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

  1. Tristan Frum
  2. Tayler M Murphy
  3. Amy Ralston  Is a corresponding author
  1. Michigan State University, United States
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Cite this article as: eLife 2018;7:e42298 doi: 10.7554/eLife.42298

Abstract

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.

https://doi.org/10.7554/eLife.42298.001

eLife digest

As an embryo develops, its cells divide, grow and migrate in specific patterns to build an organized collection of cells that go on to form our tissues and organs. One of the first steps – well before the embryo has implanted into the womb – is to allocate cells to make part of the placenta.

Once this process is complete, the remaining cells continue building the organism. These cells are pluripotent, meaning they can develop into any part of the body. Scientists think that the embryo manages to sort ‘placenta cells’ from pluripotent ones with the help of certain proteins, which the mother has packaged into her eggs.

To investigate this further, Frum et al. used genetic tools to track a specific gene called Sox2 that identifies pluripotent cells as soon as they are formed in mouse embryos. The experiments revealed that the mother places two closely related proteins known as YAP1 and WWTR1 within each egg, which help to make placenta cells different from pluripotent cells. Moreover, both proteins enable the embryo to segregate these two cell types to two different locations: placenta cells are moved to the outer layer of the embryo, while pluripotent cells are moved to the inside.

Current technologies allow researchers to create pluripotent cells in the laboratory. But these approaches often result in error, failing to replicate the embryo’s natural ability. By studying how embryos form and arrange pluripotent cells, scientists hope to advance stem cell technology (which emerge from pluripotent cells). This may help to find new ways to heal damaged tissues and organs, or to treat or even prevent many diseases.

https://doi.org/10.7554/eLife.42298.002

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, 2017). However, at this stage, pluripotency genes such as Pou5f1 (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 (Strumpf et al., 2005), 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 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 (Guo et al., 2010; Wicklow et al., 2014). The discovery of how Sox2 expression is regulated in the embryo therefore provides 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. We previously showed that 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.

The expression of Cdx2 is regulated by members of the HIPPO signaling pathway. In particular, the HIPPO pathway kinases LATS1/2 become active in unpolarized cells located deep inside the embryo, where they antagonize activity of the YAP1/WWTR1/TEAD4 transcriptional complex that is thought to promote expression of Cdx2 (Anani et al., 2014; Cockburn et al., 2013; Hirate et al., 2013; Kono et al., 2014; Korotkevich et al., 2017; Leung and Zernicka-Goetz, 2013; Lorthongpanich et al., 2013; Mihajlović and Bruce, 2016; Nishioka et al., 2009; Nishioka et al., 2008; Posfai et al., 2017; Rayon et al., 2014; Watanabe et al., 2017; Yagi et al., 2007; Zhu et al., 2017). In this way, the initially ubiquitous expression of Cdx2 becomes restricted to outer trophectoderm cells. 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.

Results

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 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 hr (Figure 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 (Figure 1B,C) as expected (Vestweber et al., 1987; Vinot et al., 2005). Additionally, SOX2 was detected only in inside cells in control embryos (Figure 1C,D). By contrast, embryos cultured in ROCKi exhibited defects in cell polarity (Figure 1B’, C’), consistent with prior studies (Kono et al., 2014). Interestingly, in ROCKi-treated embryos, we observed ectopic SOX2 expression in cells located on the outer surface of the embryo (Figure 1C’, D), indicating that ROCK1/2 participates in repressing expression of Sox2 in the trophectoderm.

To scrutinize the identity of outside-positioned SOX2-positive cells in ROCKi-treated 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 (Figure 1—figure supplement 1A) as expected at this stage (Ralston and Rossant, 2008; Strumpf et al., 2005). In ROCKi-treated embryos, CDX2 expression levels were reduced (Figure 1—figure supplement 1A’) as was the proportion of outside cells in which CDX2 was detected (Figure 1E), as previously reported (Kono et al., 2014). However, among outside cells, a substantial proportion coexpressed CDX2 and SOX2 in ROCKi-treated embryos compared with controls (Figure 1E and Figure 1—figure supplement 1A), 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 regulates 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.

Figure 1 with 1 supplement see all
ROCK1/2 and nuclear YAP1 repress expression of SOX2.

(A) Experimental design: embryos were collected at E2.5 and treated with ROCK inhibitor Y-27632 (ROCKi) or DMSO (control) for 24 hr. (B–B’) Confocal images of apical (PARD6B) and basolateral (CDH1) membrane components in control and ROCKi-treated embryos. As expected, PARD6B and CDH1 are mislocalized to the entire cell membrane of all cells in ROCKi-treated embryos, demonstrating effective ROCK inhibition (n = number of embryos examined). (C–C’) In control embryos, SOX2 is detected only in inside cells, while in ROCKi-treated embryos, SOX2 is detected in inside and outside cells (arrowheads, outside cells; n = embryos). (D) Quantification of ectopic SOX2 detected in outside cells of control and ROCKi-treated embryos (p, student’s t-test, n = embryos). (E) SOX2 and CDX2 staining in outside cells of control and ROCKi-treated embryos. ROCK-inhibitor treatment leads to outside cells with mixed lineage marker expression (CDX2+/SOX2+). (F) Experimental design: embryos were collected at E1.5 and one of two blastomeres injected with mRNAs encoding YAP1CA and GFP. Embryos were cultured for 72 hr, fixed, and then analyzed by immunofluorescence and confocal microscopy. (G) SOX2 is detected non-injected inside cells. SOX2 is not detected in YAP1CA-overexpressing inside cells (arrowheads), n = embryos. (H) Across multiple embryos, all non-injected inside cells express SOX2, whereas the vast majority of YAP1CA-injected inside cells fail to express SOX2.

https://doi.org/10.7554/eLife.42298.003

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 (YAP1CA). Substitution of alanine at serine 112 leads YAP1 to be constitutively nuclear and constitutively active (YAP1CA hereafter) (Dong et al., 2007; Nishioka et al., 2009; Zhao et al., 2007). We injected mRNAs encoding YAP1CA and GFP into one of two blastomeres at the 2-cell stage, and then cultured these to the blastocyst stage (Figure 1F). This mosaic approach to overexpression permitted comparison of Yap1CA-overexpressing 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 expectation that YAP1 would be detected in nuclei of both inside and outside cells in YAP1CA-overexpressing cells (Nishioka et al., 2009). As expected, YAP1 was observed in nuclei of all Yap1CA-overexpressing cells (Figure 1—figure supplement 1B,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 Yap1CA-overexpressing inside cells expressing detectable SOX2 (Figure 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 (Figure 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 (Figure 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 (Figure 2D), suggesting that Lats2-overexpressing cells were not only localized to the inner cell mass but also exhibited position-appropriate regulation of Sox2.

Figure 2 with 2 supplements see all
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 hr, fixed, and then analyzed by immunofluorescence and confocal microscopy. (B) Cells injected with GFP (dotted line) contributed to trophectoderm and inner cell mass, while cells injected with Lats2 and GFP (dotted line) contributed almost exclusively to the inner cell mass, leaving only cellular fragments in the trophectoderm (arrows), suggestive of cell death (n = embryos). (C) Proportion of inside, outside, and total cell populations across multiple embryos, which were comprised of non-injected cells, or cells injected with either GFP or GFP/Lats2 mRNAs. Cells injected with GFP/Lats2 were overrepresented within the inside cell population and underrepresented in the outside and total cell populations, relative to cells injected with GFP alone (P, chi-squared test). (D) Percentage of SOX2-positive cells within non-injected and GFP-injected or Lats2/GFP-injected populations observed inside and outside of the embryo. SOX2 was detected in all of the Lats2/GFP-injected inside cells, and in half of the rare, Lats2/GFP-injected outside cells (same number of embryos as in panel C) (p, student’s t-test). (E) Average number of outside and total cells per embryo. The average number of outside cells is reduced in embryos injected with Lats2/GFP, relative to GFP-injected (p, student’s t-test). (F) Proportion of GFP and Lats2/GFP-injected cells, relative to total cell number, over the course of development to the ~80-cell blastocyst (solid lines = average of indicated data point and four previous data points). (G) Data as shown in panel H, shown relative to outside cell number. (H) Data as shown in panel H, shown relative to inside cell number. (I) Contribution of injected and non-injected cells to the inside cell population, following injection with GFP or Lats2/GFP. Injection with Lats2/GFP increases the overall number of inside cells compared to injection with GFP only through increasing the number of injected cells contributing to the inside cell population, without affecting the number of non-injected cells contributing to the inside cell population (p, student’s t-test).

https://doi.org/10.7554/eLife.42298.005

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 (Figure 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 (Figure 2B, yellow arrowheads), as well as increased TUNEL staining in Lats2-overexpressing embryos compared to embryos injected with GFP mRNA only (Figure 2—figure supplement 1A–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 (Figure 2D). Therefore, LATS2 is sufficient to induce expression of SOX2 in cells regardless of their position within the embryo. We predicted that, if Lats2 overexpression drove cells to adopt inner cell mass fate by influencing YAP1 and WWTR1 activity, then co-overexpression of Yap1CA would enable Lats2-overexpressing cells to contribute to trophectoderm. Consistent with this prediction, cooverexpression of Lats2 and Yap1CA led to a significant decrease in the proportion of Lats2-overexpressing cells contributing to the inside cell position, and a concomitant increase in the proportion of Lats2-overexpressing cells remaining in the outside position (Figure Figure 2—figure supplement 2A–D). Moreover, cooverexpression of Lats2 and Yap1CA reduced the number of TUNEL positive nuclei, consistent with Yap1CA rescuing survival of outside-positioned Lats2-overexpressing cells (Figure 2—figure supplement 1C–D). Collectively, these observations strongly suggest that LATS2 promotes inside cell positioning by regulating the activities of YAP1 and, likely, the related protein WWTR1.

To pinpoint when Lats2-overexpressing cells come to occupy the inside of the embryo, we performed a time course, examining the position of injected and non-injected cells from the 16-cell to the blastocyst stage (~80 cells). Surprisingly, between the 16 and 32-cell stages, the proportion of injected and non-injected cells in the total, outside, and inside cell populations were comparable whether embryos had been injected with Lats2 and GFP or GFP mRNA alone (Figure 2F–H). In embryos injected with GFP mRNA alone, the proportion of injected and non-injected cells making up the total, outside, and inside cell populations remained constant throughout the time course. In contrast, starting around the 32-cell stage, the average proportion of Lats2-overexpressing cells making up the inside population began to increase dramatically. This increase was associated with a decrease in the proportion Lats2-overexpressing cells making up the outside population, consistent with internalization of Lats2-overexpressing cells after the 32-cell stage (Figure 2G). After the 32-cell stage, Lats2-injected cells became underrepresented as a proportion of the total cell population (Figure 2H), lending further support to the idea that Lats2-overexpressing cells that fail to internalize undergo cell death. Interestingly, the inside-skewed contribution of Lats2-overexpressing cells did not influence the ability of non-injected cells to contribute to the ICM (Figure 2I), arguing that Lats2-overexpression drives inside positioning cell-autonomously. We therefore conclude that Lats2 overexpression acts on cell position and survival around the time of blastocyst formation.

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 mass-inducing activity of LATS2 by overexpressing Lats2 in embryos lacking maternal and zygotic Sox2 (Figure 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 (Figure 3B,C). Furthermore, Lats2-overexpressing cells were equally unlikely to occupy outside position in the presence and absence of Sox2 (Figure 3D). Therefore, although Lats2 overexpression is sufficient to induce expression of Sox2, LATS2 acts on cell positioning/survival independently of Sox2.

LATS2 directs inner cell mass fate independently of Sox2 (A) Lats2 and GFP or GFP alone were overexpressed in embryos lacking maternal or maternal and zygotic Sox2.

(B) Lats2/GFP-overexpressing cells (dotted line) contribute almost exclusively to the inner cell mass in the presence or absence of Sox2 (n = embryos). (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 differences were observed based on embryo genotype (P, chi-squared test; n = embryos).

https://doi.org/10.7554/eLife.42298.008

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., 2016; Maître et al., 2015; 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 Lats2 overexpression leads to cell positioning starting around the 32-cell stage, we examined the localization of aPKCz and PARD6B in embryos just prior to the 32-cell stage. At this stage, apical membrane components PARD6B and aPKCz were detected at the apical membrane of non-injected outside cells and outside cells injected with GFP only (Figure 4A–D). By contrast, most Lats2-overexpressing outside cells lacked detectable aPKCz and PARD6B (Figure 4A–D). Therefore, LATS2 is sufficient to antagonize localization of key apical domain proteins in outside cells, providing a compelling mechanism for the observed repositioning of Lats2-overexpressing outside cells.

LATS2 antagonizes formation of the apical domain (A) In embryos at 16–32 cell stages, PARD6B is detectable in GFP-overexpressing and in non-injected cells, but not in Lats2-overexpressing cells (arrowheads, n = embryos).

(B) At 16–32 cell stages, aPKCz is detectable in GFP-overexpressing and in non-injected cells, but not in Lats2-overexpressing cells (arrowheads, n = embryos). (C) Quantification of embryos shown in panel A (p, student’s t-test). (D) Quantification of embryos shown in panel B (p, student’s t-test). (E) At 16–32 cell stages, CDH1 is localized to the basolateral membrane in both Lats2-overexpressing and non-injected cells (n = embryos). (F) At 16–32 cell stages, Phalloidin staining demonstrates that filamentous Actin is apically enriched in Lats2-overexpressing and non-injected cells (n = embryos). (G) At 16–32 cell stages, pERM is localized to the apical membrane in both Lats2-overexpressing and non-injected cells (n = embryos).

https://doi.org/10.7554/eLife.42298.009

We also examined other markers of apicobasal polarization in Lats2-overexpressing outside cells prior to the 32-cell stage. Curiously, other markers of apicobasal polarization were properly localized in all cells examined. For example, CDH1 was restricted to the basolateral membrane (Figure 4E), while filamentous Actin and phospho-ERM were restricted to the apical domain in outside cells of both Lats2-overexpressing and non-injected outside cells (Figure 4F,G). Thus, we propose that Lats2-overexpressing outside cells initially possess hallmarks of apicobasal polarization, but aPKC and PARD6B fail to properly localize, leading to the eventual depolarization and internalization of outside cells.

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 requirements 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 scrutinized.

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 Materials and methods). From these crosses, we obtained embryos lacking maternally provided Wwtr1 and Yap1 and either heterozygous or null for Wwtr1 and/or Yap1 (Supplementary file 1). At E3.25 (≤32 cells), SOX2 and CDX2 are normally mutually exclusive (Figure 5A). However, with decreasing number of wild type zygotic alleles of Wwtr1 and Yap1, we observed worsening phenotypes (Figure 5B–F). In the complete absence of Wwtr1 and Yap1, we observed a severe loss of CDX2 and expansion of SOX2 in outside cells (Figure 5D–F), phenocopying Lats2 overexpression. However, in embryos of intermediate genotypes, we observed expanded SOX2 and persistent, yet lower, expression levels of CDX2 (Figure 5C,E–F). Thus, regulation of Sox2 expression is more sensitive to Wwtr1 and Yap1 dosage than is Cdx2. Moreover, these observations indicate that intermediate doses of Wwtr1 and Yap1 produce outside cells expressing markers of mixed cell lineage at E3.25.

Wwtr1 and Yap1 are required to repress SOX2 expression in outside cells.

(A) CDX2 and SOX2 in wild type embryos at E3.25 (16–32 cell stages). CDX2 staining is more intense in outside cells than inside cells and SOX2 staining is specific to inside cells (n = embryos). (B) Embryos lacking maternal Wwtr1 and Yap1 with and heterozygous for Wwtr1 and Yap1 (which we consider to have 2 doses of WWTR1/YAP1) exhibit normal CDX2 and SOX2 expression (n = embryos). (C) Embryos lacking maternal Wwtr1 and Yap1 and heterozygous for either Wwtr1 or Yap1 (1 dose of WWTR1/YAP1) exhibit a high degree of ectopic SOX2 in outside cells (arrowheads), but continue to express CDX2, although the levels appear reduced (n = embryos). (D) Embryos lacking maternal and zygotic Wwtr1 and Yap1 (0 doses of WWTR1/YAP1) have the most severe phenotype, with a high degree of ectopic SOX2 in outside cells (arrowheads) and little or no detectable CDX2 (n = embryos). (E) Quantification of the percentage of outside cells in which ectopic SOX2 is detected in the presence of decreasing dose of Wwtr1 and Yap1 (t = student’s t-test, n = embryos). (F) Quantification of the percentage of outside cells in which CDX2 is detected in the presence of decreasing dose of Wwtr1 and Yap1 (t = student’s t-test, n = embryos).

https://doi.org/10.7554/eLife.42298.010

YAP1 and WWWTR1 maintain outside cell positioning

Based on our observations of Lats2-overexpressing embryos, we anticipated that defects in cell positioning in embryos lacking maternal and zygotic Wwtr1 and Yap1 could arise after the 32-cell stage. We therefore examined embryos lacking Wwtr1 and Yap1 at E3.75, when embryos possess more than 32 cells. Indeed, we observed skewed lineage contributions, correlating with the dosage of Wwtr1 and Yap1 (Figure 6A–D). Embryos with one or fewer wild type alleles of Wwtr1 or Yap1 exhibited an increase in the number of inside cells and a reduction in the number of outside cells (Figure 6A–B), consistent with altered cell positioning.

Figure 6 with 1 supplement see all
Positioning and epithelialization defects in embryos with Wwtr1 and Yap1 null alleles (A) Quantification of the average number of inside cells per embryo with decreasing dose of Wwtr1 and Yap1.

The number of inside cells increases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (B) Quantification of the average number of outside cells per embryo with decreasing dose of Wwtr1 and Yap1. The number of outside cells decreases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (C) Quantification of the average number of total cells per embryos with decreasing dose of wild type zygotic Wwtr1 and Yap1. The number of total cells decreases as the dose of wild type Wwtr1 and Yap1 is reduced (p, student’s t-test, n = embryos). (D) Quantification of the average ratio of inside to outside cells per embryo with decreasing dose of Wwtr1 and Yap1. The ratio of inside to outside cells increases as the dose of wild type Wwtr1 and Yap1 is reduced (p, student’s t-test, n = embryos). (E) Wild type embryos at E3.75 exhibit inner cell mass-specific expression of SOX2 (n = embryos). (E’) E3.75 embryos lacking maternal Wwtr1 and Yap1 and heterozygous for zygotic Wwtr1 and Yap1 cavitate and repress Sox2 in outside cells, leading to inner cell mass-specific expression of SOX2 similar to wild type embryos (n = embryos). (E’’) Embryos lacking maternal Wwtr1 and Yap1 but with only one wild type allele of Wwtr1 or Yap1 fail to cavitate and repress Sox2 in outside cells, leading to ectopic SOX2 in outside cells (arrowheads, n = embryos). (E’’’) Embryos lacking maternal and zygotic Wwtr1 and Yap1 fail to cavitate and repress Sox2 in outside cells, leading to ectopic SOX2 in outside cells (arrowheads, n = embryos). (F) Quantification of ectopic SOX2 detected in embryos such as those shown in panels E-E’’’. The percentage of outside cells with ectopic SOX2 increases as the dose of wild type Wwtr1 and Yap1 alleles is reduced (p, student’s t-test, n = embryos). (G) TUNEL analysis of embryos lacking maternal Wwtr1 and Yap1 heterozygous for zygotic Wwtr1 and Yap1 or lacking maternal and zygotic Wwtr1 and Yap1. Extensive TUNEL staining is observed in embryos lacking maternal and zygotic Wwtr1 and Yap1 indicative of cell death. Max projections of all confocal sections from a single embryo are shown (n = embryos). (H) aPKCz staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. aPKC is not localized to the apical membrane of embryos with no zygotic Wwtr1 and Yap1 (n = embryos). (I) ZO-1 staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. ZO-1 is disorganized in embryos with no zygotic Wwtr1 and Yap1, suggesting that formation of a mature epithelium depends on Wwtr1 and Yap1 (n = embryos). (J) pERM and CDH1 staining in embryos lacking maternal Wwtr1 and Yap1, either heterozygous for zygotic Wwtr1 and Yap1 or with no zygotic Wwtr1 and Yap1. pERM is localized to apical membranes and CDH1 to basolateral membranes regardless of the dose of wild type Wwtr1 and Yap1 alleles (n = embryos).

https://doi.org/10.7554/eLife.42298.011

Although the average total number of cells was also reduced in these embryos (Figure 6C), the reduction in total cell number did not alone account for the loss of cells on the outside of the embryo (Supplementary file 2). This observation suggested that, similar to Lats2-overexpressing 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 exhibited an increase in the ratio of inside to outside cells (Figure 6D) and an increase in cells undergoing apoptosis by TUNEL assay (Figure 6G and Figure 6—figure supplement 1A,B).

Critically, the fewer outside cells in embryos lacking Wwtr1 and Yap1, which appeared stretched over the mass of inside cells, exhibited ectopic expression of SOX2 (Figure 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 (Figure 6—figure supplement 1F,G).

Since loss of Wwtr1 and Yap1 phenocopied Lats2 overexpression in terms of Sox2 expression, cell death, and cell repositioning, we next evaluated the apical domain and cell polarization in outside cells of embryos lacking Wwtr1 and Yap1 at E3.75. As expected, observed greatly reduced aPKC at the apical membrane of outside cells in embryos with one or fewer doses of Wwtr1 or Yap1 (Figure 6H and Figure 6—figure supplement 1C). In addition, we evaluated the localization of the tight junction protein ZO-1, which suggested failure in tight junction formation in embryos with one or fewer doses of Wwtr1 and Yap1 (Figure 6I and Figure 6—figure supplement 1D). Notably, however, other markers of apicobasal polarity, such as CDH1 and pERM were correctly localized in outside cells of mutant embryos at this stage (Figure 6J and Figure 6—figure supplement 1E), consistent with some normal cell polarization. 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 yield cells that coexpress both SOX2 and CDX2 (Figure 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 initially possess intermediate doses of active YAP1/WWTR1 at this early stage. By contrast, outside cells 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 (Figure 7B).

Resolution of cell fate conflicts in the preimplantation mouse embryo.

(A) The expression of Sox2 and Cdx2 is differentially sensitive to YAP1/WWTR1 activity, leading to co-expression of both lineage markers in cells when YAP1/WWTR1 activity levels are intermediate. (B) During division from the 16 to the 32-cell stage, cells that inherit the apical membrane repress HIPPO signaling and maintain an outside position. However, cells that inherit a smaller portion of the apical membrane would initially elevate their HIPPO signaling. We propose that elevated HIPPO then feeds back onto polarity by further antagonizing PAR-aPKC complex formation, leading to a snowball effect on repression 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.

https://doi.org/10.7554/eLife.42298.013

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 (Hirate and Sasaki, 2014; 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 (Figure 7B).

We propose two mechanisms by which HIPPO signaling eliminates cells from the trophectoderm, both of which are downstream of YAP1/WWTR1 (Figure 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 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 lineage-specific 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 apical domain in 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 allocation 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.

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 cell-specific 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.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain
background
(Mus musculus)
CD-1Charles River
Laboratories
RRID:IMSR_CRL:22
Strain, strain
background
(M. musculus)
Sox2tm1.1Lan
Smith et al. (2009)
PMID:19666824
RRID:IMSR_JAX:013093mixed
background,
Sox2 null refers
to recombined
allele
Strain, strain
background
(M. musculus)
Wwtr1
conditional
allele;
Wwtr1tm1.1Eno;
Wwtr1loxp
Xin et al., 2013
Xin et al., 2013
PMID:23918388
MGI:5544289mixed
background,
Wwtr1-’
or ‘Wwtr1Δ
refers to recombined
allele
Strain, strain
background
(M. musculus)
Yap1
conditional
allele; Yap1tm1.1Eno;
Yap1loxp
Xin et al., 2011
Xin et al., 2011
PMID:22028467
MGI:5446483mixed
background,
, ‘Yap1-’ or
Yap1Δ’ refers to
recombined allele
Strain, strain
background
(M. musculus)
Tg(Zp3-cre)
93Knw; Zp3Cre
de Vries et al., 2000
de Vries et al., 2000
de Vries et al., 2000
PMID:10686600
RRID:MGI:3835503mixed
background
Strain, strain
background
(M. musculus)
129-Alpl
tm(cre)Nagy
Lomelí et al., 2000
Lomelí et al., 2000
Lomelí et al., 2000
PMID:10686602
RRID:IMSR_
JAX:008569
mixed
background
Antibodymouse
anti-CDX2
BiogenexBioGenex
Cat# AM392;
RRID:AB_2650531
(1:200)
Antibodygoat anti
-SOX2
NeuromicsNeuromics
Cat# GT15098;
RRID:AB_2195800
(1:200)
Antibodyrabbit
anti-PARD6B
Novus
Biologicals
Novus Cat#
NBP1-87337;
RRID:AB_11034389
(1:100)
Antibodyrabbit
anti-PARD6B
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Cat# sc-67393;
RRID:AB_2267889
(1:100)
Antibodymouse
anti-PKCζ
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Cat# sc-17781;
RRID:AB_628148
(1:100)
Antibodymouse
anti-YAP1
Santa Cruz
Biotechnology
Santa Cruz
Biotechnology
Cat# sc-101199;
RRID:AB_1131430
(1:200)
Antibodymouse
anti-pYAP1
Cell Signaling
Technology
Cell Signaling
Technology
Cat# 4911;
RRID:AB_2218913
(1:800)
Antibodychicken
anti-GFP
Aves LabsAves Labs
Cat# GFP-1020;
RRID:AB_10000240
(1:2000)
Antibodyrat anti-
CDH1
Sigma-
Aldrich
Sigma-Aldrich
Cat# U3254;
RRID:AB_477600
(1:500)
Antibodymouse
anti-ZO1
Thermo Fisher
Scienctific
Thermo Fisher
Scientific Cat#
33–9100;
RRID:AB_2533147
(1:1000)
Recombinant
DNA reagent
Lats2
mRNA; LATS2
Nishioka et al., 2009
Nishioka et al., 2009
Nishioka et al., 2009
PMID:19289085
pcDNA3.1
-pA83-Lats2; RIKEN: RDB12200
In Vitro
Transcription
template for
Lats2 mRNA
Recombinant
DNA reagent
Yap1CA
mRNA; YAP1CA
Nishioka et al., 2009
Nishioka et al., 2009
Nishioka et al., 2009
PMID:19289085
pcDNA3.1-pA83
-HA-Yap-S112A;
RIKEN: RDB12194
In Vitro
Transcription
template for
Yap1CA mRNA
Recombinant
DNA reagent
nls-GFP mRNA;
nls-GFP
Ariotti et al., 2015
Ariotti et al., 2015
Ariotti et al., 2015
PMID:26585296
Addgene:
Plasmid #67652
In Vitro
Transcription
template for nls-
GFP mRNA
Recombinant
DNA reagent
pCS2-EGFP;
EGFP mRNA;
GFP mRNA; GFP
Chazaud et al., 2006
PMID: 16678776
In Vitro
Transcription
template for
GFP mRNA
Commercial
assay or kit
mMessage
mMachine Sp6
Transcription Kit
Thermo Fisher
Scienctific
Thermo Fisher
Scientific
Cat# AM1340
Commercial
assay or kit
mMessage
mMachine T7
Transcription Kit
Thermo Fisher
Scienctific
Thermo Fisher
Scientific
Cat# AM1344
Commercial
assay or kit
MEGAClear
Transcription
Clean-Up Kit
Thermo Fisher
Scienctific
Thermo Fisher
Scientific
Cat# AM1908
Commercial
assay or kit
In-Situ Cell
Death Detection
Kit,
Fluorescein;
TUNEL assay
Sigma-AldrichSigma-Aldrich
Cat# 11684795910
Commercial
assay or kit
Extract-N
-Amp Kit
Sigma-
Aldrich
Sigma-
Aldrich Cat #
XNAT2
Chemical
compound, drug
Y-27632;
ROCK-inhibitor
MilliporeMillipore
Cat# SCM075
Software,
algorithm
Adobe
Photoshop
AdobeRRID:SCR_014199
Software,
algorithm
Fijihttp://fiji.scRRID:SCR_002285

Mouse strains and genotyping

Request a detailed protocol

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: Sox2tm1.1Lan (Smith et al., 2009), Yaptm1.1Eno (Xin et al., 2011), Wwtr1tm1.1Eno (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-Alpltm(cre)Nagy (Lomelí et al., 2000).

Embryo collection and culture

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Mice were maintained on a 12 hr 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 37°C in a 5% CO2 incubator under light mineral oil.

Embryo microinjection

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Lats2 and Yap1S112A (Yap1CA) mRNA was synthesized from cDNAs cloned into the pcDNA3.1-poly(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 and YAP1CA 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.

Immunofluorescence and confocal microscopy

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Embryos were fixed with 4% formaldehyde (Polysciences) for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 30 min, and then blocked with blocking solution (10% Fetal Bovine Serum (Hyclone), 0.1% Triton X-100) for 1 hr at room temperature, or overnight at 4°C. Primary Antibodies used were: mouse anti-CDX2 (Biogenex, CDX2-88), goat anti-SOX2 (Neuromics, GT15098), rabbit anti-PARD6B (Santa Cruz, sc-67393), rabbit anti-PARD6B (Novus Biologicals, NBP1-87337), mouse anti-PKCζ (Santa Cruz Biotechnology, sc-17781), rat anti-CDH1 (Sigma Aldrich, U3254), mouse anti-ZO1 (Thermo Fisher Scientific, 33–9100), mouse anti-YAP (Santa Cruz Biotechnology, sc101199), rabbit anti phospho-YAP (Cell Signaling Technologies, 4911), chicken anti-GFP (Aves, GFP-1020). Stains used were: Phallodin-633 (Invitrogen), DRAQ5 (Cell Signaling Technologies) and DAPI (Sigma Aldrich). Secondary antibodies conjugated to DyLight 488, Cy3 or Alexa Flour 647 fluorophores were obtained from Jackson ImmunoResearch. Embryos were imaged using an Olympus FluoView FV1000 Confocal Laser Scanning Microscope system with 20x UPlanFLN objective (0.5 NA) and 5x digital zoom. For each embryo, z-stacks were collected, with 5 µm intervals between optical sections. All embryos were imaged prior to knowledge of their genotypes.

Embryo analysis

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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

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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 hr at 37°C. Embryos were then washed twice with blocking solution for 10 min each, and then mounted in a 1 to 400 dilution of DRAQ5 in blocking solution to stain DNA.

Embryo genotyping

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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 (Supplementary file 3).

References

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Decision letter

  1. Elizabeth Robertson
    Reviewing Editor; University of Oxford, United Kingdom
  2. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "HIPPO provides a fail-safe for resolving embryonic cell fate conflicts during establishment of pluripotency in vivo" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that in its current state your manuscript will not be considered further for publication in eLife.

However as you will see both reviewers were very positive and feel that your genetic approach certainly has provided further insights into lineage segregation in the early embryo. However the biggest issue is that the study is a bit premature given the small numbers of embryos you've been able to analyse to date. If you can considerably improve the manuscript along the lines suggested by the reviewers, including expanding the samples sizes for critical genotypes we would be happy to see a major revision of the paper in the coming months, which would be considered as a new submission.

Reviewer #2:

In this paper, the authors investigate further the mechanism of restriction of Sox2 expression to the ICM during early mouse development. As the blastocyst forms, there is a segregation of expression of key lineage-specific transcription factors for the outer trophectoderm (TE) (Cdx2) and the pluripotent ICM (Sox2), which is regulated by the Hippo signaling pathway. They previously showed that Sox2 is activated (probably indirectly) and Cdx2 inactivated by active Lats kinase function in the inside cells that will form the ICM. Sox2 is regulated independently of Cdx2. Here they explore further this regulatory interaction by making floxed alleles of both Yap and Wwtr1, the redundant coactivators downstream of Hippo signaling. This enabled them to make a dosage series of maternal and zygotically inactivated alleles of the two proteins, demonstrating their redundant nature, the requirement for maternal protein and the fact that Sox2 is more sensitive to reduced dosage than is Cdx2. They then went on to ask whether Lats kinase acts through Yap to mediate cell position during the cleavage stages leading up to blastocyst formation. They claim that overexpressing Lats2 reduces expression of PARD6B and aPKCz in cells and leads to their internalization, alongside a reduction in the number of outside versus inside cells.

Overall this is a carefully carried out study that provides some more insights into the mechanisms of the first lineage segregation in the mouse embryo. The strength of the paper is the description of the allelic series of mutations in Yap/Wwtr1, which have not been previously described. All previous studies depended largely on dominant negative and overexpression studies, which have their limitations. What is less strong is the section proposing a direct role for Lats in regulating polarity via downregulation of PARD6B and aPKCz. This underlies their model that Hippo signaling interaction with polarity components acts as a failsafe feedback mechanism to ensure lineage segregation. As outlined below, the data as presented raise some issues that need further resolution. In addition, there is no clear molecular mechanism proposed by which Lats activity would regulate specifically PARD6B and aPKCz and not other polarity components including the actin domain and phospho-ERM. The linkage between Lats, the apical domain, ROCK, etc. is presented in a model but there are many missing links in the model and a failure to link to other models of how polarity is thought to control Hippo signaling.

1) The first section on ROCK1/2 being upstream regulators of Cdx2 and Sox2 expression is not well connected to the rest of the paper. They also did not actually look at the effects of Rocki on both Cdx2 and Sox2 in the same embryos. Are the outside cells that remain Sox2-ve expressing Cdx2? Inhibition of Rock has multiple effects on the cell- which downstream response do they consider to be the critical one? Are they proposing a direct effect on Lats- as shown in Figure 7? Or the more usually suggested effect on the cortical actin domain, thus disrupting the segregation of Lats2? Or an effect on aPKC? All might be possible, but do they have any evidence for one versus the other?

2) Much of the conclusion on the involvement of Yap/WWtr1 and Lats on changing the behavior of cells depends on the scoring of cells as inside or outside. The authors define inside cells as "appearing inside and showing uniform CDH1 over the cell surface". It is not clear exactly how these criteria were applied. What does it mean to 'appear' internal? 3D reconstructed from z stacks or just estimated from midline optical sections? I am not sure that this is a very accurate way to determine inner and outer cells and indeed they note that uniform CDH1 was not always a good predictor of position. This is a key point, because they then go on to claim that loss of Yap/WWTr1 or activation of Lats leads not just to internalization of Lats-overexpressing cells, but to a shift in the actual proportion of inside and outside cells, with loss of outside and gain of inside cells. It is hard to understand how this can occur topologically, if the total cell number is unchanged, because reduction in outside cells would presumably lead to bulging of inside cells to the outside. Or are they proposing that the reduced number of outside cells somehow stretch out over the enlarged group of inner cells? The resolution of the images provided does not really resolve this issue.

3) It is not clear to me how the apparent reduction in expression of PARD6B and aPKCz in Lats-overexpressing cells is proposed to alter polarity and contractility, leading to internalization of the cells, given that the cells actually remain polarized as judged by other markers. Are they proposing a specific phosphorylation event that would alter cell polarity and contractility? Recent work from other labs has suggested that differential contractility is key to internalization of blastomeres during cleavage (e.g. Maitre et al., 2016)- have they looked at actomyosin? What happens to other components of the Hippo signaling pathway?

4) The model proposed in Figure 7 has ROCK as the factor linking the cell membrane to Lats regulation, but no specific mechanism is proposed. This model does not include the data from the Sasaki lab suggesting that the apical actin domain in the outside cells binds Lats and segregates it from its active complex with Nf2/Angiomotin/E-cadherin, thus reducing Hippo signaling in outside cells. ROCK could be involved in regulating the cortical actin domain, but has several other roles in the cell. A more comprehensive model, including data from other groups should be developed.

Reviewer #3:

The manuscript by Frum and Ralston reports an analysis of the role of the Hippo signalling pathway during the differentiation between trophectoderm and ICM.

They first show that the exclusion of SOX2, an inner cell marker, from outer cells depends on the activity of Rock as the use of a pharmacological inhibitor causes SOX2 expression in outer cells. The ectopic expression of a constitutively activated YAP prevents SOX2 expression in inner cells, showing nuclear Yap inhibition on SOX2 expression. This data reinforces their previous findings using Tead4 loss of function.

They then analysed the loss of function of Yap1 and Wwtr genes in compound mutants. Their analysis shows that the phenotypes are linked to the number of alleles present, in a dose dependent manner, and that maternal expression partially rescues the loss of zygotic expression. The lower the dose of "Yap/Wwtr", the less CDX2 expression in outer cells and the more SOX2 expression in outer cells. They nicely show that completely removing the alleles by maternal-zygotic double deletion fully abolishes CDX2 expression and causes SOX2 expression in all the cells. Moreover, the loss of "Yap/Wwtr" is also correlated with a lower number of outside cells. Gain of function experiments with ectopic expression of Lats not only induced ectopic SOX2 expression but also decreases the number of outer cells and increases the number of inside cells. This lead them to the conclusion that ectopic Lats expression induces inside cell repositioning. Ectopic Lats expression phenotype can be partially rescued by co-expressing constitutively activated YAP. The mutation of Sox2 does not interfere with YAP expression and activity, showing that SOX2 is only a marker (an important one) but not an actor. Finally, using Lats ectopic expression in outside cells, they show that the Hippo pathway can strikingly downregulate some polarity markers (Pard6B/aPKCz) but maintain others (Cdh1, pERM).

Their results are clearly described. They report an important analysis of Yap;Wwtr compound mutants, an involvement of the Hippo pathway for cell (re-)positioning and an effect of ectopically activated Hippo in outside cells on cell polarisation.

I suggest to address the following points:

1) The number of mutants is very low even if the phenotype seems to be fully penetrant. Would it be possible to increase the numbers, at least for the MZ-double mutants (2 is very/too small).

2) What is the proportion of outside cell death in these mutant embryos?

3) It is proposed that caYAP can rescue cell survival in Lats overexpressing cells. Can cell counts confirm this (only proportions between inside and outside are given).

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo" for further consideration at eLife. Your revised article has been favorably evaluated by Marianne Bronner (Senior Editor), a Reviewing Editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.

We would recommend that you remove the experiment involving the LATS2-kinase dead construct, since the data are difficult to interpret. Removing the work will not impact on the primary conclusions of the paper, and indeed was not requested by either of the reviewers.

Reviewer #1:

The authors have carefully responded to the critiques and addressed most of the issues raised. The paper adds new information on the complexity of lineage segregation in the early embryo that will be of value to the field and also opens up new questions for investigation.

Reviewer #2:

With this revised version, the manuscript has appreciably improved with more numbers for the mutants (and more analyses) and better explanations in the analyses.

My only negative comment will concern the LATS2-kinase dead experiments that do not seem to work as YAP expression is not nuclear in inside GFP expressing cells (at least on the picture shown in Figure 6—figure supplement 1). This could be an explanation why it does not alter cell position. An appropriate embryo/section showing YAP nuclear expression in inside cells should be presented to allow concluding on the experiment.

Concerning the final interpretation, do the authors think that YAP and WWTR1 inhibit apoptosis? Could apoptosis be the result of an unresolved conflict between inside and outside (as they are still partially polarized), whereas cells moving in may have resolved the conflict by becoming inside cells?

https://doi.org/10.7554/eLife.42298.019

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

[…] As you will see both reviewers were very positive and feel that your genetic approach certainly has provided further insights into lineage segregation in the early embryo. However the biggest issue seems to be the study is a bit premature given the small numbers of embryos you've been able to analyse to date. If you can considerably improve the manuscript along the lines suggested by the reviewers, including expanding the samples sizes for critical genotypes we would be happy to see a major revision of the paper in the coming months, which would be considered as a new submission.

I am pleased to communicate that we have addressed all of the reviewers’ questions and concerns with new data. Our major changes include:

• Increased sample size for the critical genotypes, as requested.

• Additional phenotypic characterizations requested, including cell death assays.

We would like to take the opportunity to explain the novelty and significance of our study.

• No other study has examined the consequences of the combined loss of maternal and zygotic Yap1 and Wwtr1 in the mouse.

• We report new and unexpected roles for Yap1/Wwtr1 in repressing expression of Sox2, the earliest known marker of pluripotency in the embryo.

• We report new and unexpected roles for Yap1/Wwtr1 in promoting outside cell positioning.

• We identify the mechanism by which YAP1/WWTR1 promotes outside cell positioning by promoting formation of the apical domain – a process not previously defined.

Reviewer #2:

[…] Overall this is a carefully carried out study that provides some more insights into the mechanisms of the first lineage segregation in the mouse embryo. The strength of the paper is the description of the allelic series of mutations in Yap/Wwtr1, which have not been previously described. All previous studies depended largely on dominant negative and overexpression studies, which have their limitations. What is less strong is the section proposing a direct role for Lats in regulating polarity via downregulation of PARD6B and aPKCz. This underlies their model that Hippo signaling interaction with polarity components acts as a failsafe feedback mechanism to ensure lineage segregation. As outlined below, the data as presented raise some issues that need further resolution. In addition, there is no clear molecular mechanism proposed by which Lats activity would regulate specifically PARD6B and aPKCz and not other polarity components including the actin domain and phospho-ERM. The linkage between Lats, the apical domain, ROCK, etc. is presented in a model but there are many missing links in the model and a failure to link to other models of how polarity is thought to control Hippo signaling.

We thank the reviewer for the very careful and thoughtful review. We found all of the reviewer’s questions and suggestions to be extremely valuable. Accordingly, we have performed the requested experiments and analyses to increase sample sizes and solidify the mechanism. These are detailed below.

1) The first section on ROCK1/2 being upstream regulators of Cdx2 and Sox2 expression is not well connected to the rest of the paper. They also did not actually look at the effects of Rocki on both Cdx2 and Sox2 in the same embryos. Are the outside cells that remain Sox2-ve expressing Cdx2?

We have performed the requested analysis of CDX2 and SOX2 in ROCKi-treated embryos (Figure 1E and Figure 1—figure supplement 1A). In short, many outside cells in ROCKi-treated embryos coexpress SOX2 and CDX2. We have revised the text to better connect these observations to the rest of the paper and address the reviewer’s questions in the last paragraph of the subsection “Patterning of Sox2 is ROCK-dependent”.

Inhibition of Rock has multiple effects on the cell- which downstream response do they consider to be the critical one? Are they proposing a direct effect on Lats- as shown in Figure 7? Or the more usually suggested effect on the cortical actin domain, thus disrupting the segregation of Lats2? Or an effect on aPKC? All might be possible, but do they have any evidence for one versus the other?

We do not yet know the biochemical mechanisms by which ROCK influences Sox2 expression. Since ROCKi has been shown to alter YAP1 localization (Kono et al., 2014), we propose that at least part of the ROCKi effect on Sox2 is through its role in antagonizing YAP1 and WWTR1 activity, since ROCKi phenocopies Wwtr1 and Yap1 loss of function.

We now address this comment in the revised Discussion: “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.”

2) Much of the conclusion on the involvement of Yap/WWtr1 and Lats on changing the behavior of cells depends on the scoring of cells as inside or outside. the authors define inside cells as "appearing inside and showing uniform CDH1 over the cell surface". It is not clear exactly how these criteria were applied. What does it mean to 'appear' internal? 3D reconstructed from z stacks or just estimated from midline optical sections? I am not sure that this is a very accurate way to determine inner and outer cells and indeed they note that uniform CDH1 was not always a good predictor of position.

Indeed, CDH1 localization was not usually our method for determining cell position. We thank the reviewer for inviting the opportunity to clarify the methods used throughout the paper, we have added a section to the Materials and methods section entitled “Embryo Analysis.” Briefly, cell position was determined based on whether each individual cell in each section of each embryo made contact with the embryo’s external environment (outside) or whether it was surrounded by other cells (inside).

This is a key point, because they then go on to claim that loss of Yap/WWTr1 or activation of Lats leads not just to internalization of Lats-overexpressing cells, but to a shift in the actual proportion of inside and outside cells, with loss of outside and gain of inside cells. It is hard to understand how this can occur topologically, if the total cell number is unchanged, because reduction in outside cells would presumably lead to bulging of inside cells to the outside. Or are they proposing that the reduced number of outside cells somehow stretch out over the enlarged group of inner cells? The resolution of the images provided does not really resolve this issue.

The fewer outside cells appear to spread over the inner cells in these mutants. We have provided additional images and quantification of this phenotype in Figure 6, which we hope better illustrate this unusual phenotype. In addition, we describe the phenotype: “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 (Figure 6E-F).”

3) It is not clear to me how the apparent reduction in expression of PARD6B and aPKCz in Lats-overexpressing cells is proposed to alter polarity and contractility, leading to internalization of the cells, given that the cells actually remain polarized as judged by other markers. Are they proposing a specific phosphorylation event that would alter cell polarity and contractility? Recent work from other labs has suggested that differential contractility is key to internalization of blastomeres during cleavage (e.g. Maitre et al., 2016)- have they looked at actomyosin? What happens to other components of the Hippo signaling pathway?

We now discuss the reviewer’s questions: “[…] 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).”

Our lab intends to evaluate these mechanisms (including actomyosin and other HIPPO pathway members) in our future investigations.

4) The model proposed in Figure 7 has ROCK as the factor linking the cell membrane to Lats regulation, but no specific mechanism is proposed. This model does not include the data from the Sasaki lab suggesting that the apical actin domain in the outside cells binds Lats and segregates it from its active complex with Nf2/Angiomotin/E-cadherin, thus reducing Hippo signaling in outside cells. ROCK could be involved in regulating the cortical actin domain, but has several other roles in the cell. A more comprehensive model, including data from other groups should be developed.

We are grateful for the recommendation to develop a more inclusive model (Figure 7).

Reviewer #3:

[…] I suggest to address the following points:

1) The number of mutants is very low even if the phenotype seems to be fully penetrant. Would it be possible to increase the numbers, at least for the MZ-double mutants (2 is very/too small).

We have increased sample sizes as requested – the phenotypes are fully penetrant.

2) What is the proportion of outside cell death in these mutant embryos?

We have now evaluated cell death by TUNEL assay both Lats2-overexpressing (Figure 2—figure supplement 1) and Wwtr1/Yap1 double knockout embryos (Figure 6G and Figure 6—figure supplement 1A). As anticipated, we observe increased cell death in both genotypes.

3) It is proposed that caYAP can rescue cell survival in Lats overexpressing cells. Can cell counts confirm this (only proportions between inside and outside are given).

We have also addressed this question by TUNEL assay (Figure 2—figure supplement 1). We observe decreased cell death in these embryos compared with embryos overexpressing Lats2 alone, consistent with our proposal that caYAP can rescue cell survival.

[Editors' note: the author responses to the re-review follow.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

We would recommend that you remove the experiment involving the LATS2-kinase dead construct, since the data are difficult to interpret. Removing the work will not impact on the primary conclusions of the paper, and indeed was not requested by either of the reviewers.

Reviewer #2:

With this revised version, the manuscript has appreciably improved with more numbers for the mutants (and more analyses) and better explanations in the analyses.

My only negative comment will concern the LATS2-kinase dead experiments that do not seem to work as YAP expression is not nuclear in inside GFP expressing cells (at least on the picture shown in Figure 6—figure supplement 1). This could be an explanation why it does not alter cell position. An appropriate embryo/section showing YAP nuclear expression in inside cells should be presented to allow concluding on the experiment.

We have made the suggested changes and removed the LATS2-kinase dead experiments that the reviewers found to be ambiguous.

https://doi.org/10.7554/eLife.42298.020

Article and author information

Author details

  1. Tristan Frum

    Department of Biochemistry and Molecular Biology, Michigan State University, Michigan, United States
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3452-8762
  2. Tayler M Murphy

    1. Genetics Graduate Program, Michigan State University, Michigan, United States
    2. Reproductive and Developmental Biology Training Program, Michigan State University, Michigan, United States
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  3. Amy Ralston

    1. Department of Biochemistry and Molecular Biology, Michigan State University, Michigan, United States
    2. Genetics Graduate Program, Michigan State University, Michigan, United States
    3. Reproductive and Developmental Biology Training Program, Michigan State University, Michigan, United States
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration
    For correspondence
    aralston@msu.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3755-8262

Funding

National Institute of General Medical Sciences (R01 GM104009)

  • Amy Ralston

National Institute of Child Health and Human Development (T32HD087166)

  • Tayler M Murphy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Dr. Hiroshi Sasaki for providing expression constructs, to Dr. Randy L Johnson for providing mice carrying conditional alleles of Yap1 and Wwtr1, and to Dr. Jason Knott for embryo microinjection training. We also thank Dr. Ripla Arora, Dr. Julia Ganz, and members of the Ralston Lab for comments. This work was supported by NIH R01 GM104009 and the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number T32HD087166. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank anonymous reviewers for insightful questions and suggestions.

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Elizabeth Robertson, University of Oxford, United Kingdom

Publication history

  1. Received: September 27, 2018
  2. Accepted: November 12, 2018
  3. Version of Record published: December 11, 2018 (version 1)

Copyright

© 2018, Frum et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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