Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Laboratory of Biochemistry and Molecular Biology of Germ Cells Institute of Animal Physiology and Genetics Czech Academy of Sciences Liběchov Czechia

Laboratory of Early Mammalian Developmental Biology Department of Molecular Biology and Genetics Faculty of Science University of South Bohemia České Budějovice Czechia

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Asteriti I. A., De Mattia F., Guarguaglini G. (2015). Cross-talk between AURKA and Plk1 in mitotic entry and spindle assembly. Front. Oncol. 5:283. PubMed PMC

Baumgartner R., Poernbacher I., Buser N., Hafen E., Stocker H. (2010). The WW domain protein Kibra acts upstream of Hippo in Drosophila. Dev. Cell 18 309–316. 10.1016/j.devcel.2009.12.013 PubMed DOI

Bennabi I., Terret M. E., Verlhac M. H. (2016). Meiotic spindle assembly and chromosome segregation in oocytes. J. Cell Biol. 215 611–619. 10.1083/jcb.201607062 PubMed DOI PMC

Bettencourt-Dias M., Rodrigues-Martins A., Carpenter L., Riparbelli M., Lehmann L., Gatt M. K., et al. (2005). SAK/PLK4 is required for centriole duplication and flagella development. Curr. Biol. 15 2199–2207. 10.1016/j.cub.2005.11.042 PubMed DOI

Bolton H., Graham S. J. L., Van Der Aa N., Kumar P., Theunis K., Fernandez Gallardo E., et al. (2016). Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential. Nat. Commun. 7:11165. PubMed PMC

Bury L., Coelho P. A., Simeone A., Ferries S., Eyers C. E., Eyers P. A., et al. (2017). Plk4 and Aurora A cooperate in the initiation of acentriolar spindle assembly in mammalian oocytes. J. Cell Biol. 216 3571–3590. 10.1083/jcb.201606077 PubMed DOI PMC

Chazaud C., Yamanaka Y. (2016). Lineage specification in the mouse preimplantation embryo. Development 143 1063–1074. 10.1242/dev.128314 PubMed DOI

Coelho P. A., Bury L., Sharif B., Riparbelli M. G., Fu J., Callaini G., et al. (2013). Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles. Dev. Cell 27 586–597. 10.1016/j.devcel.2013.09.029 PubMed DOI PMC

Courtois A., Schuh M., Ellenberg J., Hiiragi T. (2012). The transition from meiotic to mitotic spindle assembly is gradual during early mammalian development. J. Cell Biol. 198 357–370. 10.1083/jcb.201202135 PubMed DOI PMC

Davis J. R., Tapon N. (2019). Hippo signalling during development. Development 146:dev167106. 10.1242/dev.167106 PubMed DOI PMC

Frum T., Murphy T. M., Ralston A. (2018). HIPPO signaling resolves embryonic cell fate conflicts during establishment of pluripotency in vivo. Elife 7:e42298. PubMed PMC

Frum T., Ralston A. (2015). Cell signaling and transcription factors regulating cell fate during formation of the mouse blastocyst. Trends Genet. 31 402–410. 10.1016/j.tig.2015.04.002 PubMed DOI PMC

Frum T., Watts J. L., Ralston A. (2019). TEAD4, YAP1 and WWTR1 prevent the premature onset of pluripotency prior to the 16-cell stage. Development 146. PubMed PMC

Genevet A., Wehr M. C., Brain R., Thompson B. J., Tapon N. (2010). Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev. Cell 18 300–308. 10.1016/j.devcel.2009.12.011 PubMed DOI PMC

Gruss O. J. (2018). Animal female meiosis: the challenges of eliminating centrosomes. Cells 7:73. 10.3390/cells7070073 PubMed DOI PMC

Habedanck R., Stierhof Y. D., Wilkinson C. J., Nigg E. A. (2005). The Polo kinase Plk4 functions in centriole duplication. Nat. Cell Biol. 7 1140–1146. 10.1038/ncb1320 PubMed DOI

Han Q., Kremerskothen J., Lin X., Zhang X., Rong X., Zhang D., et al. (2018). WWC3 inhibits epithelial-mesenchymal transition of lung cancer by activating Hippo-YAP signaling. Onco Targets Ther. 11 2581–2591. 10.2147/ott.s162387 PubMed DOI PMC

Hashimoto M., Sasaki H. (2019). Epiblast formation by TEAD-YAP-dependent expression of pluripotency factors and competitive elimination of unspecified cells. Dev. Cell 50:e135. PubMed

Hassold T., Hunt P. (2001). To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2 280–291. 10.1038/35066065 PubMed DOI

Hirate Y., Hirahara S., Inoue K., Kiyonari H., Niwa H., Sasaki H. (2015). Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos. Dev. Growth Differ. 57 544–556. 10.1111/dgd.12235 PubMed DOI

Horn T., Boutros M. (2010). E-RNAi: a web application for the multi-species design of RNAi reagents–2010 update. Nucleic Acids Res. 38 W332–W339. PubMed PMC

Johnson M. H., Ziomek C. A. (1981). The foundation of two distinct cell lineages within the mouse morula. Cell 24 71–80. 10.1016/0092-8674(81)90502-x PubMed DOI

Koncicka M., Tetkova A., Jansova D., Del Llano E., Gahurova L., Kracmarova J., et al. (2018). Increased expression of maturation promoting factor components speeds up meiosis in oocytes from aged females. Int. J. Mol. Sci. 19:2841. 10.3390/ijms19092841 PubMed DOI PMC

Kovarikova V., Burkus J., Rehak P., Brzakova A., Solc P., Baran V. (2016). Aurora kinase A is essential for correct chromosome segregation in mouse zygote. Zygote 24 326–337. 10.1017/s0967199415000222 PubMed DOI

Lemaire P., Garrett N., Gurdon J. B. (1995). Expression cloning of Siamois, a xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81 85–94. 10.1016/0092-8674(95)90373-9 PubMed DOI

Li W., Wang P., Zhang B., Zhang J., Ming J., Xie W., et al. (2017). Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos. Protein Cell 8 662–674. 10.1007/s13238-017-0407-5 PubMed DOI PMC

Livak K. J., Schmittgen T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402–408. 10.1006/meth.2001.1262 PubMed DOI

Lu L. Y., Wood J. L., Minter-Dykhouse K., Ye L., Saunders T. L., Yu X., et al. (2008). Polo-like kinase 1 is essential for early embryonic development and tumor suppression. Mol. Cell Biol. 28 6870–6876. 10.1128/mcb.00392-08 PubMed DOI PMC

Makuch L., Volk L., Anggono V., Johnson R. C., Yu Y., Duning K., et al. (2011). Regulation of AMPA receptor function by the human memory-associated gene KIBRA. Neuron 71 1022–1029. 10.1016/j.neuron.2011.08.017 PubMed DOI PMC

Masek T., Del Llano E., Gahurova L., Kubelka M., Susor A., Roucova K., et al. (2020). Indentifying the translatome of mouse NEBD-stage oocytes via SSP-profiling; A novel polysome fractionation method. Int. J. Mol. Sci. 21:1254. 10.3390/ijms21041254 PubMed DOI PMC

Mihajlovic A. I., Bruce A. W. (2016). Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development. Reprod. Biomed. Online 33 381–390. 10.1016/j.rbmo.2016.06.028 PubMed DOI

Mihajlovic A. I., Bruce A. W. (2017). The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity. Open Biol. 7:170210. 10.1098/rsob.170210 PubMed DOI PMC

Mihajlovic A. I., Fitzharris G. (2018). Segregating chromosomes in the mammalian oocyte. Curr. Biol. 28 R895–R907. PubMed

Mihajlovic A. I., Thamodaran V., Bruce A. W. (2015). The first two cell-fate decisions of preimplantation mouse embryo development are not functionally independent. Sci. Rep. 5:15034. PubMed PMC

Mogessie B., Scheffler K., Schuh M. (2018). Assembly and positioning of the oocyte meiotic spindle. Annu. Rev. Cell Dev. Biol. 34 381–403. 10.1146/annurev-cellbio-100616-060553 PubMed DOI

Morris S. A., Teo R. T., Li H., Robson P., Glover D. M., Zernicka-Goetz M. (2010). Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo. Proc. Natl. Acad. Sci. U.S.A. 107 6364–6369. 10.1073/pnas.0915063107 PubMed DOI PMC

Nagaoka S. I., Hassold T. J., Hunt P. A. (2012). Human aneuploidy: mechanisms and new insights into an age-old problem. Nat. Rev. Genet. 13 493–504. 10.1038/nrg3245 PubMed DOI PMC

Namgoong S., Kim N. H. (2018). Meiotic spindle formation in mammalian oocytes: implications for human infertility. Biol. Reprod. 98 153–161. 10.1093/biolre/iox145 PubMed DOI

Nguyen A. L., Drutovic D., Vazquez B. N., El Yakoubi W., Gentilello A. S., Malumbres M., et al. (2018). Genetic interactions between the aurora kinases reveal new requirements for AURKB and AURKC during oocyte meiosis. Curr. Biol. 28 3458.e–3468.e. PubMed PMC

Nguyen A. L., Schindler K. (2017). Specialize and divide (Twice): Functions of three aurora kinase homologs in mammalian oocyte meiotic maturation. Trends Genet. 33 349–363. 10.1016/j.tig.2017.03.005 PubMed DOI PMC

Nishioka N., Inoue K., Adachi K., Kiyonari H., Ota M., Ralston A., et al. (2009). The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev. Cell 16 398–410. 10.1016/j.devcel.2009.02.003 PubMed DOI

O’farrell P. H., Stumpff J., Su T. T. (2004). Embryonic cleavage cycles: how is a mouse like a fly? Curr. Biol. 14 R35–R45. PubMed PMC

Rossant J. (2016). Making the mouse blastocyst: past, present, and future. Curr. Top. Dev. Biol. 117 275–288. PubMed

Rossant J. (2018). Genetic control of early cell lineages in the mammalian embryo. Annu. Rev. Genet. 52 185–201. 10.1146/annurev-genet-120116-024544 PubMed DOI

Salles F. J., Darrow A. L., O’connell M. L., Strickland S. (1992). Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation. Genes Dev. 6 1202–1212. 10.1101/gad.6.7.1202 PubMed DOI

Sanders J. R., Jones K. T. (2018). Regulation of the meiotic divisions of mammalian oocytes and eggs. Biochem. Soc. Trans. 46 797–806. 10.1042/bst20170493 PubMed DOI PMC

Sasaki H. (2017). Roles and regulations of Hippo signaling during preimplantation mouse development. Dev. Growth Differ. 59 12–20. 10.1111/dgd.12335 PubMed DOI

Saskova A., Solc P., Baran V., Kubelka M., Schultz R. M., Motlik J. (2008). Aurora kinase A controls meiosis I progression in mouse oocytes. Cell Cycle 7 2368–2376. 10.4161/cc.6361 PubMed DOI PMC

Sathananthan A. H., Kola I., Osborne J., Trounson A., Ng S. C., Bongso A., et al. (1991). Centrioles in the beginning of human development. Proc. Natl. Acad. Sci. U.S.A. 88 4806–4810. 10.1073/pnas.88.11.4806 PubMed DOI PMC

Schatten H., Sun Q. Y. (2009). The role of centrosomes in mammalian fertilization and its significance for ICSI. Mol. Hum. Reprod. 15 531–538. 10.1093/molehr/gap049 PubMed DOI PMC

Severson A. F., Von Dassow G., Bowerman B. (2016). Oocyte meiotic spindle assembly and function. Curr. Top Dev. Biol. 116 65–98. 10.1016/bs.ctdb.2015.11.031 PubMed DOI PMC

Solc P., Baran V., Mayer A., Bohmova T., Panenkova-Havlova G., Saskova A., et al. (2012). Aurora kinase A drives MTOC biogenesis but does not trigger resumption of meiosis in mouse oocytes matured in vivo. Biol. Reprod. 87:85. PubMed PMC

Swain J. E., Ding J., Wu J., Smith G. D. (2008). Regulation of spindle and chromatin dynamics during early and late stages of oocyte maturation by aurora kinases. Mol. Hum. Reprod. 14 291–299. 10.1093/molehr/gan015 PubMed DOI PMC

Terada Y., Tatsuka M., Suzuki F., Yasuda Y., Fujita S., Otsu M. (1998). AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J. 17 667–676. 10.1093/emboj/17.3.667 PubMed DOI PMC

Virnicchi G., Bora P., Gahurova L., Susor A., Bruce A. W. (2020). Wwc2 is a novel cell division regulator during preimplantation mouse embryo lineage formation and oogenesis. bioRxiv 872366 10.1101/2019.12.12.872366 PubMed DOI PMC

Wang Q. T., Piotrowska K., Ciemerych M. A., Milenkovic L., Scott M. P., Davis R. W., et al. (2004). A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev. Cell 6 133–144. 10.1016/s1534-5807(03)00404-0 PubMed DOI

Wennmann D. O., Schmitz J., Wehr M. C., Krahn M. P., Koschmal N., Gromnitza S., et al. (2014). Evolutionary and molecular facts link the WWC protein family to Hippo signaling. Mol. Biol. Evol. 31 1710–1723. 10.1093/molbev/msu115 PubMed DOI

White M. D., Zenker J., Bissiere S., Plachta N. (2018). Instructions for assembling the early mammalian embryo. Dev. Cell 45 667–679. 10.1016/j.devcel.2018.05.013 PubMed DOI

Wicklow E., Blij S., Frum T., Hirate Y., Lang R. A., Sasaki H., et al. (2014). HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst. PLoS Genet. 10:e1004618. 10.1371/journal.pgen.1004618 PubMed DOI PMC

Xiao L., Chen Y., Ji M., Dong J. (2011a). KIBRA regulates Hippo signaling activity via interactions with large tumor suppressor kinases. J. Biol. Chem. 286 7788–7796. 10.1074/jbc.m110.173468 PubMed DOI PMC

Xiao L., Chen Y., Ji M., Volle D. J., Lewis R. E., Tsai M. Y., et al. (2011b). KIBRA protein phosphorylation is regulated by mitotic kinase aurora and protein phosphatase 1. J. Biol. Chem. 286 36304–36315. 10.1074/jbc.m111.246850 PubMed DOI PMC

Yu J., Zheng Y., Dong J., Klusza S., Deng W. M., Pan D. (2010). Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and Expanded. Dev. Cell 18 288–299. 10.1016/j.devcel.2009.12.012 PubMed DOI PMC

Zenker J., White M. D., Templin R. M., Parton R. G., Thorn-Seshold O., Bissiere S., et al. (2017). A microtubule-organizing center directing intracellular transport in the early mouse embryo. Science 357 925–928. 10.1126/science.aam9335 PubMed DOI

Zernicka-Goetz M., Pines J., Ryan K., Siemering K. R., Haseloff J., Evans M. J., et al. (1996). An indelible lineage marker for Xenopus using a mutated green fluorescent protein. Development 122 3719–3724. PubMed

Zhang L., Iyer J., Chowdhury A., Ji M., Xiao L., Yang S., et al. (2012). KIBRA regulates aurora kinase activity and is required for precise chromosome alignment during mitosis. J. Biol. Chem. 287 34069–34077. 10.1074/jbc.m112.385518 PubMed DOI PMC

Zhang Y., Yan S., Chen J., Gan C., Chen D., Li Y., et al. (2017). WWC2 is an independent prognostic factor and prevents invasion via Hippo signalling in hepatocellular carcinoma. J. Cell Mol. Med. 21 3718–3729. 10.1111/jcmm.13281 PubMed DOI PMC

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis