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Novel approaches to link apicobasal polarity to cell fate specification
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Novel approaches to link apicobasal polarity to cell fate specification Fumio Motegi1,2,5, Nicolas Plachta3,5 and Virgile Viasnoff1,2,4,5 Abstract
Understanding the development of apicobasal polarity (ABP) is a long-standing problem in biology. The molecular components involved in the development and maintenance of APB have been largely identified and are known to have ubiquitous roles across organisms. Our knowledge of the functional consequences of ABP establishment and maintenance is far less comprehensive. Recent studies using novel experimental approaches and cellular models have revealed a growing link between ABP and the genetic program of cell lineage. This mini-review describes some of the most recent advances in this new field, highlighting examples from Caenorhabditis elegans and mouse embryos, human pluripotent stem cells, and epithelial cells. We also speculate on the most interesting and challenging avenues that can be explored. Addresses 1 Department of Biological Sciences, National University of Singapore, 117583, Singapore 2 Mechanobiology Institute, National University of Singapore, 117 411, Singapore 3 Institute of Molecular and Cell Biology, ASTAR, Singapore 4 CNRS, 117411, Singapore 5 Contributed equally Corresponding author: Viasnoff, Virgile ([email protected])
interrelation with the spatial patterning of cell fate acquisition, still remain to be fully understood. The classical understanding is that epithelial genetic programs drive ABP development in the mature epithelium, and this polarity, in turn, modulates apical contractility and tissue mechanics. However, a reverse causality is described during the early development of mouse and Caenorhabditis elegans embryos. Here, it is shown that the mechanical constraints generated by actomyosin flows result in the establishment of polarity that subsequently determines cell fate. At one end of the spectrum, single-cell polarity in embryos plays a major role in cell lineage establishment. At the other end, the collective maintenance of ABP is viewed as the end point of the epithelial program. Recently, this classical understanding has been challenged, with experimental evidence pointing toward a reciprocal relation between cell lineage commitment, ABP, and mechanics. This paved the way toward a deeper understanding of how polarity development orchestrates and responds to the spatial organization of cell lineages. We review here recent results that address the interplay between ABP development and functional genetic programs. We focus on minimal models linking cell autonomous polarity, cortical dynamics, and cell fate acquisition.
Current Opinion in Cell Biology 2020, 62:78–85 This review comes from a themed issue on Cell Architecture Edited by Sandrine Etienne-Manneville and Robert Arkowitz For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.ceb.2019.09.003 0955-0674/© 2019 Elsevier Ltd. All rights reserved.
Keywords Apico-basal polarity, Single cell, Differentiation, Stem cells.
Introduction The establishment of apicobasal polarity (ABP) is essential to many morphogenetic processes. ABP results from the spatial segregation of proteins and the organization of the cytoskeleton. The main molecular complexes that are involved have been identified and are ubiquitous across species and development stages. However, the functional consequences of polarity establishment and maintenance, in particular, its Current Opinion in Cell Biology 2020, 62:78–85
C. elegans embryo, a paradigm for studying the biomechanics of single-cell polarity establishment The newly fertilized C. elegans zygote is one of the widely used model systems for studying single-cell polarity. Recent advances in imaging and micromanipulation have paved the way for understanding how cell mechanics, polarity, and fate interact to precisely orchestrate zygote development. The zygote starts as an initially unpolarized cell. The local disruption of the contractile actomyosin network at the cortex that is proximal to the sperm-donated centrosomes [1,2] leads to asymmetric cortex compartmentalization, and a highly contractile anterior domain segregates from a less contractile posterior domain. The disruption of centrosomes hinders the reorganization of the cortical actomyosin network (Figure 1a) [3e5]. In this context, Aurora-A, a mitotic kinase that stimulates centrosome maturation and cell cycle progression, was www.sciencedirect.com
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Figure 1
C. elegans zygotes during polarization. (a) Centrosomes (orange) trigger polarization of the contractile network of myosin heavy chain (cyan). (b) Cortical actomyosin contractility stimulates clustering of PAR-3 (orange) at the anterior cortex. (c) A PAR-3–containing complex mediates the establishment of a graded PAR-1 distribution (orange) at the posterior cortex. (d) PAR-1 leads to segregation of MEX-5 (cyan) and PIE-1 (orange) in the anterior and the posterior cytoplasm, respectively. All zygotes are oriented with the posterior to the right.
shown to be a key centrosome component that interferes with cortical contractility [6e8**]. Aurora-A also negatively regulates the spontaneous polarization of Par proteins and the cortical actomyosin network. Hence, by promoting symmetry breaking at the one-cell stage but suppressing its spontaneous occurrence [6e 8**], Aurora-A is essential for robust polarity development in early embryos. The local disruption of the cortical actomyosin network creates an imbalance in cortical forces, which ultimately triggers the advective flow of cortical proteins toward the anterior pole [9,10*]]. The physical basis underlying the generation of cortical flows remains unclear; however, UV laser radiation experiments and examination of cortical myosin II dynamics suggest the existence of unbalanced hydrodynamic forces [9,10*]] within the cortex that consequently lead to its flow as a viscous material. This flow further stabilizes the cortical PAR proteins and facilitates their passive transport [11**]. Cortical contractility and the resulting increase in cortical tension are essential for PAR-3, PAR-6, and atypical protein kinase C (aPKC) to form cortical clusters advected with cortical actomyosin and with slower turnover rates (Figure 1b) [11e13**]. Therefore, two types of cortical mechanics (advective flows and cortical tension) synergistically stimulate the efficient segregation of cortical PAR proteins to the anterior compartment. The advective flow of cortical proteins induces cytoplasmic streaming in an opposite orientation [14]. This, in turn, stimulates cortical flows, setting the basis for a positive feedback loop. The artificial induction of cytoplasmic streaming (by inducing a local change in the cytoplasmic temperature using infrared laser irradiation) is sufficient to induce cortical flows and to lead to the segregation of cortical PAR proteins [15*]. Such cytoplasmic streaming is also expected to enhance www.sciencedirect.com
centrosome-mediated symmetry breaking by reducing centrosomeecortex proximity (Figure 1c) [16]. Centrosomes can also contribute to the segregation of cortical PAR proteins via astral microtubuleemediated local recruitment of PAR-1 and PAR-2 to the posterior cortex, which in turn leads to the segregation of a complex comprising PAR-3, PAR-6, and aPKC to the anterior cortex [17]. Such multilayered feedback between chemical signaling and mechanical regulation supports the robust and stereotypical nature of cortical polarization. The biomechanically and biochemically induced compartmentalization of cortical PAR proteins, in turn, helps in the segregation of fate determinants in the cytoplasm, leading to a strict hierarchy in cortical polarity establishment and cell lineage commitment. PAR1 kinase controls the cytoplasmic diffusion of the CCCH zinc finger proteins MEX-5 and MEX-6, leading to their enrichment in the anterior cytoplasm [18,19]. MEX-5 and MEX-6, in turn, polarize a second set of CCCH zinc fingers, PIE-1 and POS-1, in the posterior cytoplasm (Figure 1d) [20]. The anteriorly segregated MEX5 and MEX-6 will contribute to the specification of the somatic cell, and the posteriorly enriched PIE-1 and POS-1 will specify the germ line lineage during early embryogenesis. Hence, cortical polarity establishment by PAR proteins is a robust foundation that ensures the plasticity of the segregation of germ line from the soma even under diverse genetic backgrounds and environmental conditions.
The preimplantation mouse: the interplay of polarity and cell fate specification at the multicellular level At the small cell aggregate level, the preimplantation mouse embryo serves as an excellent system to study how initially unpolarized cells establish a polarized Current Opinion in Cell Biology 2020, 62:78–85
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structure displaying tissue organization [21]. Until the 8-cell stage of development, cells in the embryo are relatively spherical. However, at the end of the 8-cell stage, they undergo a process known as compaction, wherein cells extend long filopodial protrusions (supported by F-actin, E-cadherin, and myosin-10) over the apical surface of the neighboring cells, which draws the neighboring cells closer, particularly at their apical regions (Figure 2aeb). This results in an increased celle cell contact areas and triggers initiation of ABP [22]. As is the case in the C. elegans zygote, the establishment of ABP changes the balance of cortical tension within the cells, favoring again larger cellecell contact areas [23]. Some recent studies have reported the use of laser ablation and pipette aspiration assays to probe the cortical tension in individual blastomeres during this early embryonic stage. When the 8-cell stage blastomere cells divide, they start to form the first physically segregated lineages of the embryo. This spatial segregation is regulated via the establishment of anisotropies in cortical tension [24], likely regulated by ABP [25**], and a change in the organization of microtubules within the cell (Figure 2c). After cell division, some cells remain in the outer layer of the embryo and form the trophectoderm lineage, which later gives rise to the placenta, whereas the rest of the cells become internalized to form the pluripotent inner cell mass, the lineage that later produces all cells in the body. Interestingly, the cells of the embryo lack centrosomes, and they maintain their cytokinetic bridge after cell division. This bridge then becomes positioned apically and helps to direct the transport of key proteins
required for cell polarity, such as E-cadherin, to the cell membrane [26]. A central question in the field has been how inner and outer cells acquire their distinct fate. The outer trophectoderm cells maintain ABP, whereas the inner cells lose their apical polarity and only maintain basally localized components [27]. Importantly, it is also during this stage that the inner and outer cells of the embryo start to differentially express or regulate key transcription factors that determine cell fate, such as Cdx2 (required for the trophectoderm lineage), and key pluripotency-associated transcription factors, such as Nanog, Oct4, and Sox2 [28]. The classical ‘cell polarity model’ suggests that cell polarity components are differentially inherited during cell divisions, leading to distinct regulation of the transcription factors for inner and outer cells [28,29]. The selective inheritance of apically localized proteins such as F-actin or proteins such as Protein kinase C (PKC), PARs, and Ezrin [25**] has been proposed as the key mechanism responsible for fate determination of inner and outer cells. However, recent work using live imaging of cell divisions occurring within the intact, developing embryo revealed that most of these components disassemble from the apical cortex before cell division and are not differentially inherited [27]. Instead, they are gradually reestablished at the apical cortex of outer cells after division is complete. Therefore, it still remains uncertain if some form of polarity components can be asymmetrically inherited in the mouse embryo to determine cell fate. Interestingly, when these apically localized proteins are re-established after division, they play a role in
Figure 2
The early mammalian embryo establishes diverse forms of cell polarity, which are dynamically organized to support preimplantation development. (a) The mouse embryo establishes clear ABP at the 8-cell stage. F-actin becomes enriched in the apical domain, while E-cadherin concentrates at basolateral cell–cell junctions. (b) In addition to stereotypical ABP, the cells of the embryo also extend long filopodia from the most apical part of their junctions over the apical surface of the embryo. These protrusions help cells to be pulled together to promote embryo compaction. (c) The cells of the embryo are also connected via a microtubule bridge. This structure is derived from the cytokinetic bridge, which is retained after cell division and transformed into a noncentrosomal microtubule-organizing center. The microtubules formed from this bridge drive intracellular transport to support cell polarity and also likely contribute to mechanical coordination between sister cells. (d) At the end of preimplantation development, the cells of the embryo form well-defined apical actin rings. These rings expand over the surface of the embryo and zipper with each other along the cell–cell junctions to promote junctional maturation and seal the embryo for blastocyst formation. ABP, apicobasal polarity.
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regulating the localization of the transcription factor Yap [30e34]. Apical polarity is thought to enhance the nuclear localization of Yap in outer cells, triggering the Cdx2 expression and the acquisition of trophectoderm lineage. Although studies have shown that polarity influences cell fate via the regulation of Hippo signaling and Yap localization [30,32,33**,35], it is plausible that polarity may affect the mechanical properties or forces acting within the embryo, which could also contribute to Yap regulation, as shown in other systems [36]. After the establishment of inner and outer lineages, the mammalian embryo starts to form the blastocyst. Blastocyst formation is driven via a dynamic reorganization of cell polarity components within the embryo [27]. Just before cavitation, the cells in the embryo form a prominent F-actin ring at their apical cortex (Figure 2a). This structure appears at the apical cortex as a result of the combined effects of cortical flow and a specific set of microtubules that clear F-actin from the most apical parts of the cortex after cell division. Yet unlike other Factinerich structures such as actin cables or cytokinetic furrows [37], the actin rings of the mouse embryo do not accumulate significant levels of myosin II and are consistently noncontractile. Instead, as the embryo flattens its apical surface, these rings expand over the entire surface until they come into contact with neighboring rings at the most apical regions of cellecell contacts. This triggers a rapid change in ABP, mediated by the recruitment of myosin II and many of the key components required for the formation of the epithelial permeability barrier, including adherens junction proteins such as E-cadherin and E-cadherine associated catenins and tight junction proteins such as ZO1. As the actin rings fuse or ‘zipper’ along the entire surface of the embryo, these components are distributed across the cellecell contact area, leading to junctional maturation and blastocyst formation (Figure 2d). The establishment of the epithelial permeability barrier seals the embryo and enables the accumulation of fluid between cells. This increases the pressure in the paracellular cleft and leads to the formation of the cavity during blastocyst development [38,39]. Internal osmotic forces are also likely to contribute to cell fate specification [40]. Thus, changes in apical polarity and junction maturation triggered by actin ring zippering are critical to establish the polarized molecular organization that enables the large-scale morphogenesis of the blastocyst during the cavitation process.
Epithelial polarity: novels views on autonomous vs collective development The influence of polarity on cell lineage establishment has also been revealed in vitro using 2D microcolonies of stem cells [41,42]. The segregation of growth factor receptors during polarity development appeared to modulate the spatial commitment of human embryonic www.sciencedirect.com
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stem cells plated on 2D circular Extra-cellular matrix (ECM) patterns [43**,44]. On addition of the growth factor BMP4, the outermost cells in the colony differentiated into the ectoderm, whereas an intermediate belt of cells and the cells in the colony center became mesoderm-like and endoderm-like cells, respectively. It recapitulated the morphogenetic events taking place during gastrula and primitive streak formation in human embryos. At the colony center, high cell density and ABP led to the localization of Bone morphogenic protein (BMP) receptors at the lateral junctions [43**], whereas the receptors were more apically localized in cells at the colony edge. The density gradient of cells resulted in a localization gradient in NOGGIN, an antagonist of BMP, contributing to the spatial patterning of cell lineage. These experiments questioned the role of mechanical cues such as ABP and cortical tension in cell fate acquisition. Interestingly, a recent report shows that human pluripotent stem cells can autonomously develop single-cell polarity independently of the surrounding environment [45]. They present intracellular vesicles cultured in suspension with an apical-like membrane, microvilli, and primary cilia (Figure 3). These intracellular compartments, termed apicosomes, shared similarities with apical-like vesicles found in isolated blastocysts [41,45*,46]. The apicosomes tend to fuse with the cortical membranes when the cells form aggregates to generate the apical intraluminal membrane of a multicellular cyst. The development of ABP in an epithelium or a cyst is often pictured as a collective process. Symmetry breaking in protein distribution is shown to take place along an axis determined by cellematrix adhesion (basal pole) and cellecell contacts (lateral poles) in cystforming cells [47,48]. Most in vitro models MadinDarbin canin kidney (MDCK and EPH4) studied polarity development on symmetric division of mature epithelial cells. They implicitly considered a synchronous development of APB in all neighboring cells. However, recent observations, such as the one described previously, demonstrate the ability of cells to selfpolarize independently of the response of their neighboring cells. This observation is further supported by similar findings from several other studies. For example, the overactivation of LKB1 in intestinal epithelial cells (Figure 3) [49] leads to the development of an apical pole in individual cells plated on an ECM-coated substrate. Similarly, single primary rat hepatocytes autonomously polarized when they were individually cultured in artificial microniches [50,51**]] comprising a rigid polymeric cavity coated differentially with fibronectin and cadherin. The microniche mimics the minimal environmental cues for a liver cell (ECM and cadherin density) that prompt the development of an apical secretory hemilumen (bile canaliculi-like) at the Current Opinion in Cell Biology 2020, 62:78–85
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Figure 3
Cellular models to study the interplay between apicobasal polarity, cellular mechanics, and cell fate acquisition. Classical models: cysts and epithelial monolayers, bile canaliculi in hepatocytes. Embryonic models: single-cell C. elegans zygote and preimplantation mouse embryo. Newly observed models with single-cell polarity: circulating tumor cells, human pluripotent stem cells, colon cells with overactivated LKB1, and hepatocytes grown in microniches. The polarity markers present at the apical pole are noted for each model. ERM: Ezrin Radixin Moesin; PDX: podocalixin, aPKC: atypical protein kinase C, ECM: extracellular matrix.
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interface between the cell and substrate (Figure 3) [51**]. Such an approach allows unprecedented imaging resolution of the lumenogenesis process. Single mature hepatocytes were also seen to polarize when they came in contact with another epithelial cell (MDCK, CaCo, or EPH4 cell) [51**]. In this case, the spatial orientation of the ECM around the cellular doublets strongly influenced the lumen shape [52], suggesting that the anisotropic intercellular tension preexisting in the lateral contacts guided lumenogenesis at the two-cell level. These converging observations demonstrate that epithelial ABP can develop independently of the neighboring cells’ response, once triggered by minimal external cues (ECM and cadherin adhesion). Such mechanical induction results from both homotypic and heterotypic contacts alike. In the near future, we hope these experimental systems can help in determining how the establishment of polarity between multipotent cells synchronizes cell fate establishment and contribute to the zonation of cell lineage during development.
development can function as a local coordinating mechanism for spatially controlling cell fate, not just during early embryogenesis but throughout development is currently emerging. We propose that the new experimental approaches (involving a small number of cells) described in this article can provide a deeper understanding of the interrelation and between polarity, mechanics, and cell lineage could have during development. Furthermore, we argue that experimental observations need to be complemented by in silico modeling and computer simulations. For instance, a recent study [56**] used in silico modeling techniques to predict how luminal changes shape under different conditions of apical and planar polarity development. In silico organ approaches could now include the extra complexity of polarity to account and predict tissue shape [56**,57]. From an experimental point of view, live imaging of organoid models grown in controlled biochemical and biophysical conditions could provide valuable insights to address similar questions in other organ development scenarios.
Finally, a key morphogenetic process that is affected by the interaction of polarity with genetic expression programs in mature epithelial cells is epithelial-to-mesenchymal transition. During epithelial-to-mesenchymal transition, cells are believed to fully loose their ABP. However, a substantial fraction of circulating tumor cells (CTCs) in suspension appears to retain their apical poles [53*]. The metastatic potential of the cancer correlated with the fraction of apically polarized CTCs as the presence of this pole enhanced the adhesiveness of CTCs and favored their intravasation into distant tissues. Therefore, the partial maintenance of ABP in CTCs appeared to be an aggravating factor for cancer dissemination. On the other hand, stable maintenance of ABP can regulate cancer cell response to antidrug treatment [54]. The maintenance of polarity in mouse mammary organoids was found to lead to SNAI1 inactivation via a Par3-aPKCemediated mechanism [55*]. In turn, SNAI1 inactivation results in the stabilization of adherens junctions and the maintenance of the epithelial phenotype. Oppositely, disruption of polarity in Caco2 organoids led to SNAI1 activation and an enhanced transition to mesenchymal types [55*].
Conflict of interest statement Nothing declared.
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Growing evidence demonstrates how ABP is not only an end point of cell lineage commitment but also a cause to the specification of lineages. It is also largely involved in cellular rearrangements driven by biomechanics. It suggests the development and maintenance of polarity, and the cellular state of the neighboring cells is likely coupled to mechanical feedback loops. It is now widely recognized that a given genetic background can give rise to various phenotypes, depending on the environmental conditions of the cell. The notion that polarity
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40. Chan CJ, Costanzo M, Ruiz-Herrero T, Mönke G, Petrie RJ, Mahadevan L, Hiiragi T: Hydraulic control of embryo size, tissue shape and cell fate. bioRxiv 2018:389619. 41. Deglincerti A, Croft GF, Pietila LN, Zernicka-Goetz M, Siggia ED, Brivanlou AH: Self-organization of the in vitro attached human embryo. Nature 2016, 533:251–254. 42. Morgani SM, Metzger JJ, Nichols J, Siggia ED, Hadjantonakis AK: Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning. Elife 2018, 7. 43. Etoc F, Metzger J, Ruzo A, Kirst C, Yoney A, Ozair MZ, * * Brivanlou AH, Siggia ED: A balance between secreted inhibitors and edge sensing controls gastruloid self-organization. Dev Cell 2016, 39:302–315. This paper describes the mechanism by wich cell density can regualte the stem cell fate development when submitted to soluble BMP4. It proposes a mechanism for the formation of the primitive streak. 44. Martyn I, Brivanlou AH, Siggia ED: A wave of WNT signaling balanced by secreted inhibitors controls primitive streak formation in micropattern colonies of human embryonic stem cells. Development 2019:146. 45. Taniguchi K, Shao Y, Townshend RF, Cortez CL, Harris CE, * Meshinchi S, Kalantry S, Fu J, O’Shea KS, Gumucio DL: An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells. J Cell Biol 2017, 216:3981–3990. This work reports the existence of Apical comptartment inside individual hPSCs that fuse to the cell membrane to create lumen in Embryoid bodies. 46. Bedzhov I, Zernicka-Goetz M: Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 2014, 156:1032–1044. 47. Bryant DM, Roignot J, Datta A, Overeem AW, Kim M, Yu W, Peng X, Eastburn DJ, Ewald AJ, Werb Z, et al.: A molecular switch for the orientation of epithelial cell polarization. Dev Cell 2014, 31:171–187. 48. Akhtar N, Streuli CH: An integrin-ILK-microtubule network orients cell polarity and lumen formation in glandular epithelium. Nat Cell Biol 2013, 15:17–27. 49. Baas AF, Kuipers J, van der Wel NN, Batlle E, Koerten HK, Peters PJ, Clevers HC: Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 2004, 116:457–466.
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50. Stoecklin C, Yue Z, Chen WW, de Mets R, Fong E, Studer V, Viasnoff V: A new approach to design artificial 3D microniches with combined chemical, topographical, and rheological cues. Advanced Biosystems 2018, 2:1700237. 51. Zhang Y, de Mets R, Monzel C, Toh P, Van Hul N, Ng SS, Tamir * * Rashid S, Viasnoff V: Autonomous induction of hepatic polarity to construct single cell liver. bioRxiv 2019:636654. This work describes how singel hepatocytes can be polarized in contact with cadherin coated substrates in an ECM scaffold. The paper demsontrate the ability of hepatocytes to polarize in absence of neighbor response and to form heterotypic apical lumens 52. Li Q, Zhang Y, Pluchon P, Robens J, Herr K, Mercade M, Thiery JP, Yu H, Viasnoff V: Extracellular matrix scaffolding guides lumen elongation by inducing anisotropic intercellular mechanical tension. Nat Cell Biol 2016, 18:311–318. 53. Lorentzen A, Becker PF, Kosla J, Saini M, Weidele K, Ronchi P, * Klein C, Wolf MJ, Geist F, Seubert B, et al.: Single cell polarity in liquid phase facilitates tumour metastasis. Nat Commun 2018, 9:887. This article describes the maintenance of ABP in suspended circualting tumor cells. They relate the occurrence of the apical patch to the invasivity of the metastatic potential of the cancer cells. 54. Ashley N, Ouaret D, Bodmer WF: Cellular polarity modulates drug resistance in primary colorectal cancers via orientation of the multidrug resistance protein ABCB1. J Pathol 2019, 247:293–304. 55. Jung H-Y, Fattet L, Tsai JH, Kajimoto T, Chang Q, Newton AC, * Yang J: Apical–basal polarity inhibits epithelial–mesenchymal transition and tumour metastasis by PAR-complex-mediated SNAI1 degradation. Nat Cell Biol 2019, 21:359–371. 56. Nissen SB, Ronhild S, Trusina A, Sneppen K: Theoretical tool * * bridging cell polarities with development of robust morphologies. Elife 2018, vol. 7. This work proposes asimulation methods that include apico-basal polarity,planar polarity and cell mechanics to predict lumen shape development. 57. Okuda S, Takata N, Hasegawa Y, Kawada M, Inoue Y, Adachi T, Sasai Y, Eiraku M, Okuda S, Takata N, et al.: Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis. Science Advances 2018, 4.
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Novel approaches to link apicobasal polarity to cell fate specification Fumio Motegi1,2,5, Nicolas Plachta3,5 and Virgile Viasnoff1,2,4,5 Abstract
Understanding the development of apicobasal polarity (ABP) is a long-standing problem in biology. The molecular components involved in the development and maintenance of APB have been largely identified and are known to have ubiquitous roles across organisms. Our knowledge of the functional consequences of ABP establishment and maintenance is far less comprehensive. Recent studies using novel experimental approaches and cellular models have revealed a growing link between ABP and the genetic program of cell lineage. This mini-review describes some of the most recent advances in this new field, highlighting examples from Caenorhabditis elegans and mouse embryos, human pluripotent stem cells, and epithelial cells. We also speculate on the most interesting and challenging avenues that can be explored. Addresses 1 Department of Biological Sciences, National University of Singapore, 117583, Singapore 2 Mechanobiology Institute, National University of Singapore, 117 411, Singapore 3 Institute of Molecular and Cell Biology, ASTAR, Singapore 4 CNRS, 117411, Singapore 5 Contributed equally Corresponding author: Viasnoff, Virgile ([email protected])
interrelation with the spatial patterning of cell fate acquisition, still remain to be fully understood. The classical understanding is that epithelial genetic programs drive ABP development in the mature epithelium, and this polarity, in turn, modulates apical contractility and tissue mechanics. However, a reverse causality is described during the early development of mouse and Caenorhabditis elegans embryos. Here, it is shown that the mechanical constraints generated by actomyosin flows result in the establishment of polarity that subsequently determines cell fate. At one end of the spectrum, single-cell polarity in embryos plays a major role in cell lineage establishment. At the other end, the collective maintenance of ABP is viewed as the end point of the epithelial program. Recently, this classical understanding has been challenged, with experimental evidence pointing toward a reciprocal relation between cell lineage commitment, ABP, and mechanics. This paved the way toward a deeper understanding of how polarity development orchestrates and responds to the spatial organization of cell lineages. We review here recent results that address the interplay between ABP development and functional genetic programs. We focus on minimal models linking cell autonomous polarity, cortical dynamics, and cell fate acquisition.
Current Opinion in Cell Biology 2020, 62:78–85 This review comes from a themed issue on Cell Architecture Edited by Sandrine Etienne-Manneville and Robert Arkowitz For a complete overview see the Issue and the Editorial
https://doi.org/10.1016/j.ceb.2019.09.003 0955-0674/© 2019 Elsevier Ltd. All rights reserved.
Keywords Apico-basal polarity, Single cell, Differentiation, Stem cells.
Introduction The establishment of apicobasal polarity (ABP) is essential to many morphogenetic processes. ABP results from the spatial segregation of proteins and the organization of the cytoskeleton. The main molecular complexes that are involved have been identified and are ubiquitous across species and development stages. However, the functional consequences of polarity establishment and maintenance, in particular, its Current Opinion in Cell Biology 2020, 62:78–85
C. elegans embryo, a paradigm for studying the biomechanics of single-cell polarity establishment The newly fertilized C. elegans zygote is one of the widely used model systems for studying single-cell polarity. Recent advances in imaging and micromanipulation have paved the way for understanding how cell mechanics, polarity, and fate interact to precisely orchestrate zygote development. The zygote starts as an initially unpolarized cell. The local disruption of the contractile actomyosin network at the cortex that is proximal to the sperm-donated centrosomes [1,2] leads to asymmetric cortex compartmentalization, and a highly contractile anterior domain segregates from a less contractile posterior domain. The disruption of centrosomes hinders the reorganization of the cortical actomyosin network (Figure 1a) [3e5]. In this context, Aurora-A, a mitotic kinase that stimulates centrosome maturation and cell cycle progression, was www.sciencedirect.com
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Figure 1
C. elegans zygotes during polarization. (a) Centrosomes (orange) trigger polarization of the contractile network of myosin heavy chain (cyan). (b) Cortical actomyosin contractility stimulates clustering of PAR-3 (orange) at the anterior cortex. (c) A PAR-3–containing complex mediates the establishment of a graded PAR-1 distribution (orange) at the posterior cortex. (d) PAR-1 leads to segregation of MEX-5 (cyan) and PIE-1 (orange) in the anterior and the posterior cytoplasm, respectively. All zygotes are oriented with the posterior to the right.
shown to be a key centrosome component that interferes with cortical contractility [6e8**]. Aurora-A also negatively regulates the spontaneous polarization of Par proteins and the cortical actomyosin network. Hence, by promoting symmetry breaking at the one-cell stage but suppressing its spontaneous occurrence [6e 8**], Aurora-A is essential for robust polarity development in early embryos. The local disruption of the cortical actomyosin network creates an imbalance in cortical forces, which ultimately triggers the advective flow of cortical proteins toward the anterior pole [9,10*]]. The physical basis underlying the generation of cortical flows remains unclear; however, UV laser radiation experiments and examination of cortical myosin II dynamics suggest the existence of unbalanced hydrodynamic forces [9,10*]] within the cortex that consequently lead to its flow as a viscous material. This flow further stabilizes the cortical PAR proteins and facilitates their passive transport [11**]. Cortical contractility and the resulting increase in cortical tension are essential for PAR-3, PAR-6, and atypical protein kinase C (aPKC) to form cortical clusters advected with cortical actomyosin and with slower turnover rates (Figure 1b) [11e13**]. Therefore, two types of cortical mechanics (advective flows and cortical tension) synergistically stimulate the efficient segregation of cortical PAR proteins to the anterior compartment. The advective flow of cortical proteins induces cytoplasmic streaming in an opposite orientation [14]. This, in turn, stimulates cortical flows, setting the basis for a positive feedback loop. The artificial induction of cytoplasmic streaming (by inducing a local change in the cytoplasmic temperature using infrared laser irradiation) is sufficient to induce cortical flows and to lead to the segregation of cortical PAR proteins [15*]. Such cytoplasmic streaming is also expected to enhance www.sciencedirect.com
centrosome-mediated symmetry breaking by reducing centrosomeecortex proximity (Figure 1c) [16]. Centrosomes can also contribute to the segregation of cortical PAR proteins via astral microtubuleemediated local recruitment of PAR-1 and PAR-2 to the posterior cortex, which in turn leads to the segregation of a complex comprising PAR-3, PAR-6, and aPKC to the anterior cortex [17]. Such multilayered feedback between chemical signaling and mechanical regulation supports the robust and stereotypical nature of cortical polarization. The biomechanically and biochemically induced compartmentalization of cortical PAR proteins, in turn, helps in the segregation of fate determinants in the cytoplasm, leading to a strict hierarchy in cortical polarity establishment and cell lineage commitment. PAR1 kinase controls the cytoplasmic diffusion of the CCCH zinc finger proteins MEX-5 and MEX-6, leading to their enrichment in the anterior cytoplasm [18,19]. MEX-5 and MEX-6, in turn, polarize a second set of CCCH zinc fingers, PIE-1 and POS-1, in the posterior cytoplasm (Figure 1d) [20]. The anteriorly segregated MEX5 and MEX-6 will contribute to the specification of the somatic cell, and the posteriorly enriched PIE-1 and POS-1 will specify the germ line lineage during early embryogenesis. Hence, cortical polarity establishment by PAR proteins is a robust foundation that ensures the plasticity of the segregation of germ line from the soma even under diverse genetic backgrounds and environmental conditions.
The preimplantation mouse: the interplay of polarity and cell fate specification at the multicellular level At the small cell aggregate level, the preimplantation mouse embryo serves as an excellent system to study how initially unpolarized cells establish a polarized Current Opinion in Cell Biology 2020, 62:78–85
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structure displaying tissue organization [21]. Until the 8-cell stage of development, cells in the embryo are relatively spherical. However, at the end of the 8-cell stage, they undergo a process known as compaction, wherein cells extend long filopodial protrusions (supported by F-actin, E-cadherin, and myosin-10) over the apical surface of the neighboring cells, which draws the neighboring cells closer, particularly at their apical regions (Figure 2aeb). This results in an increased celle cell contact areas and triggers initiation of ABP [22]. As is the case in the C. elegans zygote, the establishment of ABP changes the balance of cortical tension within the cells, favoring again larger cellecell contact areas [23]. Some recent studies have reported the use of laser ablation and pipette aspiration assays to probe the cortical tension in individual blastomeres during this early embryonic stage. When the 8-cell stage blastomere cells divide, they start to form the first physically segregated lineages of the embryo. This spatial segregation is regulated via the establishment of anisotropies in cortical tension [24], likely regulated by ABP [25**], and a change in the organization of microtubules within the cell (Figure 2c). After cell division, some cells remain in the outer layer of the embryo and form the trophectoderm lineage, which later gives rise to the placenta, whereas the rest of the cells become internalized to form the pluripotent inner cell mass, the lineage that later produces all cells in the body. Interestingly, the cells of the embryo lack centrosomes, and they maintain their cytokinetic bridge after cell division. This bridge then becomes positioned apically and helps to direct the transport of key proteins
required for cell polarity, such as E-cadherin, to the cell membrane [26]. A central question in the field has been how inner and outer cells acquire their distinct fate. The outer trophectoderm cells maintain ABP, whereas the inner cells lose their apical polarity and only maintain basally localized components [27]. Importantly, it is also during this stage that the inner and outer cells of the embryo start to differentially express or regulate key transcription factors that determine cell fate, such as Cdx2 (required for the trophectoderm lineage), and key pluripotency-associated transcription factors, such as Nanog, Oct4, and Sox2 [28]. The classical ‘cell polarity model’ suggests that cell polarity components are differentially inherited during cell divisions, leading to distinct regulation of the transcription factors for inner and outer cells [28,29]. The selective inheritance of apically localized proteins such as F-actin or proteins such as Protein kinase C (PKC), PARs, and Ezrin [25**] has been proposed as the key mechanism responsible for fate determination of inner and outer cells. However, recent work using live imaging of cell divisions occurring within the intact, developing embryo revealed that most of these components disassemble from the apical cortex before cell division and are not differentially inherited [27]. Instead, they are gradually reestablished at the apical cortex of outer cells after division is complete. Therefore, it still remains uncertain if some form of polarity components can be asymmetrically inherited in the mouse embryo to determine cell fate. Interestingly, when these apically localized proteins are re-established after division, they play a role in
Figure 2
The early mammalian embryo establishes diverse forms of cell polarity, which are dynamically organized to support preimplantation development. (a) The mouse embryo establishes clear ABP at the 8-cell stage. F-actin becomes enriched in the apical domain, while E-cadherin concentrates at basolateral cell–cell junctions. (b) In addition to stereotypical ABP, the cells of the embryo also extend long filopodia from the most apical part of their junctions over the apical surface of the embryo. These protrusions help cells to be pulled together to promote embryo compaction. (c) The cells of the embryo are also connected via a microtubule bridge. This structure is derived from the cytokinetic bridge, which is retained after cell division and transformed into a noncentrosomal microtubule-organizing center. The microtubules formed from this bridge drive intracellular transport to support cell polarity and also likely contribute to mechanical coordination between sister cells. (d) At the end of preimplantation development, the cells of the embryo form well-defined apical actin rings. These rings expand over the surface of the embryo and zipper with each other along the cell–cell junctions to promote junctional maturation and seal the embryo for blastocyst formation. ABP, apicobasal polarity.
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regulating the localization of the transcription factor Yap [30e34]. Apical polarity is thought to enhance the nuclear localization of Yap in outer cells, triggering the Cdx2 expression and the acquisition of trophectoderm lineage. Although studies have shown that polarity influences cell fate via the regulation of Hippo signaling and Yap localization [30,32,33**,35], it is plausible that polarity may affect the mechanical properties or forces acting within the embryo, which could also contribute to Yap regulation, as shown in other systems [36]. After the establishment of inner and outer lineages, the mammalian embryo starts to form the blastocyst. Blastocyst formation is driven via a dynamic reorganization of cell polarity components within the embryo [27]. Just before cavitation, the cells in the embryo form a prominent F-actin ring at their apical cortex (Figure 2a). This structure appears at the apical cortex as a result of the combined effects of cortical flow and a specific set of microtubules that clear F-actin from the most apical parts of the cortex after cell division. Yet unlike other Factinerich structures such as actin cables or cytokinetic furrows [37], the actin rings of the mouse embryo do not accumulate significant levels of myosin II and are consistently noncontractile. Instead, as the embryo flattens its apical surface, these rings expand over the entire surface until they come into contact with neighboring rings at the most apical regions of cellecell contacts. This triggers a rapid change in ABP, mediated by the recruitment of myosin II and many of the key components required for the formation of the epithelial permeability barrier, including adherens junction proteins such as E-cadherin and E-cadherine associated catenins and tight junction proteins such as ZO1. As the actin rings fuse or ‘zipper’ along the entire surface of the embryo, these components are distributed across the cellecell contact area, leading to junctional maturation and blastocyst formation (Figure 2d). The establishment of the epithelial permeability barrier seals the embryo and enables the accumulation of fluid between cells. This increases the pressure in the paracellular cleft and leads to the formation of the cavity during blastocyst development [38,39]. Internal osmotic forces are also likely to contribute to cell fate specification [40]. Thus, changes in apical polarity and junction maturation triggered by actin ring zippering are critical to establish the polarized molecular organization that enables the large-scale morphogenesis of the blastocyst during the cavitation process.
Epithelial polarity: novels views on autonomous vs collective development The influence of polarity on cell lineage establishment has also been revealed in vitro using 2D microcolonies of stem cells [41,42]. The segregation of growth factor receptors during polarity development appeared to modulate the spatial commitment of human embryonic www.sciencedirect.com
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stem cells plated on 2D circular Extra-cellular matrix (ECM) patterns [43**,44]. On addition of the growth factor BMP4, the outermost cells in the colony differentiated into the ectoderm, whereas an intermediate belt of cells and the cells in the colony center became mesoderm-like and endoderm-like cells, respectively. It recapitulated the morphogenetic events taking place during gastrula and primitive streak formation in human embryos. At the colony center, high cell density and ABP led to the localization of Bone morphogenic protein (BMP) receptors at the lateral junctions [43**], whereas the receptors were more apically localized in cells at the colony edge. The density gradient of cells resulted in a localization gradient in NOGGIN, an antagonist of BMP, contributing to the spatial patterning of cell lineage. These experiments questioned the role of mechanical cues such as ABP and cortical tension in cell fate acquisition. Interestingly, a recent report shows that human pluripotent stem cells can autonomously develop single-cell polarity independently of the surrounding environment [45]. They present intracellular vesicles cultured in suspension with an apical-like membrane, microvilli, and primary cilia (Figure 3). These intracellular compartments, termed apicosomes, shared similarities with apical-like vesicles found in isolated blastocysts [41,45*,46]. The apicosomes tend to fuse with the cortical membranes when the cells form aggregates to generate the apical intraluminal membrane of a multicellular cyst. The development of ABP in an epithelium or a cyst is often pictured as a collective process. Symmetry breaking in protein distribution is shown to take place along an axis determined by cellematrix adhesion (basal pole) and cellecell contacts (lateral poles) in cystforming cells [47,48]. Most in vitro models MadinDarbin canin kidney (MDCK and EPH4) studied polarity development on symmetric division of mature epithelial cells. They implicitly considered a synchronous development of APB in all neighboring cells. However, recent observations, such as the one described previously, demonstrate the ability of cells to selfpolarize independently of the response of their neighboring cells. This observation is further supported by similar findings from several other studies. For example, the overactivation of LKB1 in intestinal epithelial cells (Figure 3) [49] leads to the development of an apical pole in individual cells plated on an ECM-coated substrate. Similarly, single primary rat hepatocytes autonomously polarized when they were individually cultured in artificial microniches [50,51**]] comprising a rigid polymeric cavity coated differentially with fibronectin and cadherin. The microniche mimics the minimal environmental cues for a liver cell (ECM and cadherin density) that prompt the development of an apical secretory hemilumen (bile canaliculi-like) at the Current Opinion in Cell Biology 2020, 62:78–85
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Figure 3
Cellular models to study the interplay between apicobasal polarity, cellular mechanics, and cell fate acquisition. Classical models: cysts and epithelial monolayers, bile canaliculi in hepatocytes. Embryonic models: single-cell C. elegans zygote and preimplantation mouse embryo. Newly observed models with single-cell polarity: circulating tumor cells, human pluripotent stem cells, colon cells with overactivated LKB1, and hepatocytes grown in microniches. The polarity markers present at the apical pole are noted for each model. ERM: Ezrin Radixin Moesin; PDX: podocalixin, aPKC: atypical protein kinase C, ECM: extracellular matrix.
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interface between the cell and substrate (Figure 3) [51**]. Such an approach allows unprecedented imaging resolution of the lumenogenesis process. Single mature hepatocytes were also seen to polarize when they came in contact with another epithelial cell (MDCK, CaCo, or EPH4 cell) [51**]. In this case, the spatial orientation of the ECM around the cellular doublets strongly influenced the lumen shape [52], suggesting that the anisotropic intercellular tension preexisting in the lateral contacts guided lumenogenesis at the two-cell level. These converging observations demonstrate that epithelial ABP can develop independently of the neighboring cells’ response, once triggered by minimal external cues (ECM and cadherin adhesion). Such mechanical induction results from both homotypic and heterotypic contacts alike. In the near future, we hope these experimental systems can help in determining how the establishment of polarity between multipotent cells synchronizes cell fate establishment and contribute to the zonation of cell lineage during development.
development can function as a local coordinating mechanism for spatially controlling cell fate, not just during early embryogenesis but throughout development is currently emerging. We propose that the new experimental approaches (involving a small number of cells) described in this article can provide a deeper understanding of the interrelation and between polarity, mechanics, and cell lineage could have during development. Furthermore, we argue that experimental observations need to be complemented by in silico modeling and computer simulations. For instance, a recent study [56**] used in silico modeling techniques to predict how luminal changes shape under different conditions of apical and planar polarity development. In silico organ approaches could now include the extra complexity of polarity to account and predict tissue shape [56**,57]. From an experimental point of view, live imaging of organoid models grown in controlled biochemical and biophysical conditions could provide valuable insights to address similar questions in other organ development scenarios.
Finally, a key morphogenetic process that is affected by the interaction of polarity with genetic expression programs in mature epithelial cells is epithelial-to-mesenchymal transition. During epithelial-to-mesenchymal transition, cells are believed to fully loose their ABP. However, a substantial fraction of circulating tumor cells (CTCs) in suspension appears to retain their apical poles [53*]. The metastatic potential of the cancer correlated with the fraction of apically polarized CTCs as the presence of this pole enhanced the adhesiveness of CTCs and favored their intravasation into distant tissues. Therefore, the partial maintenance of ABP in CTCs appeared to be an aggravating factor for cancer dissemination. On the other hand, stable maintenance of ABP can regulate cancer cell response to antidrug treatment [54]. The maintenance of polarity in mouse mammary organoids was found to lead to SNAI1 inactivation via a Par3-aPKCemediated mechanism [55*]. In turn, SNAI1 inactivation results in the stabilization of adherens junctions and the maintenance of the epithelial phenotype. Oppositely, disruption of polarity in Caco2 organoids led to SNAI1 activation and an enhanced transition to mesenchymal types [55*].
Conflict of interest statement Nothing declared.
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Growing evidence demonstrates how ABP is not only an end point of cell lineage commitment but also a cause to the specification of lineages. It is also largely involved in cellular rearrangements driven by biomechanics. It suggests the development and maintenance of polarity, and the cellular state of the neighboring cells is likely coupled to mechanical feedback loops. It is now widely recognized that a given genetic background can give rise to various phenotypes, depending on the environmental conditions of the cell. The notion that polarity
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