO control (green) and Dox-induced cells + 6 μM CHIR versus DMSO control (purple) using endoderm-related gene sets from embryo single-cell RNA-seq experiments. Cells were differentiated with 10 ng/ml Activin A. (I) Heat maps for a similar comparison as in (H) using a definitive endoderm signature gene set ( Pijuan-Sala et al., 2019 ). n ≥ 3 biologically independent experiments. Data in B-G are presented as mean ± SEM. Statistical significance in B was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Statistical significance in E was evaluated with one-way ANOVA followed by Dunnett’s test for multiple comparisons relative to WT. Statistical significance in F was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. To confirm the time dependency of WNT signaling in DE specification at 75 ng/ml Activin A, WNT signaling was inhibited on either Day 3 or Day 4 during differentiation before DE progenitors emerge ( Figure 1H , WT cells). Inhibition of WNT signaling by adding the XAV939 (XAV) compound on Day 3 but not on Day 4 represses DE specification ( Figure 4E ). To test the hypothesis that GATA factors repress WNT and thereby program progenitors to be WNT independent, Gata6 was induced with Dox in cells that were simultaneously inhibited for WNT with XAV at Day 3. Strikingly, induction of Gata6 expression at either low or high concentrations of Activin A rescues DE specification when WNT is inhibited, indicating that progenitors already expressing Gata6 no longer depend on WNT signaling ( Figure 4F ). The qPCR analysis of Axin2 transcript levels confirmed the inhibitory effect of XAV in both -Dox and +Dox conditions ( Figure S4E ). A similar rescue of DE fate caused by WNT inhibition is observed with the induction of Gata4 ( Figure S4F ). To test if the ability to repress WNT is necessary for the specification of DE by GATA factors, activation of the WNT pathway was enforced with increasing doses of CHIR. DE progenitor specification using a high dose of Activin A without Dox is inhibited by concentrations of CHIR as low as 1 μM. In contrast, with Dox-induction of Gata6 ( Figure 4G and S5D ), at least 6 μM CHIR is required to inhibit DE specification significantly. The qPCR analysis of Axin2 levels indicates that cells respond to the activation of the pathway but require a higher concentration of CHIR to achieve the same level of Axin2 expression compared to cells not induced by Dox ( Figure S4G ). RNAseq analysis confirmed down-regulation of the DE gene regulatory network directed by GATA6 after adding 6 μM CHIR ( Figure 4H and 4I ). Taken together, these results show that GATA6 negatively regulates WNT signaling as progenitors exit the mesendoderm state towards DE fate, and GATA6 expression renders these progenitors WNT independent during commitment to the DE lineage. Gata6 Regulation of WNT Signaling Transcriptional Output is a Key for Mesoderm Subtype differentiation Like DE, ME development requires signaling from multiple pathways, including NODAL and WNT/β-Catenin ( Salehin et al., 2022 ; Stemple, 2001 ). Because activation of WNT signaling, one of the main drivers of ME development ( Cadigan and Nusse, 1997 ; Dunty et al., 2008 ; Galceran et al., 1999 ), repressed DE specification ( Figure 4G ), we tested if DE fate was being changed to ME by the activation of WNT. In contrast to DE, there is no single marker or combination of markers to distinguish all ME progenitors. Thus, we quantified the expression of DLL1, FLK1, and PDGFRα. DLL1 is a marker of paraxial ME ( Bettenhausen et al., 1995 ); FLK1 is a marker of lateral plate, cardiovascular, and hematopoietic ME ( Kataoka et al., 1997 ; Yamaguchi et al., 1993 ); and PDGFRα is a marker for a broad range of ME types, including early paraxial and lateral plate, cardiovascular progenitors, and skeletal ME progenitors ( Kataoka et al., 1997 ; Loh et al., 2016 ; Orr-Urtreger et al., 1992 ). Using these markers, we defined two ME subtypes: DLL1-positive ME (DLL1+/PDGFRα+ and DLL1+/PDGFRα-) or DLL1-negative (non-DLL) ME (PDGFRα+/FLK1+, PDGFRα+/FLK1- and PDGFRα-/FLK1+). In all our experiments, no significant amount of DLL1+/FLK1+ cells were found. Using Activin A alone, we found that 15-20% of DLL1+ or PDGFRα+ ME cells are produced at low or high concentrations of Activin A, respectively, reflecting different subtypes induced in the primitive streak ( Figure S5A ). Results for each subtype in all different conditions in which Activin A, WNT, and Gata6 are manipulated are found in Figure S5C . After adding CHIR in -Dox conditions, low doses of Activin A efficiently induce DLL1+ progenitors, whereas high concentrations of Activin A are required to generate non-DLL1 progenitors ( Figures 5A and 5B ). Additionally, DLL1+ progenitors are generated in a CHIR dose-dependent manner, whereas non-DLL1 progenitors are induced by 1 or 3 μM CHIR but not by 6 or 9 μM. Importantly, in cultures without Dox, endogenous Gata6 expression level is associated with concentrations of Activin and CHIR that generate a high yield of DE or non-DLL1 progenitors ( Figure S5B ). Strikingly, after Dox-induction of Gata6 , DLL1+ ME is largely abrogated, with only a small percentage generated at 9 μM CHIR in low-Activin A conditions ( Figure 5A, 5B , and S5C ). Furthermore, at high concentrations of Activin A, Dox-induction of Gata6 completely blocks the generation of DLL1+ ME and results in progenitors that are FLK1+ (positive or negative for PDGFRα) ( Figure 5B and S5C ). The qPCR analysis further confirmed that WNT activation (using CHIR) in the absence of Gata6 expression (-Dox conditions) strongly down-regulates DE markers and upregulates paraxial ME markers ( Figure S5D ). In contrast, dose-dependent WNT activation and Dox-induced Gata6 expression strongly increase levels for markers of lateral plate ME while repressing paraxial ME marker expression levels, indicating that ME progenitors directed by Gata6 resemble lateral plate ME at the expense of paraxial ME ( Figure S5D ). Download figure Open in new tab Figure 5. GATA6 cooperates with WNT signaling to specify mesoderm sub-type. (A) Flow cytometry using antibodies to quantify DLL1 subtype mesoderm (DLL1+ regardless of PDGFRα expression) or non-DLL subtype of mesoderm (combining PDGFRα+/FLK1+, PDGFRα+/FLK1-, and PDGFRα-/FLK+) with cells induced (+Dox) or uninduced (-Dox) for Gata6 expression and cultured in 10 ng/ml Activin A with 0, 1, 3, 6, or 9 μM CHIR to modulate the WNT signaling pathway. Samples were analyzed at day 4.75. (B) Flow cytometry quantification for specification of mesoderm sub-type as in (A) except with culture in the presence of 75 ng/ml Activin A. (C) Volcano plot mapping changes in transcript levels at day 4 comparing RNA-seq of cells induced to express Gata6 and exposed to DMSO or 6 μM CHIR at day 3. Note WNT-mediated down-regulation of DE markers and up-regulation of mesoderm markers. (D) Volcano plot mapping changes in transcript levels at day 4 comparing RNA-seq of cells exposed to 6 μM CHIR that were (+Dox) or were not (-Dox) induced to express Gata6 . Note Gata6 -mediated up-regulation of lateral plate mesoderm and down-regulation of paraxial and somitic mesoderm markers. (E) Comparison of the RNA-seq data from the samples described in (D) for the gene sets shown to be co-regulated by Activin and WNT signaling by Estaras et al., 2015. The pie chart shows that 90/117 (37 genes were not expressed or had less than 30 base mean counts) are significantly changed for expression levels by induction of Gata6 and these patterns are illustrated by heat maps on the right. (F) Flow cytometry was used to quantify relative levels of non-DLL1 (top) or DLL1+ mesoderm sub-types at day 4 comparing WT or Gata6 -/- cells cultured in 75 ng/ml Activin A and exposed at day 3 to 0 (DMSO), 3 or 6 μM CHIR (CH3, CH6, respectively). (G) Gene expression analysis by RT-qPCR to measure relative transcript levels at day 4 of representative paraxial (top) or lateral (bottom) mesoderm markers for WT or Gata6 -/- cells cultured as in (F). Data in F and G are presented as mean ± SEM. Statistical significance in F and G was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons Comparison of the transcriptome of cells under +Dox/6 μM CHIR or +Dox/DMSO conditions confirms the WNT-mediated down-regulation of DE markers and the upregulation of ME markers ( Figure 5C ). Additionally, analysis of cells in +Dox/6 μM CHIR and -Dox/6μM CHIR reveals that Dox-induced expression of Gata6 , when WNT is activated, upregulates markers associated with the lateral plate ME ( Figure 5D ). These markers include Eomes , Mixl1 , and FoxH1 , which are known to drive a highly conserved regulatory program for lateral plate ME ( Prummel et al., 2019 ). Importantly, consistent with the flow cytometry data, Gata6 down-regulates the expression levels of several drivers and markers of paraxial ME ( Tbx6 , Msgn1 , FoxC2 , Lfng , and Aldh1A2 ), which are otherwise enhanced by WNT activation ( Figure 5D ). GSEA analysis also reveals upregulation of multiple gene signatures associated with lateral plate ME and its derivatives following Dox-induced expression of Gata6 and WNT stimulation. In contrast, several paraxial and somitic ME signatures are down-regulated ( Figure S5E ). Analysis of previously categorized WNT target gene sets demonstrates the significant regulatory role of Gata6 in WNT-controlled networks, as evidenced by extensive transcriptional changes in genes either directly induced or repressed by WNT ( Zhang et al., 2013 ) ( Figure S5F and S5G ). Furthermore, the expression analysis of 84 components of the WNT pathway, including ligands, receptors, effectors, positive and negative regulators, co-activators, and co-repressors, revealed that 54 of these components (64.3%) exhibited significant changes following Gata6 expression ( Figure S5H ). These changes include the upregulation of Fzd7 and the downregulation of Fzd2 , genes shown to direct lateral or paraxial ME differentiation, respectively ( Chidiac et al., 2025 ). Notably, while the induction of WNT direct targets by Gata6 is associated with the induction of lateral plate ME but not DE ( Figure S5I ), the simultaneous induction of both DE and lateral plate ME by Gata6 correlates with increased expression of Nodal and its direct targets ( Figure S5J ). Additionally, Gata6 regulates the expression of genes co-regulated by Activin A and WNT ( Estarás et al., 2015 ) ( Figure 5E ). Together, these results indicate that the role of Gata6 in ME patterning arises from its simultaneous transcriptional regulation of WNT and NODAL signaling pathways. The dual addition of CHIR and XAV has been shown to inhibit the nuclear transcriptional activity of β-Catenin while preserving its cytoplasmic functions ( Kim et al., 2013 ). Using this combination, we found that ME specification, as well as transcription of Axin2 and ME genes, are down-regulated in cells with or without Dox-induction of Gata6 ( Figure S6A-S6D ), indicating that ME generation of cells expressing Gata6 is also dependent on the transcriptional activity of β-Catenin. In addition, adding XAV with CHIR restored the DE population induced by Gata6 , further confirming that Gata6 renders DE progenitors independent of WNT signaling ( Figure S6E ). Taken together, these results indicate that expression of Gata6 not only represses the WNT canonical pathway but also modifies its transcriptional output when WNT signaling activity is increased. We showed that WNT signaling activity in the absence of Gata6 persists at a high level during Activin-induced DE differentiation, as indicated by the expression levels of Axin2 ( Figure 4C ). To examine the response to ME induction by WNT stimulation in the absence of Gata6 , we analyzed the response of Gata6 -/- cells to CHIR. As expected, based on our results with WT cells, increasing concentrations of CHIR resulted in a decrease in DE specification and an increase in ME progenitors ( Figure S6F ). In addition, the higher the CHIR concentration, the higher the yield of paraxial ME ( Figure S6F ). Notably, the type of ME progenitor generated by 3 μM or 6 μM CHIR differs between WT and Gata6 -/- cells. While DLL1+ ME is significantly increased in Gata6 -/- cells at 3 μM or 6 μM CHIR, non-DLL1 ME is significantly decreased, suggesting that WNT activation in the absence of Gata6 biases fate toward paraxial ME ( Figure 5F and S6G ). Analysis of paraxial and lateral ME markers by qPCR confirmed the upregulation of markers of paraxial ME and down-regulation of markers associated with lateral plate ME in Gata6 -/- cells after WNT activation ( Figure 5G ). These results show that Gata6 regulates WNT signaling during ME specification to promote lateral versus paraxial ME fate. Gata6 Regulates Eomes and T/ Bra Expression during both Endoderm and Mesoderm Specification Although expression of Eomes and T / Bra overlaps initially in the primitive streak during mesendoderm formation, during lineage specification, Eomes antagonizes T /Bra to ensure the specification of DE and anterior ME ( Schüle et al., 2023 ). Subsequently, the lack of expression of Eomes in more posterior regions allows T/Bra to drive axial, paraxial, and other posterior ME fates ( Schüle et al., 2023 ). As Gata4/6 expression induced a rapid increase in Eomes expression and decrease in T / Bra expression and modulates the gene networks regulated by both transcription factors ( Figure 3D-G , S3D , and S3G ), we hypothesized that Gata6 regulates lineage segregation in cooperation with NODAL and WNT signaling through regulation of Eomes and T / Bra . The expression levels of Eomes and T / Bra were therefore evaluated by qPCR under conditions that induce DE (+Dox/DMSO), paraxial ME (-Dox/6 μM CHIR), or lateral ME (+Dox/6μM CHIR) ( Figure 4G , 5A-B , and S5C-D ). Increased expression levels of Eomes are associated with conditions that generate DE or lateral plate ME, while increased expression level of T/Bra is associated with both paraxial and lateral ME ( Figure 6A and 6B ). Notably, paraxial ME is associated with a faster and higher increase in T/Bra expression levels that subsided by 24 hr, at which point the expression level is similar as seen for lateral plate ME. In addition, we used flow cytometry to evaluate the effect of Dox-induced Gata6 expression on BRACHYURY expression after WNT activation with CHIR at 3 μM or 6 μM. In cells without Dox, 3 μM or 6 μM CHIR generates paraxial ME ( Figure 5A-B and S5C-D ), and BRACHYURY expression is induced to similar levels ( Figure S7A ). In contrast, in +Dox conditions, BRACHYURY expression reaches similar levels to -Dox cells only in the presence of 6 μM CHIR (which in these conditions induces lateral plate-like ME) but not at 3 μM ( Figure S7A ), conditions in which Gata6 instead promotes DE progenitors ( Figure 4G , 5A-B , and S5C-D ). This further confirms that Gata6 inhibition of T/Bra is associated with the capacity to direct specification of DE. Download figure Open in new tab Figure 6. Gata6 regulates both Eomes and T / Bra during endoderm and mesoderm specification. (A, B) Gene expression analysis by RT-qPCR at indicated time points to measure relative levels of Eomes (A) or T/Bra (B) in cells cultured in 10 ng/ml Activin A and induced by Gata6 to specify DE (+Dox/DMSO), paraxial ME (-Dox/6 μM CHIR, CH), or lateral ME (+Dox/6 μM CHIR, CH), compared to those uninduced for Gata6 or WNT (- Dox, DMSO). (C-E) Quantification by flow cytometry for the percentage of double-positive EOMES+/BRACHYURY+ cells (C), single-positive EOMES+ cells (D) and single-positive BRACHURY+ cells (E) at indicated time points following induction for Gata6 expression at day 3 with Dox (or controls without Dox) and addition of either DMSO or 6 μM CHIR. MFI for each marker in positive populations is shown in associated graphs at day 3.5 (C) or day 4 (D, E). (F) Quantification by flow cytometry at day 4 as described for (C-E) except with or without addition of the WNT inhibitor XAV at day 3. Color of the bar represents predominant fate generated by each condition: endoderm (red), lateral mesoderm (green) and paraxial mesoderm (purple). White represents undefined lineage. (G, H) Heat maps based on RNA-seq at day 4 following addition of CHIR to activate WNT signaling in the absence (-Dox) or presence (+Dox) of Dox to induce expression of Gata6 compared to Dox-induction of Gata6 alone (+Dox, DMSO) evaluating the expression of genes positively regulated by BRACHYURY (G) and EOMES (H) as defined previously ( Tsaytler et al., 2023 ) (I, J) Flow cytometry to quantify EOMES+ and BRACHYURY+ cells at day 4 or day 5 in WT or Gata6 -/- cells induced for DE specification with 75 ng/ml Activin A. Quantification is provided for EOMES+ High /BRA+ Low (I) or EOMES+ Low /BRA+ High (J). (K, L) Flow cytometry as in (I, J) except including 0 (DMSO) or 0.5, 1, 3, or 6 μM CHIR (CH). Cells were analyzed at day 4 (L) and day 5 (K). n ≥ 3 biologically independent experiments. Data in A-F and I-L are presented as mean ± SEM. Statistical significance in I and J was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Statistical significance in B-F and K-L was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons and significance for all these tests and comparisons is found in Table S3 . View this table: View inline View popup Table S1. RT-qPCR primer sequences used in the study View this table: View inline View popup Table S2. Gene sets compiled from literature for GSEA analysis and heatmaps View this table: View inline View popup Table S3. Statistical analysis for Figure 6. We also evaluated the co-expression of BRACHYURY and EOMES by flow cytometry. At day 3, before Dox-induced Gata6 expression or CHIR addition, approximately 50% of cells co-express BRACHYURY and EOMES. Without Dox or CHIR, these progenitors transition to a double negative population by Day 4 ( Figure 6C and S7B ). 12 hr after Dox-induced Gata6 expression (without CHIR), the percentage of EOMES + /BRACHYURY + (E+/B+) double-positive cells is slightly reduced, and they display high levels of EOMES and low levels of BRACHYURY, as indicated by Median Fluorescence Intensity (MFI) ( Figure 6C and S7B ). 12 hr later (24 hr after Dox addition), most progenitors have transitioned to an EOMES + /BRACHYURY - (E+/B-) single positive population expressing even higher EOMES levels ( Figure 6D and S7B ). In contrast, in 6 μM CHIR conditions, there is an initial increase in the number of E+/B+ cells at 12 hr regardless of Dox that is maintained up to 24 hr only in cells induced with Dox to express Gata6 ( Figure 6C ). Of note, at 12 hr, the E+/B+ progenitors in -Dox/6 μM CHIR conditions have higher levels of BRACHYURY, while the same progenitors in +Dox/6 μM CHIR have lower levels ( Figure 6C and S7B ). By 24 hr, most progenitors in -Dox/6 μM CHIR conditions are EOMES - /BRACHYURY + (E-B+) single-positive cells expressing the highest BRACHYURY levels for any conditions tested ( Figure 6E ). In contrast, at 24 hr in +Dox/6 μM CHIR conditions, around 80% of the population expresses EOMES, with half also expressing BRACHYURY ( Figure 6C and 6D ). One important distinction is that at 24 hr, the E+/B-cells in +Dox/6 μM CHIR expressed lower levels of EOMES compared to E+/B-cells in +Dox/DMSO conditions ( Figure 6D and S7B ). Strikingly, when a WNT inhibitor is added, the E+/B+ population induced by +Dox/6 μM CHIR was replaced by an E+/B-population that expresses higher levels of EOMES ( Figure 6F and S7C ) and correlates with restoration of DE fate and repression of lateral ME ( Figure S6C-S6E ). Similarly, WNT inhibition in the -Dox/CHIR 6 μM conditions results in a significant decrease in the number of E-/B+ cells and reduced BRACHYURY MFI ( Figure 6F and S7D ), which correlates with the loss of paraxial ME specification observed ( Figure S6B-S6D ). Next, we compared the expression of positively regulated targets of EOMES and BRACHYURY in different ME types that were previously described ( Faial et al., 2015 ; Harland et al., 2021 ; Koch et al., 2017 ; Tsaytler et al., 2023 ). WNT activation alone enhances the expression levels of multiple well-known drivers and markers of paraxial ME ( Wnt3a Msgn1 , Cdx2 , Alh1A2 , Dll1 ). In contrast, WNT activation along with Dox-induced expression of Gata6 represses the expression of these targets and activates instead the expression of the lateral plate ME markers Bmp4 , Hand1 , Tbx3 , FoxF1 , and Mesp1 ( Figure 6G , 6H and S7E-S7G ). These data indicate that Gata6 promotes Eomes expression and represses T/Bra expression to specify DE. In contrast, WNT activation plays an opposing role, promoting T/Bra expression and repressing Eomes to drive the generation of paraxial ME. Furthermore, simultaneous activation of Gata6 expression with WNT activation promotes Eomes and T/Bra co-expression to promote lateral-like ME fate. Finally, to explore if this represents a normal function of Gata6 , we compared the dynamics of EOMES and BRACHYURY expression from Day 2 to Day 5 during DE specification with a high dose of Activin A in the presence or absence of endogenous GATA6. In WT cells, EOMES expression initiates around Day 3 and increases to generate a population with high levels of EOMES and low levels of BRACHYURY (E+ High /B+ Low) maintained up to Day 5 ( Figure 6I and S8A ). In contrast, although the number of E+ High /B+ Low progenitors is not significantly different at day 4, a significantly lower number of E+ High /B+ Low progenitors are derived by Gata6 -/- cells at Day 5 ( Figure 6I ad S8A), indicating a failure to maintain the expression of Eomes in the absence of GATA6 during the transition to DE fate. Furthermore, a distinct population expressing high levels of BRACHYURY and low levels of EOMES (E+ Low / B+ High ) was increased in the absence of GATA6 ( Figure 6J and S8A ). While the percentage of E+ High /B+ Low cells on day 5 corresponds roughly to the same number of DE progenitors evaluated simultaneously in the experiment (DMSO in Figure S8E ), the number of E+ Low / B+ High cells on Day 4 or Day 5 does not correlate to the number of ME progenitors ( Figure S8B ). Despite this, Gata6 -/- cells display an increase in the expression of multiple ME markers, most of which are characteristic of paraxial ME ( Figure S8C ). Although the level of expression of ME markers is low when compared to conditions that directly specify ME ( Figure 5G ), the observation suggests that misregulation of T/Bra in the absence of GATA6 may underlie the differentiation bias of Gata6 -/- cells toward paraxial ME fate in response to WNT activation ( Figure 5F , 5G and S6F-G ). To test this, we evaluated the levels of EOMES and BRACHYURY in Gata6 -/- cells in response to WNT activation with CHIR. Increasing concentrations of CHIR are associated with a decrease in the E+ High /B+ Low population with a concomitant increase in the E+ Low / B+ High population ( Figure S8D ). The number of E+ High /B+ Low cells peaks at Day 5 and correlates with the number of DE progenitors that decrease as the CHIR concentration increases ( Figure S8E ). In contrast, the E+ Low / B+ High population, which peaks at Day 4, correlates with the amount of ME generated ( Figure S8F ). Compared to WT, Gata6 -/- cells generate a significantly lower percentage of E+ High /B+ Low cells at all CHIR concentrations ( Figure 6K and S8E ). On the other hand, CHIR induces a higher percentage of E+ Low / B+ High cells at Day 4 in Gata6 -/- cells correlating with the increased percentage of paraxial ME cells ( Figure 5F , 6L , and S8F ), demonstrating that the absence of GATA6 enhances the expression of T/Bra in response to WNT activation. Altogether, our results indicate that during DE specification, GATA6 is necessary for the upregulation and maintenance of Eomes and the downregulation of T/Bra . Furthermore, defective regulation of T/Bra in the absence of GATA6 is associated with a loss of lateral mesoderm capacity and a differentiation bias toward paraxial ME specification. DISCUSSION The wide cellular diversity of a developing triplobastic organism arises from the three primary germ layers specified at the onset of gastrulation when the NODAL, WNT, and BMP signaling pathways converge to induce the PS ( Tam and Loebel, 2007 ). During PS induction, these signaling pathways define a transient mesendoderm stage that will segregate to form the DE and ME lineages. How these mesendoderm progenitors developing in close spatiotemporal proximity interpret the same signaling cues to segregate into multiple fates remains a fundamental but unresolved question in early development. Using Gata6 -inducible mouse embryonic stem cells, we demonstrate that Gata6 expression during mesendoderm exit drives the DE GRN while simultaneously repressing the ME gene network regulated by WNT signaling. Unexpectedly, we found that upon activation of the WNT signaling pathway, GATA6 drives transcriptional changes in the expression of multiple components of the WNT pathway and a switch in the transcriptional output to promote lateral plate ME fate at the expense of paraxial ME fate. The different fate outcomes are correlated with the regulation of Eomes and T/Bra by GATA6 and WNT such that in DE, GATA6 upregulates EOMES and downregulates T expression and WNT signaling; in lateral plate ME, Gata6 upregulates Eomes and WNT upregulates T ; and in posterior ME high WNT activity over-rides GATA6 and NODAL. Furthermore, we found that the absence of GATA6 results in a failure to deploy the DE GRN and produces a bias in ME differentiation towards paraxial ME. Thus, we define a new role for Gata6 in the dual regulation of the transition from mesendoderm to both DE and ME, which helps explain the pleiotropic impact of patients with GATA6 mutations ( Škorić-Milosavljević et al., 2019 ; Tuhan et al., 2015 ; Yasuhara et al., 2024 ). Our study suggests that loss of Gata6 disrupts a positive feedback loop between EOMES and GATA6 that is integral to both DE and ME GRNs. Furthermore, our evidence indicates that GATA6 mediates some functions previously attributed to EOMES. Two distinct functions have been proposed for EOMES early in development. The initial activity of EOMES during exit from pluripotency and entry into the mesendoderm stage does not require transcriptional activity and involves de novo establishment of the accessible mesendoderm enhancer landscape ( Schröder et al., 2023 ; Schüle et al., 2023 ). A subsequent lineage specification role for EOMES relies on transcriptional activity regulated by NODAL and WNT and is co-regulated by GATA6 ( Chia et al., 2019 ; Schüle et al., 2023 ). Additionally, GATA6 and EOMES were found to interact and co-regulate genes required for precardiac ME patterning in the anterior lateral ME ( Bisson et al., 2024 ). Using an Activin differentiation protocol similar to the one used in our study, it was shown that EOMES indirectly represses T/Bra to drive anterior fates ( Schüle et al., 2023 ) through additional unidentified factors ( Schüle et al., 2023 ). Our finding that Gata6 is essential for maintaining Eomes expression and downregulating T/Bra to progress through the mesendoderm stage helps explain this proposed EOMES function. The role of GATA6 in T/Bra downregulation and ME repression during DE differentiation is also supported by recent reports that GATA6 promotes DE lineage commitment in part by restricting accessibility at enhancers linked to alternative lineages ( Heslop et al., 2022 ). It has been proposed that NODAL initially induces both DE and ME genes, while both lineages are segregated by mutually repressive gene networks ( Zorn and Wells, 2009 ). Our findings about the role of Gata6 in DE and ME differentiation enhance our understanding of how the NODAL and WNT signaling pathways interact to drive the networks that segregate lineage fate. During DE specification, Gata6 reinforces NODAL signaling to maintain and enhance Eomes expression and create a positive feedback loop that contributes to the deployment of the full DE GRN. Additionally, GATA6 negatively regulates WNT to repress the ME GRN. Furthermore, Gata6 expression marks the point of WNT inhibition independence in DE differentiation, which was proposed to occur when mesendoderm progenitors downregulate T/Bra before the overt expression of Sox17 in DE progenitors ( Pour et al., 2022 ). During mesendoderm exit toward ME fate, high WNT activity represses the expression of Nodal and its targets, including Eomes and Gata6 . This repression in turn releases inhibition of T/Bra expression, as has been shown in vivo ( Schüle et al., 2023 ), leading to establishment of the paraxial ME GRN ( Mariani et al., 2021 ). Our inducible ESC model allowed us to show that lateral plate ME is favored by sustained activity of both NODAL/GATA6 and WNT, which maintains the expression of Eomes and T/Bra , respectively. We envision that EOMES cooperates with GATA6 and BRACHYURY to modify the transcriptional outputs of NODAL and WNT and deploy the lateral plate ME GRN, including Mesp1 , Hand1 , Tbx3 , Isl1 , and FoxF1 . This mechanism is supported by the extensive literature documenting the cooperation of BRACHYURY and EOMES, GATA6 and EOMES and all these transcription factors with SMAD effectors of the NODAL signaling pathway ( Bisson et al., 2024 ; Chia et al., 2019 ; Faial et al., 2015 ; Kumar et al., 2024 ; Liu et al., 2011 ; Prummel et al., 2019 ; Schüle et al., 2023 ; Slagle et al., 2011 ; Teo et al., 2011 ; Tosic et al., 2019 ; Tsaytler et al., 2023 ). During subsequent organogenesis, regulation of the WNT pathway by Gata6 has been described in heart and lung development ( Afouda et al., 2008 ; Alexandrovich et al., 2006 ; Bisson et al., 2024 ; Lee and Evans, 2019 ; Novikov and Evans, 2013 ; Tian et al., 2010 ; Weidenfeld et al., 2002 ; Zhang et al., 2008 ). During lung development and airway regeneration after injury, loss of Gata6 leads to an increase in canonical WNT signaling, impairing the balance between progenitor expansion and epithelial differentiation ( Zhang et al., 2008 ). Additionally, regulation of WNT signaling by Gata6 has emerged as an important factor promoting pancreatic and colon cancer, and Gata6 is a critical contributor to WNT independence in a pathway involved in the switch from classical subtype to basal-like/squamous subtype of pancreatic cancer ( Seino et al., 2018 ; Tsuji et al., 2014 ; Whissell et al., 2014 ; Zhong et al., 2011 , 2022 ). Recapitulation of critical developmental pathways in the regulation of progenitor cell expansion is required for tissue regeneration and is altered in tumorigenesis; thus, our findings that Gata6 is a regulator of fate through modulation of WNT signaling helps explain how Gata6 may also regulate similar processes in regeneration and disease. EXPERIMENTAL PROCEDURES Detailed methods can be found in the supplemental information and are briefly described below. Cell lines, culture, and differentiation Mouse embryonic stem cell lines engineered to allow conditional expression of Gata4 or Gata6 were maintained as previously described ( Turbendian et al., 2013 ). ESCs were dissociated and aggregated for differentiation as embryoid bodies (EBs) as previously described ( Gouon-Evans et al., 2006 ). The Gata4-inducible mesendoderm reporter was generated by targeting the Gata4 cDNA into the tet-regulated promoter locus of the AinV/GFP-Bry/CD4-Foxa2 ESC line as previously described ( Cheng et al., 2008 ; Holtzinger et al., 2010 ). For liver differentiation and mid-hindgut differentiation, day 5 EBs were dissociated with Accutase and plated at a ratio of 1:2, and differentiation was performed as previously described ( Gouon-Evans et al., 2006 ; Loh et al., 2014) with modifications as detailed in the supplemental information. RNA isolation, RT-qPCR analysis, and Bulk RNA sequencing Detailed methods for processing and analysis methods, as well as primer sequences, can be found in supplemental information and Table S1 . Flow cytometry For extracellular markers, EBs were harvested, dissociated, and stained with antibodies before being stained with Sytox blue for viability. For intracellular antigens, cells were fixed with 2% PFA before staining with antibodies. Samples were analyzed on an Attune Nxt (Thermofisher). See supplemental information for details on the staining and antibodies used. Western blotting Proteins were extracted using RIPA buffer supplemented with 1x protease and phosphatase inhibitors (Pierce), and benzonase (Millipore). Lysates were centrifuged at 15,000 rpm for 5 min at 4°C. Total protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce 23225). See supplemental methods for more details. Quantification and statistical analysis All data is presented as mean ± SEM of independent biological replicates with n indicated in each figure legend. GraphPad Prism software was used for statistical analysis with two-tailed Student’s t -test when comparing two groups and one-way or two-way ANOVA when comparing three or more groups, as indicated in figure legends. Statistically significant p values were indicated in figures as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Non-significant comparisons are not indicated in figures. RESOURCE AVAILABILITY Lead contact Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Todd Evans ( tre2003{at}med.cornell.edu ). Materials availability All unique/stable reagents generated in this study are available from the lead contact. Data and code availability The accession number for the RNA-seq data reported in this paper is deposited at the Gene Expression Omnibus (GEO) Database: GSEXXXXX. AUTHOR CONTRIBUTIONS MG designed and performed the experiments, analyzed the data, and wrote the manuscript. RW and NdS performed experiments, KB helped analyze data. TE contributed with funding acquisition, experimental design, interpretation of the data, and wrote the manuscript. DECLARATION OF INTERESTS TE is a co-founder of OncoBeat, LLC. Supplemental Figures and Text Download figure Open in new tab Figure S1. Expression of GATA4 or GATA6 can drive murine definitive endoderm specification, and GATA6 is required. Related to Figure 1. (A) Schematic diagram of the definitive endoderm (DE) directed differentiation protocol. Embryoid bodies (EBs) were set up on day 0 and reaggregated on day 2 in the presence of 75 ng/ml Activin A. (B) Representative gating strategy for identification of progenitors after differentiation. ESCs cultured without doxycycline (in experiments with inducible cell lines) or WT cells (in experiments with GATA KO cells) at day 5 of differentiation were used to define the gating strategy used throughout all DE flow cytometry experiments. To exclude debris, cells were gated from all events by analyzing FSC-H and SSC-H. Then, cells were analyzed by FSC-A and FSC-H scatter to exclude doublets. After gating on singlets, live cells for analysis of surface markers were gated as the Sytox Blue negative population. (C) Flow cytometry to quantify DE specification at day 5 comparing cKIT+/CXCR4+ or EpCAM+/CXCR4+ populations at different Activin A concentrations. (D) RT-qPCR analysis for key DE markers at the indicated day (D) of differentiation using various concentrations of Activin A showing activation at and above 25 ng/ml. (E) GSEA analysis of the transcriptome from day 4 RNA-seq data comparing 10 or 75 ng/ml Activin A shows enrichment with 75 ng/ml Activin A of DE signatures derived from single-cell RNA-seq of mouse embryos. (F) Temporal expression analysis by RT-qPCR of key TFs of the DE Gene Regulatory Network at the indicated day of differentiation using 75 ng/ml Activin A showing 3 waves of activation around day 3 ( FoxA2 , Eomes , Mixl1 ), day 4 ( Gata6 , Sox17 , FoxH1 , Lhx1 ), and day 5 ( Hesx1 , Gata4 , Hhex , FoxA3 ). Data points represent the mean normalized to the maximum expression of each gene. (G) Flow cytometry analysis of DE progenitors at day 5 using the Gata4 -inducible ESC line and culture conditions in either 10 ng/ml or 75 ng/ml Activin A and either DMSO or Dox added at day 3. (H) RT-qPCR analysis for key DE markers at day 5 using the Gata4 -inducible ESC line cultured in 10 ng/ml Activin A and either DMSO or Dox added at day 3. (I) Gene list analysis of the Gata6 -induced transcriptome using Enrichr shows a high enrichment of genes associated with DE development. (J, K) Following induced expression of Gata4 , day 5 EBs were replated in hepatoblast (J) or mid-hindgut (K) differentiation conditions and evaluated by RT-qPCR analysis after 4 days (J) or 3 days (K) for expression of liver (J) or hindgut (K) markers. Statistical significance in H, J, and K was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Statistical significance in G was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. Download figure Open in new tab Figure S2. GATA6 reinforces the NODAL pathway. Related to Figure 2. (A, B) Enrichment analysis of the Gata6 -induced transcriptome using RNA-seq data highlights gene sets associated with the TGFβ/NODAL signaling pathway (A) and SMAD2/3/4 (B). (C) RT-qPCR analysis for known direct SMAD2/3/4 target genes at the indicated day of differentiation using various concentrations (ng/ml) of Activin A. (D) RT-qPCR analysis for cells cultured in 10 ng/ml Activin A collected 24h after they were induced (+Dox) or not (-Dox) for expression of Gata4 . Statistical significance was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. (E) RT-qPCR analysis at day 5 for key NODAL pathway regulators in WT or GATA mutant cells cultured in 75 ng/ml Activin A. Statistical significance was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. No statistically significant differences were found. (F) As quantified by flow cytometry at day 5, specification of DE by induction of Gata6 expression (in 75 ng/ml Activin A) requires active Activin signaling, as it is blocked by addition of the A83-01 ALK4/5/7 type I receptor inhibitor. Download figure Open in new tab Figure S3. GATA factors drive mesendoderm cells to definitive endoderm fate. Related to Figure 3. (A) GSEA using RNA-seq data of cells at day 4 with or without Dox-induced expression of Gata4 at low concentration of Activin A shows suppression of gene sets for mesoderm. (B) Gene expression analysis by RT-qPCR for key mesoderm drivers on day 4 of differentiation following Dox-induction of Gata4 and culture in 10 ng/ml Activin A. (C) Gene expression analysis by RT-qPCR at day 4 of differentiation following Dox-induction of Gata4 and culture in 10 ng/ml Activin A shows enhanced expression of FoxA2 and down-regulation of T/Bra . (D) Flow cytometry to quantify the percentage of cells expressing BRACHURY at defined time points shows Dox-induction of Gata4 expression reduces BRA+ cells. (E) Schematic illustrating of proposed lineage progression and expression of FOXA2 and BRACHYURY during the mesoderm and endoderm differentiation from mesendoderm progenitors (Gadue et al., 2006). (F, G) Flow cytometry results using the dual reporter line to follow BRA-GFP and FOXA2-CD4 expression when cultured in 10 ng/ml Activin A and induced (or not) with Dox at day 3 showing BRA-/FOXA2+ DE (F) and BRA+/FOXA2+ mesendoderm (G). Data are presented as mean ± SEM. Statistical significance in B and C was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Download figure Open in new tab Figure S4. GATA6 negatively regulates WNT signaling and grants WNT independence during DE specification. Related to Figures 3 and 4. (A) GO terms and pathway enrichment analysis of differentially regulated genes at day 4 following Dox-induced Gata4 expression shows WNT/β-catenin signaling as one of the main down-regulated pathways. (B) Gene expression analysis by RT-qPCR shows relative down-regulation of the canonical WNT early target gene Axin2 at day 4 in cells induced to express Gata4 during differentiation using 10 ng/ml Activin A. (C) Gene expression analysis by RT-qPCR to measure relative Axin2 transcript levels at indicated time points in cells lacking GATA4, GATA6, or both proteins compared to WT cells during differentiation using 75 ng/ml Activin A. Only GATA6 is required to repress Axin transcript levels. Related to Figure 3C . (D) Gene expression analysis by RT-qPCR at indicated time points to measure relative Axin2 transcript levels in WT cells induced by different concentrations of Activin A. Related to Figure 4D . (E) RT-qPCR analysis of Axin2 transcript levels confirms the inhibitory effect of XAV in both - Dox and +Dox conditions and at 10 or 75 ng/ml Activin A. (F) Flow cytometry was used to measure DE specification at day 5 showing that induction of Gata4 expression is sufficient to rescue DE fate from XAV-mediated WNT inhibition. (G) RT-qPCR analysis of Axin2 transcript levels indicates that cells induced to express Gata6 respond to the activation of the pathway but require a higher concentration of CHIR to achieve the same level of Axin2 expression compared to cells not induced by Dox. Data in B-G are presented as mean ± SEM. n ≥ 3 biologically independent experiments. Statistical significance in B and G was evaluated by unpaired, two-tailed Student’s t test for two-group comparisons. Statistical significance in C was evaluated with one-way ANOVA followed by Dunnett’s test for multiple comparisons relative to WT. Statistical significance in D-F was evaluated with two-way ANOVA followed by Tukey’s test for multiple comparisons. Download figure Open in new tab Figure S5. GATA6 cooperates with WNT signaling to specify mesoderm subtype. Related to Figure 5. (A) Quantification of flow cytometry at day 4.75 to measure the percentage of mesoderm sub-types based on PDGFRα, DLL1, and FLK1 expression, as impacted by the concentration of Activin A (5, 10, or 75 ng/ml). (B) WT cells were cultured in 10 ng/ml (top) or 75 ng/ml (bottom) Activin A and various concentrations of WNT activator CHIR99021, as indicated. Graphs chart the relative level of Gata6 expression (based on RT-qPCR) correlating with relative percent of DE, DLL1+ mesoderm, or non-DLL mesoderm (based on flow cytometry), showing that Gata6 levels are associated with Activin/CHIR that generates either a high yield of DE or non-DLL1 progenitors. Flow cytometry samples were collected at day 4.75 and RNA samples were collected at day 4. (C) Analysis of flow cytometry data at day 4.75 as in (B) for mesoderm sub-types (PDGFRα and FLK1, top; PDGFRα and DLL1, bottom) when cells were cultured with low (10 ng/ml) or high (75 ng/ml) Activin A plus increasing concentrations of CHIR and either treated at day 3 with DMSO (-Dox) or with Dox to induce expression of Gata6 . With induction of Gata6 , DLL1+ mesoderm is largely abrogated, but at high concentration of Activin A promotes generation of FLK1+ progenitors. (D) Analysis as in (B) except that cells were harvested at day 4 for RT-qPCR to evaluate markers for DE, paraxial mesoderm, and lateral plate mesoderm, for which relative expression levels are shown on the heat maps. Expression of Gata6 promotes DE but with high concentrations of WNT (activated by CHIR at 6 or 9 μM) promotes lateral plate mesoderm at the expense of paraxial mesoderm. (E) GSEA based on RNA-seq at day 4 for cells cultured in 6 μM CHIR comparing those uninduced or induced (+Dox) to express Gata6 , showing promotion of lateral plate mesoderm and repression of paraxial or somitic mesoderm signatures. (F, G) Heat maps show RNA-seq profiles from samples as in (E) for gene sets that are induced (F) or repressed (G) by WNT, showing that Gata6 widely impacts WNT-controlled gene networks. (H) Heat map shows RNA-seq profiles for components of the WNT signaling pathway for which 54/84 are altered significantly by expression of Gata6 . (I) Gene expression evaluated as normalized counts from RNA-seq at day 4 for known direct targets of WNT signaling. (J) Gene expression evaluated as normalized counts from RNA-seq at day 4 for known direct targets of NODAL signaling. Download figure Open in new tab Figure S6. GATA6 cooperates with WNT signaling to specify mesoderm subtype. Related to Figure 5. (A-D) RT-qqPCR assays (A, C, D) or flow cytometry (B) were performed after differentiation in DMSO, XAV, 6 μM CHIR, or both XAV and CHIR, either without (-Dox) or with Dox-induction (+Dox) of Gata6 . Assays quantified levels of WNT target Axin2 (A), mesoderm sub-types (B) and mesoderm sub-type markers in 10 ng/l Activin A (C) or 75 ng/ml Activin A (D). (E) Flow cytometry to measure DE specification at day 5 showing that expression of Gata6 (+Dox) promotes DE fate when XAV is added in cultures with 6 μM CHIR. (F) Flow cytometry was used to measure at day 5 the specification of DE (red) and DLL1+ (blue) or non-DLL mesoderm subtypes using WT or Gata6 -/- (KO) cells treated with 0, 0.5, 1, 3, or 6 μM CHIR as indicated. In cells lacking GATA6, CHIR promotes specification of DLL1+ mesoderm. (G) Flow cytometry data to quantify mesoderm sub-types comparing WT and Gata6 -/- cells cultured in various concentrations of CHIR, as indicated. Download figure Open in new tab Figure S7. Gata6 regulates Eomes and T / Bra during both endoderm and mesoderm specification. Related to Figure 6. (A) Flow cytometry was used to quantify the percentage of BRA+ cells every 6 hours from day 3 to day 4 for cells cultured in 10 ng/ml Activin A and 0, 3, or 6 μM CHIR and either uninduced (-Dox) or induced with Dox (+Dox) at day 3 to express Gata6 . (B) Flow cytometry was used to quantify percentage of cells expressing EOMES and/or BRACHYURY initially at day 3 (left panel) and then at day 3.5 or 4 middle panels) following culture in 10 ng/ml Activin A, without or with 6 μM CHIR and without (-Dox) or with induction (+Dox) to express Gata6 . Graphs on the far-right associate each of the four conditions with the generation of DE, DLL1+ mesoderm, and non-DLL1 mesoderm. Expression of Gata6 alone drives DE (EOMES up, BRA down), CHIR alone drives DLL1+ mesoderm (EOMES down, BRA up), and Gata6 expression in the presence of CHIR drives predominantly non-DLL mesoderm (EOMES and BRA both sustained). (C, D) MFI data for EOMES in EOMES+/BRA-cells (C) or BRACHYURY in EOMES-/BRA+ cells (D) quantified in Figure 6F . Color of the bar represents predominant fate generated by each condition: endoderm (red), lateral mesoderm (green) and paraxial mesoderm (purple). White represents undefined lineage. (E-G) Heat maps show transcriptomic profiles from RNA-seq data at day 4 for cells cultured in 6 μM CHIR (or DMSO) and induced with Dox at day 3 to express Gata6 (or without Dox) to compare impact on genes regulated by BRACHYURY as defined by (E) Koch et al. (2017) and (F) Faial et al. (2015) or EOMES targets as defined by (G) Harland et al. 2021 . Download figure Open in new tab Figure S8. Gata6 regulates Eomes and T / Bra during both endoderm and mesoderm specification. Related to Figure 6. (A) Flow cytometry was used to quantify EOMES and BRACHURY expressing cells at day 2-5 as indicated for WT or Gata6 -/- cells cultured in 75 ng/ml Activin A. Shown are representative flow plots. (B) BRA+ cells from (A) at day 4 or day 5 are shown relative to the percentage of DLL1+ and non-DLL mesoderm progenitors from the same samples at day 5 (ME). (C) RT-qPCR assays were used to quantify levels of ME markers in day 5 samples from (A) showing primarily increased levels of paraxial markers in the Gata6 -/- cells. (D) Flow cytometry was used to quantify in WT cells the percentage of EOMES and BRACHURY expressing cells at day 4 following culture in 75 ng/ml Activin A (as in A) except with the addition at day 3 of 0 (DMSO), 0.5, 1, 3, or 6 μM CHIR. Higher levels of CHIR substantially shift the progenitor population to BRA+ and EOMES-fate. (E) Flow cytometry data demonstrates a correlation between loss of EOMES+ cells and DE as cells are exposed in 75 ng/ml Activin A to increasing levels of CHIR, which is much enhanced in the absence of GATA6. (F) Flow cytometry data for mesoderm sub-types in WT or Gata6 -/- cells in 75 ng/ml Activin A following addition of 0 (DMSO), 0.5, 1, 3, or 6 μM CHIR. CHIR drives specification to ME fate and in the absence of GATA6 this is almost entirely DLL1+ paraxial mesoderm that is also BRA+. SUPPLEMENTAL METHODS Cell lines, culture, and differentiation All cells were cultured under humidified normoxic conditions at 37°C and 5% CO 2 in an air-jacketed incubator. Mouse embryonic stem cell lines engineered to allow conditional expression of Gata4 and Gata6 and the Gata4 -/- , Gata6 -/- , and Gata4 -/- ;Gata6 -/- cell lines have been previously described ( Holtzinger et al., 2010 ; Molkentin et al., 1997 ; Turbendian et al., 2013 ; Zhao et al., 2008 , 2005 ). The Gata4 inducible GFP-Bry/CD4-FoxA2 ES cell line was generated by targeting Gata4 cDNA into the tet-regulated promoter near the HPRT locus of the AinV/GFP-Bry/CD4-Foxa2 cells as previously described ( Cheng et al., 2008 ; Holtzinger et al., 2010 ). Stably transfected cells were selected using increasing concentrations of G418 (Cellgro; up to 300 μg/ml). Individual clonal lines were selected by picking colonies. Proper integration of the transgene was confirmed by PCR analysis of genomic DNA using the following primers: fwd: 5′-CTAGATCTCGAAGGATCTGGAG-3′; rev: 5′-ATACTTTCTCGGCAGGAGCA-3′. All cell lines were maintained on plates coated with 0.2% gelatin in 2i+LIF media composed of 25% DMEM (Corning 15-018-CV), 25% Ham’s F12 (Corning 10-080-CV), and 50% Neurobasal media (Gibco 21103049) supplemented with 0.5x N-2 (Gibco, 17502048), 0.5x B-27 (Gibco,17504044), 0.05% BSA (Gibco 15260-037), 100U/mL Penicillin and 0.1 mg/ml Streptomycin (Corning 30-002-CI), 1000 U/ml recombinant mouse LIF (Millipore ESG1106), 3 mM CHIR 99021 (STEMCELL Technologies 72054), 1 mM PD0325901 (STEMCELL Technologies 72184), 1.5×10 -4 M 1-Thioglycerol (Sigma M6145) and 2 mM L-glutamine (Corning 25-005-CI). After thawing, cells were passaged once before differentiation and were not maintained in culture in 2i+LIF media for more than three passages. B-27 batches were tested to select only batches that maintained colonies with good morphology. For differentiation, ES cells were dissociated with Accutase (Biolegend 423201) and plated in 100 x 15 mm Petri dishes (Falcon 351029) at a density of 40,000 cells/ml in 12.5ml serum-free differentiation media (SFD). SFD is composed of 75% IMDM (Corning 10-016-CM), 25% Ham’s F12 (Corning 10-080-CV), 0.5x N-2 (Gibco 17502048), 0.5x B-27 (Gibco 17504044), 0.05% BSA (Gibco 15260-037), 0.5mM Ascorbic Acid (Sigma A4544), 2mM Glutamine (Corning 25-005-CI), 0.45 mM 1-Thioglycerol (Sigma M6145), 100U/mL Penicillin and 0.1 mg/ml Streptomycin (Corning 30-002-CI). After two days, embryoid bodies (EBs) were dissociated with Accutase and resuspended at a density of 80,000 cells/ml in 4.5 ml SFD with Activin A (R&D Systems 338-AC) at the indicated concentration in Petri dishes (60 x 15 mm dishes, Falcon 351007). The differentiation efficiency was dependent on B-27 batches, so batches were tested to give higher than 50% DE progenitor at a concentration of 75 ng/ml of Activin A. For induction of Gata4 or Gata6, doxycycline (Sigma D9891) was added as a single dose of 30 ng/ml on day 3 (24 hours after Activin A supplementation). Stocks of doxycycline were maintained at −20 °C for no more than 3 months. Where indicated, WNT signaling activity was induced with CHIR 99021 (STEMCELL Technologies 72054) at the concentrations indicated. To inhibit Wnt signaling, 5 mM XAV-939 (Sigma-Aldrich X3004) was used. For liver differentiation, day 5 EBs were dissociated with Accutase and plated at a ratio of 1:2 in SFD containing 50 ng/ml BMP4 (R&D Systems 314-BP), 10 ng/ml FGF2 (R&D Systems 233-FB), 50 ng/ml Activin A, and 10 ng/ml VEGF (R&D Systems 293-VE). After 24 hours, media was changed to SFD containing 50 ng/ml BMP4 (R&D systems), 10 ng/ml EGF (Peprotech 315-09), 10 ng/ml FGF2, 20 ng/ml HGF (Peprotech 100-39), 20 ng/ml TGFα (Peprotech 100-16A), 10 ng/ml VEGF and 10 −7 M dexamethasone (Sigma D8893). Cells were maintained in this media for 2 days with daily media change. For mid-hindgut differentiation, day 5 EBs were dissociated with Accutase and plated at a ratio of 1:2 in SFD supplemented with 10 ng/ml BMP4, 100 ng/ml FGF2, and 3 μM CHIR99021. Cells were maintained in the same media for 3 days with daily media changes. RNA isolation and RT-qPCR analysis Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN 74136), and 1 μg RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen 11754250) following the manufacturer’s instructions. Quantitative PCR was carried out in triplicate with the LUNA universal qPCR Master mix (NEB M3003E) on a LightCycler 480 II instrument (Roche). Gene expression was normalized to endogenous Tbp. Primer sequences can be found in Table S1 . RNA-seq Total RNA was extracted from EBs 24h after treatment with Dox, CHIR, or DMSO with the RNeasy Plus Mini Kit (QIAGEN 74136) from three independent biological replicates per sample. Concentration was measured using a Nanodrop (Thermo Scientific), and RNA integrity was evaluated using a 2100 Bioanalyzer (Agilent Technologies). All processed samples had a greater than 9 RIN. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, USA), following the manufacturer’s protocol for use with the NEBNext Poly(A) mRNA Magnetic Isolation Module. Libraries were sequenced on the Illumina NovaSeq X Plus platform with paired-end 100 base pair reads at the Weill Cornell Medicine Genomics Resources Core Facility. Raw sequencing data in BCL format were converted to FASTQ files and demultiplexed using bcl2fastq v2.20 (Illumina). Reads from FASTQ files were aligned to the mouse genome (GRCm38.p6) and analyzed for differential expression using Basepair software with a Compare Gene Expression Levels using the DESeq2 pipeline ( basepair.com ). Differentially expressed genes (DEG) were defined as genes with a log 2 fold change > 1 and p-adj < 0.05. Gene Set Enrichment Analysis (GSEA) was performed using GSEA 4.3.3 software (Broad Institute, Inc. and Regents of the University of California) ((Subramanian et al., 2005) on RNA-seq gene lists in which genes with a base mean of less than 50 counts. The comparison was performed using the mouse collections of the Molecular Signatures Database (MSigDB) mouse as a reference (Castanza et al., 2023) or custom gene lists compiled from literature as described in supplementary Table S2 . Enrichment was considered significant for gene sets with a normalized enrichment score (NES) with a nominal p-value < 0.05 and a False Discovery Rate (FDR) q-value < 0.25. Heatmaps were generated from normalized counts from selected gene lists using SRplot ( bioinformatics.com.cn/srplot ) (Tang et al., 2023). Functional enrichment gene list analysis for transcription factors, pathways, and Gene Ontology (GO) terms was performed using the Enrichr web tool ( https://maayanlab.cloud/Enrichr/ ) ( Chen et al., 2013 ). For this analysis, the gene lists included only DEGs with a base mean of more than 100 counts. Flow cytometry Live cells were used to detect cell surface markers. EBs were dissociated with Accutase, rinsed with FACS buffer (PBS containing 2mM EDTA and 1.5% IgG-free, Protease-Free BSA [Jackson ImmunoResearch 001-000-162]), and then incubated for 30 minutes on ice with primary antibodies diluted in FACS buffer. Subsequently, cells were rinsed twice with FACS buffer and stained for 5 mins at RT with SYTOX Blue Dead Cell Stain for flow cytometry (Invitrogen S34857) diluted 1:1000 in FACS buffer. Antibodies used for DE progenitors identification were CD117 (c-Kit) Monoclonal Antibody (2B8), PerCP-eFluor 710 conjugated (eBioscience 46-1171-82); CD184 (CXCR4) Monoclonal Antibody (2B11), APC conjugated (eBioscience 17-9991-82); and CD326 (EpCAM) Monoclonal Antibody (G8.8), PE-conjugated (eBioscience 12-5791-81). Antibodies against mesoderm markers were Dll1 Monoclonal Antibody (HMD1-3), PE-conjugated (Biolegend 128307); CD309 (VEGFR2, Flk-1) Monoclonal Antibody (Avas12), PE/Cyanine7 conjugated (Biolegend 136414); and CD140a (PDGFRA) Monoclonal Antibody (APA5), APC conjugated (eBioscience 17-1401-81). For detection of Eomes and Brachyury, after EB dissociation, the cells were rinsed with PBS and then fixed with 2% PFA (Electron microscopy Sciences 15710) for 30 mins on ice. After centrifugation, cells were washed with PBS, resuspended in FACS buffer, and stored for up to a week at 4oC. Permeabilization and washes were done with the FoxP3 Transcription Factor Staining Buffer Set (eBioscience 00-5523-00) according to manufacturer instructions. After blocking with 5% FBS in permeabilization buffer for 15 minutes at RT, cells were incubated with PE-conjugated BRACHYURY polyclonal antibody (R&D Systems IC2085P), and Eomes Monoclonal antibody (W17001A), Alexa Fluor 647 conjugated (Biolegend 157703) for two hours at RT. After washing twice, cells were resuspended in FACS buffer and analyzed. All flow data was collected using an Attune NxT Flow Cytometer (ThermoFisher). Western Blotting EBs were collected, washed with cold PBS, and lysed using RIPA buffer composed of 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 1x protease and phosphatase inhibitors (Pierce A32961) and 25 U/ml benzonase (Millipore 70664) on ice. After 10 minutes, SDS and EDTA were added to the lysate to a final concentration of 0.1% SDS and 2 mM EDTA, respectively. After an additional 20 min incubation on ice, the lysate was centrifuged at 15,000 × g for 15 min at 4°C. Aliquots were maintained at −80°C until analysis. The protein content was measured using the bicinchoninic acid protein assay kit (Pierce 23225). Proteins were then separated using NuPAGE 4 to 12% Bis-Tris Gels (Invitrogen NP0335BOX) before transferring to a PVDF membrane (Bio-Rad 162-0177). The membrane was blocked for 1 hour at room temperature using 5% IgG-free, Protease-Free BSA (Jackson ImmunoResearch 001-000-162) in 1x Tris-buffered saline plus 1% Tween 20 (TBST). After blocking, the blot was incubated with a primary antibody in 5% IgG-free BSA overnight at 4°C with gentle shaking. The next day, the membrane was washed 3 times using TBST. The secondary antibody was incubated for 1 hour at room temperature in TBST. Then, three washes were performed, and the membrane was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific 34577) and scanned using a chemiluminescence Licor’s C-DiGit imaging system. Primary antibodies used were GATA6 (Cell signaling technology 5851), GATA4 (Cell signaling technology 36966), SOX17 (R&D Systems AF1924), EOMES (Abcam ab23345), OCT3/4 (BD Transduction Laboratories 611202), and BRACHYURY (R&D Systems AF2085). Secondary antibodies used were Goat anti-rabbit IgG (H+L)-HRP Conjugate (BioRad 1706515), Goat Anti-Mouse IgG (H + L)-HRP Conjugate (BioRad 1706516), and Rabbit Anti-Sheep IgG (H+L)-HRP Conjugate (BioRad 1721017). ACKNOWLEDGEMENTS We thank Gordon M. Keller from the University Health Network in Toronto and Valerie Gouon-Evans from Boston University for sharing the AinV/GFP-Bry/CD4-Foxa2 cell line. Stephen A. 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