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TEAD4, YAP1 and WWTR1 Prevent the Premature Onset of Pluripotency Prior to the 16-cell Stage

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TEAD4, YAP1 and WWTR1 Prevent the Premature Onset of Pluripotency Prior to the 16-cell Stage

Tristan Frum et al. Development.

Abstract

In mice, pluripotent cells are thought to derive from cells buried inside the embryo around the 16-cell stage. Sox2 is the only pluripotency gene known to be expressed specifically within inside cells at this stage. To understand how pluripotency is established, we therefore investigated the mechanisms regulating the initial activation of Sox2 expression. Surprisingly, Sox2 expression initiated normally in the absence of both Nanog and Oct4 (Pou5f1), highlighting differences between embryo and stem cell models of pluripotency. However, we observed precocious ectopic expression of Sox2 prior to the 16-cell stage in the absence of Yap1, Wwtr1 and Tead4 Interestingly, the repression of premature Sox2 expression was sensitive to LATS kinase activity, even though LATS proteins normally do not limit activity of TEAD4, YAP1 and WWTR1 during these early stages. Finally, we present evidence for direct transcriptional repression of Sox2 by YAP1, WWTR1 and TEAD4. Taken together, our observations reveal that, while embryos are initially competent to express Sox2 as early as the four-cell stage, transcriptional repression prevents the premature expression of Sox2, thereby restricting the pluripotency program to the stage when inside cells are first created.

Keywords: HIPPO signaling; Preimplantation; Stem cell progenitors.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Nanog and Oct4 are required for the maintenance, but not the initiation, of Sox2. (A) Immunostaining for SOX2, E-cadherin (ECAD) and DNA in non-mutant and Nanog;Oct4 null embryos at the 16-cell stage (E3.0). (B) SOX2, ECAD and DNA in non-mutant and Nanog;Oct4 null embryos at the 32-cell stage (E3.25). (C) SOX2 and DNA in non-mutant and Nanog;Oct4 null embryos at E3.5. (D) Manual counting of the percentage of inside cells, across all embryos, exhibiting intense SOX2 staining in non-mutant and Nanog;Oct4 null embryos at E3.5 (see Materials and Methods for details). Data are mean±s.d., Student's t-test. (E) NANOG-GFP, SOX2 and DNA in non-mutant and Nanog null embryos at E3.75. (F) NANOG, SOX2 and DNA in non-mutant and Oct4 null embryos at E3.75. (G) NANOG-GFP, SOX2 and DNA in non-mutant and Nanog null embryos at E4.25. (H) NANOG, SOX2 and DNA in non-mutant and Oct4 null embryos at E4.25. For all panels, n indicates number of embryos examined. Dashed white lines demarcate non-epiblast/presumptive primitive endoderm cells.
Fig. 2.
Fig. 2.
TEAD4/WWTR1/YAP1 represses precocious Sox2 expression at the eight-cell stage. (A) Immunostaining for SOX2 in non-mutant, Tead4 zygotic (z) null and Tead4 maternal-zygotic (mz) null embryos at the eight-cell stage. (B) SOX2 in embryos lacking m Wwtr1 and Yap1 at the eight-cell stage, with indicated zygotic genotypes. (C) SOX2 in embryos of indicated genotypes at the four-cell stage. (D) SOX2 in embryos of indicated genotypes at the four-cell stage. n indicates number of embryos examined.
Fig. 3.
Fig. 3.
YAP1 localization and Sox2 expression are sensitive to LATS2 kinase. (A) Experimental approach: both blastomeres of two-cell stage embryos were injected with either 500 ng/µl NLS-GFP mRNA, which encodes GFP bearing a nuclear localization sequence (NLS), or 500 ng/µl Lats2 mRNA, and were then cultured to the four- or eight-cell stages. (B) YAP1 and SOX2 immunostaining in four-cell stage embryos injected with NLS-GFP mRNA or Lats2 mRNA. (C) YAP1 and SOX2 in eight-cell stage embryos injected with NLS-GFP mRNA or Lats2 mRNA. (D) The percentage of SOX2-positive cells per embryo (each value obtained is indicated by a dot) at the indicated stages. Data are mean±s.d. n indicates number of embryos examined.
Fig. 4.
Fig. 4.
TEAD4/WWTR1/YAP1 repress Sox2 expression through a direct mechanism. (A) Models for indirect and direct repression of Sox2 by TEAD4/WWTR1/YAP1 and predicted effect of Tead4EnR and Tead4VP16 on Sox2 expression. (B) Experimental approach: a single blastomere of each four-cell embryo was injected with 150 ng/µl NLS-GFP mRNA and either 150 ng/µl Tead4VP16 or Tead4EnR mRNA, and then cultured to the eight-cell stage. (C) GFP and SOX2 immunostaining in embryos injected with Tead4VP16 or Tead4EnR. (D) The percentage of NLS-GFP-positive, SOX2-positive cells per embryo (each value obtained is indicated by a dot) injected with Tead4VP16 or Tead4EnR. Student's t-test, n indicates number of embryos examined. (E) Model for regulation of Sox2 at indicated developmental stages. ICM, inner cell mass; TE, trophectoderm; EPI, epiblast; PE, primitive endoderm.

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