The transcriptional effector of the Hippo signalling pathway, YAP, regulates the first lineage specification in mouse preimplantation embryos. However, how YAP undergoes dephosphorylation specifically in the trophectoderm (TE) but not in the inner cell mass (ICM) remains unresolved. Here, we discovered that the serine/threonine phosphatase PPP1CC exhibits uniform distribution prior to blastocyst formation but becomes specifically localised to the TE during the blastocyst stage. Through mediating YAP dephosphorylation in the outer cells of mouse morula, PPP1CC facilitates YAP nuclear translocation, thereby ultimately driving TE lineage specification. Importantly, the spatially restricted localisation of PPP1CC in TE is achieved via its interaction with the long non-coding RNA GAS5, which localises to the subcortical region throughout early mouse embryonic development. Knockdown of GAS5 phenocopies PPP1CC deficiency, causing developmental arrest at the morula stage accompanied by impaired YAP dephosphorylation in outer cells. Moreover, overexpression of GAS5 in one blastomere of the 2-cell stage biases its descendants predominantly towards the TE fate. In summary, our study identifies the GAS5-PPP1CC-YAP axis as a central regulator of first lineage specification during mouse preimplantation development, highlighting its critical role in reversible phosphorylation during early embryogenesis.
| [1] |
K. F. Harvey, C. M. Pfleger, and I. K. Hariharan, “The Drosophila Mst Ortholog, Hippo, Restricts Growth and Cell Proliferation and Promotes Apoptosis,” Cell 114 (2003): 457–467.
|
| [2] |
R. S. Udan, M. Kango-Singh, R. Nolo, C. Tao, and G. Halder, “Hippo Promotes Proliferation Arrest and Apoptosis in the Salvador/Warts Pathway,” Nature Cell Biology 5 (2003): 914–920.
|
| [3] |
K. F. Harvey, X. Zhang, and D. M. Thomas, “The Hippo Pathway and Human Cancer,” Nature Reviews Cancer 13 (2013): 246–257.
|
| [4] |
F. X. Yu and K. L. Guan, “The Hippo Pathway: Regulators and Regulations,” Genes & Development 27 (2013): 355–371.
|
| [5] |
S. Ma, Z. Meng, R. Chen, and K. L. Guan, “The Hippo Pathway: Biology and Pathophysiology,” Annual Review of Biochemistry 88 (2019): 577–604.
|
| [6] |
D. Pan, “The Hippo Signaling Pathway in Development and Cancer,” Developmental Cell 19 (2010): 491–505.
|
| [7] |
F. X. Yu, B. Zhao, and K. L. Guan, “Hippo Pathway in Organ Size Control, Tissue Homeostasis, and Cancer,” Cell 163 (2015): 811–828.
|
| [8] |
X. Sun, Z. Ren, Y. Cun, et al., “Hippo-Yap Signaling Controls Lineage Differentiation of Mouse Embryonic Stem Cells Through Modulating the Formation of Super-Enhancers,” Nucleic Acids Research 48 (2020): 7182–7196.
|
| [9] |
E. H. Chan, M. Nousiainen, R. B. Chalamalasetty, et al., “The ste20-Like Kinase mst2 Activates the Human Large Tumor Suppressor Kinase lats1,” Oncogene 24 (2005): 2076–2086.
|
| [10] |
Z. Meng, T. Moroishi, V. Mottier-Pavie, et al., “Map4k Family Kinases Act in Parallel to mst1/2 to Activate lats1/2 in the Hippo Pathway,” Nature Communications 6 (2015): 8357.
|
| [11] |
J. S. Mo, H. W. Park, and K. L. Guan, “The Hippo Signaling Pathway in Stem Cell Biology and Cancer,” EMBO Reports 15 (2014): 642–656.
|
| [12] |
E. Yildirim, G. Bora, T. Onel, N. Talas, and A. Yaba, “Cell Fate Determination and Hippo Signaling Pathway in Preimplantation Mouse Embryo,” Cell and Tissue Research 386 (2021): 423–444.
|
| [13] |
T. Frum and A. Ralston, “Cell Signaling and Transcription Factors Regulating Cell Fate During Formation of the Mouse Blastocyst,” Trends in Genetics 31 (2015): 402–410.
|
| [14] |
R. Yagi, M. J. Kohn, I. Karavanova, et al., “Transcription Factor tead4 Specifies the Trophectoderm Lineage at the Beginning of Mammalian Development,” Development (Cambridge, England) 134 (2007): 3827–3836.
|
| [15] |
A. Ralston, B. J. Cox, N. Nishioka, et al., “Gata3 Regulates Trophoblast Development Downstream of tead4 and in Parallel to cdx2,” Development (Cambridge, England) 137 (2010): 395–403.
|
| [16] |
N. Nishioka, K. Inoue, K. Adachi, et al., “The Hippo Signaling Pathway Components Lats and Yap Pattern tead4 Activity to Distinguish Mouse Trophectoderm From Inner Cell Mass,” Developmental Cell 16 (2009): 398–410.
|
| [17] |
M. Zhu, M. Meglicki, A. Lamba, et al., “Tead4 and tfap2c Generate Bipotency and a Bistable Switch in Totipotent Embryos to Promote Robust Lineage Diversification,” Nature Structural & Molecular Biology 31 (2024): 964–976.
|
| [18] |
R. Zhou, Q. Wu, M. Wang, et al., “The Protein Phosphatase ppm1a Dephosphorylates and Activates Yap to Govern Mammalian Intestinal and Liver Regeneration,” PLoS Biology 19 (2021): e3001122.
|
| [19] |
Q. Deng, D. Ramsköld, B. Reinius, and R. Sandberg, “Single-Cell Rna-Seq Reveals Dynamic, Random Monoallelic Gene Expression in Mammalian Cells,” Science (New York, N.Y.) 343 (2014): 193–196.
|
| [20] |
B. Zhao, X. Wei, W. Li, et al., “Inactivation of Yap Oncoprotein by the Hippo Pathway Is Involved in Cell Contact Inhibition and Tissue Growth Control,” Genes & Development 21 (2007): 2747–2761.
|
| [21] |
M. DeRan, J. Yang, C. H. Shen, et al., “Energy Stress Regulates Hippo-Yap Signaling Involving Ampk-Mediated Regulation of Angiomotin-Like 1 Protein,” Cell Reports 9 (2014): 495–503.
|
| [22] |
J. S. Mo, Z. Meng, Y. C. Kim, et al., “Cellular Energy Stress Induces Ampk-Mediated Regulation of Yap and the Hippo Pathway,” Nature Cell Biology 17 (2015): 500–510.
|
| [23] |
W. Wang, Z. D. Xiao, X. Li, et al., “Ampk Modulates Hippo Pathway Activity to Regulate Energy Homeostasis,” Nature Cell Biology 17 (2015): 490–499.
|
| [24] |
B. Zhao, X. Ye, J. Yu, et al., “Tead Mediates Yap-Dependent Gene Induction and Growth Control,” Genes & Development 22 (2008): 1962–1971.
|
| [25] |
A. Vassilev, K. J. Kaneko, H. Shu, Y. Zhao, and M. L. DePamphilis, “Tead/Tef Transcription Factors Utilize the Activation Domain of yap65, a Src/Yes-Associated Protein Localized in the Cytoplasm,” Genes & Development 15 (2001): 1229–1241.
|
| [26] |
H. Zhang, H. A. Pasolli, and E. Fuchs, “Yes-Associated Protein (Yap) Transcriptional Coactivator Functions in Balancing Growth and Differentiation in Skin,” Proceedings of the National Academy of Sciences of the United States of America 108 (2011): 2270–2275.
|
| [27] |
H. Ceulemans and M. Bollen, “Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button,” Physiological Reviews 84 (2004): 1–39.
|
| [28] |
R. Chakrabarti, D. Kline, J. Lu, J. Orth, S. Pilder, and S. Vijayaraghavan, “Analysis of ppp1cc-Null Mice Suggests a Role for pp1gamma2 in Sperm Morphogenesis,” Biology of Reproduction 76 (2007): 992–1001.
|
| [29] |
A. F. Ferreira, J. Santiago, J. V. Silva, P. F. Oliveira, and M. Fardilha, “Pp1, pp2a and pp2b Interplay in the Regulation of Sperm Motility: Lessons From Protein Phosphatase Inhibitors,” International Journal of Molecular Sciences 23 (2022): 23.
|
| [30] |
T. Hirose, G. Virnicchi, A. Tanigawa, et al., “Neat1 Long Noncoding Rna Regulates Transcription via Protein Sequestration Within Subnuclear Bodies,” Molecular Biology of the Cell 25 (2014): 169–183.
|
| [31] |
J. Wang, L. Wang, G. Feng, et al., “Asymmetric Expression of Lincget Biases Cell Fate in Two-Cell Mouse Embryos,” Cell 175 (2018): 1887–1901.e18.
|
| [32] |
X. Wei, F. Liu, X. Jiang, X. Xu, T. Zhou, and C. Kang, “Yy1 Promotes Telomerase Activity and Laryngeal Squamous Cell Carcinoma Progression Through Impairment of gas5-Mediated p53 Stability,” Frontiers in Oncology 11 (2021): 692405.
|
| [33] |
W. Yi, J. Li, X. Zhu, et al., “Crispr-Assisted Detection of Rna-Protein Interactions in Living Cells,” Nature Methods 17 (2020): 685–688.
|
| [34] |
P. S. Ribeiro, F. Josué, A. Wepf, et al., “Combined Functional Genomic and Proteomic Approaches Identify a pp2a Complex as a Negative Regulator of Hippo Signaling,” Molecular Cell 39 (2010): 521–534.
|
| [35] |
Y. Zheng, B. Liu, L. Wang, H. Lei, K. D. Pulgar Prieto, and D. Pan, “Homeostatic Control of Hpo/Mst Kinase Activity Through Autophosphorylation-Dependent Recruitment of the Stripak pp2a Phosphatase Complex,” Cell Reports 21 (2017): 3612–3623.
|
| [36] |
R. Chen, R. Xie, Z. Meng, S. Ma, and K. L. Guan, “Stripak Integrates Upstream Signals to Initiate the Hippo Kinase Cascade,” Nature Cell Biology 21 (2019): 1565–1577.
|
| [37] |
C. Y. Liu, X. Lv, T. Li, et al., “Pp1 Cooperates With aspp2 to Dephosphorylate and Activate Taz,” Journal of Biological Chemistry 286 (2011): 5558–5566.
|
| [38] |
X. B. Lv, C. Y. Liu, Z. Wang, et al., “Pard3 Induces Taz Activation and Cell Growth by Promoting lats1 and pp1 Interaction,” EMBO Reports 16 (2015): 975–985.
|
| [39] |
W. Wang, J. Huang, X. Wang, et al., “Ptpn14 Is Required for the Density-Dependent Control of yap1,” Genes & Development 26 (2012): 1959–1971.
|
| [40] |
Y. Q. Su, K. Sugiura, F. Sun, et al., “Marf1 Regulates Essential Oogenic Processes in Mice,” Science (New York, N.Y.) 335 (2012): 1496–1499.
|
| [41] |
P. Gu, X. Qi, Y. Zhou, Y. Wang, and X. Gao, “Generation of ppp2ca and ppp2cb Conditional Null Alleles in Mouse,” Genesis 50 (2012): 429–436.
|
| [42] |
D. Bebbere, D. F. Albertini, G. Coticchio, A. Borini, and S. Ledda, “The Subcortical Maternal Complex: Emerging Roles and Novel Perspectives,” Molecular Human Reproduction 27 (2021): 27.
|
| [43] |
L. Li, B. Baibakov, and J. Dean, “A Subcortical Maternal Complex Essential for Preimplantation Mouse Embryogenesis,” Developmental Cell 15 (2008): 416–425.
|
| [44] |
X. J. Yu, Z. Yi, Z. Gao, et al., “The Subcortical Maternal Complex Controls Symmetric Division of Mouse Zygotes by Regulating f-Actin Dynamics,” Nature Communications 5 (2014): 4887.
|
| [45] |
Z. Gao, X. Zhang, X. Yu, et al., “Zbed3 Participates in the Subcortical Maternal Complex and Regulates the Distribution of Organelles,” Journal of Molecular Cell Biology 10 (2018): 74–88.
|
| [46] |
C. Xu, D. Qin, X. Lu, et al., “The Subcortical Maternal Complex Safeguards Mouse Oocyte-To-Embryo Transition by Preventing Nuclear Entry of spin1,” Nature Structural & Molecular Biology 32 (2025): 1358–1372.
|
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2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.
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