bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 TEAD4/YAP1/WWTR1 prevent the premature onset of pluripotency prior to the 16- 2 cell stage 3 4 Tristan Frum1, Jennifer Watts2,3, and Amy Ralston1,3# 5 6 1Department of Biochemistry and Molecular Biology, Michigan State University, East 7 Lansing, Michigan, 48824, United States. 8 9 2Physiology Graduate Program, Michigan State University, East Lansing, Michigan, 10 48824, United States. 11 12 3Reproductive and Developmental Biology Training Program, Michigan State University, 13 East Lansing, Michigan, 48824, United States. 14 15 #) Correspondence: [email protected] 16 17 Running title: Mechanism preventing premature Sox2 18 19 Key words: preimplantation, stem cell progenitors, HIPPO signaling 20

1 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

21 Abstract 22 In the mouse embryo, pluripotent cells arise inside the embryo around the 16-cell stage. 23 During these early stages, Sox2 is the only whose expression is known to be 24 induced specifically within inside cells as they are established. To understand how 25 pluripotent cells are created, we investigated the mechanisms regulating the initial 26 activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the 27 absence of both Nanog and Oct4, highlighting differences between embryo and stem 28 cell models of pluripotency. However, we observed precocious, ectopic expression of 29 Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1, and Tead4. Interestingly, 30 the repression of premature Sox2 expression was sensitive to LATS1/2 activity, even 31 though it normally does not limit TEAD4/YAP1/WWTR1 activity during these early 32 stages. Finally, we present evidence for direct transcriptional repression of Sox2 by 33 YAP1/WWTR1/TEAD4. Taken together, our observations reveal that, while embryos are 34 initially competent to express Sox2 as early as the 4-cell stage, transcriptional 35 repression prevents the premature expression of Sox2, thereby restricting the 36 pluripotency program to the stage when inside cells are first created. 37 38 Introduction 39 Pluripotency describes the developmental potential to produce all adult cell types. 40 However, in mammals, the establishment of pluripotency takes place in the context of 41 lineage decisions that separate pluripotent cells of the fetus from cells that give rise to 42 extraembryonic tissues such as the placenta. Thus, in mammals, the onset of 43 pluripotency is initially delayed as the blastomeres transition from totipotency to adopt 44 the more specialized pluripotent and extraembryonic states. 45 46 The mouse embryo has provided an invaluable tool to understand the molecular 47 mechanisms that initially create pluripotent cells, which are also the progenitors of 48 embryonic stem cells. While much progress has been made in understanding how 49 pluripotency is maintained once pluripotent cells are established, the mechanisms 50 driving the initial establishment of pluripotency remain relatively obscure. 51

2 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

52 In the mouse embryo, pluripotent cells emerge from cells positioned inside the embryo, 53 which occurs around the 16-cell stage, and continues as the inside cells form the inner 54 cell mass of the blastocyst. The inner cell mass will go on to differentiate into either 55 pluripotent epiblast or non-pluripotent primitive endoderm. As the epiblast matures, it 56 gradually acquires a more embryonic stem cell-like transcriptional signature (Boroviak et 57 al., 2014; Boroviak et al., 2015). 58 59 While studies in mammalian embryos and embryonic stem cells have developed an 60 extensive catalog of transcription factors that promote pluripotency, the only 61 pluripotency-promoting transcription factor known to distinguish inside cells as they form 62 at the 16-cell stage is Sox2 (Guo et al., 2010; Wicklow et al., 2014). Therefore, 63 understanding how Sox2 expression is regulated at the 16-cell stage can provide insight 64 into how pluripotency is first established. 65 66 Here, we use genetic approaches to test mechanistic models of the initial activation 67 Sox2 expression. We investigate the contribution, at the 16-cell stage and prior, of 68 factors and pathways that are known to regulate expression of Sox2 at later 69 preimplantation stages and in embryonic stem cells. We show that embryos are 70 competent to express Sox2 as early as the four-cell stage, although they normally do 71 not do so. Finally, we uncover the molecular mechanisms that ensure that Sox2 72 expression remains repressed until the developmentally appropriate stage. 73 74 Results and Discussion 75 76 The initiation of Sox2 expression is Nanog- and Oct4-independent 77 To identify mechanisms contributing to the onset of Sox2 expression in the embryo, we 78 first focused on the role of transcription factors that are required for Sox2 expression in 79 embryonic stem cells. The core pluripotency Nanog and Oct4 (Pou5f1) are 80 required for Sox2 expression in embryonic stem cells (Chambers et al., 2003; Mitsui et 81 al., 2003; Niwa et al., 2005) and are expressed at the 8-cell stage (Dietrich and Hiiragi, 82 2007; Palmieri et al., 1994; Rosner et al., 1990; Strumpf et al., 2005), prior to the onset

3 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

83 of Sox2 expression at the 16-cell stage, suggesting that NANOG and OCT4 could 84 activate the initial expression of Sox2. 85 86 We previously showed that the initiation of Sox2 expression is Oct4-independent, as 87 embryos lacking Oct4 have normal levels of Sox2 expression at E3.5 (Frum et al., 88 2013). We therefore hypothesized that Nanog and Oct4 could act redundantly to initiate 89 Sox2 expression. To test this hypothesis, we bred mice carrying the null allele Nanog- 90 GFP (Maherali et al., 2007) with mice carrying an Oct4 null allele (Kehler et al., 2004) to 91 generate Nanog;Oct4 null embryos (Fig. S1A). In wild-type embryos, Sox2 is first 92 detected in inside cells at the 16-cell stage, with increasing robustness in inside cells of 93 the 32-cell stage embryo (Guo et al., 2010; Wicklow et al., 2014). In Nanog;Oct4 null 94 embryos, SOX2 was detectable at the 16-cell (E3.0) and 32-cell (E3.25) stages (Fig. 95 1A-B). We observed no differences in the proportions of SOX2-expressing cells at the 96 16- and 32-cell stages between non-mutant embryos and embryos lacking Nanog, Oct4, 97 or both (Fig. S1B,C). These observations indicate that Nanog and Oct4 do not regulate 98 initial Sox2 expression. 99 100 Nanog and Oct4 are individually required to maintain Sox2 expression 101 To investigate a role for Nanog and Oct4 in maintaining expression of Sox2, we 102 evaluated double null embryos at a later time point. By E3.5, SOX2 appeared weak or 103 undetectable in most cells of Nanog;Oct4 null embryos (Fig. 1C). Moreover, the 104 proportion of cells expressing the wild type level of SOX2 was significantly lower in 105 Nanog;Oct4 null embryos (Fig. 1D), but not in embryos lacking Nanog or Oct4 only (Fig. 106 S1D). We therefore conclude that Nanog and Oct4 work together to maintain Sox2 107 expression and can compensate for the loss of one another up to at least E3.5. 108 109 To evaluate whether Nanog and Oct4 cooperatively maintain Sox2 expression at later 110 preimplantation stages, we examined SOX2 expression in embryos lacking either 111 Nanog or Oct4 at later developmental stages. At E3.75, SOX2 levels were 112 indistinguishable between non-mutant, Nanog null and Oct4 null embryos (Fig 1E,F). In 113 fact, the only difference between non-mutant, Oct4 null and Nanog null embryos at this

4 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

114 timepoint was the previously reported failure of Nanog null embryos to undergo primitive 115 endoderm differentiation by downregulating Nanog expression in a subset of inner mass 116 cells (Frankenberg et al., 2011; Messerschmidt and Kemler, 2010). 117 118 By contrast, both Nanog null and Oct4 null embryos exhibited defects in SOX2 by 119 E4.25. Nanog null embryos exhibited the more severe SOX2 expression phenotype, 120 with almost undetectable SOX2 (Fig. 1G). Oct4 null embryos exhibited a less severe 121 phenotype, with reduced, but detectable SOX2 (Fig. 1H). These observations indicate 122 that, while the initial phase of Sox2 expression is independent of Nanog and Oct4, this 123 is followed by a period during which Nanog and Oct4 act redundantly to maintain Sox2 124 expression, which then gives way to a phase during which Nanog and Oct4 are 125 individually required to achieve maximal Sox2 expression. 126 127 TEAD4/WWTR1/YAP1 regulate the onset of Sox2 expression 128 Having observed that the initiation of Sox2 expression is Nanog- and Oct4-independent, 129 we next examined the role of other factors in regulating initial Sox2 expression. TEAD4 130 and its co-factors WWTR1 and YAP1 repress Sox2 expression in outside cells starting 131 around the 16-cell stage (Frum et al., 2018; Wicklow et al., 2014). However, YAP1 is 132 detected within nuclei as early as the 4-cell stage (Nishioka et al., 2009), suggesting 133 that the complex is active prior to the 16-cell stage. Recent studies have highlighted the 134 roles and regulation of TEAD4/WWTR1/YAP1 in promoting outside cell maturation to 135 trophectoderm during blastocyst formation (Anani et al., 2014; Cao et al., 2015; 136 Cockburn et al., 2013; Hirate et al., 2013; Kono et al., 2014; Leung and Zernicka-Goetz, 137 2013; Lorthongpanich et al., 2013; Nishioka et al., 2009; Nishioka et al., 2008; Posfai et 138 al., 2017; Rayon et al., 2014; Shi et al., 2017; Yagi et al., 2007; Yu et al., 2016). Yet, the 139 developmental requirement for TEAD4/WWTR1/YAP1 prior to the 16-cell stage has not 140 been investigated. We therefore hypothesized that TEAD4/WWTR1/YAP1 repress Sox2 141 expression prior to the 16-cell stage. 142 143 To test this hypothesis, we examined SOX2 in embryos lacking Tead4. Consistent with 144 our hypothesis, Tead4 null embryos exhibited precocious SOX2 at the 8-cell stage (Fig.

5 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

145 2A,C). Notably, this phenotype that was not exacerbated by elimination of maternal 146 Tead4 (Fig. S2A and Fig. 2A,C), consistent with the absence of detectable Tead4 in 147 oocytes (Yagi et al., 2007). By contrast, deletion of maternal Wwtr1 and Yap1 (Fig. S2B) 148 led to precocious SOX2 at the 8-cell stage (Fig. 2B,D). The presence of wild-type, 149 paternal alleles of Wwtr1 and/or Yap1 did not rescue SOX2 in the maternally null 150 embryos (Fig. 2B, D). Therefore, maternally provided WWTR1/YAP1 and zygotically 151 expressed TEAD4 repress Sox2 expression at the 8-cell stage. 152 153 We next evaluated SOX2 in embryos lacking maternal Wwtr1;Yap1 at the 4-cell stage. 154 We observed that 4-cell embryos lacking maternal Wwtr1 and Yap1 occasionally 155 exhibited weak ectopic SOX2 (Fig. S2D,E). However, SOX2 was never detected in 4- 156 cell embryos lacking Tead4 (Fig. S2C). These observations suggest that Wwtr1 and 157 Yap1 partner with other factors to regulate the onset of Sox2 expression at the 4-cell 158 stage and point to a requirement for other TEAD that are expressed at the 4- 159 cell stage (Nishioka et al., 2008). 160 161 The premature onset of Sox2 expression in embryos lacking Tead4 or Wwtr1 and Yap1 162 demonstrates that preimplantation mouse embryos are capable of expressing markers 163 of inside cell identity as early as the 4-cell stage and reveals an earlier than expected 164 role for TEAD4/WWTR1/YAP1 in repressing the expression of Sox2 until the formation 165 of inside cells, thus permitting the establishment of discrete trophectoderm and inner 166 cell mass gene expression. Furthermore, the appearance of Sox2 expression prior to 167 the formation of inside cells argues that no cues specific to inside-position are required 168 for Sox2 expression beyond regulated TEAD4/WWTR1/YAP1 activity. Rather, our 169 results suggest that the mechanism regulating the onset of Sox2 expression is that 170 constitutive repression of Sox2 by TEAD4/WWTR1/YAP1 is relieved once cells are 171 positioned inside the embryo at the 16-cell stage. 172 173 Repression of Sox2 at the 4- and 8-cell stage is sensitive to LATS2 kinase 174 In many contexts, TEAD4/WWTR1/YAP1 activity is repressed by the HIPPO pathway 175 LATS1/2 kinases, which repress nuclear localization of WWTR1/YAP1 (Nishioka et al.,

6 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

176 2009; Zhao et al., 2010; Zhao et al., 2007). To evaluate the role of HIPPO signaling in 177 regulating initial Sox2 expression, we therefore examined whether Sox2 expression is 178 LATS1/2-sensitive prior to the 16-cell stage. 179 180 We injected mRNA encoding Lats2 into both blastomeres of 2-cell stage embryos, 181 which is sufficient to inactivate the TEAD4/WWTR1/YAP1 complex during blastocyst 182 formation (Nishioka et al., 2009; Wicklow et al., 2014), and then evaluated SOX2 at 4- 183 and 8-cell stages (Fig. 3A). As anticipated, Lats2 mRNA injection, but not injection of 184 Green Fluorescent mRNA, greatly reduced YAP1 nuclear localization at 4- and 185 8-cell stages (Fig. 3B,C). In addition, we observed precocious SOX2 in embryos 186 overexpressing Lats2 (Fig 3B,C). Therefore, LATS kinases can prevent 187 TEAD4/WWTR1/YAP1 from repressing expression of Sox2 prior to the 16-cell stage, 188 but LATS kinases must not normally do so, since Sox2 is usually repressed during early 189 development. 190 191 We observed in published RNA-seq data sets that Lats1 and Lats2 are expressed in 4- 192 cell and 8-cell stage embryos (Tang et al., 2010; Wu et al., 2016), opening an 193 interesting future direction of discovering how TEAD4/WWTR1/YAP1 escape inhibition 194 by LATS kinases prior to the 16-cell stage, which is not currently understood. 195 196 TEAD4/WWTR1/YAP1 may repress Sox2 expression through a direct mechanism 197 While the TEAD4/WWTR1/YAP1 complex is widely recognized as a transcriptional 198 activator, it has more recently been shown to act as a transcriptional repressor (Beyer et 199 al., 2013; Kim et al., 2015). Therefore, we considered two mechanisms by which 200 TEAD4/WWTR1/YAP1 could repress Sox2 expression (Fig. 4A): an indirect model, in 201 which TEAD4/WWTR1/YAP1 promote transcription of a Sox2 repressor, and a direct 202 model, in which TEAD4/WWTR1/YAP1 themselves act as the Sox2 repressor. 203 204 To test these models, we employed variants of Tead4 in which the WWTR1/YAP1 205 interaction domain has been replaced with either the transcriptional activator VP16 206 (Tead4VP16) or the transcriptional repressor engrailed (Tead4EnR). These variants

7 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

207 have previously been used in preimplantation embryos to provide evidence that 208 TEAD4/WWTR1/YAP1 promote Cdx2 expression through a direct mechanism (Nishioka 209 et al., 2009). We reasoned that if TEAD4/WWTR1/YAP1 represses Sox2 indirectly, then 210 overexpression of Tead4EnR would induce Sox2 expression. Alternatively, if 211 TEAD4/WWTR1/YAP1 represses Sox2 directly, then Tead4VP16 would induce Sox2 212 expression. We injected mRNA encoding Tead4VP16 or Tead4EnR into a single 213 blastomere of 4-cell stage embryos and tracked progeny of the injected blastomere at 214 the 8-cell stage with co-injection of GFP (Fig. 4B). We observed that overexpression of 215 Tead4VP16, but not Tead4EnR induced SOX2 (Fig. 4C,D). These observations are 216 consistent with the direct repression of Sox2 by TEAD4/WWTR1/YAP1. 217 218 This study highlights distinct phases of Sox2 regulation occurring during the 219 establishment of pluripotency in mouse development. As early as the 4-cell stage, 220 blastomeres are competent to express Sox2, but this is overridden by 221 TEAD/WWTR1/YAP1 (Fig. 4E, box 1). Initiation of Sox2 expression is independent of 222 Nanog and Oct4 and does not require cues associated with inside cell position or 223 developmental time. Instead, LATS1/2 activity in inside cells relieves repression of 224 TEAD4/WWTR1/YAP1 on Sox2 (Fig. 4E, box 2). After blastocyst formation, NANOG 225 and OCT4 work together ensure that Sox2 expression is maintained (Fig 4E, box 3). 226 Finally, as the embryo approaches implantation, Nanog and Oct4 become individually 227 required to sustain Sox2 expression (Fig. 4E, box 4). 228 229 Figure Legends 230 231 Figure 1. Nanog and Oct4 are required for the maintenance, but not the initiation 232 of Sox2. (A) Immunostaining for SOX2, E-Cadherin (ECAD), and DNA in non-mutant 233 and Nanog;Oct4 null embryos at the 16-cell stage (E3.0). (B) SOX2, ECAD, and DNA in 234 non-mutant and Nanog;Oct4 null embryos at the 32-cell stage (E3.25). (C) SOX2 in 235 non-mutant and Nanog;Oct4 null embryos at E3.5. (D) Quantification of the percentage 236 of inside cells, across all embryos, with intense SOX2 staining in non-mutant and 237 Nanog;Oct4 null embryos at E3.5. Columns = mean, error bars = standard deviation, p

8 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

238 = Student’s t-test. (E) NANOG-GFP, SOX2, and DNA in non-mutant and Nanog null 239 embryos at E3.75. (F) NANOG, SOX2, and DNA in non-mutant and Oct4 null embryos 240 at E3.75. (G) NANOG-GFP, SOX2, and DNA in non-mutant and Nanog null embryos at 241 E4.25. (H) NANOG, SOX2, and DNA in non-mutant and Oct4 null embryos at E4.25. 242 For all panels, n = number of embryos examined. Dashed white lines demarcate non- 243 epiblast/presumptive primitive endoderm cells. 244 245 Figure 2. TEAD4/WWTR1/YAP1 repress precocious Sox2 expression at the 8-cell 246 stage. (A) Immunostaining for SOX2 in non-mutant, Tead4 zygotic (z) null and Tead4 247 maternal-zygotic (mz) null embryos at the 8-cell stage. (B) SOX2 in embryos lacking m 248 Wwtr1 and Yap1 at the 8-cell stage, with indicated zygotic genotypes. (C) The 249 percentage of SOX2-positive cells per embryo (dots) in non-mutant, Tead4 z null and 250 Tead4 mz null embryos. (D) The percentage of SOX2-positive cells per embryo (dots) in 251 Wwtr1/Yap1 m null embryos with indicated zygotic genotype. Dashed line = mean, p = 252 one-way ANOVA with Tukey post-hoc test, n = number of embryos examined. 253 254 Figure 3. YAP1 localization and Sox2 expression are sensitive to LATS2 kinase. 255 (A) Experimental approach: both blastomeres of 2-cell stage embryos were injected with 256 either 500 ng/µl NLS-GFP mRNA, which encodes GFP bearing a nuclear localization 257 sequence (NLS), or 500 ng/µl Lats2 mRNA, and were then cultured to the 4- or 8-cell 258 stages. (B) YAP1 and SOX2 immunostaining in 4-cell stage embryos injected with NLS- 259 GFP mRNA or Lats2 mRNA. (C) YAP1 and SOX2 in 8-cell stage embryos injected with 260 NLS-GFP mRNA or Lats2 mRNA. (D) Dot-plot of the percentage of SOX2-positive cells 261 per embryo (dots) at the indicated stages. The means and standard deviations are 262 represented by columns and error bars, while n = number of embryos examined. 263 264 Figure 4. TEAD4/WWTR1/YAP1 repress Sox2 expression through a direct 265 mechanism. (A) Models for indirect and direct repression of Sox2 by 266 TEAD4/WWTR1/YAP1 and predicted effect of Tead4EnR and Tead4VP16 on Sox2 267 expression. (B) Experimental approach: a single blastomere of each 4-cell embryo was 268 injected with 150 ng/µl NLS-GFP mRNA and either 150 ng/µl Tead4VP16 or Tead4EnR

9 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

269 mRNA, and then cultured to the 8-cell stage. (C) GFP and SOX2 immunostaining in 270 embryos injected with Tead4VP16 or Tead4EnR. (D) Quantification of the percentage of 271 NLS-GFP-positive, SOX2-positive cells per embryo (dots) injected with Tead4VP16 and 272 Tead4EnR. p = student’s t-test, n = number of embryos examined. (E) Model for 273 regulation of Sox2 at indicated developmental stages. ICM = inner cell mass, TE = 274 trophectoderm, EPI = epiblast, PE = primitive endoderm. 275 276 Materials and Methods 277 278 Mouse strains 279 Animal care and husbandry was performed in accordance with the guidelines 280 established by the Institutional Animal Care and Use Committee at Michigan State 281 University. Wild type embryos were generated by mating CD-1 mice (Charles River). 282 Female mice used in this study were between six weeks and six months of age and 283 males were used from eight weeks to nine months. Alleles and transgenes used in this 284 study were maintained on a CD-1 background and include: Nanogtm1.1Hoch (Maherali et 285 al., 2007), Pou5f1tm1Scho (Kehler et al., 2004), Tead4tm1Bnno (Yagi et al., 2007), 286 Yap1tm1.1Eno (Xin et al., 2011), Wwtr1tm1.1Eno (Xin et al., 2013), Tg(Zp3-cre)93Knw (De 287 Vries et al., 2004). Conditional, floxed alleles were recombined to generate null alleles 288 by breeding mice carrying conditional alleles to Alpltm(cre)Nagy (Lomelí et al., 2000) mice. 289 290 Embryo collection and culture 291 Embryos were collected from naturally mated mice by flushing dissected oviducts or

292 uteri with M2 medium (Millipore-Sigma). All embryos were cultured in 5% CO2 at 37ºC 293 under ES cell grade mineral oil (Millipore-Sigma). Prior to embryo culture, KSOM 294 medium (Millipore-Sigma) was equilibrated overnight in the embryo incubator. 295 296 Embryo microinjection 297 cDNAs encoding Lats2, Tead4VP16, and Tead4EnR (Nishioka et al., 2009) cloned into 298 the pcDNA3.1 poly(A)83 plasmid (Yamagata et al., 2005) were linearized, and then 299 used as a template to generate mRNAs for injection by the mMessage mMachine T7

10 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

300 transcription kit (Invitrogen). NLS-GFP mRNA was synthesized from linearized NLS- 301 GFP plasmid (Ariotti et al., 2015) using the mMessage mMachine Sp6 transcription kit 302 (Invitrogen). Prior to injection, synthesized mRNAs were cleaned and concentrated 303 using the MEGAclear Transcription Clean-up Kit (Invitrogen). Lats2 and NLS-GFP 304 mRNAs were injected into both blastomeres of 2-cell stage embryos at a concentration 305 of 500 ng/µl. Tead4VP16 or Tead4EnR mRNAs were injected into a single blastomere 306 of 4-cell stage embryos at a concentration of or 150 ng/µl each. NLS-GFP mRNA was 307 included in 4-cell stage injections at a concentration of 150 ng/µl to trace the progeny of 308 the injected blastomere. All mRNAs were diluted in 10 mM Tris-HCl (pH 7.4), 0.1 mM 309 EDTA. Injections were performed using a Harvard Apparatus PL-100A microinjector.

310 Immunofluorescence and Confocal Microscopy 311 Embryos were fixed in 4% formaldehyde (Polysciences) for 10 minutes, permeabilized 312 with 0.5% Triton X-100 (Millipore-Sigma) for 30 minutes and blocked with 10% FBS, 313 0.1% Triton X-100 for at least 1 hour at room temperature or longer at 4ºC. Primary 314 antibody incubation was performed at 4ºC overnight using the following antibodies: goat 315 anti-SOX2 (Neuromics, GT15098, 1:2000), rabbit anti-NANOG (Reprocell, RCAB002P- 316 F, 1:400) mouse anti-Tead4 (Abcam, ab58310, 1:1000), mouse anti-YAP (Santa Cruz, 317 sc101199, 1:200), and rat anti-ECAD (Millipore-Sigma, U3254, 1:500). Anti-SOX2, anti- 318 TEAD4 and anti-YAP antibodies were validated by the absence of positive staining on 319 embryos homozygous for null alleles encoding antibody target Nuclei were labelled 320 with either DRAQ5 (Cell Signaling Technology) or DAPI (Millipore-Sigma). Antibodies 321 raised against IgG and coupled to Dylight 488, Cy3 or Alexa Fluor 647 (Jackson 322 ImmunoResearch) were used to detect primary antibodies. Embryos were imaged on 323 an Olympus FluoView FV1000 Confocal Laser Scanning Microscope using a 20x 324 UPlanFLN objected (0.5 NA) and 5x digital zoom. Each embryo was imaged in entirety 325 using 5 µm optical section thickness. 326 327 Image Analysis 328 Z-stacks obtained from confocal microscopy were analyzed using ImageJ (Schneider et 329 al., 2012). Each nucleus was identified by DNA stain and then scored for the presence 330 or absence of SOX2. In Fig. 1A and B, cells were classified as inside or outside on the

11 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

331 basis of ECAD localization. For analysis of Nanog;Oct4 null embryos in Fig. 1C,D and 332 Fig. S1D, SOX2 staining intensity was categorized as intense or weak. Intense SOX2 333 staining was defined as the level observed in non-mutant embryos, which was uniform 334 among inside cells. In Fig. 1, S1, 2, and S2, embryo genotypes were not known prior to 335 analysis. In Fig. 3 and 4 embryos were grouped according to injection performed, and 336 therefore the researcher was not blind to embryo treatment. 337 338 Embryo Genotyping 339 For embryos at the 8-cell stage or older, DNA was extracted from fixed embryos after 340 imaging using the Extract-N-Amp kit (Millipore-Sigma) in a total volume of 10 µl. For 341 embryos at the 4-cell stage, DNA was extracted from fixed embryos in a total volume of 342 5 µl. 1 µl of extracted DNA sw useda as template, w ith allele specific primers (Table 343 S1). 344 345 Acknowledgements 346 We thank members of the Ralston Lab for thoughtful discussion. This work was 347 supported by National Institutes of Health (R01 GM104009 and R35 GM131759 to A.R. 348 and T32HD087166 to J.W.). 349 350 Competing Interests 351 No competing interests declared. 352 353 References 354 355 Anani, S., Bhat,, HonS. ma-Yamanaka, N., Krawchuk, D. and Yamanaka, Y. (2014). 356 Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre- 357 implantation mouse embryo. Development 141, 2813–2824.

358 Ariotti, N., Hall, T. E., Rae, J., Ferguson, C., McMahon, K.-A., Martel, N., Webb, R. 359 E., Webb, R. I., Teasdale, R. D. and Parton, R. G. (2015). Modular Detection of 360 GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and 361 Organisms. Developmental Cell 35, 513–525.

12 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

362 Beyer, T. A., Weiss, A., Khomchuk, Y., Huang, K., Ogunjimi, A. A., Varelas, X. and 363 Wrana, J. L. (2013). Switch enhancers interpret TGF-β and Hippo signaling to 364 control cell fate in human embryonic stem cells. CellReports 5, 1611–1624.

365 Boroviak, T., Loos, R., Bertone, P., Smith, A. and Nichols, J. (2014). The ability of 366 inner-cell-mass cells to self-renew as embryonic stem cells is acquired following 367 epiblast specification. Nat Cell Biol 16, 516–528.

368 Boroviak, T., Loos, R., Lombard, P., Okahara, J., Behr, R., Sasaki, E., Nichols, J., 369 Smith, A. and Bertone, P. (2015). Lineage-Specific Profiling Delineates the 370 Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis. 371 Developmental Cell 35, 366–382.

372 Cao, Z., Carey, T. S., Ganguly, A., Wilson, C. A., Paul, S. and Knott, J. G. (2015). 373 Transcription factor AP-2 induces early Cdx2 expression and represses HIPPO 374 signaling to specify the trophectoderm lineage. Development 142, 1606–1615.

375 Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and Smith, 376 A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor 377 in embryonic stem cells. Cell 113, 643–655.

378 Cockburn, K., Biechele, S., Garner, J. and Rossant, J. (2013). The Hippo Pathway 379 Member Nf2 Is Required for Inner Cell Mass Specification. Current Biology 23, 380 1195–1201.

381 De Vries, W. N., Evsikov, A. V., Haac, B. E., Fancher, K. S., Holbrook, A. E., 382 Kemler, R., Solter, D. and Knowles, B. B. (2004). Maternal beta-catenin and E- 383 cadherin in mouse development. Development 131, 4435–4445.

384 Dietrich, J. E. and Hiiragi, T. (2007). Stochastic patterning in the mouse pre- 385 implantation embryo. Development 134, 4219–4231.

386 Frankenberg, S., Gerbe, F., Bessonnard, S., Belville, C., Pouchin, P., Bardot, O. 387 and Chazaud, C. (2011). Primitive Endoderm Differentiates via a Three-Step

13 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

388 Mechanism Involving Nanog and RTK Signaling. Developmental Cell 21, 1005– 389 1013.

390 Frum, T., Halbisen, M. A., Wang, C., Amiri, H., Robson, P. and Ralston, A. (2013). 391 Oct4 Cell-Autonomously Promotes Primitive Endoderm Development in the Mouse 392 Blastocyst. Developmental Cell 25, 610–622.

393 Frum, T., Murphy, T. M. and Ralston, A. (2018). HIPPO signaling resolves embryonic 394 cell fate conflicts during establishment of pluripotency in vivo. Elife 7, 347.

395 Guo, G., Huss, M., Tong, G. Q., Wang, C., Sun, L. L., Clarke, N. D. and Robson, P. 396 (2010). Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression 397 Analysis from Zygote to Blastocyst. Developmental Cell 18, 675–685.

398 Hirate, Y., Hirahara, S., Inoue, K.-I., Suzuki, A., Alarcon, V. B., Akimoto, K., Hirai, 399 T., Hara, T., Adachi, M., Chida, K., et al. (2013). Polarity-Dependent Distribution of 400 Angiomotin Localizes Hippo Signaling in Preimplantation Embryos. Current Biology 401 23, 1181–1194.

402 Kehler, J., Tolkunova, E., Koschorz, B., Pesce, M., Gentile, L., Boiani, M., Lomelí, 403 H., Nagy, A., McLaughlin, K. J., Schöler, H. R., et al. (2004). Oct4 is required for 404 primordial germ cell survival. EMBO Rep 5, 1078–1083.

405 Kim, M., Kim, T., Johnson, R. L. and Lim, D.-S. (2015). Transcriptional co-repressor 406 function of the hippo pathway transducers YAP and TAZ. CellReports 11, 270–282.

407 Kono, K., Tamashiro, D. A. A. and Alarcon, V. B. (2014). Inhibition of RHO-ROCK 408 signaling enhances ICM and suppresses TE characteristics through activation of 409 Hippo signaling in the mouse blastocyst. Developmental Biology 394, 142–155.

410 Leung, C. Y. and Zernicka-Goetz, M. (2013). Angiomotin prevents pluripotent lineage 411 differentiation in mouse embryos via Hippo pathway-dependent and -independent 412 mechanisms. Nature Communications 4, 1–11.

14 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

413 Lomelí, H., Ramos-Mejía, V., Gertsenstein, M., Lobe, C. G. and Nagy, A. (2000). 414 Targeted insertion of Cre recombinase into the TNAP gene: excision in primordial 415 germ cells. genesis 26, 116–117.

416 Lorthongpanich, C., Messerschmidt, D. M., Chan, S. W., Hong, W., Knowles, B. B. 417 and Solter, D. (2013). Temporal reduction of LATS kinases in the early 418 preimplantation embryo prevents ICM lineage differentiation. Genes & Development 419 27, 1441–1446.

420 Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., 421 Yachechko, R., Tchieu, J., Jaenisch, R., et al. (2007). Directly reprogrammed 422 fibroblasts show global epigenetic remodeling and widespread tissue contribution. 423 Cell Stem Cell 1, 55–70.

424 Messerschmidt, D. M. and Kemler, R. (2010). Nanog is required for primitive 425 endoderm formation through a non-cell autonomous mechanism. Developmental 426 Biology 1–27.

427 Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., 428 Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Nanog is 429 required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 430 631–642.

431 Nishioka, N., Inoue, K.-I., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta, N., 432 Hirahara, S., Stephenson, R. O., Ogonuki, N., et al. (2009). The Hippo Signaling 433 Pathway Components Lats and Yap Pattern Tead4 Activity to Distinguish Mouse 434 Trophectoderm from Inner Cell Mass. Developmental Cell 16, 398–410.

435 Nishioka, N., Yamamoto, S., Kiyonari, H., Sato, H., Sawada, A., Ota, M., Nakao, K. 436 and Sasaki, H. (2008). Tead4 is required for specification of trophectoderm in pre- 437 implantation mouse embryos. Mech. Dev. 125, 270–283.

15 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

438 Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. and 439 Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 Determines 440 Trophectoderm Differentiation. Cell 123, 917–929.

441 Palmieri, S. L., Peter, W., Hess, H. and Scholer, H. R. (1994). Oct-4 transcription 442 factor is differentially expressed in the mouse embryo during establishment of the 443 first two extraembryonic cell lineages involved in implantation. Developmental 444 Biology 166, 259–267.

445 Posfai, E., Petropoulos, S., de Barros, F. R. O., Schell, J. P., Jurisica, I., Sandberg, 446 R., Lanner, F. and Rossant, J. (2017). Position- and Hippo signaling-dependent 447 plasticity during lineage segregation in the early mouse embryo. Elife 6, e22906.

448 Rayon, T., Menchero, S., Nieto, A., Xenopoulos, P., Crespo, M., Cockburn, K., 449 Cañon, S., Sasaki, H., Hadjantonakis, A.-K., la Pompa, de, J. L., et al. (2014). 450 Notch and hippo converge on Cdx2 to specify the trophectoderm lineage in the 451 mouse blastocyst. Developmental Cell 30, 410–422.

452 Rosner, M. H., Vigano, M. A., Ozato, K., Timmons, P. M., Poirier, F., Rigby, P. W. 453 and Staudt, L. M. (1990). A POU-domain transcription factor in early stem cells and 454 germ cells of the mammalian embryo. Nature 345, 686–692.

455 Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 456 years of image analysis. Nat Meth 9, 671–675.

457 Shi, X., Yin, Z., Ling, B., Wang, L., Liu, C., Ruan, X., Zhang, W. and Chen, L. (2017). 458 Rho differentially regulates the Hippo pathway by modulating the interaction 459 between Amot and Nf2 in the blastocyst. Development.

460 Strumpf, D., Mao, C.-A., Yamanaka, Y., Ralston, A., Chawengsaksophak, K., Beck, 461 F. and Rossant, J. (2005). Cdx2 is required for correct cell fate specification and 462 differentiation of trophectoderm in the mouse blastocyst. Development 132, 2093– 463 2102.

16 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

464 Tang, F., Barbacioru, C., Bao, S., Lee, C., Nordman, E., Wang, X., Lao, K. and 465 Surani, M. A. (2010). Tracing the Derivation of Embryonic Stem Cells from the Inner 466 Cell Mass by Single-Cell RNA-Seq Analysis. Stem Cell 6, 468–478.

467 Wicklow, E., Blij, S., Frum, T., Hirate, Y., Lang, R. A., Sasaki, H. and Ralston, A. 468 (2014). HIPPO pathway members restrict SOX2 to the inner cell mass where it 469 promotes ICM fates in the mouse blastocyst. PLoS Genetics 10, e1004618.

470 Wu, J., Huang, B., Chen, H., Yin, Q., Liu, Y., Xiang, Y., Zhang, B., Liu, B., Wang, Q., 471 Xia, W., et al. (2016). The landscape of accessible chromatin in mammalian 472 preimplantation embryos. Nature 534, 652–657.

473 Xin, M., Kim, Y., Sutherland, L. B., Murakami, M., Qi, X., McAnally, J., Porrello, E. 474 R., Mahmoud, A. I., Tan, W., Shelton, J. M., et al. (2013). Hippo pathway effector 475 Yap promotes cardiac regeneration. Proc. Natl. Acad. Sci. U.S.A. 110, 13839– 476 13844.

477 Xin, M., Kim, Y., Sutherland, L. B., Qi, X., McAnally, J., Schwartz, R. J., 478 Richardson, J. A., Bassel-Duby, R. and Olson, E. N. (2011). Regulation of 479 insulin-like signaling by Yap governs cardiomyocyte proliferation and 480 embryonic heart size. Sci Signal 4, ra70–ra70.

481 Yagi, R., Kohn, M. J., Karavanova, I., Kaneko, K. J., Vullhorst, D., DePamphilis, M. 482 L. and Buonanno, A. (2007). Transcription factor TEAD4 specifies the 483 trophectoderm lineage at the beginning of mammalian development. Development 484 134, 3827–3836.

485 Yamagata, K., Yamazaki, T., Yamashita, M., Hara, Y., Ogonuki, N. and Ogura, A. 486 (2005). Noninvasive visualization of molecular events in the mammalian zygote. 487 genesis 43, 71–79.

488 Yu, C., Ji, S.-Y., Dang, Y.-J., Sha, Q.-Q., Yuan, Y.-F., Zhou, J.-J., Yan, L.-Y., Qiao, J., 489 Tang, F. and Fan, H.-Y. (2016). Oocyte-expressed yes-associated protein is a key 490 activator of the early zygotic genome in mouse. Cell Res 26, 275–287.

17 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

491 Zhao, B., Li, L., Tumaneng, K., Wang, C.-Y. and Guan, K.-L. (2010). A coordinated 492 phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP). 493 Genes & Development 24, 72–85.

494 Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., 495 Li, L., et al. (2007). Inactivation of YAP oncoprotein by the Hippo pathway is 496 involved in cell contact inhibition and tissue growth control. Genes & Development 497 21, 2747–2761.

18 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

498

19 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Frum et al., 2019 Figure 1 A. SOX2 ECAD DNA B. SOX2 ECAD DNA C. SOX2 SOX2 DNA non-mutant non-mutant non-mutant E3.0 n = 14 E3.25 n = 22 E3.5 n = 21 null null null Nanog; Oct4 Nanog; Oct4 Nanog; Oct4 n = 3 n = 4 n = 5

D.E3.5 E. F. p = 0.003 GFP NANOG Nanog-GFP SOX2 SOX2 DNA NANOG SOX2 SOX2 DNA 100 non-mutant 50 Nanog +/GFP E3.75 n = 15 E3.75 n = 9 n = 5 n = 21

0 Oct4 null Nanog null % inside cells with intense SOX2 n = 4 n = 3 non-mutantNanog;Oct4 null G. GFP H. NANOG Nanog-GFP SOX2 SOX2 DNA NANOG SOX2 SOX2 DNA non-mutant Nanog +/GFP E4.25 n = 9 E4.25 n = 13 Oct4 null Nanog null n = 4 n = 5 bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Frum et al., 2019 Figure 2 A. SOX2 DNA B. SOX2 DNA

non-mutant n = 16 +/- Wwtr1 +/-; Yap1 n = 5 Tead4 z null Tead4 n = 6 n = 5 -/- Wwtr1 +/-; Yap1 m null Wwtr1 / Yap1 Tead4 mz null Tead4 n = 6 -/- Wwtr1 -/-; Yap1 n = 4

C. p = 0.001 D. p = 0.001 p = 0.299 p > 0.50 100 100

50 50 (8-cell stage) (8-cell stage) % of cells SOX2-positive % of cells SOX2-positive x16 0 0 non- Tead4 Tead4 Wwtr1+/- Wwtr1+/- Wwtr1-/- Wwtr1-/- mutant z null mz null Yap1+/- Yap1-/- Yap1+/- Yap1-/- Wwtr1/Yap1 m null bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Frum et al., 2019 Figure 3 A. 500 ng/µl Lats2 or B. YAP1 SOX2 DNA 500 ng/µl NLS-GFP mRNA mRNA 24 hrs 16 hrs NLS-GFP n = 5 Lats2 mRNA Fig. 3B,D Fig. 3C,D n = 14 C. D. YAP1 SOX2 DNA 100 mRNA

NLS-GFP 50 n = 3 Lats2

mRNA 0 % of cells SOX2-positive n = 10 4-cell stage 8-cell stage bioRxiv preprint doi: https://doi.org/10.1101/663005; this version posted June 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Frum et al., 2019 Figure 4 C. A. B.150 ng/µl NLS-GFP + NLS-GFP SOX2 DNA Indirect Model: 150 ng/µl Tead4VP16 or Tead4EnR

WWTR1/YAP1 TEAD4 Repressor Sox2

Prediction: Tead4EnR will turn on Sox2 Tead4VP16 12 hrs 8-cell stage n = 7 Direct Model:

WWTR1/YAP1 TEAD4 Sox2 Tead4EnR Prediction: Tead4VP16 will turn on Sox2 Fig. 4C,D 8-cell stage n = 5

D. p < 0.001 E. 1. 2. 3. 4.

100 LATS1/2 WWTR1/YAP1 Nanog Nanog TEAD4 Sox2 WWTR1/YAP1 or Oct4 and Oct4 all cells TEAD4 Sox2 Sox2 Sox2 ICM cells inside cells EPI cells

50 ICM

EPI outside cells PE

0 % injected cells SOX2-positive WWTR1/YAP1 TEAD4 Sox2 TE

Tead4VP16 Tead4EnR 4- to 8-cell stage 16-cell stage early blastocyst stage late blastocyst stage + NLS-GFP + NLS-GFP