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ASPP2/PP1 Complexes Maintain the Integrity of Pseudostratified Epithelia Undergoing Remodelling During Morphogenesis

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ASPP2/PP1 Complexes Maintain the Integrity of Pseudostratified Epithelia Undergoing Remodelling During Morphogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.366906; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ASPP2/PP1 complexes maintain the integrity of pseudostratified epithelia undergoing remodelling during morphogenesis Christophe Royer1*, Elizabeth Sandham1, Elizabeth Slee2, Jonathan Godwin1,3, Nisha Veits1, Holly Hathrell1, Felix Zhou2, Karolis Leonavicius1,4, Jemma Garratt1,6, Tanaya Narendra1,6, Anna Vincent5, Celine Jones6, Tim Child5,6, Kevin Coward6, Chris Graham6, Xin Lu2 and Shankar Srinivas1* 1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom. 2Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom. 3Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. 4Current address: Institute of Biotechnology, Vilnius University, Vilnius, Lithuania. 5Oxford Fertility, Institute of Reproductive Sciences, Oxford Business Park North, Oxford, OX4 2HW, United Kingdom. 6Nuffield Department of Women’s and Reproductive Health, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom. *e-mail: [email protected]; [email protected] ABSTRACT primitive streak as they push their cell body basally to During development, pseudostratified epithelia eventually delaminate into the underlying mesoderm 4–7 undergo large scale morphogenetic events cell layer or when the ectoderm is reshaped to form associated with increased mechanical stress. the head folds. The combined mechanical strains The molecular mechanisms that maintain tissue due to IKNM and morphogenetic events poses an integrity in this context are poorly understood. incredible challenge for pseudostratified epithelia to Using a variety of genetic and imaging approaches, maintain tissue integrity during development. However, we uncover that the ASPP2/PP1 complex ensures the molecular mechanisms that allow them to cope with proper epiblast and proamniotic cavity architecture increased mechanical stress are poorly defined. via a mechanism that specifically prevents the most During these morphogenetic events, epithelial cells apical daughter cells from delaminating apically continually rely on apical constrictions involving specific following cell division events. The ASPP2/PP1 F-actin cytoskeleton organisation and actomyosin 8,9 complex achieves this by maintaining the integrity contractility to modify tissue shape and organisation . and organisation of the F-actin cytoskeleton at the As cells apically constrict, the coupling of apical apical surface of dividing cells. ASPP2/PP1 is also junctions to the actomyosin network is essential in 10 essential during gastrulation in the primitive streak, transmitting forces across tissues . Reciprocally, in somites and in the head fold region, suggesting as apical constrictions reshape the apical domain that this complex is required across a wide range of epithelial cells, apical junctions must be able to of pseudostratified epithelia during morphogenetic withstand the forces generated to maintain tissue events that are accompanied by intense tissue integrity. Apical-basal polarity components, such as 11 remodelling and high cell proliferation. Finally, Par3, are vital for apical constrictions and the integrity 12 our study also suggests that the interaction of apical junctions . However, it remains unknown how between ASPP2 and PP1 is essential to the tumour components of the apical-basal polarity machinery suppressor function of ASPP2 which may be maintain tissue integrity in conditions of increased particularly relevant in the context of tissues that mechanical stress as morphogenetic events occur. are subject to increased mechanical stress. ASPP2 is a Par3 interactor and component of the apical junctions13,14. Here, using a variety of genetic and INTRODUCTION imaging approaches, we show that during morphogenetic Pseudostratified epithelia are common building events crucial for the normal development of the early blocks and organ precursors throughout embryonic post-implantation embryo, ASPP2 maintains epithelial development in a wide array of organisms1. As in other integrity in pseudostratified epithelia under increased epithelia, their cells establish and maintain apical- mechanical stress. ASPP2 is required for proamniotic basal polarity. However, their high nuclear density, cavity formation, the maintenance of primitive streak high proliferation rate and nuclei movement during architecture, somite structure and head fold formation. interkinetic nuclear migration (IKNM) make them In the proamniotic cavity, ASPP2 maintains epithelial unique. As IKNM proceeds, mitotic cells round up at architecture by preventing apical daughter cells from the apical surface of the epithelium before dividing. escaping the epiblast. Mechanistically, we show that During this process, mitotic cells generate enough this is achieved via the ability of ASPP2 to directly force to locally distort the shape of the epithelium2 recruit protein phosphatase 1 and through its essential or accelerate invagination3. During development, role in maintaining F-actin cytoskeleton organisation at pseudostratified epithelia are also subject to large the apical junctions. Our results show that ASPP2 is an scale morphogenetic events that dramatically affect essential component of a system that maintains tissue their shape and organisation. This is particularly true integrity under conditions of increased mechanical during gastrulation when cells apically constrict in the stress in a broad range of tissues. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.366906; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. a similar way across mammals (Figure S1B). RESULTS A Previous study revealed that ASPP2 may play a role during early embryogenesis, as ASPP2-mutant The phosphatase and polarity function of ASPP2 embryos in which exons 10-17 were deleted could not are not required for trophectoderm development be recovered at E6.523. However, the phenotype of ASPP2 can regulate both apical-basal cell polarity these embryos was not described, and earlier stages and the phosphorylation status of YAP/TAZ through its were not examined. To investigate the role of ASPP2 interaction with Par313,14 and PP115,16 respectively. Both during preimplantation development we generated cell polarity and the phosphorylation of YAP and TAZ ASPP2-null embryos in which exon 4 was deleted are crucial to trophectoderm (TE) development17–21. We (ASPP2ΔE4/ΔE4) resulting in a frameshift and early stop therefore started with the hypothesis that ASPP2 may codons. To be able to distinguish between phenotypes be important for outside cell polarisation and TE fate relating to ASPP2’s PP1 regulatory function and other determination during preimplantation development. functions, we also generated embryos homozygous for In support of this, we found that ASPP2 could start a mutant form of ASPP2 that was specifically unable to be detected as early as E2.5 at cell-cell junctions to associate with PP1 (ASPP2RAKA/RAKA)24. ASPP2ΔE4/ (Figure S1A). As seen in other examples of polarised ΔE4 and ASPP2RAKA/RAKA blastocysts appeared to be epithelia13,14,16,22, ASPP2 was strongly localised to the morphologically normal with properly formed blastocyst apical junction in the TE from the 32-cell stage onwards cavities (Figure 1B and S1C). YAP was clearly (Figure 1A). This localisation pattern was similar in nuclear in the TE and cytoplasmic in the inner cell human blastocysts, suggesting that ASPP2 behaves in mass (ICM) suggesting that the ICM and TE lineage ASPP2 F-actin ASPP2 F-actin AJ A ASPP2 DAPI F-actin DAPI ASPP2 DAPI DAPI DAPI B AJ B 1500 cross section 1000 E3.5 500 Signal Intensity (grey levels) (grey Intensity Signal 0 0 2000 4000 6000 Scale (nm) 3D opacity ASPP2 F-actin B YAP Par3 DAPI C ASPP2 DAPI YAP ASPP2 DAPI ASPP2 YAP wild type Control siRNA RAKA/RAKA ASPP2 siRNA ASPP2 cross section MIP cross section 3D opacity Figure 1: The ASPP2/PP1 complex is not required during preimplantation development. A. ASPP2 was detected by indirect immunofluorescence in E3.5 embryos at the early blastocyst stage to analyse its localisation pattern. A cross section through the equatorial plane of a representative embryo is shown (top row), as well as a 3D opacity rendering of the same embryo (bottom row). The F-actin cytoskeleton and nuclei were visualised using Phalloidin and DAPI respectively. A magnified image of the dashed area is shown on the right. Note how ASPP2 colocalises with F-actin at the apical junctions in cells of the trophectoderm (TE) (white arrowheads). This is quantified in the juxtaposed graph showing ASPP2 and F-actin signal intensity along the apical-basal axis of cell-cell junctions. AJ: apical junction; B: base of the trophectoderm. Scale bars: 20 μm and 5 μm (for the magnification). B. The localisation pattern of YAP and Par3 were analysed in wild type and ASPP2RAKA/RAKA embryos by indirect immunofluorescence. A cross section of representative embryos through the equatorial plane shows the localisation of YAP in the nuclei of the TE in both wild type and ASPP2RAKA/RAKA
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