Dev Biol. 331:210-21, 2009

Active cell movements coupled to positional induction are involved in lineage segregation in the mouse blastocyst.

In the mouse blastocyst, some cells of the inner cell mass (ICM) develop into primitive endoderm (PE) at the surface, while deeper cells form the epiblast. It remained unclear whether the position of cells determines their fate, such that gene expression is adjusted to cell position, or if cells are pre-specified at random positions and then sort. We have tracked and characterised dynamics of all ICM cells from the early to late blastocyst stage. Time-lapse microscopy in H2B-EGFP embryos shows that a large proportion of ICM cells change position between the surface and deeper compartments. Most of this cell movement depends on actin and is associated with cell protrusions. We also find that while most cells are precursors for only one lineage, some give rise to both, indicating that lineage segregation is not complete in the early ICM. Finally, changing the expression levels of the PE marker Gata6 reveals that it is required in surface cells but not sufficient for the re-positioning of deeper cells. We provide evidence that Wnt9A, known to be expressed in the surface ICM, facilitates re-positioning of Gata6-expressing cells. Combining these experimental results with computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.

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●Blastcyst内の細胞の動きをtime-lapseでモニターし、epi-PEのsegrigationを説明する3つのモデル（Positional, Salt&Pepper, Cell sorting+positonal induction）のうち、最後のモデルに妥当性があることをコンピューターシミュレーションを用いて示す。
●Gata6+細胞はICMの表層側に来るが、この移動にはWnt9aが必要

●4/7数理ゼミで議論された論文

Dev Cell. 16:398-410, 2009

The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass.

Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

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CDBより。
●Tead4-Cdx2 pathwayによるTE分化制御について
●Tead4の発現はpan-embryonicなのに、なぜTEでのみ機能するか？

●Cell-cell contacts→Hippo→Lats1/2---|Yorkie/Yap1→Tead4経路活性化

●活性化型Tead4として、Tead4VP16を使用（いいのか？）
●Tead4haCdx2を含むTE細胞分化にかかわる因子群を制御（Fig1～2）
●核内YapとCdx2の発現がcorrelate、かつYapでCdx2の発現が制御される（Fig.3，4)
●Wwtr1（Yap-related)がcompensativeに機能
●Yap/Wwtr1のkinaseであるLats1/2がYapのリン酸化を介して核外排出を促進
●E-cadherin阻害剤のECCD1を用いると、Yapの核外排出とリン酸化が阻害されることから、ICMのcell-cell interactionが（Hippoを介して）Yapのリン酸化+核外排出に重要（Fig6)
●胚をばらしてreaggregateしたもの/immunosurgeryでICMだけ出してcultureしたもので、cell positionによってYapの核局在が規定されていることを証明（Fig7）
●モデル（Fig7)

Immunosurgeryについての文献は。
http://www.pnas.org/content/72/12/5099.full.pdf
http://media.wiley.com/product_data/excerpt/68/04700335/0470033568.pdf

PLoS ONE 4(12):e8530, 2009

SNAI1 and SNAI2 Are Asymmetrically Expressed at the 2-Cell Stage and Become Segregated to the TE in the Mouse Blastocyst

SNAI1 and SNAI2 are transcription factors that initiate Epithelial-to-Mesenchymal cell transitions throughout development and in cancer metastasis. Here we show novel expression of SNAI1 and SNAI2 throughout mouse preimplantation development revealing asymmetrical localization of both SNAI1 and SNAI2 in individual blastomeres beginning at the 2-cell stage through to the 8-cell stage where SNAI1 and SNAI2 are then only detected in outer cells and not inner cells of the blastocyst. This study implicates SNAI1 and SNAI2 in the lineage segregation of the trophectoderm and inner cell mass, and provides new insight into these oncogenes.

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Trophectoderm(TE)の分化に重要？な転写因子であるSNAI1/2の発現パターンを見た論文。

予備知識として、
SNAI1/2→E-cadherin/Na-K ATPase→TE

SNAI1KO→neural crestの発育以上
SNAI2KO→survive at birth
compensatoryなメカニズムか？

Nature Cell Biology 10:429 - 436, 2008

Tensile forces govern germ-layer organization in zebrafish

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells1. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFbeta-signalling (transforming growth factor beta), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

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細胞表面のcortical tensionが細胞の種類によって異なっていることをAFMを用いて証明。
それがprogenitor-cell sortingの主要なfactorであることを示す。

なかなか面白い仮説。

MCB 26:7539-7549, 2006

The Grb2/Mek Pathway Represses Nanog in Murine Embryonic Stem Cells

The homeobox gene Nanog is a key intrinsic determinant of self renewal in embryonic stem (ES) cells, and its repression leads ES cells to selectively differentiate into primitive endoderm. Although Nanog repression occurs at the outermost layer of ES cell aggregates independent of the leukemia inhibitory factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and intracellular signals cause the event. Of interest, addition of the tyrosine phosphatase inhibitor, sodium vanadate, selectively repressed Nanog transcription without any detectable changes in upstream transcriptional regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive endoderm differentiation, even in the inner cells of ES cell aggregates. Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also increased and decreased by the addition of sodium vanadate, respectively, but these changes were eliminated by exogenous Nanog expression. The effects of sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES cell aggregation as well. Furthermore, transfection of a constitutive active Mek mutant into ES cells induced Nanog repression and primitive endoderm differentiation. These data indicate that the Grb2/Mek pathway primarily mediates Nanog gene repression upon ES cell differentiation into primitive endoderm.

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タイトル通りの内容。

●SU5402→FGFR inhibitor
●Fig8にまとめた図あり

IntroにFGF-PI3K-AKT経路の寄与について言及あり（Ref.）

Development 137:715-24, 2010

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.

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初期胚の細胞分化の様子やシグナルの関与を細かくみた秀作。
IntroやDisの情報もよくreviewされてて有用。

●8cell stageの細胞の一つにH2B-RFPとmembrane-RFPを入れて、その後の分裂の様子をライブでフォロー（Fig1, Table1)。primary inside cellとsecondary inside cellとの寄与度は差がないことを証明。

● FGF/MAPKシグナルのinhibitorをE2.5～4.5の間のさまざまな時間でtreatして、どこからが不可逆なポイントの検討、PE/EPIへの分化の影響などを検討。（Fig2-4）→E4.5で最終的にplasciticyが失われるが、それまではNanog/Gata6のどちらかをふらふらしている。
Gata4の発現がcritical?（Discussion）

●High-doseなFGF4（+heparin）がEPI formationのブロックに十分であることを証明（Fig4, 5）

●PE(Gata6+)/EPI(Nanog+)の細胞が"pepper-salt pattern"でICM内に散在していることから、stochasticなメカニズムでヘテロな細胞集団が生じた後、どちらかのlineageにcommitすると考えられる（Discussion）←ここの決定機構は？？

●inner cell-outer cellのcontactについて、cell表面のcortical tensionの寄与？（Fig1D）(Ref.)

●FGFR/MEK inhibitorがtrophoectodermの細胞運命に影響を与えていない。
ICM内のFGF4の発現はOct4/Sox2による（Ref.1&2）

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過去の報告のまとめ

●ICM内の細胞でのtranscriptomeの揺らぎ、position independent？

●NanogとGata6の発現は、mutually exclusive

●FGF4、FGFR2、Grb2、MAPKなどのシグナルの寄与、過去文献（Hamazaki et al. MCB 2006など、次の記事でまとめ）→Ffg4, Fgfr2, Grb2KOマウスはPEの形成が完全に失われてEPIになる、DN-FGFRをESに入れるとPE形成能が失われる、一方でexogenous FGFはPE形成をenhanceしない（今回の論文と結論が異なるので条件が違う？）、PTPase inhibitor(vanadate)やCA-MAPKはPE形成を促進する、など

●8細胞期のpolarizationと8-16細胞期のsymmetric-asymmetric division (Ref.)

●平均のouter:inner cellの割合は10.8:5.2

Li et al. PNAS 107:1402-1407, 2010

 Stk40 links the pluripotency factor Oct4 to the Erk/MAPK pathway and controls extraembryonic endoderm differentiation

Self-renewal and differentiation of embryonic stem cells (ESCs) are controlled by intracellular transcriptional factors and extracellular factor-activated signaling pathways. Transcription factor Oct4 is a key player maintaining ESCs in an undifferentiated state, whereas the Erk/MAPK pathway is known to be important for ESC differentiation. However, the manner in which intracellular pluripotency factors modulate extracellular factor-activated signaling pathways in ESCs is not well understood. Here, we report identification of a target gene of Oct4, serine/threonine kinase 40 (Stk40), which is able to activate the Erk/MAPK pathway and induce extraembryonic--endoderm (ExEn) differentiation in mouse ESCs. Interestingly, cells overexpressing Stk40 exclusively contribute to the ExEn layer of chimeric embryos when injected into host blastocysts. In contrast, deletion of Stk40 in ESCs markedly reduces ExEn differentiation in vitro. Mechanistically, Stk40 interacts with Rcn2, which also activates Erk1/2 to induce ExEn specification in mouse ESCs. Moreover, Rcn2 proteins are specifically located in the cytoplasm of the ExEn layer of early mouse embryos. Importantly, knockdown of Rcn2 blocks Stk40-activated Erk1/2 and ESC differentiation. Therefore, our study establishes a link between the pluripotency factor Oct4 and the Erk/MAPK signaling pathway, and it uncovers cooperating signals in the Erk/MAPK activation that control ExEn differentiation.

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 　Oct4 --| Skt40 (kinase)-Rcn2　→　Ras/MAPK　→　ExEn
                                                          （機序不明）

一応Skt40はkinaseのようだが。

Oct4によって制御される遺伝子群

Molecular Systems Biology 6 Article number: 354  doi:10.1038/msb.2010.9
Published online: 9 March 2010

Zebrafish Pou5f1-dependent transcriptional networks in temporal control of early development

Pou5f1/Oct4の下流遺伝子を、ゼブラフィッシュ胚を用いて解析。

●マウスなど他種のターゲットとかぶっていて、マウスOct4はゼブラPou5f1mutantをレスキューできる。

●Pou5f1/Oct4の転写制御に関する数学的モデルを提唱。

Early Embryogenesisの障害を引き起こす薬剤

Early embryonic losses in mice induced by diethylstilbestrol

Congenital Anomalies　49:269-273, 2010

Estrogens cause embryonic lethality and the disturbance of early placental development in mice. Diethylstilbestrol (DES) at 1, 10, or 100 ?g/kg was orally administered to Institute of Cancer Research mice on gestational days (GD) 4 through 8, and the uterus and placenta were examined histopathologically on GD 9. Decidua of DES-treated mice showed insufficient development, and the uterine lumen at the implantation site did not effectively minimize. The trophoblast giant cell layer was not separated from the uterine lumen by the decidua capsularis, and hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted at the peripheral part of the decidua basalis. The results of the present study suggest that decidual hypoplasia and subsequent placental hemorrhage causes fetal death due to the administration of DES during the early stage of pregnancy.