Abstract
In the mouse embryo, pluripotent cells arise inside the embryo around the 16-cell stage. During these early stages, Sox2 is the only gene whose expression is known to be induced specifically within inside cells as they are established. To understand how pluripotent cells are created, we investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4, highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious, ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1, and Tead4. Interestingly, the repression of premature Sox2 expression was sensitive to LATS1/2 activity, even though it normally does not limit TEAD4/YAP1/WWTR1 activity during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1/WWTR1/TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the 4-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.
Introduction
Pluripotency describes the developmental potential to produce all adult cell types. However, in mammals, the establishment of pluripotency takes place in the context of lineage decisions that separate pluripotent cells of the fetus from cells that give rise to extraembryonic tissues such as the placenta. Thus, in mammals, the onset of pluripotency is initially delayed as the blastomeres transition from totipotency to adopt the more specialized pluripotent and extraembryonic states.
The mouse embryo has provided an invaluable tool to understand the molecular mechanisms that initially create pluripotent cells, which are also the progenitors of embryonic stem cells. While much progress has been made in understanding how pluripotency is maintained once pluripotent cells are established, the mechanisms driving the initial establishment of pluripotency remain relatively obscure.
In the mouse embryo, pluripotent cells emerge from cells positioned inside the embryo, which occurs around the 16-cell stage, and continues as the inside cells form the inner cell mass of the blastocyst. The inner cell mass will go on to differentiate into either pluripotent epiblast or non-pluripotent primitive endoderm. As the epiblast matures, it gradually acquires a more embryonic stem cell-like transcriptional signature (Boroviak et al., 2014; Boroviak et al., 2015).
While studies in mammalian embryos and embryonic stem cells have developed an extensive catalog of transcription factors that promote pluripotency, the only pluripotency-promoting transcription factor known to distinguish inside cells as they form at the 16-cell stage is Sox2 (Guo et al., 2010; Wicklow et al., 2014). Therefore, understanding how Sox2 expression is regulated at the 16-cell stage can provide insight into how pluripotency is first established.
Here, we use genetic approaches to test mechanistic models of the initial activation Sox2 expression. We investigate the contribution, at the 16-cell stage and prior, of factors and pathways that are known to regulate expression of Sox2 at later preimplantation stages and in embryonic stem cells. We show that embryos are competent to express Sox2 as early as the four-cell stage, although they normally do not do so. Finally, we uncover the molecular mechanisms that ensure that Sox2 expression remains repressed until the developmentally appropriate stage.
Results and Discussion
The initiation of Sox2 expression is Nanog- and Oct4-independent
To identify mechanisms contributing to the onset of Sox2 expression in the embryo, we first focused on the role of transcription factors that are required for Sox2 expression in embryonic stem cells. The core pluripotency genes Nanog and Oct4 (Pou5f1) are required for Sox2 expression in embryonic stem cells (Chambers et al., 2003; Mitsui et al., 2003; Niwa et al., 2005) and are expressed at the 8-cell stage (Dietrich and Hiiragi, 2007; Palmieri et al., 1994; Rosner et al., 1990; Strumpf et al., 2005), prior to the onset of Sox2 expression at the 16-cell stage, suggesting that NANOG and OCT4 could activate the initial expression of Sox2.
We previously showed that the initiation of Sox2 expression is Oct4-independent, as embryos lacking Oct4 have normal levels of Sox2 expression at E3.5 (Frum et al., 2013). We therefore hypothesized that Nanog and Oct4 could act redundantly to initiate Sox2 expression. To test this hypothesis, we bred mice carrying the null allele Nanog-GFP (Maherali et al., 2007) with mice carrying an Oct4 null allele (Kehler et al., 2004) to generate Nanog;Oct4 null embryos (Fig. S1A). In wild-type embryos, Sox2 is first detected in inside cells at the 16-cell stage, with increasing robustness in inside cells of the 32-cell stage embryo (Guo et al., 2010; Wicklow et al., 2014). In Nanog;Oct4 null embryos, SOX2 was detectable at the 16-cell (E3.0) and 32-cell (E3.25) stages (Fig. 1A-B). We observed no differences in the proportions of SOX2-expressing cells at the 16- and 32-cell stages between non-mutant embryos and embryos lacking Nanog, Oct4, or both (Fig. S1B,C). These observations indicate that Nanog and Oct4 do not regulate initial Sox2 expression.
Nanog and Oct4 are individually required to maintain Sox2 expression
To investigate a role for Nanog and Oct4 in maintaining expression of Sox2, we evaluated double null embryos at a later time point. By E3.5, SOX2 appeared weak or undetectable in most cells of Nanog;Oct4 null embryos (Fig. 1C). Moreover, the proportion of cells expressing the wild type level of SOX2 was significantly lower in Nanog;Oct4 null embryos (Fig. 1D), but not in embryos lacking Nanog or Oct4 only (Fig. S1D). We therefore conclude that Nanog and Oct4 work together to maintain Sox2 expression and can compensate for the loss of one another up to at least E3.5.
To evaluate whether Nanog and Oct4 cooperatively maintain Sox2 expression at later preimplantation stages, we examined SOX2 expression in embryos lacking either Nanog or Oct4 at later developmental stages. At E3.75, SOX2 levels were indistinguishable between non-mutant, Nanog null and Oct4 null embryos (Fig 1E,F). In fact, the only difference between non-mutant, Oct4 null and Nanog null embryos at this timepoint was the previously reported failure of Nanog null embryos to undergo primitive endoderm differentiation by downregulating Nanog expression in a subset of inner mass cells (Frankenberg et al., 2011; Messerschmidt and Kemler, 2010).
By contrast, both Nanog null and Oct4 null embryos exhibited defects in SOX2 by E4.25. Nanog null embryos exhibited the more severe SOX2 expression phenotype, with almost undetectable SOX2 (Fig. 1G). Oct4 null embryos exhibited a less severe phenotype, with reduced, but detectable SOX2 (Fig. 1H). These observations indicate that, while the initial phase of Sox2 expression is independent of Nanog and Oct4, this is followed by a period during which Nanog and Oct4 act redundantly to maintain Sox2 expression, which then gives way to a phase during which Nanog and Oct4 are individually required to achieve maximal Sox2 expression.
TEAD4/WWTR1/YAP1 regulate the onset of Sox2 expression
Having observed that the initiation of Sox2 expression is Nanog- and Oct4-independent, we next examined the role of other factors in regulating initial Sox2 expression. TEAD4 and its co-factors WWTR1 and YAP1 repress Sox2 expression in outside cells starting around the 16-cell stage (Frum et al., 2018; Wicklow et al., 2014). However, YAP1 is detected within nuclei as early as the 4-cell stage (Nishioka et al., 2009), suggesting that the complex is active prior to the 16-cell stage. Recent studies have highlighted the roles and regulation of TEAD4/WWTR1/YAP1 in promoting outside cell maturation to trophectoderm during blastocyst formation (Anani et al., 2014; Cao et al., 2015; Cockburn et al., 2013; Hirate et al., 2013; Kono et al., 2014; Leung and Zernicka-Goetz, 2013; Lorthongpanich et al., 2013; Nishioka et al., 2009; Nishioka et al., 2008; Posfai et al., 2017; Rayon et al., 2014; Shi et al., 2017; Yagi et al., 2007; Yu et al., 2016). Yet, the developmental requirement for TEAD4/WWTR1/YAP1 prior to the 16-cell stage has not been investigated. We therefore hypothesized that TEAD4/WWTR1/YAP1 repress Sox2 expression prior to the 16-cell stage.
To test this hypothesis, we examined SOX2 in embryos lacking Tead4. Consistent with our hypothesis, Tead4 null embryos exhibited precocious SOX2 at the 8-cell stage (Fig. 2A,C). Notably, this phenotype that was not exacerbated by elimination of maternal Tead4 (Fig. S2A and Fig. 2A,C), consistent with the absence of detectable Tead4 in oocytes (Yagi et al., 2007). By contrast, deletion of maternal Wwtr1 and Yap1 (Fig. S2B) led to precocious SOX2 at the 8-cell stage (Fig. 2B,D). The presence of wild-type, paternal alleles of Wwtr1 and/or Yap1 did not rescue SOX2 in the maternally null embryos (Fig. 2B, D). Therefore, maternally provided WWTR1/YAP1 and zygotically expressed TEAD4 repress Sox2 expression at the 8-cell stage.
We next evaluated SOX2 in embryos lacking maternal Wwtr1;Yap1 at the 4-cell stage. We observed that 4-cell embryos lacking maternal Wwtr1 and Yap1 occasionally exhibited weak ectopic SOX2 (Fig. S2D,E). However, SOX2 was never detected in 4-cell embryos lacking Tead4 (Fig. S2C). These observations suggest that Wwtr1 and Yap1 partner with other factors to regulate the onset of Sox2 expression at the 4-cell stage and point to a requirement for other TEAD proteins that are expressed at the 4-cell stage (Nishioka et al., 2008).
The premature onset of Sox2 expression in embryos lacking Tead4 or Wwtr1 and Yap1 demonstrates that preimplantation mouse embryos are capable of expressing markers of inside cell identity as early as the 4-cell stage and reveals an earlier than expected role for TEAD4/WWTR1/YAP1 in repressing the expression of Sox2 until the formation of inside cells, thus permitting the establishment of discrete trophectoderm and inner cell mass gene expression. Furthermore, the appearance of Sox2 expression prior to the formation of inside cells argues that no cues specific to inside-position are required for Sox2 expression beyond regulated TEAD4/WWTR1/YAP1 activity. Rather, our results suggest that the mechanism regulating the onset of Sox2 expression is that constitutive repression of Sox2 by TEAD4/WWTR1/YAP1 is relieved once cells are positioned inside the embryo at the 16-cell stage.
Repression of Sox2 at the 4- and 8-cell stage is sensitive to LATS2 kinase
In many contexts, TEAD4/WWTR1/YAP1 activity is repressed by the HIPPO pathway LATS1/2 kinases, which repress nuclear localization of WWTR1/YAP1 (Nishioka et al., 2009; Zhao et al., 2010; Zhao et al., 2007). To evaluate the role of HIPPO signaling in regulating initial Sox2 expression, we therefore examined whether Sox2 expression is LATS1/2-sensitive prior to the 16-cell stage.
We injected mRNA encoding Lats2 into both blastomeres of 2-cell stage embryos, which is sufficient to inactivate the TEAD4/WWTR1/YAP1 complex during blastocyst formation (Nishioka et al., 2009; Wicklow et al., 2014), and then evaluated SOX2 at 4- and 8-cell stages (Fig. 3A). As anticipated, Lats2 mRNA injection, but not injection of Green Fluorescent Protein mRNA, greatly reduced YAP1 nuclear localization at 4- and 8-cell stages (Fig. 3B,C). In addition, we observed precocious SOX2 in embryos overexpressing Lats2 (Fig 3B,C). Therefore, LATS kinases can prevent TEAD4/WWTR1/YAP1 from repressing expression of Sox2 prior to the 16-cell stage, but LATS kinases must not normally do so, since Sox2 is usually repressed during early development.
We observed in published RNA-seq data sets that Lats1 and Lats2 are expressed in 4-cell and 8-cell stage embryos (Tang et al., 2010; Wu et al., 2016), opening an interesting future direction of discovering how TEAD4/WWTR1/YAP1 escape inhibition by LATS kinases prior to the 16-cell stage, which is not currently understood.
TEAD4/WWTR1/YAP1 may repress Sox2 expression through a direct mechanism
While the TEAD4/WWTR1/YAP1 complex is widely recognized as a transcriptional activator, it has more recently been shown to act as a transcriptional repressor (Beyer et al., 2013; Kim et al., 2015). Therefore, we considered two mechanisms by which TEAD4/WWTR1/YAP1 could repress Sox2 expression (Fig. 4A): an indirect model, in which TEAD4/WWTR1/YAP1 promote transcription of a Sox2 repressor, and a direct model, in which TEAD4/WWTR1/YAP1 themselves act as the Sox2 repressor.
To test these models, we employed variants of Tead4 in which the WWTR1/YAP1 interaction domain has been replaced with either the transcriptional activator VP16 (Tead4VP16) or the transcriptional repressor engrailed (Tead4EnR). These variants have previously been used in preimplantation embryos to provide evidence that TEAD4/WWTR1/YAP1 promote Cdx2 expression through a direct mechanism (Nishioka et al., 2009). We reasoned that if TEAD4/WWTR1/YAP1 represses Sox2 indirectly, then overexpression of Tead4EnR would induce Sox2 expression. Alternatively, if TEAD4/WWTR1/YAP1 represses Sox2 directly, then Tead4VP16 would induce Sox2 expression. We injected mRNA encoding Tead4VP16 or Tead4EnR into a single blastomere of 4-cell stage embryos and tracked progeny of the injected blastomere at the 8-cell stage with co-injection of GFP (Fig. 4B). We observed that overexpression of Tead4VP16, but not Tead4EnR induced SOX2 (Fig. 4C,D). These observations are consistent with the direct repression of Sox2 by TEAD4/WWTR1/YAP1.
This study highlights distinct phases of Sox2 regulation occurring during the establishment of pluripotency in mouse development. As early as the 4-cell stage, blastomeres are competent to express Sox2, but this is overridden by TEAD/WWTR1/YAP1 (Fig. 4E, box 1). Initiation of Sox2 expression is independent of Nanog and Oct4 and does not require cues associated with inside cell position or developmental time. Instead, LATS1/2 activity in inside cells relieves repression of TEAD4/WWTR1/YAP1 on Sox2 (Fig. 4E, box 2). After blastocyst formation, NANOG and OCT4 work together ensure that Sox2 expression is maintained (Fig 4E, box 3). Finally, as the embryo approaches implantation, Nanog and Oct4 become individually required to sustain Sox2 expression (Fig. 4E, box 4).
Materials and Methods
Mouse strains
Animal care and husbandry was performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at Michigan State University. Wild type embryos were generated by mating CD-1 mice (Charles River). Female mice used in this study were between six weeks and six months of age and males were used from eight weeks to nine months. Alleles and transgenes used in this study were maintained on a CD-1 background and include: Nanogtm1.1Hoch (Maherali et al., 2007), Pou5f1tm1Scho (Kehler et al., 2004), Tead4tm1Bnno (Yagi et al., 2007), Yap1tm1.1Eno (Xin et al., 2011), Wwtr1tm1.1Eno (Xin et al., 2013), Tg(Zp3-cre)93Knw (De Vries et al., 2004). Conditional, floxed alleles were recombined to generate null alleles by breeding mice carrying conditional alleles to Alpltm(cre)Nagy (Lomelí et al., 2000) mice.
Embryo collection and culture
Embryos were collected from naturally mated mice by flushing dissected oviducts or uteri with M2 medium (Millipore-Sigma). All embryos were cultured in 5% CO2 at 37°C under ES cell grade mineral oil (Millipore-Sigma). Prior to embryo culture, KSOM medium (Millipore-Sigma) was equilibrated overnight in the embryo incubator.
Embryo microinjection
cDNAs encoding Lats2, Tead4VP16, and Tead4EnR (Nishioka et al., 2009) cloned into the pcDNA3.1 poly(A)83 plasmid (Yamagata et al., 2005) were linearized, and then used as a template to generate mRNAs for injection by the mMessage mMachine T7 transcription kit (Invitrogen). NLS-GFP mRNA was synthesized from linearized NLS-GFP plasmid (Ariotti et al., 2015) using the mMessage mMachine Sp6 transcription kit (Invitrogen). Prior to injection, synthesized mRNAs were cleaned and concentrated using the MEGAclear Transcription Clean-up Kit (Invitrogen). Lats2 and NLS-GFP mRNAs were injected into both blastomeres of 2-cell stage embryos at a concentration of 500 ng/μl. Tead4VP16 or Tead4EnR mRNAs were injected into a single blastomere of 4-cell stage embryos at a concentration of or 150 ng/μl each. NLS-GFP mRNA was included in 4-cell stage injections at a concentration of 150 ng/μl to trace the progeny of the injected blastomere. All mRNAs were diluted in 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA. Injections were performed using a Harvard Apparatus PL-100A microinjector.
Immunofluorescence and Confocal Microscopy
Embryos were fixed in 4% formaldehyde (Polysciences) for 10 minutes, permeabilized with 0.5% Triton X-100 (Millipore-Sigma) for 30 minutes and blocked with 10% FBS, 0.1% Triton X-100 for at least 1 hour at room temperature or longer at 4°C. Primary antibody incubation was performed at 4°C overnight using the following antibodies: goat anti-SOX2 (Neuromics, GT15098, 1:2000), rabbit anti-NANOG (Reprocell, RCAB002P-F, 1:400) mouse anti-Tead4 (Abcam, ab58310, 1:1000), mouse anti-YAP (Santa Cruz, sc101199, 1:200), and rat anti-ECAD (Millipore-Sigma, U3254, 1:500). Anti-SOX2, anti-TEAD4 and anti-YAP antibodies were validated by the absence of positive staining on embryos homozygous for null alleles encoding antibody target Nuclei were labelled with either DRAQ5 (Cell Signaling Technology) or DAPI (Millipore-Sigma). Antibodies raised against IgG and coupled to Dylight 488, Cy3 or Alexa Fluor 647 (Jackson ImmunoResearch) were used to detect primary antibodies. Embryos were imaged on an Olympus FluoView FV1000 Confocal Laser Scanning Microscope using a 20x UPlanFLN objected (0.5 NA) and 5x digital zoom. Each embryo was imaged in entirety using 5 μm optical section thickness.
Image Analysis
Z-stacks obtained from confocal microscopy were analyzed using ImageJ (Schneider et al., 2012). Each nucleus was identified by DNA stain and then scored for the presence or absence of SOX2. In Fig. 1A and B, cells were classified as inside or outside on the basis of ECAD localization. For analysis of Nanog;Oct4 null embryos in Fig. 1C,D and Fig. S1D, SOX2 staining intensity was categorized as intense or weak. Intense SOX2 staining was defined as the level observed in non-mutant embryos, which was uniform among inside cells. In Fig. 1, S1, 2, and S2, embryo genotypes were not known prior to analysis. In Fig. 3 and 4 embryos were grouped according to injection performed, and therefore the researcher was not blind to embryo treatment.
Embryo Genotyping
For embryos at the 8-cell stage or older, DNA was extracted from fixed embryos after imaging using the Extract-N-Amp kit (Millipore-Sigma) in a total volume of 10 μl. For embryos at the 4-cell stage, DNA was extracted from fixed embryos in a total volume of 5 μl. 1 μl of extracted DNA was used as template, with allele specific primers (Table S1).
Competing Interests
No competing interests declared.
Acknowledgements
We thank members of the Ralston Lab for thoughtful discussion. This work was supported by National Institutes of Health (R01 GM104009 and R35 GM131759 to A.R. and T32HD087166 to J.W.).