Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 51 (11), 135

Loss of Mob1a/b Impairs the Differentiation of Mouse Embryonic Stem Cells Into the Three Germ Layer Lineages

Affiliations

Loss of Mob1a/b Impairs the Differentiation of Mouse Embryonic Stem Cells Into the Three Germ Layer Lineages

June Sung Bae et al. Exp Mol Med.

Abstract

The Hippo pathway plays a crucial role in cell proliferation and apoptosis and can regulate stem cell maintenance and embryonic development. MOB kinase activators 1A and 1B (Mob1a/b) are key components of the Hippo pathway, whose homozygous deletion in mice causes early embryonic lethality at the preimplantation stage. To investigate the role of Mob1a/b in stem cell maintenance and differentiation, an embryonic stem cell (ESC) clone in which Mob1a/b could be conditionally depleted was generated and characterized. Although Mob1a/b depletion did not affect the stemness or proliferation of mouse ESCs, this depletion caused defects in differentiation into the three germ layers. Yap knockdown rescued the in vitro and in vivo defects in differentiation caused by Mob1a/b depletion, suggesting that differentiation defects caused by Mob1a/b depletion were Yap-dependent. In teratoma experiments, Yap knockdown in Mob1a/b-depleted ESCs partially restored defects in differentiation, indicating that hyperactivation of Taz, another effector of the Hippo pathway, inhibited differentiation into the three germ layers. Taken together, these results suggest that Mob1a/b or Hippo signaling plays a critical role in the differentiation of mouse ESCs into the three germ layers, which is dependent on Yap. These close relationship of the Hippo pathway with the differentiation of stem cells supports its potential as a therapeutic target in regenerative medicine.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Mob1a/b has little effect on the maintenance of stemness and proliferation in mouse ESCs.
a Immunoblot analysis for Mob1a/b and p-Yap (S112) in lysates from Mob1af/−:Mob1bf/f:CAGGCre-ERTM mouse ESCs harvested at the indicated times after 4-OHT treatment. Gapdh served as a loading control. b Immunoblot analysis for Mob1a/b, Oct4, Nanog, and Yap in lysates from Mob1af/−:Mob1bf/f:CAGGCre-ERTM mESCs harvested at the indicated times after 4-OHT treatment. GAPDH served as a loading control. c Semiquantitative PCR for Mob1a, pluripotency markers (Oct4, Sox2, and Nanog), and differentiation markers (Gata6, Gata4, Sox17, Eomes, T-brachyury, Nestin, and Fgf5) in Mob1af/−:Mob1bf/f:CAGGCre-ERTM ESCs. d Representative images of alkaline phosphatase staining of Mob1af/−:Mob1bf/f:CAGGCre-ERTM ESCs on the seventh day after 4-OHT treatment. Scale bars, 200 μm. e Cell cycle analysis of Mob1af/−:Mob1bf/f:CAGGCre-ERTM mouse ESCs on the seventh day after 4-OHT treatment
Fig. 2
Fig. 2. Mob1a/b is essential for the differentiation of mouse ESCs.
a Schematic diagram of EB formation and representative images of EB outgrowth on tissue culture dishes on days 7 and 8 after EB formation. Dotted lines indicate EB outgrowth. Scale bar, 400 μm b, c Quantitative PCR for Mob1a/b and pluripotency markers (Oct4, Sox2, and Nanog) in mESCs and EBs. ES (−), ESCs without 4-OHT treatment; ES (+), 4-OHT-treated ESCs; EB (−), EB without 4-OHT treatment; EB (+), 4-OHT-treated EB. Data are presented as the mean ± SEM. ***P < 0.001
Fig. 3
Fig. 3. Mob1a/b knockout causes a defect in the differentiation of mouse ESCs.
a Quantitative PCR for germ layer markers (endoderm: Gata6, Gata4, Sox17; mesoderm: T-brachyury, MixL1, Mesp1, Hand1; ectoderm: Nestin; trophectoderm: Cdx2, Eomes) in mouse ESCs and EBs. bd Quantitative PCR for germ layer markers at different stages of EB formation. (b, endoderm; c, mesoderm; d, ectoderm). ES, ESCs before EB formation; EB D2, EB after 2-day culture (hanging drop stage); EB D4, EB after 4-day culture (in bacteriological-grade Petri dish); EB D6, EB after 6-day culture (in cell and tissue culture dish). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4. The function of Mob1a/b in the differentiation of mouse ESCs is dependent on Yap activity.
a Immunoblot analysis for Yap, Mob1a/b, and Oct4 in lysates from Mob1af/f:Mob1bf/f:CAGGCre-ERTM ESCs after 4-OHT and shYap treatment. GAPDH served as a loading control. b Representative images of EB outgrowth on tissue culture dishes eight days after EB formation. Dotted lines indicate EB outgrowth. Scale bar, 100 μm c, d Quantitative PCR for Mob1a/b, Yap, and pluripotency markers (Oct4, Sox2, and Nanog) on day 6 after EB formation. e Quantitative PCR for germ layer markers (endoderm, Gata6; mesoderm, T-brachyury; ectoderm, Otx2) at the different stages of EB formation. (−) shCon, shControl ESCs without 4-OHT treatment; (+) shCon, 4-OHT-treated shControl ESCs; (−) shYap ESCs without 4-OHT treatment; (+) shYap, 4-OHT-treated shYap ESCs. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5. Mob1a/b is required for the differentiation of mouse ESCs in vivo.
a, b Representative images of hematoxylin and eosin staining images of a teratoma originating from Mob1af/f:Mob1bf/f:CAGGCre-ERTM ESCs. Teratomas were recovered by dissection with the surrounding tissue 6 weeks after mouse ESC injection. Scale bar, 200 μm. c Immunoblot analysis for Yap and Taz in lysates of Mob1af/f:Mob1bf/f:CAGGCre-ERTM ESCs after shYap treatment. Gapdh served as a loading control. d Representative images of immunohistochemical staining of a teratoma using an anti-Taz antibody. EC, ectodermal tissue; ME, mesodermal tissue; EN, endodermal tissue. Scale bar, 200 μm

Similar articles

See all similar articles

References

    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156. - PubMed
    1. Fatehullah A, Tan SH, Barker N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 2016;18:246–254. - PubMed
    1. Young RA. Control of the embryonic stem cell state. Cell. 2011;144:940–954. - PMC - PubMed
    1. Chambers I, Smith A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene. 2004;23:7150–7160. - PubMed
    1. Avilion AA, et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 2003;17:126–140. - PMC - PubMed
Feedback