Yojiro Yamanaka

Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst

Blastocyst formation marks the segregation of the first two cell lineages in the mammalian preimplantation embryo: the inner cell mass (ICM) that will form the embryo proper and the trophectoderm (TE) that gives rise to the trophoblast lineage. Commitment to ICM lineage is attributed to the function of the two transcription factors, Oct4 (encoded by Pou5f1) and Nanog. However, a positive regulator of TE cell fate has not been described. The T-box protein eomesodermin (Eomes) and the caudal-type homeodomain protein Cdx2 are expressed in the TE, and both Eomes and Cdx2 homozygous mutant embryos die around the time of implantation. A block in early TE differentiation occurs in Eomes mutant blastocysts. However, Eomes mutant blastocysts implant, and Cdx2 and Oct4 expression are correctly restricted to the ICM TE. Blastocoel formation initiates in Cdx2 mutants but epithelial integrity is not maintained and embryos fail to implant. Loss of Cdx2 results in failure to downregulate Oct4 and Nanog in outer cells of the blastocyst and subsequent death of those cells. Thus, Cdx2 is essential for segregation of the ICM and TE lineages at the blastocyst stage by ensuring repression of Oct4 and Nanog in the TE.

Two Signals Are Necessary for Cell Proliferation Induced by a Cytokine Receptor gp130: Involvement of STAT3 in Anti-Apoptosis

gp130 is a common signal transducer for the interleukin-6-related cytokines. To delineate the gp130-mediated growth signal, we established a series of pro-B cell lines expressing chimeric receptors composed of the extracellular domain of the granulocyte colony-stimulating factor receptor and the transmembrane and cytoplasmic domains of gp130. The second tyrosine (from the membrane) of gp130, which was required for the tyrosine phosphorylation of SHP-2, its association with GRB2, and activation of a MAP kinase, was essential for mitogenesis, but not for anti-apoptosis. On the other hand, the tyrosine in the YXXQ motifs essential for STAT3 activation was required for bcl-2 induction and anti-apoptosis. Furthermore, dominant-negative STAT3 inhibited anti-apoptosis. These data demonstrate that two distinct signals, mitogenesis and anti-apoptosis, are required for gp130-induced cell growth and that STAT3 is involved in anti-apoptosis.

FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst

Primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Recent studies showed that EPI and PE progenitors expressing the lineage-specific transcriptional factors Nanog and Gata6, respectively, arise progressively as the ICM develops. Subsequent sorting of the two progenitors during blastocyst maturation results in the ormation of morphologically distinct EPI and PE layers at E4.5. It is, however, unknown how the initial differences between the two populations become established in the E3.5 blastocyst. Because the ICM cells are derived from two distinct rounds of polarized cell divisions during cleavage, a possible role for cell lineage history in promoting EPI versus PE fate has been proposed. We followed cell lineage from the eight-cell stage by live cell tracing and could find no clear linkage between developmental history of individual ICM cells and later cell fate. However, modulating FGF signaling levels by inhibition of the receptor/MAP kinase pathway or by addition of exogenous FGF shifted the fate of ICM cells to become either EPI or PE, respectively. Nanog- or Gata6-expressing progenitors could still be shifted towards the alternative fate by modulating FGF signaling during blastocyst maturation, suggesting that the ICM progenitors are not fully committed to their final fate at the time that initial segregation of gene expression occurs. In conclusion, we propose a model in which stochastic and progressive specification of EPI and PE lineages occurs during maturation of the blastocyst in an FGF/MAP kinase signal-dependent manner.

Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase

ABSTRACT Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation.

Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts

The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages, respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst – extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.

Cell and molecular regulation of the mouse blastocyst.

Animals use diverse strategies to specify tissue lineages during development. A common strategy is to partition maternally supplied and localized lineage determinants into progenitor cells. The mouse embryo appears to use a different, more regulative strategy to specify the first three lineages: the epiblast (EPI: future embryo), the trophectoderm (TE: future placenta), and the primitive endoderm (PE: future yolk sac). These lineages are specified during two successive differentiation steps leading to formation of the blastocyst. Here, we review classic and contemporary models of early lineage specification in the mouse, and describe recent efforts to understand the molecular regulation of these events. We describe evidence that trophectoderm differentiation bears resemblance to the process of epithelialization and describe the importance of apical/basal protein complexes in regulating this process. Next, we present a revised model of PE specification, and describe evidence that PE cells in the inner cell mass sort out to occupy their ultimate position on the surface of the EPI. Finally, we describe factors that reinforce these lineages and three distinct stem cell types that can be isolated from them. Together, these mechanisms guide the differentiation of the first lineages of the mouse and thereby set up tissues that will be important for the first steps of embryonic body patterning. Developmental Dynamics 235:2301–2314, 2006.

An alternative pathway for STAT activation that is mediated by the direct interaction between JAK and STAT.

JAK is believed to be an essential tyrosine kinase that mediates signals from the cytokine receptor to its downstream events. JAK associates with the cytoplasmic domain of the type I cytokine receptor superfamily and upon the ligand stimulation it can be activated, resulting in the receptor phosphorylation. In signaling from gp130, a common signal transducer for the IL-6 family cytokines, STAT3, a transcription factor that contains an SH2 domain, is recruited by phosphotyrosines on gp130 and is subsequently phosphorylated by gp130-associated JAKs. In this study, we attempted to find a new target for JAK that is directly activated by JAK, independent of gp130 tyrosine phosphorylation, by using a yeast two-hybrid system. In the process we found that the JH2 domain of JAK1, JAK2 or JAK3 could specifically associate with the carboxy-terminal portion of STAT5, but not with STAT3 or STAT1. The interaction was confirmed using both a transient expression system in a cell line and a GST-fusion protein binding assay. Furthermore, we showed that the activation of STAT5 via gp130 did not need any phosphotyrosines on gp130 while that of STAT3 strictly depended on phosphotyrosines on gp130. Mutations of STAT5 that eliminated the interaction with JAK1 reduced the activation of STAT5 upon the gp130 stimulation, although such mutants could be still activated through erythropoietin receptor. These results indicate that STATs are activated through cytokine receptors by two distinct mechanisms, one dependent on receptor tyrosine phosphorylation and the other mediated by the JAK – STAT direct interaction.

Krüppel-like factor 5 is essential for blastocyst development and the normal self-renewal of mouse ESCs.

The transcription factor Klf4 has demonstrated activity in the reprogramming of somatic cells to a pluripotent state, but the molecular mechanism of this process remains unknown. It is, therefore, of great interest to understand the functional role of Klf4 and related genes in ESC regulation. Here, we show that homozygous disruption of Klf5 results in the failure of ESC derivation from ICM cells and early embryonic lethality due to an implantation defect. Klf5 KO ESCs show increased expression of several differentiation marker genes and frequent, spontaneous differentiation. Conversely, overexpression of Klf5 in ESCs suppressed the expression of differentiation marker genes and maintained pluripotency in the absence of LIF. Our results also suggest that Klf5 regulates ESC proliferation by promoting phosphorylation of Akt1 via induction of Tcl1. These results, therefore, provide new insights into the functional and mechanistic role of Klf5 in regulation of pluripotency.

AUTOREGULATION OF THE STAT3 GENE THROUGH COOPERATION WITH A CAMP-RESPONSIVE ELEMENT-BINDING PROTEIN

STAT3 (signal transducer and activator of transcription 3) is a key transcription factor mediating the signals for a variety of cytokines, including interleukin-6 (IL-6). The Stat3 gene itself is activated by IL-6 signals. We show that the region of the signal-transducing subunit, gp130, essential for STAT3 activation, is also required for activation of the Stat3 gene. To elucidate the mechanisms activating the Stat3 gene, we identified an IL-6 response element (IL-6RE) in the Stat3gene promoter containing both a low affinity STAT3-binding element and a cAMP-responsive element (CRE). Electrophoretic mobility shift assays showed that IL-6 induced a slowly migrating complex on the IL-6RE containing a STAT3 homodimer and an unidentified CRE-binding protein. With the combination of transient transfection assays using mutantStat3 promoter-reporter constructs and electrophoretic mobility shift assays, we found that the formation of a slowly migrating complex was required for full activation of theStat3 gene. Thus, STAT3 activates the Stat3gene in cooperation with an unidentified CRE-binding protein. This regulatory mechanism is similar to that of the junB gene, which is activated by IL-6 through the junB IL-6RE, which contains a low affinity STAT3-binding site and a CRE-like site.

Disorganized epithelial polarity and excess trophectoderm cell fate in preimplantation embryos lacking E-cadherin

The first two cell lineages in the mouse, the surface trophectoderm (TE) and inner cell mass (ICM), are morphologically distinguishable by E3.5, with the outer TE forming a polarized epithelial layer enclosing the apolar ICM. We show here that in mouse embryos completely lacking both maternal and zygotic E-cadherin (cadherin 1), the normal epithelial morphology of outside cells is disrupted, but individual cells still initiate TE- and ICM-like fates. A larger proportion of cells than normal showed expression of TE markers such as Cdx2, suggesting that formation of an organized epithelium is not necessary for TE-specific gene expression. Individual cells in such embryos still generated an apical domain that correlated with elevated Cdx2 expression. We also show that repolarization can occur in isolated early ICMs from both wild-type and Cdx2 mutant embryos, indicating that Cdx2 is not required for initiating polarity. The results demonstrate that epithelial integrity mediated by E-cadherin is not required for Cdx2 expression, but is essential for the normal allocation of TE and ICM cells. They also show that Cdx2 expression is strongly linked to apical membrane polarization.