HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo

During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.


Introduction
During embryogenesis cells gradually differentiate, adopting distinct gene expression profiles and fates. In mammals, the first cellular differentiation is the segregation of trophectoderm and inner cell mass. The trophectoderm, which comprises the polarized outer surface of the blastocyst, will mainly produce cells of the placenta, while the inner cell mass will produce pluripotent cells, which are progenitors of both fetus and embryonic stem cells. Understanding how pluripotent inner cell mass cells are segregated from non-pluripotent cells therefore reveals how pluripotency is induced in a naturally occurring setting.
Progenitors of inner cell mass are first morphologically apparent at the 16-cell stage as unpolarized cells residing inside the morula (reviewed in Frum and Ralston, 2018).
However, at this stage, pluripotency genes such as Oct4 and Nanog, do not specifically label inside cells (Dietrich and Hiiragi, 2007;Niwa et al., 2005;Palmieri et al., 1994;Strumpf et al., 2005). Thus, the first cell fate decision has been studied mainly from the perspective of trophectoderm specification because the transcription factor CDX2, which is essential for trophectoderm development , is expressed specifically in outer cells of the 16-cell embryo (Ralston and Rossant, 2008), and has provided a way to distinguish future trophectoderm cells from non-trophectoderm cells.
Knowledge of CDX2 as a marker of trophectoderm cell fate enabled the discovery of mechanisms that sense cellular differences in polarity and position in the embryo, and then respond by regulating expression of Cdx2 (Nishioka et al., 2009). However, the exclusive study of Cdx2 regulation does not provide direct knowledge of how 4 pluripotency is established because the absence of Cdx2 expression does not necessarily indicate acquisition of pluripotency. As such, our understanding of the first cell fate decision in the early mouse embryo is incomplete.
In contrast to other markers of pluripotency, Sox2 is expressed specifically in inside cells at the 16-cell stage, and is therefore the first marker of pluripotency in the embryo Wicklow et al., 2014). The discovery of how Sox2 expression is regulated in the embryo provide therefore provide unique insight into how pluripotency is first established in vivo. Genes promoting expression of Sox2 in the embryo have been described (Cui et al., 2016;Wallingford et al., 2017). However, it is currently unclear how expression of Sox2 becomes restricted to inside cells. Interestingly, Sox2 is restricted to inside cells by a Cdx2-independent mechanism (Wicklow et al., 2014), which differs from Oct4 and Nanog, which are restricted to the inner cell mass by CDX2 (Niwa et al., 2005;Strumpf et al., 2005). Thus, Sox2 and Cdx2 are regulated in parallel, leading to complementary inside/outside expression patterns. However, it is not known whether Sox2 is regulated by the same pathway that regulates Cdx2 or whether a distinct pathway could be in use.
However, the specific requirements for Yap1 and Wwtr1 in the regulation of Cdx2 has been inferred from overexpression of wild type and dominant-negative variants, neither of which provide the standard of gene expression analysis that null alleles can provide.
Nonetheless, the roles of Yap1 and Wwtr1 in regulating expression of Sox2 have not been investigated. Here, we evaluate the roles of maternal and zygotic YAP1/WWTR1 in regulating expression of Sox2 and cell fate during blastocyst formation.

Patterning of Sox2 is ROCK-dependent
To identify the mechanisms regulating Sox2 expression during blastocyst formation, we focused on how Sox2 expression is normally repressed in the trophectoderm to achieve inside cell-specific expression. We previously showed that SOX2 is specific to inside cells in the absence of the trophectoderm factor CDX2 (Wicklow et al., 2014), suggesting that mechanisms that repress Sox2 in the trophectoderm act upstream of Cdx2. Rho-associated, coiled-coil containing protein kinases (ROCK1 and 2) are thought to act upstream of Cdx2 because embryos developing in the presence of a ROCK-inhibitor (Y-27632, ROCKi) exhibit reduced Cdx2 expression (Kono et al., 2014).
Additionally, quantitative RT-PCR showed that Sox2 mRNA levels are elevated in ROCKi-treated embryos (Kono et al., 2014), suggesting that ROCK1/2 activity leads to 6 transcriptional repression of Sox2. However, the role of ROCK1/2 in regulating the spatial expression of Sox2 has not been investigated.
To evaluate the roles of ROCK1/2 in patterning Sox2 expression, we collected 8-cell stage embryos prior to embryo compaction (E2.5), and then cultured these either in control medium or in the presence of ROCKi for 24 hours (Fig. 1A). Embryos cultured in control medium exhibited normal cell polarity, evidenced by the apical localization of PARD6B and basolateral localization of E-cadherin (CDH1) in outside cells (Fig. 1B,C) as expected (Vestweber et al., 1987;Vinot et al., 2005). Additionally, SOX2 was detected only in inside cells in control embryos (Fig. 1C, D). By contrast, embryos cultured in ROCK inhibitor exhibited defects in cell polarity (Fig. 1B', C'), consistent with prior studies (Kono et al., 2014). Interestingly, in ROCK inhibitor-treated embryos, we observed ectopic SOX2 expression in cells located on the outer surface of the embryo ( Fig. 1C', D), indicating that ROCK1/2 participates in the pathway responsible for repressing expression of Sox2 in the trophectoderm.
To scrutinize the identity of outside positioned SOX2-positive cells in ROCK-inhibited embryos, we co-stained an additional cohort of control and ROCKi-treated embryos with CDX2 and SOX2 and compared the overlap of lineage marker expression. In control embryos, CDX2 was detected only in outside cells (Fig. S1A) as expected at this stage (Ralston and Rossant, 2008;Strumpf et al., 2005). In ROCKi-treated embryos, CDX2 expression levels were reduced (Fig. S1A') as was the proportion of outside cells in which CDX2 was detected (Fig. S1B), as previously reported (Kono et al., 2014). 7 However, among outside cells, a substantial proportion coexpressed CDX2 and SOX2 in ROCK-inhibited embryos compared with controls ( Fig. 1E and S1A), suggesting that ROCK inhibition leads to an increase in outside cells of mixed lineage. Since SOX2 expression does not regulate expression of CDX2 (Wicklow et al., 2014), these observations suggest that ROCK1/2 activity regulate these genes through parallel mechanisms. We next sought to identify mediators that act downstream of ROCK1/2 to repress expression of Sox2 in the trophectoderm.

YAP1 is sufficient to repress expression of SOX2 in the inner cell mass
Several direct and indirect targets of ROCK1/2 kinases in the early embryo have been described (Alarcon and Marikawa, 2018;Shi et al., 2017). Among these is YAP1, a transcriptional partner of TEAD4 (Nishioka et al., 2009), since ROCK activity is required for the nuclear localization of YAP1 (Kono et al., 2014). Notably, Tead4 is required to repress expression of Sox2 in the trophectoderm (Wicklow et al., 2014), consistent with the possibility that YAP1 partners with TEAD4 to repress Sox2 expression in the trophectoderm. To test this hypothesis, we overexpressed a constitutively active variant of YAP1 (YAP1 CA ). Substitution of alanine at serine 112 leads YAP1 to be constitutively nuclear and constitutively active (YAP1 CA hereafter) (Dong et al., 2007;Nishioka et al., 2009;Zhao et al., 2007). We injected mRNAs encoding YAP1 CA and GFP into one of two blastomeres at the 2-cell stage, and then cultured these to the blastocyst stage (Fig.   1F). This mosaic approach to overexpression permitted comparison of YAP1 CAoverexpressing with non-injected cells, which served as internal negative controls. We first examined localization of YAP1 in these embryos at the morula stage, with the 8 expectation that YAP1 would be detected in nuclei of both inside and outside cells in YAP1 CA -overexpressing cells (Nishioka et al., 2009). As expected, YAP1 was observed in nuclei of all YAP1 CA -overexpressing cells (Fig. S1B, C). We next evaluated the consequences of ectopic nuclear YAP1 on expression of SOX2 in inside cells. We observed a conspicuous decrease in the proportion of YAP1 CA -overexpressing inside cells lacking detectable SOX2 (Fig. 1G, H). Therefore, nuclear YAP1 is sufficient to repress Sox2 expression in the inner cell mass, indicative of a likely role for YAP1 in repressing expression of Sox2 in the trophectoderm downstream of ROCK1/2.

LATS kinase is sufficient to induce inside cell positioning
To functionally test of the role of YAP1 in repressing expression of Sox2, we injected one of two blastomeres with mRNA encoding LATS2 kinase, which inactivates YAP1 and, presumably, the related protein WWTR1 by phosphorylation, causing their cytoplasmic retention (Nishioka et al., 2008). We then examined expression of SOX2 after culturing embryos to the blastocyst stage ( Fig. 2A), predicting that LATS2 kinase would induce the ectopic expression of Sox2 in outside cells. Surprisingly, we observed that almost all Lats2-overexpressing cells ended up within the inner cell mass by the blastocyst stage (Fig. 2B, C), in contrast to cells injected with GFP mRNA only, which contributed to both inner cell mass and trophectoderm. Notably, SOX2 was detected in all Lats2-overexpressing cells observed within the inner cell mass (Fig. 2D), suggesting that Lats2-overexpressing cells were not only localized to the inner cell mass but also position-appropriate regulation of Sox2. 9 The strikingly increased prevalence of Lats2-overexpressing cells in the inner cell mass was also associated with a stark decrease in the number of Lats2-overexpressing cells detected within the trophectoderm and a decrease in the number of outside cells compared to embryos injected with GFP mRNA alone (Fig. 2C, E), suggesting that Lats2-overexpressing outside cells either internalize or undergo cell death. Furthermore, we observed cellular fragments within the trophectoderm of Lats2-overexpressing embryos (Fig. 2B, yellow arrowheads), as well as increased TUNEL staining in Lats2overexpressing embryos compared to embryos injected with GFP mRNA only ( Fig.   S2A-B, D), consistent with increased death of Lats2-overexpressing cells.
In addition to detecting SOX2 in all Lats2-overexpressing cells located inside the embryo, SOX2 was also detected in rare Lats2-overexpressing cells that remained on the embryo surface (Fig. 2D). Therefore, LATS2 is sufficient to induce expression of SOX2 in cells regardless of their position within the embryo. Importantly, the kinasedead variant of LATS2 (Nishioka et al., 2009), did not alter cell positioning, survival, or SOX2 expression (Fig. S3A, B), consistent with a previous report (Posfai et al., 2017).
Thus, overexpressed LATS2 influences cell position and gene expression by modulating the activity of YAP1 and possibly WWTR1. We predicted that, if Lats2 overexpression drove cells to adopt inner cell mass fate by influencing YAP1 and WWTR1 activity, then co-overexpression of Yap1 CA would enable Lats2-overexpressing cells to contribute to trophectoderm. Consistent with this prediction, cooverexpression of

LATS2 induces positional changes independent of Sox2
Our observation that Lats2-overexpression induces both the expression of SOX2 and cell repositioning to inner cell mass prompted us to investigate whether SOX2 itself drives cell repositioning downstream of Lats2. In support of this hypothesis, SOX2 activity has been proposed to bias inner cell mass fate (Goolam et al., 2016;White et al., 2016). We therefore investigated whether Sox2 is required for the inner cell massinducing activity of LATS2 by overexpressing Lats2 in embryos lacking maternal and zygotic Sox2 (Fig. 3A), as previously described (Wicklow et al., 2014). However, we observed that Lats2-overexpressing cells were equally likely to occupy inside position in the presence and absence of Sox2 (Fig. 3B, C). Furthermore, Lats2-overexpressing cells were equally unlikely to occupy outside position in the presence and absence of Sox2 (Fig. 3D). Therefore, although Lats2 overexpression is sufficient to induce expression of Sox2, LATS2 acts on cell positioning/survival independently of Sox2.

LATS2 antagonizes formation of the apical domain
Trophectoderm cell fate has been proposed to be determined by apically localized membrane components that maintain the position of future trophectoderm cells on the embryo surface (Anani et al., 2014;Korotkevich et al., 2017;Maître et al., 2016Maître et al., , 2015 12 Samarage et al., 2015;Zenker et al., 2018). For example, the apical membrane components aPKC and PARD6B are required for maintaining outside cell position and trophectoderm fate (Alarcon, 2010;Dard et al., 2009;Hirate et al., 2015;Plusa et al., 2005). Because Lats2 overexpression led cells to adopt an inside position, this raised the testable possibility that LATS2 antagonizes localization of aPKC and PARD6B. Since

YAP1 and WWTR1 restrict Sox2 expression to the inner cell mass
Our overexpression data suggested that the activities of YAP1 and WWTR1 are important for regulating cell fate and gene expression. Next, we aimed to test the requirement for Yap1 and Wwtr1 in embryogenesis. Yap1 null embryos survive until E9.0 (Morin- Kensicki et al., 2006), suggesting that oocyte-expressed (maternal) Yap1 (Yu et al., 2016), or the Yap1 paralogue Wwtr1 (Varelas et al., 2010) are important for preimplantation development. However, embryos lacking maternal and zygotic Wwtr1 and Yap1 have not been analyzed.
To generate embryos lacking maternal and zygotic Wwtr1 and Yap1, we deleted Wwtr1 and Yap1 from the female germ line using mice carrying conditional alleles of Wwtr1 and Yap1 (Xin et al., 2013(Xin et al., , 2011 and the female germ line-specific Zp3Cre (de Vries et al., 2000). We then crossed these females to males heterozygous for deleted alleles of Wwtr1 and Yap1 (see Methods). From these crosses, we obtained embryos lacking maternally provided Wwtr1 and Yap1 and either heterozygous or null for Wwtr1 and/or Yap1 (Table S1). At E3.25 (≤32 cells), SOX2 and CDX2 are normally mutually exclusive Although the average total number of cells was also reduced in these embryos ( Fig.   6C), the reduction in total cell number did not alone account for the loss of cells on the outside of the embryo (Table S2). This observation suggested that, similar to Lats2overexpressing cells, cells with reduced Wwtr1 and Yap1 exhibit an increased frequency of outside cell death, in addition to increased outside cell internalization.
Consistent with this, embryos with one or fewer wild type alleles of Wwtr1 or Yap1 15 exhibited an increase in the ratio of inside to outside cells (Fig. 6D) and an increase in cells undergoing apoptosis by TUNEL assay (Fig. 6G and S6A, B).
Critically, the fewer outside cells apparent in embryos lacking Wwtr1 and Yap1, which appeared stretched over the mass of inside cells, exhibited ectopic expression of SOX2 ( Fig. 6E-F). Therefore, Wwtr1/Yap1 repress inner cell mass fate, downstream of LATS kinases. Intriguingly, our data also indicate that WWTR1 is a more potent repressor of Sox2 at E3.75 than YAP1 since embryos with a single wild type allele of Wwtr1 had significantly fewer cells expressing ectopic SOX2 then embryos with a single wild type allele of Yap1 (Fig. S4).
Since loss of Wwtr1 and Yap1 phenocopied Lats2 overexpression in terms of Sox2 expression, cell death, and cell repositioning, we next evaluated apical domain and cell polarization in outside cells of embryos lacking Wwtr1 and Yap1 at E3.75. We observed greatly reduced aPKC at the apical membrane of outside cells in embryos with one or fewer doses of Wwtr1 or Yap1 ( Fig. 6H and S6C). In addition, we evaluated the localization of the tight junction protein ZO-1, which suggested failure in tight junction formation in embryos with 1 or fewer doses of Wwtr1 and Yap1 ( Fig. 6I and S6D).
Notably, however, other markers of apicobasal polarity, such as CDH1 and pERM were correctly localized in outside cells of mutant embryos at this stage ( Fig. 6J and S6E).
Our observations indicate that WWTR1 and YAP1 play a crucial role in the formation of the apical domain and maintaining the positioning and survival of outside cells while repressing expression of Sox2.

Discussion
During preimplantation development, lineage-specific transcription factors are commonly expressed in 'noisy' domains before refining to a lineage-appropriate pattern (Simon et al., 2018). For example, Oct4 and Nanog are expressed in both inner cell mass and trophectoderm until after blastocyst formation (Dietrich and Hiiragi, 2007;Strumpf et al., 2005). Similarly, CDX2 is detected in inner cell mass, as well as trophectoderm, until blastocyst stages (McDole and Zheng, 2012;Ralston and Rossant, 2008;Strumpf et al., 2005). In striking contrast to these genes, SOX2 is never detected in outside cells (Wicklow et al., 2014), indicating that robust mechanisms must exist to minimize noise and prevent its aberrant expression in trophectoderm. Here, we identify YAP1/WWTR1 as key components that repress Sox2 expression in outside cells of the embryo. Notably, manipulations known to antagonize YAP1/WWTR1 activity, including chemical inhibition of ROCK and overexpression of LATS2 lead to ectopic expression of SOX2 in outside cells, reinforcing the notion that YAP1/WWTR1 activity are crucial for repression of Sox2 in outside cells.
Additionally, we find that Sox2 expression is more sensitive than is Cdx2 to YAP1/WWTR1 activity, since intermediate doses of active YAP1/WWTR1 yields cells that coexpress both SOX2 and CDX2 (Fig. 7A). This observation is consistent with the fact that CDX2 is initially detected in inside cells of the embryo during blastocyst formation (Dietrich and Hiiragi, 2007;McDole and Zheng, 2012;Ralston and Rossant, 2008), where SOX2 is also expressed (Wicklow et al., 2014). Thus, inside cells could 17 initially possess intermediate doses of active YAP1/WWTR1 at this early stage. By contrast, outside cells would have greatly reduced YAP1/WWTR1 activity, owing to elevated LATS activity. In this way, the HIPPO pathway ensures robust developmental transitions, by rapidly nudging SOX2-expressing cells into their correct and final positions inside the embryo (Fig. 7B).
Consistent with our proposed model, the timing of HIPPO-induced cell internalization coincides with loss of cell fate plasticity around the 32-cell stage (Posfai et al., 2017).
This timing also coincides with the formation of mature tight-junctions among outside cells (Sheth et al., 1997), which reinforce and intensify differences in HIPPO signaling activity between inside and outside compartments of the embryo Leung and Zernicka-Goetz, 2013). Our observations indicate that HIPPO signaling can, in turn, interfere with trophectoderm epithelialization. Therefore, we propose that HIPPO engages in a negative feedback loop with cell polarity components (Fig. 7B).
We propose two mechanisms by which HIPPO signaling eliminates cells from the trophectoderm, both of which are downstream of YAP1/WWTR1 (Fig. 7C). First, a small proportion of conflicted cells undergo cell death. This is in line with the observed increase in the level of apoptosis detected after the 32-cell stage (Copp, 1978). We showed that cell lethality due to elevated HIPPO can be rescued by increasing levels of nuclear YAP1, suggesting that YAP1 activity normally provides a pro-survival signal to trophectoderm cells, consistent with the proposed role of YAP1 in promoting 18 proliferation in non-eutherian mammals (Frankenberg, 2018). Moreover, deletion of Sox2 did not rescue survival of outside cells in which HIPPO signaling was artificially elevated, arguing that HIPPO resolves cell fate conflicts independently of lineagespecific genes.
The second way that conflicted cells are eliminated from the trophectoderm is that cells with elevated HIPPO signaling drive their own internalization. This is consistent with the observation that cells in which Tead4 has been knocked down become internalized (Mihajlović et al., 2015). However, in contrast to Tead4 loss of function, which preserves the polarization of outside cells (Mihajlović et al., 2015;Nishioka et al., 2008), we observed that Yap1/Wwtr1 loss of function leads loss of apical PARD6D/aPKC. These observations suggest that YAP1/WWTR1 could partner with proteins other than TEAD4 to regulate apical domain formation. Consistent with this proposal, TEAD1 has been proposed to play an essential role in the early embryo (Sasaki, 2017). Nevertheless, since PARD6B/aPKC are essential for outside cell positioning (Dard et al., 2009;Hirate et al., 2015;Plusa et al., 2005), the loss of the apical domain could affect cell positioning in several ways. For instance, loss of PARD6B/aPKC would eventually lead to cell depolarization (Alarcon, 2010), which could influence any of the processes normally governing the formation of inside cells, such as oriented cleavage, cell contractility, or apical constriction (Korotkevich et al., 2017;Maître et al., 2016;Samarage et al., 2015). Identifying the downstream mechanisms by which HIPPO drives cells to inner cell mass will be a stimulating topic of future study. 19 Our studies also revealed that SOX2 does not play a role in cell positioning. This observation sheds light on a recent study, which showed that SOX2 dwells longer in select nuclei of four-cell stage embryos that are destined to contribute to the inner cell mass (White et al., 2016). We propose that SOX2 is associated with future pluripotent state but does not alone contribute to all aspects of pluripotency, such as inside positioning. It is therefore still unclear why it is important to establish the inside cellspecific SOX2 expression during embryogenesis. Identification pathways that function downstream of YAP1/WWTR1 and in parallel to SOX2 to promote formation of pluripotent cells will provide meaningful insights into the natural origins of mammalian pluripotent stem cell progenitors.

Mouse strains and genotyping
All animal research was conducted in accordance with the guidelines of the Michigan State University Institutional Animal Care and Use Committee. Wild type embryos were derived from CD-1 mice (Charles River). The following alleles or transgenes were used in this study, and maintained in a CD-1 background: Sox2 tm1.1Lan (Smith et al., 2009), Yap tm1.1Eno (Xin et al., 2011), Wwtr1 tm1.1Eno (Xin et al., 2013), Tg(Zp3-cre)93Knw (de Vries et al., 2000). Null alleles were generated by breeding mice carrying floxed alleles and mice carrying ubiquitously expressed Cre, 129-Alpl tm(cre)Nagy (Lomelí et al., 2000). 20 Mice were maintained on a 12-hour light/dark cycle. Embryos were collected by flushing the oviduct or uterus with M2 medium (Millipore). For embryo culture, KSOM medium (Millipore) was equilibrated overnight prior to embryo collection. Y-27632 (Millipore) was included in embryo culture medium at a concentration of 80 µM with 0.4% DMSO, or 0.4% DMSO as control, where indicated. Embryos were cultured at 37ºC in a 5% CO2 incubator under light mineral oil.

Embryo microinjection
LATS2 and YAPS112A mRNA was synthesized from cDNAs cloned into the pcDNA3.1poly(A)83 plasmid (Yamagata et al., 2005) using the mMESSAGE mMACHINE T7 transcription kit (Invitrogen). EGFP or nls-GFP mRNA were synthesized from EGFP cloned into the pCS2 plasmid or the nls-GFP plasmid (Ariotti et al., 2015) using the mMESSAGE mMACHINE SP6 transcription kit (Invitrogen). mRNAs were cleaned and concentrated prior to injection using the MEGAclear Transcription Clean-Up Kit (Invitrogen). Lats2, Lats2 KD and YAP CA mRNAs were injected into one blastomere of two-cell stage embryos at a concentration of 500 ng/µl, mixed with 350 ng/µl EGFP or nls-GFP mRNA diluted in 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA.

Embryo Analysis
For each embryo, z-stacks were analyzed using Photoshop or Fiji, which enabled the virtual labeling, based on DNA stain, of all individual cell nuclei. Using this label to identify individual cells, each cell in each embryo was then assigned to relevant phenotypic categories, without knowledge of embryo genotype. Phenotypic categories included marker expression (e.g., SOX2 or CDX2 positive or negative), protein localization (e.g., aPKC or CDH1 apical, basal, absent, or unlocalized), and cell position, where cells making contact with the external environment were considered 'outside' and cells surrounded by other cells were considered 'inside' cells.

TUNEL Assay
Embryos were fixed, permeabilized, and blocked as described for immunofluorescence.
Zonae pellucida were removed using Tyrode's Acid treatment prior to performing the TUNEL assay (In Situ Cell Death Detection Kit, Fluorescein, Millipore-Sigma). Embryos were incubated in 200 µl of a 1:10 dilution of enzyme in label solution for 2 hours at 37 ºC. Embryos were then washed twice with blocking solution for 10 minutes each, and then mounted in a 1 to 400 dilution of DRAQ5 in blocking solution to stain DNA.

Embryo Genotyping
To determine embryo genotypes, embryos were collected after imaging and genomic DNA extracted using the Extract-N-Amp kit (Sigma) in a final volume of 10 µl. Genomic extracts (1-2 µl) were then subjected to PCR using allele-specific primers (Table S3).

Figure 2. LATS2 kinase is sufficient to direct cells to inner cell mass fate.
A) Embryos were collected at E1.5 and one of two blastomeres was injected with mRNAs encoding Lats2 and GFP or GFP alone. Embryos were cultured for 72 hours, fixed, and then analyzed by immunofluorescence and confocal microscopy.

C) Proportion of non-injected cells and cells injected with
Lats2/GFP mRNAs contributing to inner cell mass in the indicated genetic backgrounds. No significant differences were observed based on embryo genotype, indicating that Sox2 is dispensable for inside positioning by Lats2-overexpression (P, chi-squared test; n = embryos).

D) Proportion of non-injected cells and cells injected with the indicated mRNAs
contributing to trophectoderm in the indicated genetic backgrounds. No significant 37 differences were observed based on embryo genotype (P, chi-squared test; n = embryos).    of Sox2 expression, and thus ensuring that SOX2 is never detected in outside cells because these cells are rapidly internalized or apoptosed. C) A closeup of the boxed region in panel B. In most outside cells, low LATS2 activity enables high levels of YAP1/WWTR1 activity, which repress Sox2 and apoptosis and promote Cdx2 expression and apical localization of aPKC and PARD6B, which in turn repress the HIPPO pathway. In rare outside cells, LATS2 activity becomes elevated, leading to lower activity of YAP1/WWTR1, which then leads these cells to become internalized or to undergo apoptosis.

Figure S1. Effect of ROCK1/2 inhibition on Cdx2 expression and effect Yap1 CA overexpression on YAP1 localization and phosphorylation
A) Confocal images of CDX2 and SOX2 in control and embryos treated with ROCKinhibitor for 24 hours starting at E2.5. In control embryos, CDX2 is specific to outside cells and SOX2 is specific to inside cells (n = embryos).
A') Treatment with ROCKi leads to ectopic SOX2 in outside cells which is often coexpressed with CDX2 (arrowheads, n = embryos). B) YAP1 CA was injected into one of two blastomeres at the 2-cell stage and evaluated 48 hours later. C) In non-injected cells, YAP1 is exclusively nuclear in outside cells while pYAP1 is exclusively cytoplasmic in inside cells. By contrast, YAP1 is detected in the nucleus of YAP1 CA -injected cells, regardless of their position, demonstrating that YAP1 CA is constitutively nuclear. Additionally, analysis of pYAP1 in YAP1 CA -injected cells shows 42 that YAP1 CA can still be phosphorylated on non-mutated residues, but this is not sufficient to alter YAP1 nuclear localization (n = embryos).