HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog

SOX2 is restricted to ICM progenitors by HIPPO pathway members and not by CDX2.

<p>A) Immunofluorescent analysis of SOX2 and NANOG shows that SOX2 is detected specifically in ICM cells at the 16-cell stage and later, while NANOG is detected in all cells at these stages. B) SOX2 is not upregulated in the TE of <i>Cdx2</i> null embryos at early or late blastocyst stages, indicating that CDX2 does not restrict SOX2 to the ICM. C) SOX2 is ectopically expressed in outside cells of embryos lacking the HIPPO pathway member <i>Tead4</i> (asterisk  =  SOX2-positive outside cell). TE cells are defined both by outside position and by basolateral localization of E-CADHERIN (ECAD). D) Either <i>Lats2</i> or <i>β-Globin</i> mRNAs were injected into both cells of 2-cell embryos, and embryos were then cultured to blastocyst stage. E) The proportion of outside cells in which SOX2 was ectopically expressed was significantly increased in both <i>Tead4</i> null embryos, and in embryos overexpressing the HIPPO pathway member <i>Lats2</i>, relative to controls (p-value calculated by t-test). F) Overexpression of <i>Lats2</i>, which prevents nuclear YAP localization, causes ectopic expression of SOX2 in outside cells (indicated by asterisk). In all panels, bar  = 20 µm.</p

<i>Sox2</i> is required for the initial expression of PE genes in the ICM.

<p>A) At E3.75, NANOG is detected in <i>Sox2</i> null embryos, but SOX17 is not detected in most <i>Sox2</i> null embryos (arrowhead  =  ICM cell expressing neither NANOG nor SOX17). B) At E3.75, the average proportion of ICM cells in which NANOG is elevated is equivalent among all genotypes examined, indicating that <i>Sox2</i> is not required for expression of NANOG in the ICM. However, the average proportion of ICM cells in which SOX17 is detected is significantly reduced, and the proportion of ICM cells in which neither NANOG nor SOX17 are detected is significantly increased, in the absence of either Z or MZ <i>Sox2</i>. C) At E3.75, <i>Sox2</i> is required for high levels of GATA6 in PE cells. D) Quantification of immunofluorescent results showing that the proportion of ICM cells in which high levels of GATA6 are detected is significantly lower <i>Sox2</i> null embryos at E3.75, while the proportion of ICM cells expressing both NANOG and low levels of GATA6 is significantly higher. E) Expression of PDGFRA in the ICM depends on <i>Sox2</i>. F) Expression of GATA4 in the ICM depends on <i>Sox2</i>. Bar  = 20 µm, p-value calculated by t-test in B, and by Chi-squared test in E; n.s.  = p>0.05.</p

The roles and regulation of SOX2 during blastocyst formation.

<p>A) At the 16-cell stage, when ICM progenitors first arise, HIPPO pathway members regulate expression of TE (<i>Cdx2</i>) and ICM (<i>Sox2</i>) genes in parallel. At this stage, OCT4 and NANOG are still expressed ubiquitously. B) In the blastocyst, <i>Sox2</i> expression is restricted to EPI cells by FGFR/MEK signaling. In EPI cells, SOX2 helps promote expression of <i>Fgf4</i>, which signals to neighboring cells to induce expression of PE genes. In PE cells, MAPK promotes PE gene expression in an <i>Oct4</i>-dependent manner <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004618#pgen.1004618-Frum1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004618#pgen.1004618-Aksoy1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004618#pgen.1004618-LeBin1" target="_blank">[59]</a>, and represses expression of <i>Sox2</i> and <i>Nanog</i> in PE cells either directly or indirectly. C) In the late blastocyst, SOX2 helps maintain expression of pluripotency genes, and FGF4, or other signals from EPI, maintain expression of PE genes in neighboring cells.</p