Article b-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/ SMAD3 and OCT4 Graphical Abstract Authors Nina S. Funa, Karen A. Schachter, ..., Klaus Hansen, Henrik Semb Correspondence [email protected] In Brief In this article, Funa, Schachter, and colleagues et al. provide mechanistic insights into how Wnt signaling controls early cell lineage decisions. b-catenin occupies regulatory regions in numerous PS and neural crest genes, and direct interactions between b-catenin and SMAD2/SMAD3 in proximity to OCT4 binding are required to specifically activate PS genes. Highlights Accession Numbers d Wnt signaling induces primitive streak (PS) gene expression GSE58476 in hESCs d b-catenin binds upstream regulatory regions in most PS and neural crest genes d Direct interactions between b-catenin and SMAD2/SMAD3 mediate PS gene activation d OCT4 binds in proximity to b-catenin and SMAD2/SMAD3 sites to induce PS genes Funa et al., 2015, Cell Stem Cell 16, 639–652 June 4, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2015.03.008 Cell Stem Cell Article b-Catenin Regulates Primitive Streak Induction through Collaborative Interactions with SMAD2/SMAD3 and OCT4 Nina S. Funa,1,2,5 Karen A. Schachter,1,5 Mads Lerdrup,3 Jenny Ekberg,2 Katja Hess,1,2 Nikolaj Dietrich,3 Christian Honore´ ,4 Klaus Hansen,3 and Henrik Semb1,2,* 1The Danish Stem Cell Centre (DanStem), University of Copenhagen, 2200 Copenhagen N, Denmark 2Lund Stem Cell Center, Faculty of Medicine, Lund University, 22100 Lund, Sweden 3Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, University of Copenhagen, Copenhagen Biocenter, 2200 Copenhagen N, Denmark 4Department of Stem Cell Biology, Novo Nordisk A/S, 2760 Maaloev, Denmark 5Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.03.008 SUMMARY various signaling pathways, including Wnt, Nodal, and BMP (bone morphogenetic protein). It is well known that the trans- Canonical Wnt and Nodal signaling are both required forming growth factor (TGF)-b family member Nodal promotes for induction of the primitive streak (PS), which anterior-posterior axis patterning in the gastrulating mouse guides organization of the early embryo. The Wnt embryo; high levels promote anterior fates, whereas a graded effector b-catenin is thought to function in these response to lower levels allows regional specification toward early lineage specification decisions via transcrip- different mesodermal derivatives (Dunn et al., 2004; Kinder tional activation of Nodal signaling. Here, we demon- et al., 1999). Wnt3 helps maintain high levels of Nodal expression through direct regulation of the proximal epiblast enhancer in the strate a broader role for b-catenin in PS formation by Nodal locus (Norris and Robertson, 1999; Vincent et al., 2003). analyzing its genome-wide binding in a human em- Nodal mutant embryos in which the proximal epiblast enhancer b bryonic stem cell model of PS induction. -catenin element has been deleted lack anterior structures as a result of a occupies regulatory regions in numerous PS and failure in specification of definitive endoderm (DE), which also neural crest genes, and direct interactions between works as a signaling modulator for the anterior neuroectoderm b-catenin and the Nodal effectors SMAD2/SMAD3 (Norris and Robertson, 1999; Vincent et al., 2003). In addition, are required at these regions for PS gene activation. mice deficient in activators of the Wnt signaling pathway, such Furthermore, OCT4 binding in proximity to these as b-catenin, the Wnt co-receptors Lrp5/Lrp6, and the processor sites is likewise required for PS induction, suggesting of the Wnt molecule Porcupine fail to form PS and mesoderm a collaborative interaction between b-catenin and (Biechele et al., 2011; Fu et al., 2009; Hsieh et al., 2003; Huelsken OCT4. Induction of neural crest genes by b-catenin et al., 2000; Kelly et al., 2004; Liu et al., 1999). Re-examination of an epiblast-specific Wnt3 mutant mouse shows that PS is is repressed by SMAD2/SMAD3, ensuring proper induced, but not maintained, in these animals, highlighting the lineage specification. This study provides mecha- need of sustained Wnt signaling in the epiblast for gastrulation nistic insight into how Wnt signaling controls early (Tortelote et al., 2013). Thus, canonical Wnt and Nodal signaling cell lineage decisions. are required for PS formation. However, whether b-catenin is simply required for regulating Nodal expression or targets several key transcriptional regulators of PS is unclear. INTRODUCTION Whereas Wnt and Nodal/TGFb signaling work together in the regulation of PS formation, they play opposite roles during neural During gastrulation, induction of the three germ layers is regu- induction. High levels of Wnt signaling promote the expression of lated by signaling cues from the emerging primitive streak (PS). Gbx2, Pax3, and Tfap2a, which are essential for specification of PS formation is spatially initiated at the prospective posterior posterior neural fates such as neural crest progenitor (NCp) cells end of the mouse embryo and is characterized by regionalized (Kiecker and Niehrs, 2001; Li et al., 2009). In contrast, TGFb expression of lineage-specific markers (Tam et al., 2006). signaling inhibits neural induction (Hemmati-Brivanlou and Through an epithelial-mesenchymal transition, epiblast cells Melton, 1994; Surmacz et al., 2012). In fact, dual blockage of that will give rise to endoderm and mesoderm lineages invagi- BMP and NODAL signaling has been used to direct differentia- nate through the streak and migrate anteriorly, while cells re- tion of human embryonic stem cells (hESCs) into neuroectoderm maining in the epiblast give rise to surface ectoderm and neural and neural crest progenitors in vitro (Menendez et al., 2011). ectoderm (Tam and Behringer, 1997). Here, we study how b-catenin regulates differentiation of Early stages of embryonic development are specified both hESCs. Initial activation of Wnt signaling induces expression of temporally and spatially through the coordinated activities of direct targets of b-catenin, among them markers of NCp. Cell Stem Cell 16, 639–652, June 4, 2015 ª2015 Elsevier Inc. 639 (legend on next page) 640 Cell Stem Cell 16, 639–652, June 4, 2015 ª2015 Elsevier Inc. b-catenin’s activation also promotes a sustained increase in and S1B). To validate the functionality of the CHIR-induced PS, NODAL levels and SMAD2/SMAD3 activity, resulting in downre- we followed the conversion of PS into DE by adding ACTIVIN A gulation of NCp gene expression and induction of PS genes. (AA) for 2 days after the 24-hr CHIR treatment (Figure 1A). One- Genome-wide analysis of b-catenin binding sites revealed direct day treatment with AA after CHIR removal was sufficient for effi- binding of b-catenin to regulatory regions in both PS and NCp cient induction of DE markers, such as SOX17, FOXA2, CXCR4, genes. Transcriptional regulation of PS genes requires binding and CERBERUS (CER)(Figure S1F). Approximately 95% of the of b-catenin, OCT4, and SMAD2/SMAD3 to these regulatory cells expressed SOX17 at the end of the CHIR-AA 3-day protocol regions; removal of OCT4 or suppression of NODAL signaling (Figure S1C). Notably, the expression levels of SOX17 and prevents b-catenin-mediated PS induction. Subsequent differ- CXCR4 on day 3 were much higher in cells initially treated with entiation into DE is also blocked, while expression of NCp CHIR, compared to high WNT3A or WNT3A+AA (Figures S1D markers is restored. We also show that it is the SMAD2/ and S1E), suggesting a relationship between high initial b-catenin SMAD3 activation levels that determine cell fate choices beyond activity and high DE induction. These results were reproduced in the PS state. Our findings provide mechanistic information on an independent hESC culture system (Figures S2A–S2C). Finally, how hESCs coordinate inputs from the Wnt and Nodal pathways as a sign of the authenticity of the putative DE cells, we show that to regulate early lineage-specific differentiation. they are capable of differentiating into multipotent pancreatic progenitors (75% PDX1+/NKX6.1+ cells; Figure S1G) (Hald RESULTS et al., 2008, Jennings et al., 2013). Binding of NODAL to the ALK4/ALK7 receptors leads to phos- Canonical Wnt Signaling Regulates NODAL Expression phorylation, activation, and nuclear translocation of SMAD2/ and PS Specification SMAD3—a prerequisite for transcriptional regulation of its target To study how activation of Wnt signaling influences PS induction genes. As expected, CHIR-mediated increase in NODAL expres- in hESCs, we used CHIR99021 (CHIR), an inhibitor of glycogen sion led to increased receptor-mediated phosphorylation as well synthase kinase (GSK-3), for 24 hr (day 1) (Figure 1A). Canonical as nuclear localization of SMAD2/SMAD3 (Figures 1G and 1H). Wnt signaling requires the stabilization and activation of b-cate- Addition of the ALK4/ALK5/ALK7 inhibitor SB431542 (SB) nin in order to induce target gene expression. The stability of completely blocked endogenous NODAL signaling as early as b-catenin is regulated through phosphorylation by glycogen syn- 3 hr after treatment as well as CHIR-induced NODAL expression, thase kinase 3 (GSK-3). Upon engagement of Wnt signaling, SMAD2/SMAD3 activation, and PS gene expression (Figures 1D– GSK-3 is inactivated, allowing unphosphorylated b-catenin to 1H). It has been reported that GSK-3b regulates SMAD3 protein accumulate, translocate to the nucleus, and activate transcrip- stability through phosphorylation-induced proteosomal degra- tion by associating with the transcription factor/lymphoid dation (Guo et al., 2008). CHIR-induced NODAL signaling may enhancer-binding factor (TCF/LEF) transcription factors (Aberle therefore result from direct modulation of SMAD2 activity rather et al., 1997; Novak and Dedhar, 1999). Although GSK-3 pos- than through b-catenin activation. However, we observed no sesses multiple substrates (Forde and Dale, 2007), inhibition change in SMAD2 protein levels in CHIR-treated cells (Figure 1H).