ABSTRACT
Cell junctions play key roles in epithelial integrity. During development, when epithelia undergo extensive morphogenesis, these junctions must be remodeled in order to maintain mechanochemical barriers and ensure the cohesion of the tissue. In this Review, we present a comprehensive and integrated description of junctional remodeling mechanisms in epithelial cells during development, from embryonic to adult epithelia. We largely focus on Drosophila, as quantitative analyses in this organism have provided a detailed characterization of the molecular mechanisms governing cell topologies, and discuss the conservation of these mechanisms across metazoans. We consider how changes at the molecular level translate to tissue-scale irreversible deformations, exploring the composition and assembly of cellular interfaces to unveil how junctions are remodeled to preserve tissue homeostasis during cell division, intercalation, invagination, ingression and extrusion.
Introduction
Animals are composed of hundreds to trillions of cells that assemble into a rich variety of tissues. Epithelia are the most abundant tissues and sculpt a diverse collection of architectures with specialized functions, from flat, stratified layers flanking the body's entrances to branched tubular monolayers regulating metabolite exchange between inner and external environments.
Strikingly, the vast repertoire of epithelial tissues stems from the same fundamental biological units: polarized epithelial cells. These cells exhibit apicobasal polarity (see Glossary, Box 1) and assemble junctional complexes that mediate adhesion with neighboring cells and the extracellular matrix (ECM). Eumetazoans undergo gastrulation, a complex process of tissue reorganization during which two (ectoderm and endoderm) or three (ectoderm, endoderm and mesoderm) individual germ layers are formed. Each layer exhibits distinct junctional complex localization patterns, and this variation promotes the formation of the highly specialized tissues and organs that compartmentalize the organism. Examples of ectoderm-derived tissues include the skin and the central nervous system, whereas the intestine and lungs originate from the endoderm. If present, the mesoderm gives rise to structures including the connective tissue and muscles. This Review mainly focuses on ectodermal tissues because they have been the more extensively characterized epithelia during morphogenesis.
Adherens junctions (AJs). Cell-cell junctions that physically couple neighboring epithelial cells to ensure the integrity of the epithelial sheet throughout development. They are usually located apically to septate junctions and contain E-cadherins (E-cad), transmembrane proteins that homodimerize in a calcium-dependent manner. E-cad proteins interact with intracellular catenins, thus mediating contact with the actomyosin cytoskeleton.
Apicobasal polarity. A type of cell polarity specific to epithelial cells that forms upon targeted delivery of polarity complexes to the cell periphery. It delimits specialized domains and promotes compartmentalization of the organism. The apical domain of the membrane faces the external environment (which may be the surface of the body or internal lumen cavities), whereas the basolateral domain is located on the opposite side, away from the lumen. Both domains are physically separated and are required to assemble junctional complexes.
Bicellular junctions (BCJs). Cell-cell junctions formed at the contact interface between two adjacent cells that function to mechanically (AJs) and chemically (SJs, TJs) seal the epithelium and connect tricellular junctions from adjacent cells.
Epithelial-to-mesenchymal transition (EMT). The process by which epithelial cells transform into mesenchymal cells upon signaling induction, via changes in cell morphology, the progressive suppression of epithelial characteristics and acquisition of new properties such as enhanced cell motility and invasiveness. In a pathological context, tumorigenic growths with malignant invasive behavior undergo EMT to deviate from the tissue, escape tissue clearance strategies and potentially migrate towards distal tissues.
Extrusion. The process by which caspase-activated cells are seamlessly removed from the epithelium. This may be part of normal developmental programs or used to remove potentially hazardous cells from the tissue.
Finger-like protrusions (FLPs). Formed by membrane deformations between dividing epithelial cells and their neighbors at the site of cytokinetic ring ingression.
Focal adhesions (FAs). Also known as cell-matrix adhesions, FAs are integrin-containing, multi-protein assemblies that provide the mechanical link between intracellular actin bundles and the extracellular matrix (ECM).
Four-way junctions. Transient multicellular reorganizations that form during cell intercalation, once a single bicellular junction has undergone shrinkage.
Ingression. The movement of living cells out of an epithelium as part of normal developmental programs. Cells can ingress collectively (EMT processes), such as the presumptive mesoderm during gastrulation, or as a single cell, such as neural stem cells during neurogenesis.
Intercalation. The process by which neighboring epithelial cells exchange position in an epithelium.
Invagination. The infolding of an epithelial sheet. This process starts with a tissue indentation that propagates across the cell layer, resulting in the formation of a cavity, pouch or tube. The prototype of epithelial tissue invagination occurs during gastrulation.
Junctional non-muscle type-II Myosin (junctional Myo-II). The pool of Myo-II located apically at the cortex of epithelial cells. It is linked to AJs via Catenin-mediated adhesion and to medial Myo-II via the actomyosin network, integrating external and internal mechanical stimuli to ensure force balance and coordinate cell behavior.
Medial non-muscle type-II Myosin (medial Myo-II). The pool of Myo-II present in the middle of the cell, responsible for generating cell contractility upon activation via phosphorylation of its regulatory light chain. Medial Myo-II is connected to the junctional Myo-II meshwork and both pools act together to drive tissue morphogenesis and preserve homeostasis.
Midbody. A transient structure that forms in the dividing cell as an end product of cytokinetic actomyosin ring constriction. It connects the two daughter cells towards the end of cytokinesis.
Rosettes. Multicellular transient arrangements that form during cell intercalation as a result of simultaneous shrinking of several bicellular junctions. They appear at the apical and basal domains of epithelial cells and are resolved via lengthening of the newly formed bicellular junctions.
Septate junctions (SJs). Occluding junctions found in invertebrates. They are located basally to AJs in the ectoderm and apically to AJs in the endoderm. SJs establish the paracellular diffusion barrier to control the diffusion of solutes within the intercellular space and provide structural support to epithelia. SJs are considered somewhat analogous to the (vertebrate) tight junctions.
Tight junctions (TJs). Occluding junctions found in vertebrates and located apically to adherens junctions. TJs function in a similar way to septate junctions, preventing the leakage of solutes and promoting epithelial sealing.
Tricellular junctions (TCJs). These form at the interface of three adjacent epithelial cells. In invertebrates, tricellular adherens junctions (tAJs) are found in the apical plane and are labelled by Sidekick, a homophilic adhesion molecule with immunoglobulin and fibronectin repeats. Tricellular septate junctions (tSJs) form basally to TAJs and contain Anakonda, Gliotactin and M6. TCJs act as signaling hubs, spatial landmarks and sites of high tension in epithelia.
Cell junctions establish robust mechanochemical barriers that are maintained during junction remodeling to preserve tissue homeostasis. This allows the coordination of multicellular, tissue-scale rearrangements such as cell division, intercalation and extrusion. The molecular mechanisms that govern junctional remodeling have been extensively studied in Drosophila. Multidisciplinary pioneering studies combining powerful genetic tools, precise functional manipulation and in vivo quantitative imaging have produced a rich literature of biological strategies driving morphogenesis in Drosophila and across metazoans (Box 2).
Vertebrates and invertebrates both possess adherens junctions (AJs) and focal adhesions (FAs), the composition of which has been conserved throughout evolution (Abedin and King, 2010). Also common to vertebrates and invertebrates, but not discussed in this Review, are gap junctions. Gap junctions are 2-4 nm gaps composed of innexins in invertebrates and of connexins and pannexins (functional homologues of innexins) in vertebrates (Abedin and King, 2010). They form a channel called a connexon through which small molecules, ions and electrical impulses can travel (Beyer and Berthoud, 2018). Invertebrates also have septate junctions (SJs), which are functionally equivalent to vertebrate tight junctions (TJs). The position and composition of TJs and SJs differ; TJs appear apically to AJs, whereas ectodermal SJs are located basally to AJs (Tepass and Hartenstein, 1994). Both SJs and TJs contain transmembrane proteins called claudins that regulate permeability (Garcia et al., 2018). However, TJs are distinguishable from SJs by the presence of the transmembrane protein Occludin, junctional adhesion molecules (JAMs), cytosolic scaffold proteins such as membrane-associated guanylate kinases (MAGUKs) that link transmembrane proteins to cell signaling molecules and the cytoskeleton, and the intracellular zonula occludens like ZO-1 (also known as Tjp1) (the ortholog of which localizes at AJs in invertebrates) (Garcia et al., 2018). TJ proteins must interact with the actin cytoskeleton to maintain TJ structure (Van Itallie and Anderson, 2014). Vertebrate epithelia also possess desmosomes, disc-shaped anchoring junctions composed of two proteins from the cadherin family, desmocollin and desmoglein, that ensure calcium-dependent homophilic adhesion between cells (Rübsam et al., 2018). They are linked to the intermediate filaments via desmoplakin and plakoglobin (Rübsam et al., 2018). Desmosomes enable the formation of intercellular cytokeratin networks that contribute to the architectural cohesion of the epithelial tissue and enable the transmission and damping of mechanical forces exerted on the epithelial cells (Garcia et al., 2018). Interestingly, components of TJs, including JAMs and ZO-1, are required for lumen specification (Iden et al., 2012) and vertebrate neurulation (Yang et al., 2009), respectively, and connexins are required for cardiac morphogenesis (Zheng-Fischhöfer et al., 2006), suggesting that junctional complexes may contribute to morphogenesis in addition to their barrier function.
In this Review, we compile a comprehensive overview of the fundamental mechanisms and latest advances in epithelial dynamics. For the reasons described above, we primarily focus on Drosophila and briefly discuss similarities shared across the animal kingdom. We hope that this article appeals to the multidisciplinary scientific community and sparks new discussions at the junctional interface, the functional crossroad of genetic, mechanical and signaling developmental programs.
Establishment and composition of epithelial cell junctions
Epithelial cell polarity contributes to directed intracellular transport, collective movements and overall epithelia integrity (Buckley and St Johnston, 2022; Rodriguez-Boulan and Macara, 2014). The establishment of structural and functional asymmetries along the apicobasal cell polarity axis results from selective vesicular trafficking and mutual exclusion of protein complexes, known as polarity modules, which have been extensively reviewed (Buckley and St Johnston, 2022; Campanale et al., 2017; Flores-Benitez and Knust, 2016; Pickett et al., 2019; Tepass, 2012; Vasquez et al., 2021). These polarity modules include: the apical Crumbs complex, formed by Crumbs, Stardust, Patj and Lin-7 (also known as Veli) (Crb/Sdt/Patj/Lin-7); the apical Par complex, formed by the apical atypical protein kinase C (aPKC), Par-6, Cdc42 and junctional Par-3 (Baz); and the basolateral Scribble complex, composed of Scribble, Discs large (Dlg1) and Lethal giant larvae [L(2)gl] (Scrib/Dlg/Lgl) (Fig. 1). Biochemical crosstalk between these canonical polarity complexes governs polarization in most epithelia. In addition, together with the FERM protein Yurt, septate junction components including Coracle (Cora), Neurexin IV (Nrx-IV) and Na+/K+ ATPase form a group of epithelial polarity proteins that acts as a negative regulatory component of the Crb complex and stabilizes lateral membranes (Laprise et al., 2006, 2009; Laprise and Tepass, 2011). In embryonic development, the Yurt/Cora group regulates epithelial polarity during organogenesis, although it is not essential when epithelial polarity is first established nor during terminal differentiation (Laprise et al., 2009). In a similar way, in the endoderm-derived Drosophila intestine, the canonical polarity complexes are not required for the establishment of apicobasal polarity and, instead, ECM-mediated adhesion contributes to polarization (Chen et al., 2018).
Junctional complexes are found at epithelial cell-cell and cell-ECM interfaces. Schematic showing the junctions between three epithelial cells and the extracellular matrix (ECM). The components and relative locations of these different junctions are detailed in black text to the right of the schematic. The locations of the polarity complexes are indicated in blue text. Epithelial cells exhibit apicobasal polarity, which arises from the mutual exclusion of different polarity complexes along the plasma membrane. The apical Crumbs polarity complex is formed by Crb/Sdt/Patj/Lin-7 and interacts with core determinants of the apical Par complex formed by aPKC/Par-6/Cdc42 and junctional Par-3. aPKC/Par-6/Cdc42 acts in a mutually antagonistic manner with the basolateral Scribble polarity complex formed by Scrib/Dlg/Lgl. Upon establishment of apicobasal polarity, junctional complexes are assembled at specific locations in the plasma membrane. Bicellular junctions (BCJs) include apical bicellular adherens junctions (blue rectangles), formed by E-cad-Catenin complexes, and basolateral bicellular septate junctions (red ribbons), formed by proteins including Coracle, Nervana 2 and Neurexin IV. At vertices, tricellular junctions (TCJs) include apical tricellular adherens junctions (blue circle) marked with Sidekick and basolateral tricellular septate junctions (red circles) assembled by the interplay between Anakonda, Gliotactin and M6. Focal adhesions (pink pawns) mediate mechanotransduction and signaling via heterodimeric integrin receptors that link the intracellular actin cytoskeleton to the ECM.
Junctional complexes are found at epithelial cell-cell and cell-ECM interfaces. Schematic showing the junctions between three epithelial cells and the extracellular matrix (ECM). The components and relative locations of these different junctions are detailed in black text to the right of the schematic. The locations of the polarity complexes are indicated in blue text. Epithelial cells exhibit apicobasal polarity, which arises from the mutual exclusion of different polarity complexes along the plasma membrane. The apical Crumbs polarity complex is formed by Crb/Sdt/Patj/Lin-7 and interacts with core determinants of the apical Par complex formed by aPKC/Par-6/Cdc42 and junctional Par-3. aPKC/Par-6/Cdc42 acts in a mutually antagonistic manner with the basolateral Scribble polarity complex formed by Scrib/Dlg/Lgl. Upon establishment of apicobasal polarity, junctional complexes are assembled at specific locations in the plasma membrane. Bicellular junctions (BCJs) include apical bicellular adherens junctions (blue rectangles), formed by E-cad-Catenin complexes, and basolateral bicellular septate junctions (red ribbons), formed by proteins including Coracle, Nervana 2 and Neurexin IV. At vertices, tricellular junctions (TCJs) include apical tricellular adherens junctions (blue circle) marked with Sidekick and basolateral tricellular septate junctions (red circles) assembled by the interplay between Anakonda, Gliotactin and M6. Focal adhesions (pink pawns) mediate mechanotransduction and signaling via heterodimeric integrin receptors that link the intracellular actin cytoskeleton to the ECM.
Upon apicobasal polarity establishment, bicellular and tricellular junctions arise (BCJs and TCJs, respectively; see Glossary, Box 1) at the interface of adjacent cells. Epithelial cells present ubiquitous adherens (AJs) and septate (SJs) junctions and, wherever ECM is present, focal adhesions (FAs) (Fig. 1) (see Glossary, Box 1).
Adherens junctions ensure the mechanical barrier function
AJs are cell-cell junctions that physically couple neighboring epithelial cells to ensure the integrity of the epithelial sheet throughout development (Harris and Tepass, 2010; Oda and Takeichi, 2011). In ectodermally-derived epithelia, AJs are located apically, below the Crumbs complex, where they form an adhesive belt around the cell circumference (Fig. 1) (Harris and Tepass, 2010). AJs are formed by E-cadherin (E-cad)-Catenin complexes. The extracellular domains of the E-cads interact in trans with the extracellular domain of neighboring E-cads and in cis with E-cads in the same cell to provide adhesion at the surface of neighboring cells (Brasch et al., 2012; Troyanovsky, 2023). On the cytosolic side, E-cad binds to β-catenin; in turn, β-catenin binds to α-catenin, and α-catenin binds to the actomyosin cytoskeleton (Harris and Tepass, 2010; Yonemura, 2011).
Septate junctions establish the paracellular diffusion barrier
SJs are located at the lateral domain of epithelial cells and establish the paracellular diffusion barrier in invertebrate epithelia. This barrier provides a seal between epithelial cells by preventing leakage of solutes and is an essential component of host defense (Tepass et al., 2001). SJs are functionally equivalent to, but morphologically and molecularly different from, vertebrate tight junctions (see Glossary, Box 1). SJs appear as ladder-like structures under electron microscopy (Hand and Gobel, 1972) (Fig. 1). SJ components and their localization patterns differ in tissues derived from different germ layers; ectodermal tissues contain pleated SJs, located below the AJ belt (Tepass and Hartenstein, 1994), whereas smooth SJs are found in endoderm-derived tissues, above the AJs (Banerjee et al., 2006; Tepass and Hartenstein, 1994). Here, we focus on pleated SJs (henceforth referred to simply as SJs), as they are more abundant and better characterized than smooth SJs.
SJ components are biochemically diverse and can be grouped into core proteins, resident proteins and accessory proteins (Rice et al., 2021). Core proteins form a highly stable multiprotein complex, as evidenced by fluorescence recovery after photobleaching (FRAP) experiments that revealed the slow kinetics of these proteins (Oshima and Fehon, 2011). Highly immobile core proteins include cell-cell adhesion proteins such as Neuroglian, Fasciclin 2 and 3 (Fas2 and Fas3), Nrx-IV, Claudin-like proteins such as Kune-Kune, subunits of ion transporter channels such as ATPα and Nervana 2, and cytoplasmic adaptors such as Cora (Fig. 1) (Fehon et al., 1994; Laprise et al., 2009; Oshima and Fehon, 2011; Rice et al., 2021). By contrast, resident proteins are located at the SJ level and are highly mobile, not being required for the stability of the core protein complex, and comprise members of the Scribble polarity complex Dlg, Scrib and Lgl (Izumi and Furuse, 2014; Laprise et al., 2009; Oshima and Fehon, 2011). Accessory proteins contribute to SJ integrity but do not reside at the junction and include the Ly6 proteins Coiled (Cold), Crooked (Crok) and Boudin (Bou) (Hijazi et al., 2009; Nilton et al., 2010; Tempesta et al., 2017).
As discussed above, SJ core components Cora, Nrx-IV and ATPα act in concert with Yurt to help stabilize the basolateral membrane and are required for epithelial polarity during organogenesis (Laprise et al., 2009; Ward et al., 2001), whereas Dlg contributes to cellular growth in larval development (Po et al., 2008; Ward et al., 2001). This suggests that SJ function may expand beyond the formation of the paracellular diffusion barrier. This idea is further explored in the following sections.
Focal adhesions mediate mechanosensory crosstalk with the extracellular matrix
FAs are electron-dense structures found basal to SJs at ECM adhesion sites (Fig. 1). They regulate mechanosensory crosstalk between the ECM and the basal F-actin stress fibers of the cell. FAs are formed by multi-protein assemblies containing integrins (Kanchanawong and Calderwood, 2022). Integrins are heterodimeric transmembrane receptors formed by a specific combination of α and β subunits that are assembled upon substrate-specific interaction with ECM components such as collagen and fibrinogen (Zaidel-Bar et al., 2007). They interact via their intracellular domains with other FA components, namely Talin (Rhea) and Paxillin, two adaptor proteins that in turn mediate linkage with the actin cytoskeleton and are required for integrin function (Chastney et al., 2021). Additional components, such as Vinculin, α-Actinin and the Focal adhesion kinase (FAK) act as cortical adaptors, actin regulators and mechano-signaling modules. Altogether, FAs govern dynamic bi-directional signal transduction between internal cues, such as cytoskeleton organization, and external cues, such as rigidity of the underlying ECM (Yamaguchi and Knaut, 2022). Moreover, they do so in a context-specific manner, and modulate mechanochemical signaling during morphogenesis in response to internal and external biomechanical stimuli (Yamaguchi and Knaut, 2022). The initial assembly of nascent adhesions is followed by their maturation; this process is guided by ECM stimuli and cytoskeleton reorganization, progressively stabilizing the adhesions and forming mature FAs (Chastney et al., 2021; Pines et al., 2012).
Dynamics of epithelial cell junctions
During junction assembly, newly synthesized AJ, SJ and FA transmembrane proteins are transported from the endoplasmic reticulum to the Golgi apparatus and sorted at the trans-Golgi network (Fig. 2). Post-Golgi vesicles are then delivered to the plasma membrane via the exocyst complex (Fig. 2, arrow 1). Directed trafficking is facilitated by the establishment of cell polarity and is essential for the formation of junctions (for reviews, see Buckley and St Johnston, 2022; Rodriguez-Boulan and Macara, 2014). Photoconversion experiments have revealed that SJ components are delivered between AJs and mature SJs (Babatz et al., 2018; Daniel et al., 2018). As the flux of SJ proteins, newly synthesized or recycled, continues to be trafficked towards the plasma membrane, SJ components are displaced in an apical-to-basal direction, ultimately assembling mature SJs (Babatz et al., 2018; Daniel et al., 2018).
Selective vesicular trafficking governs junctional remodeling in polarized epithelial cells. The secretory pathway (1, red arrows) delivers transmembrane proteins to the plasma membrane via the exocyst complex, formed by eight evolutionarily conserved subunits, one of which interacts with Rab11. The endocytic pathway (2) modulates the dynamic internalization of junctional complexes. It requires adaptor proteins to promote the budding of the plasma membrane, forming non-Clathrin-coated (orange arrow) or Clathrin-coated (green arrow) vesicles that undergo Dynamin-mediated scission from the plasma membrane, forming Rab5-coated early endosomes (3, yellow arrow). Based on environmental stimuli and cargo nature, regulator proteins redirect vesicles toward recycling or degradation pathways that exhibit distinct turnover kinetics. Rab4 mediates fast protein recycling (4, purple arrow), bypassing the sorting endosome, whereas Rab11 (5, blue arrow) and the retromer complex (6, pink arrow) regulate slow recycling of cargo. The retromer complex is composed of two subcomplexes formed by sorting nexin (SNX) proteins and vacuole protein sorting (VPS) components, namely VPS26, VPS29 and VPS35. The ESCRT machinery is composed of four complexes acting in tandem (0, I, II, III) that, together with the ATPase Vacuolar protein sorting 4 (Vps4), regulate lysosomal degradation (7, grey arrow) of cargo, ultimately forming Rab7-coated late endosomes (LE) and multivesicular bodies (MVB). ER, endoplasmic reticulum.
Selective vesicular trafficking governs junctional remodeling in polarized epithelial cells. The secretory pathway (1, red arrows) delivers transmembrane proteins to the plasma membrane via the exocyst complex, formed by eight evolutionarily conserved subunits, one of which interacts with Rab11. The endocytic pathway (2) modulates the dynamic internalization of junctional complexes. It requires adaptor proteins to promote the budding of the plasma membrane, forming non-Clathrin-coated (orange arrow) or Clathrin-coated (green arrow) vesicles that undergo Dynamin-mediated scission from the plasma membrane, forming Rab5-coated early endosomes (3, yellow arrow). Based on environmental stimuli and cargo nature, regulator proteins redirect vesicles toward recycling or degradation pathways that exhibit distinct turnover kinetics. Rab4 mediates fast protein recycling (4, purple arrow), bypassing the sorting endosome, whereas Rab11 (5, blue arrow) and the retromer complex (6, pink arrow) regulate slow recycling of cargo. The retromer complex is composed of two subcomplexes formed by sorting nexin (SNX) proteins and vacuole protein sorting (VPS) components, namely VPS26, VPS29 and VPS35. The ESCRT machinery is composed of four complexes acting in tandem (0, I, II, III) that, together with the ATPase Vacuolar protein sorting 4 (Vps4), regulate lysosomal degradation (7, grey arrow) of cargo, ultimately forming Rab7-coated late endosomes (LE) and multivesicular bodies (MVB). ER, endoplasmic reticulum.
Once assembled, selective intracellular trafficking of junctional components can promote junctional remodeling (Sigismund et al., 2021). In response to mechanochemical stimuli, junctional complexes may be either locally reinforced, or disassembled and internalized (Iyer et al., 2019; Kanchanawong and Calderwood, 2022; Pinheiro and Bellaïche, 2018). For example, in AJs, E-cad mechanosensitive turnover takes place: under low tension conditions, p120 and β-catenin bind directly to E-cad to promote its stability, whereas mechanical stress induction promotes E-cad endocytosis (Bulgakova et al., 2013; Iyer et al., 2019). E-cad endocytosis is well characterized and has been proposed to promote the disruption of homophilic E-cad interaction in trans (de Beco et al., 2009; Troyanovsky et al. 2006). However, this issue is not entirely elucidated as these E-cad trans interactions could, on the contrary, inhibit endocytosis (West and Harris, 2016).
During the disassembly of junctional complexes, proteins take different endocytic routes to be ultimately recycled back to the plasma membrane or targeted for degradation. Endocytic routes (Fig. 2, arrow 2) include Clathrin-dependent pathways, used by AJ, SJ and FA components, and Clathrin-independent routes, used mainly by FA components (Baum and Georgiou, 2011; Ezratty et al., 2009; Tiklová et al., 2010). Both routes require a variety of adaptor proteins that sort the rich diversity of internalized cargo. These adaptors form cages around plasma membrane invaginations to stabilize the budding vesicles and help recruit Dynamin, an ATPase that mediates vesicle scission (Sigismund et al., 2021).
The fate of these newly formed endocytic vesicles and their cargo is regulated by a diverse set of lipids (Pepperl et al., 2013) and proteins, including small Rho GTPases and membrane-associated Ras-like GTPases (Ellis and Mellor, 2000). Endocytic internalized vesicles are initially coated with regulatory Ras in brain (Rab) proteins, namely Rab5 (Fig. 2, arrow 3). Rab5-coated vesicles fuse with early endosomes (EEs) to sort cargoes and direct them towards recycling or degradation pathways (Rink et al., 2005). Rab4-positive endocytic vesicles undergo fast recycling (Fig. 2, arrow 4) and return cargo back to the plasma membrane, assisting the weakening of cell-cell contacts and facilitating epithelial remodeling during organ morphogenesis and cell migration (de Madrid et al., 2015). Meanwhile, Rab11-coated sorting endosomes (SEs) mediate slow cargo recycling (Fig. 2, arrow 5) of junctional proteins back to the plasma membrane during cell division and migration (Powelka et al., 2004). Recycling of Claudins is mediated by the retromer retrieval system (Fig. 2, arrow 6) via the trans-Golgi network (Pannen et al., 2020). If the SE cargo is to be degraded, the endosomal sorting complex required for transport (ESCRT) (Fig. 2, arrow 7) progressively forms late endosomes and multivesicular bodies fated to lysosomal degradation (Gatta and Carlton, 2019; Pannen et al., 2020; Seaman, 2021).
ESCRT components have been classified as neoplastic tumor suppressor genes and their mutation leads to tumor formation and invasion (reviewed by Hariharan and Bilder, 2006). The molecular mechanisms leading to this malignant behavior have recently been established. Loss of ESCRT components mis-localizes the retromer machinery, disrupting trafficking of SJ components, therefore impairing diffusion barrier functions (Pannen et al., 2020) and promoting massive cell signaling deregulation and tumor invasiveness (Moberg et al., 2005; Thompson et al., 2005; Vaccari and Bilder, 2005). This suggests that SJ components may play additional roles in epithelia homeostasis, beyond forming the diffusion barrier. Indeed, loss of SJ components in early- and mid-embryogenesis (from gastrulation to organogenesis) results in defective morphogenesis and embryonic lethality (Baumgartner et al., 1996; Behr et al., 2003; Fehon et al., 1994; Genova and Fehon, 2003; Perrimon, 1988). Lethality results from aberrant cell shape, deregulated expression and localization of both AJ and actomyosin components and defective cell adhesion. This suggests that SJs may in fact be required to coordinate cohesive cell rearrangements required for embryogenesis (Rice et al., 2021; Sawyer et al., 2011). There also appears to be an interplay between apical and basolateral junctional complexes during morphogenesis, as basal loss of FAs during embryogenesis alters both apical Myosin dynamics and AJ-mediated adhesion, whereas aberrant cell-cell adhesion promotes FA assembly (Goodwin et al., 2016, 2017).
In summary, epithelial cells exhibit different types of cell junctions that engage in distinct cortical behaviors to organize cell packing geometry and orchestrate tissue morphogenesis. These junctions are dynamic, and we explore next how junctions are remodeled during cell division, intercalation and extrusion.
Junctional remodeling during cell division
Epithelial cell division requires extensive and dynamic junctional remodeling to sequentially drive disassembly and de novo assembly of junctional complexes while preserving cell polarity and mechanochemical barriers (Ragkousi and Gibson, 2014). Tissue integrity in proliferating epithelia is challenged at every round of division, with cytokinesis rendering the tissue highly susceptible to mechanical stress. Electron microscopy studies in the Drosophila embryonic ectoderm show that gaps form at the site of furrow ingression (Guillot and Lecuit, 2013), and disrupting junction-cytoskeleton interactions during mitotic junctional remodeling in early gastrulation forms large gaps in the epithelium (Manning et al., 2019). Similarly, in the Drosophila follicular epithelium, disrupting cell polarity produces gaps between the dividing and neighboring cells, possibly due to a deregulation of cortical tension and adhesion (Osswald et al., 2022).
A characteristic feature of epithelial cytokinesis is the formation of a new adhesive interface between daughter cells. This multistep process involves cell shape changes such as cell rounding and membrane ingression, together with de novo junction formation and regulation of the final length of the newly formed interface (Fig. 3A-I). In the Drosophila embryonic ectoderm, follicular epithelium and pupal notum, live-imaging and genetic studies have revealed that, in the dividing cell, the cytokinetic actomyosin ring is anchored to AJs during cell rounding (Fig. 3F) (Founounou et al., 2013; Guillot and Lecuit, 2013; Herszterg et al., 2013; Morais-De-Sá and Sunkel, 2013). Pulling forces exerted at this location are sufficient to override opposing tensile forces exerted by neighboring cells, promoting the cleavage of the dividing cell (Founounou et al., 2013; Guillot and Lecuit, 2013; Herszterg et al., 2013; Morais-De-Sá and Sunkel, 2013) (Fig. 3G). During this process, neighboring cells remain connected to the dividing cell, resulting in the ingression of their membrane towards the center of the dividing cell and the formation of finger-like protrusions (FLPs) (Fig. 3G; see Glossary, Box 1) (Daniel et al., 2018; Wang et al., 2018). Cell junctions between the dividing cell and its neighbors are progressively remodeled, with constriction of the cytokinetic ring in the dividing cell acting as a mechanical signal that promotes local dismantling of E-cad-Catenin complexes, and thus AJs, at the ingressing membrane (Pinheiro et al., 2017) (Fig. 3G). Concomitantly, in neighboring cells, junctional Myo-II (see Glossary, Box 1) locally detaches from the cortex and accumulates at the base of FLPs (Founounou et al., 2013; Herszterg et al., 2013). A self-organized retrograde flow of Myo-II in the neighboring cells moves from the tip towards the base of FLPs, triggering the local recruitment of the Rho guanine exchange factor Cysts (RhoGEF Cysts) (Fig. 3G) (di Pietro et al., 2023; Pinheiro and Bellaïche, 2018). RhoGEF Cysts promotes local activation of the Rho small GTPase Rho1, which activates the Myo-II at the base of FLPs to locally increase cortical tension (Fig. 3G) (di Pietro et al., 2023). Activated junctional Myo-II accumulation acts as a mechanical clamp and keeps the interface of daughter cells juxtaposed (Founounou et al., 2013; Guillot and Lecuit, 2013; Herszterg et al., 2013) (Fig. 3G). In the dividing cell, the Rho guanine exchange factor RhoGEF4 localizes at the midbody (see Glossary, Box 1) and along the daughter cell interface (Fig. 3H). RhoGEF4 locally activates the Rho small GTPase Rac, which drives Arp2/3-dependent F-actin polymerization along the membranes of the daughter cells (Fig. 3H) (di Pietro et al., 2023; Herszterg et al., 2013; Pinheiro and Bellaïche, 2018). Withdrawal of FLPs leads to the apposition of daughter cell membranes and initiates de novo AJ formation, which in turn contribute to regulating the final length of the newly formed interface between daughter cells (Fig. 3I) (di Pietro et al., 2023; Founounou et al., 2013; Herszterg et al., 2013).
Remodeling junctional complexes during cytokinesis ensures tissue growth and preserves tissue integrity. (A-M) Schematic representations of epithelial cells during cytokinesis in pupal (A-I) and larval (J-M) epithelia across time, depicting adherens junctions (AJs; blue rectangles) and septate junctions (SJs; red ribbons). Tricellular AJs are represented by blue circles and tricellular SJs are represented by pink circles. (A-I) In the pupal notum, AJs anchor the actomyosin cytokinetic ring (C and F, green oval) to the plasma membrane. During division, AJs also ensure adhesion between the dividing (E, pink hexagon) and neighboring (E, grey pentagons) cells. Cytokinetic ring constriction promotes membrane ingression and the formation of finger-like protrusions (FLPs) in the plane of the SJs (G). Neighboring cells remain attached to the dividing cell and a flux of myosin (G, black arrows) is generated from the tip to the base of the FLPs. Myo-II and Cysts are recruited to the base of the FLPs, where Cysts activates Rho1. Rho1, in turn, activates Myo-II, forming a mechanical clamp (G, yellow boxes) in the neighboring cells that allows the neighboring cell membrane and the dividing cell membrane to be apposed along FLPs (G). In the dividing cell, RhoGEF4 (H, orange boxes) is recruited along the daughter cell membrane interface, activating Rac and Arp2/3-Wasp signaling pathways. The resulting polymerized and branched F-actin generate a force that promotes withdrawal of the neighbor cell membranes, enabling de novo AJ assembly between daughter cells (H,I). The final length of the daughter cell interface is tightly regulated (I). (J-M) In the columnar epithelium of the larval wing imaginal disc, mitotic cell rounding results in the basal-to-apical movement of the dividing cell. During this process, contact to the basement membrane is maintained by the formation of a basal foot (K). This foot may function to retain cell adhesion to the extracellular matrix (orange) and help the reintegration of the daughter cells into the epithelial layer (L, black arrow) once division is completed (M).
Remodeling junctional complexes during cytokinesis ensures tissue growth and preserves tissue integrity. (A-M) Schematic representations of epithelial cells during cytokinesis in pupal (A-I) and larval (J-M) epithelia across time, depicting adherens junctions (AJs; blue rectangles) and septate junctions (SJs; red ribbons). Tricellular AJs are represented by blue circles and tricellular SJs are represented by pink circles. (A-I) In the pupal notum, AJs anchor the actomyosin cytokinetic ring (C and F, green oval) to the plasma membrane. During division, AJs also ensure adhesion between the dividing (E, pink hexagon) and neighboring (E, grey pentagons) cells. Cytokinetic ring constriction promotes membrane ingression and the formation of finger-like protrusions (FLPs) in the plane of the SJs (G). Neighboring cells remain attached to the dividing cell and a flux of myosin (G, black arrows) is generated from the tip to the base of the FLPs. Myo-II and Cysts are recruited to the base of the FLPs, where Cysts activates Rho1. Rho1, in turn, activates Myo-II, forming a mechanical clamp (G, yellow boxes) in the neighboring cells that allows the neighboring cell membrane and the dividing cell membrane to be apposed along FLPs (G). In the dividing cell, RhoGEF4 (H, orange boxes) is recruited along the daughter cell membrane interface, activating Rac and Arp2/3-Wasp signaling pathways. The resulting polymerized and branched F-actin generate a force that promotes withdrawal of the neighbor cell membranes, enabling de novo AJ assembly between daughter cells (H,I). The final length of the daughter cell interface is tightly regulated (I). (J-M) In the columnar epithelium of the larval wing imaginal disc, mitotic cell rounding results in the basal-to-apical movement of the dividing cell. During this process, contact to the basement membrane is maintained by the formation of a basal foot (K). This foot may function to retain cell adhesion to the extracellular matrix (orange) and help the reintegration of the daughter cells into the epithelial layer (L, black arrow) once division is completed (M).
The midbody is initially located in the AJ plane before being displaced basally in the SJ plane (Daniel et al., 2018; Wang et al., 2018). This apical-to-basal displacement is not yet well understood, and it remains unclear whether the midbody is first anchored exclusively to AJs, or whether it interacts with both AJ and SJ components. This raises the question as to how junctional complexes are remodeled in regards to each other. AJs and SJs have distinct turnover kinetics, with SJs being far less dynamic, hence more stable, than AJs (Oshima and Fehon, 2011). This suggests that SJs may impose the pace at which cell division progresses in order to ensure barrier function maintenance. It has been reported that apical-to-basal displacement of the midbody is concomitant to de novo AJ and SJ assembly and to SJ maturation (Daniel et al., 2018; Wang et al., 2018). FRAP experiments have revealed that SJ components found in FLPs correspond to ‘old’ SJs assembled before the onset of division, whereas de novo SJs assemble above the midbody (Bruelle et al., 2023; Daniel et al., 2018). Loss-of-function studies indicate that the basal displacement of the midbody is driven by SJ assembly and maturation (Daniel et al., 2018; Wang et al., 2018). Once FLPs are resolved, the midbody is released and the physical separation of daughter cells occurs (Bruelle et al., 2023; Daniel et al., 2018; Wang et al., 2018).
The contribution of SJs to the progression of cell division may not be restricted to bicellular interfaces. Anakonda (Aka; also known as Bark), which localizes at tricellular SJs in interphase cells, is recruited at the apical side of FLPs, acting perhaps as a spatial landmark for de novo TCJ assembly (Esmangart de Bournonville and Le Borgne, 2020). Interestingly, the loss of Aka halts midbody basal displacement and prevents FLP resolution between daughter cells and their neighbors (Daniel et al., 2018; Wang et al., 2018). This suggests that the interplay between SJs at BCJs and TCJs recently described in interphase cells (Esmangart de Bournonville and Le Borgne, 2020) may also be required to successfully complete cytokinesis.
Although the impact of AJ and SJ remodeling during cytokinesis is being decrypted, the contribution of FAs and basal interfaces to this process remains poorly understood. In isolated vertebrate cells, transport of integrins to and from the cleavage furrow was reported to be required for cytokinesis completion (Pellinen et al., 2008). A recent study using atomic force microscopy in human cell culture revealed that, in mitosis, integrins actually reduce cell-ECM contact while strengthening cell-cell adhesion, indicative of an interplay between AJs and FAs to ensure integrity of the cell layer (Huber et al., 2023). Surprisingly, to our knowledge, the localization and role of Drosophila integrins during cytokinesis are unknown. In the Drosophila midgut, during intestinal stem cell division integrin-mediated adhesion to the basal ECM orchestrates cell-intrinsic asymmetric segregation of Par-3, Par-6 and aPKC towards the apical domain in daughter cells (Goulas et al., 2012). In the Drosophila wing disc (Fig. 3J-M), en-face bloc scanning electron microscopy revealed that a basal foot remains in the dividing cell, preserving contact with the underlying ECM (Fig. 3K,L) (Daniel et al., 2018). The role of this foot and its relation to FA-mediated ECM contact is currently unknown. The basal foot has also been observed in vertebrates, where it has been proposed to act as a cue and ensure divided cells remain in the epithelium after cytokinesis completion (Guevara-Garcia et al., 2023). Further work is required to elucidate the possible contribution of FAs to cell division and to establish a hierarchy of junctional remodeling during cytokinesis.
Junctional remodeling during cell intercalation
Planar-polarized cell intercalation (see Glossary, Box 1) is the process of neighbor interchange in the plane of the epithelium (Fig. 4). It requires junctional remodeling together with intricate interplay between adhesion, force generation and force transmission to preserve tissue cohesiveness during collective cell movements. This process has been extensively studied across species, in both vertebrates and invertebrates, revealing convergent-extension movements to be essential and, in some cases, sufficient for the elongation of the embryo body axis (Irvine and Wieschaus, 1994). A combination of live imaging and quantitative high throughput analyses in the Drosophila embryo have revealed how the complementary subcellular organization of AJs and the actomyosin contractile molecular machinery govern cell intercalation at the molecular level (Gilmour et al., 2017; Paré and Zallen, 2020).
Cell intercalation requires junctional remodeling along the apicobasal axis. (A) In pupal nota, progressive shrinkage of a bicellular junction (BCJ) between neighboring cells (light blue) forms transient four-way junctions preceding de novo BCJ assembly and elongation between previously non-neighboring cells (dark blue). This type of cell intercalation is known as T1 transition. The inset shows how the small GTPase Rho1 activates the Rho kinase (Rok) at the contractile interface, increasing junctional Myo-II accumulation and contractility to drive junction shortening. Rok also prevents Par-3 stabilization at the contractile interface, causing Par-3 to become enriched along the adhesive interface. Par-3 reinforces AJ-mediated adhesion by reducing turnover of E-cad-Catenin complexes. (Bi,Bii) A second type of cell intercalation has been described in the pupal nota. Non-adjacent cells undergo neighbor exchange by forming multicellular transient apical and basal rosettes. Apical rosette formation takes place after basal rosette formation via an independent process. (Bi) On the apical plane, several apical BCJs shrink upon contraction of a multicellular actomyosin cable and an apical rosette forms. The apical rosette is resolved upon elongation of new apical BCJs perpendicular to the previously disassembled apical BCJs. (Bii) Wedge-like basolateral protrusions form in non-adjacent cells as multiple basal BCJs shrink and cells move towards each other. As a result, a transient multicellular reorganization known as a basal rosette is formed, which resolves upon basal BCJs elongation. Note that medial Myo-II distribution has been omitted to highlight the formation of basal and apical rosettes upon junction shrinkage and the subsequent resolution of these rosettes upon de novo junction elongation. (C) Basal BCJ shrinkage is driven by small Rho GTPase Rac1, which activates Src42A (pSrc) to promote F-actin polymerization at the cell cortex. (D) Apical BCJ shrinkage is driven by Rho1, which activates Rok to promote the accumulation of junctional Myo-II at the cell cortex.
Cell intercalation requires junctional remodeling along the apicobasal axis. (A) In pupal nota, progressive shrinkage of a bicellular junction (BCJ) between neighboring cells (light blue) forms transient four-way junctions preceding de novo BCJ assembly and elongation between previously non-neighboring cells (dark blue). This type of cell intercalation is known as T1 transition. The inset shows how the small GTPase Rho1 activates the Rho kinase (Rok) at the contractile interface, increasing junctional Myo-II accumulation and contractility to drive junction shortening. Rok also prevents Par-3 stabilization at the contractile interface, causing Par-3 to become enriched along the adhesive interface. Par-3 reinforces AJ-mediated adhesion by reducing turnover of E-cad-Catenin complexes. (Bi,Bii) A second type of cell intercalation has been described in the pupal nota. Non-adjacent cells undergo neighbor exchange by forming multicellular transient apical and basal rosettes. Apical rosette formation takes place after basal rosette formation via an independent process. (Bi) On the apical plane, several apical BCJs shrink upon contraction of a multicellular actomyosin cable and an apical rosette forms. The apical rosette is resolved upon elongation of new apical BCJs perpendicular to the previously disassembled apical BCJs. (Bii) Wedge-like basolateral protrusions form in non-adjacent cells as multiple basal BCJs shrink and cells move towards each other. As a result, a transient multicellular reorganization known as a basal rosette is formed, which resolves upon basal BCJs elongation. Note that medial Myo-II distribution has been omitted to highlight the formation of basal and apical rosettes upon junction shrinkage and the subsequent resolution of these rosettes upon de novo junction elongation. (C) Basal BCJ shrinkage is driven by small Rho GTPase Rac1, which activates Src42A (pSrc) to promote F-actin polymerization at the cell cortex. (D) Apical BCJ shrinkage is driven by Rho1, which activates Rok to promote the accumulation of junctional Myo-II at the cell cortex.
The molecular actors discussed below are specific to cell intercalation in the Drosophila embryo during germband extension, when cells converge along the dorso-ventral axis to promote tissue elongation (Miao and Blankenship, 2020; Rauzi, 2020). However, the process of planar cell intercalation is stereotyped and seems in part conserved across species (reviewed by Walck-Shannon and Hardin, 2014). Intercalation can occur between two or more previously non-neighboring cells and takes place in three main steps: 1) the shrinking of the contractile interface between neighboring cells; 2) junction shortening, which promotes the formation of a transient multicellular organization between neighbors and previously separated cells; 3) junction elongation of the newly established adhesive interface between previously non-neighboring cells (Fig. 4). The first step, junction shrinkage, requires the recruitment of junctional Myo-II and the disassembly of AJs at the contractile interface (Fig. 4A). Junctional Myo-II recruitment is mediated by the activation of Rho1 in intercalating cells, which generates polarized medial Myo-II (see Glossary, Box 1) flows towards the shrinking BCJ(s) and promotes the polarized distribution of the Rho kinase Rok (Fig. 4A) (Garcia De Las Bayonas et al., 2019; Rauzi et al., 2010). Rok instructs polarized enrichment of junctional Myo-II (Fig. 4A) (Levayer and Lecuit, 2012). Rok also promotes polarized localization of Par-3 by preventing its stabilization at the contractile interface(s) (Fig. 4A) (Simões et al., 2010). In turn, Par-3 reinforces the polarized distribution of junctional Myo-II and promotes the stabilization of E-cadherin, α-catenin and β-catenin at the non-contractile adhesive cell interface(s) (Fig. 4A) (Davey and Moens, 2017; Kasza et al., 2014; Simões et al., 2010; Tetley et al., 2016; Vichas and Zallen, 2011). Subsequent activation of the polarized junctional Myo-II promotes shortening of the contractile interface(s), and the unidirectionality of this process is ensured by Rab35-, Clathrin- and Dynamin-dependent endocytosis of E-cad (Jewett et al., 2017; Levayer et al., 2011; Rauzi et al., 2010).
As a result of junction(s) shrinkage, two different types of transient cell reorganizations are formed. When a single junction shrinks and four cells appear to converge at a single point, the rearrangement is termed a four-way junction (see Glossary, Box 1) and the process of cell intercalation is referred to as a T1-transition (Fig. 4A) (Bertet et al., 2004). The four-way junction is not a junction per se, as E-cad complexes mediate homophilic interaction and adhesion exclusively between two cells, making convergent contact between dorso-ventral and anterior-posterior cells mutually exclusive (Rauzi, 2020). If, on the other hand, not one but multiple junctions shrink and converge simultaneously, the multicellular rearrangement that arises is known as a rosette (Fig. 4B; see Glossary, Box 1) (Blankenship et al., 2006). At sites of elevated mechanical tension, junctional Myo-II is recruited and then activated, promoting further enrichment of activated junctional Myo-II and ultimately increasing mechanical tension at contractile interfaces (Fernandez-Gonzalez et al., 2009). AJ-mediated force transmission between neighboring cells forms a multicellular actomyosin cable that, upon contraction, results in rosette formation (Fig. 4Bi). In addition to Rok activity, rosette formation requires the activity of Abelson (Abl) a tyrosine kinase localizing along contractile interfaces. Abl promotes AJ remodeling at contractile interfaces by increasing β-catenin turnover which, together with Myo-II activity, progressively shortens convergent junctions (Tamada et al., 2012).
Both four-way junctions and rosettes are resolved via cycles of junctional Myo-II dis/assembly induced by medial actomyosin contractile pulses (Bertet et al., 2004; Blankenship et al., 2006). Endocytosis of E-cad at the contractile interface(s) and de novo assembly of BCJs and TCJs at the adhesive interface(s) result in neighbor exchange and tissue elongation (Iyer et al., 2019; Jewett et al., 2017; Levayer et al., 2011; Shaye et al., 2008; Warrington et al., 2013). A new interface(s) of contact between previously non-neighboring cells emerges along the orthogonal axis and the final length of contact is tightly regulated by junctional Myo-II stabilization (Herszterg et al., 2013). Interplay between contractile and adhesive machinery effectively drives higher order tissue-scale deformations such as tissue elongation through cell intercalation, showcasing the major role junction remodeling plays in morphogenesis (Fernandez-Gonzalez et al., 2009; Yu and Zallen, 2020).
Relative contribution of apical and basal junctional remodeling during intercalation
Although AJ remodeling has a well-established role in cell intercalation (Cavanaugh et al., 2020; Lecuit and Yap, 2015; Magie et al., 2002; Sumi et al., 2018; Yap et al., 2018), the contribution of basolateral membrane surfaces is not so well characterized. A recent in vivo study in the prospective ectoderm of the Drosophila embryo revealed that wedge-like basolateral protrusions and basal rosettes (Fig. 4Bii) form before apical rosettes and behave independently of the apical contractility machinery located in the AJ plane (Sun et al., 2017). Time-lapse imaging using fluorescently tagged probes for actin binding partners and specific phospholipids known to be enriched in actin-polymerization sites showed that basolateral protrusions are enriched in actin at the migrating front (Sun et al., 2017). This is due to the activation of small Rho GTPase Rac1 (Krause and Gautreau, 2014). Rac1 activates Src42A (pSrc), a non-specific protein tyrosine kinase that, in turn, induces actin polymerization and the formation of wedge-like basolateral protrusions (Fig. 4C) (Sun et al., 2017). These basolateral protrusions have also been observed during intercalation in other organisms including vertebrates, suggesting they have an important role in this process (Munro and Odell, 2002; Shih and Keller, 1992; Williams-Masson et al., 1998). Basolateral protrusions migrate towards each other and form a transient basal rosette, which becomes resolved upon extension of newly formed basal BCJs (Fig. 4Bii). Meanwhile, at the apical pole of intercalating cells, Rho1 activates Rok, inducing accumulation of junctional Myo-II at the apical cell cortex (Garcia De Las Bayonas et al., 2019) (Fig. 4D). Junction shrinkage of apical BCJs results in formation of an apical rosette, which is resolved upon lengthening of de novo apical BCJs (Fig. 4Bi). Sun et al. (2017) focused on the period from gastrulation to germ band extension, when SJ components are expressed but not yet assembled into stable SJs and the diffusion barrier is not yet functional. As extensive topological rearrangements are required for embryogenesis, this observation raises the possibility that SJs are kept as immature, mobile complexes to enable cell intercalation. In this line, once assembled into stable, mature complexes, SJs could act as regulators of cell movements, preventing or hampering unwanted topological remodeling in order to preserve tissue homeostasis.
The Drosophila egg chamber is also a useful model for exploring the relative contribution of basal and apical surfaces to cell intercalation, as it presents FAs but lacks mature SJs until late vittelogenesis (Isasti-Sanchez et al., 2021). In this tissue, FA components including integrins, Talin and Paxillin are required to promote assembly of junctional Myo-II on basal F-actin stress fibers via downstream effectors Parvin and Integrin-linked kinase (ILK), ultimately generating oscillatory pulses of Myo-II that drive oogenesis (He et al., 2010; Keramidioti et al., 2022). Depleting ECM components or integrins results in defects in cell intercalation, suggesting that basal interfaces play an important role in this process (Lovegrove et al., 2019; Van De Bor et al., 2021). Interestingly, Fas2 and Fas3, two SJ components, are expressed along all cell contacts in the germarium but are downregulated during cell intercalation of stalk cells, a group of cells separating egg chambers at different developmental stages (Lovegrove et al., 2019). This observation suggests alternative roles for SJ components beyond their barrier functions and a requirement for active SJ remodeling during cell intercalation (Keramidioti et al., 2022; Lovegrove et al., 2019).
Basolateral junctional remodeling also appears to be essential during intercalation in mature epithelia. Epithelia found at larval and later stages of development, such as the wing imaginal disc, contain mature AJs, SJs and FAs with an enveloping ECM, and exhibit a polarity axis that spans across the tissue (Tripathi and Irvine, 2022). In the pupal wing, ECM remodeling mediates the actomyosin machinery responsible for driving cell intercalation (Diaz-de-la-Loza et al., 2018; Lecuit and Yap, 2015).
The role of tricellular junctions in intercalation
TCJs have become a major subject of study in the intercalation field and are now regarded as active molecular remodeling hubs during cell intercalation rather than passive geometric entities (Vanderleest et al., 2018). Proteins spatially restricted to TCJs, such as M6, or enriched at TCJs in a context-dependent manner, such as Canoe and Ajuba in high-tension environments, contribute to reorganize the cell cortex during cell intercalation (Ikawa et al., 2023). In the prospective ectoderm of the Drosophila embryo, the tyrosine kinase Ajuba specifically phosphorylates Canoe, an actin-binding protein linked to AJs (Yu and Zallen, 2020). This promotes the enrichment of Canoe at TCJs, which is important for the correct resolution of cell intercalation (Yu and Zallen, 2020). In the Drosophila pupal wing epithelium, M6, a proteolipid component of tricellular SJs, reduces Ajuba levels at TCJs (Ikawa et al., 2023). This, in turn, leads to reduced levels of Canoe at TCJs and thus weakens the link between AJs and the cytoskeleton to help resolve transient four-way junctions and promote proper intercalation (Ikawa et al., 2023). This suggests that, even in mature epithelia, such as that found in the pupal wing, tricellular SJs may contribute to epithelial homeostasis in ways that go beyond their paracellular diffusion barrier function (Ikawa et al., 2023).
Apical TCJ components also play important roles in cell intercalation. It appears that the apical TCJ component Sidekick (Sdk) governs later steps of neighbor exchange. In the pupal eye, an epithelium with mature AJs, SJs, FAs and ECM (Finegan et al., 2019; Letizia et al., 2019; Uechi and Kuranaga, 2019), Sdk is required to specifically localize actin and junctional Myo-II at de novo TCJs, promoting force transmission from the actomyosin network to the cell periphery and ultimately regulating junction lengthening (Uechi and Kuranaga, 2019). In both pupal eyes and the adult nota, Sdk directly associates with either Polychaetoid (to favor actin binding and cell contraction) or with the WAVE regulatory complex (to promote actin branching and cell extension), which also contributes to regulating the final length of the interface (Finegan et al., 2019; Letizia et al., 2019; Malin et al., 2022; Uechi and Kuranaga, 2019).
Junctional remodeling during tissue invagination, cell ingression and cell extrusion
The deviation of a cell or group of cells from the epithelial layer requires coordinated cell behaviors to preserve barrier functions (Fig. 5). Distinct modes of epithelial cell movements named invagination, ingression and extrusion (see Glossary, Box 1) have been well described during embryogenesis (Miao and Blankenship, 2020). To illustrate how cell-cell contacts are remodeled during epithelial morphogenesis, we have chosen to focus on three distinct processes which are well characterized at the molecular, cellular and tissue level: 1) the invagination of the mesoderm primordium during gastrulation, which involves the ventral folding of a subset of living cells and their subsequent detachment and migration to form a new germ layer (Font-Noguera et al., 2021); 2) the ingression (often also described as delamination) of neural stem cells (named neuroblasts in Drosophila), which deviate out of the neuroectoderm on the embryo surface and migrate to the embryo interior, where they go on to form the central nervous system (An et al., 2017; Simões et al., 2017); 3) cell extrusion, which is the removal of unwanted or dying cells from the epithelium and induction into cell death as part of developmental programs and tissue clearance strategies (Ambrosini et al., 2017; Villars and Levayer, 2022).
Tissue invagination, cell ingression and cell extrusion occur across development and require extensive junctional remodeling. (A-D) In the Drosophila embryo, epithelial-to-mesenchymal transition (EMT) is a type of invagination that occurs during gastrulation. (A) The presumptive mesoderm (red) is a subset of cells expressing twist and snail. Fog activation in this region promotes apical constriction (B), progressively driving tissue invagination (C). Loss of polarity and AJs in invaginating cells precedes cell migration and formation of the new germ layer (the mesoderm) (D), with the ectoderm (blue) undergoing tissue healing. (E) In the Drosophila neuroectoderm, neuroblast (pink polygon) ingression is the result of a progressive apical constriction beginning with cell-autonomous medial Myo-II pulses (yellow lines) that drive Myo-II flow towards vertical BCJs, resulting in junctional Myo-II (red lines) accumulation at a BCJ with a neighboring, non-ingressing cell (grey polygon). This generates a force imbalance between the neuroblast and its neighboring cells and causes BCJ shrinkage and reduction in the number of TCJs (blue dots). This is a stepwise process in which one BCJ shrinks at a time; in this schematic, the process is repeated three times and, in the last step, neuroblasts ingress at the BCJ between two remaining neighboring cells. Ultimately, these neuroblasts will go on to form the central nervous system. (F) In the Drosophila pupal notum, an increase in mechanical tension due to tissue compaction drives single-cell extrusion of less fit, caspase-activated cells (pink polygon). Caspase effectors promote disassembly of the medio-apical microtubule meshwork (brown lines) in the extruding cell before apical constriction. Junctional Myo-II (red lines) is enriched in the extruding cell, forming an actomyosin ring, and AJ-mediated mechanotransduction promotes the formation of a supracellular actomyosin cable (green lines) in the neighboring cells (grey polygons) that contracts and pushes the dying cell out of the epithelium. The extruded cell is then engulfed and eliminated via phagocytosis.
Tissue invagination, cell ingression and cell extrusion occur across development and require extensive junctional remodeling. (A-D) In the Drosophila embryo, epithelial-to-mesenchymal transition (EMT) is a type of invagination that occurs during gastrulation. (A) The presumptive mesoderm (red) is a subset of cells expressing twist and snail. Fog activation in this region promotes apical constriction (B), progressively driving tissue invagination (C). Loss of polarity and AJs in invaginating cells precedes cell migration and formation of the new germ layer (the mesoderm) (D), with the ectoderm (blue) undergoing tissue healing. (E) In the Drosophila neuroectoderm, neuroblast (pink polygon) ingression is the result of a progressive apical constriction beginning with cell-autonomous medial Myo-II pulses (yellow lines) that drive Myo-II flow towards vertical BCJs, resulting in junctional Myo-II (red lines) accumulation at a BCJ with a neighboring, non-ingressing cell (grey polygon). This generates a force imbalance between the neuroblast and its neighboring cells and causes BCJ shrinkage and reduction in the number of TCJs (blue dots). This is a stepwise process in which one BCJ shrinks at a time; in this schematic, the process is repeated three times and, in the last step, neuroblasts ingress at the BCJ between two remaining neighboring cells. Ultimately, these neuroblasts will go on to form the central nervous system. (F) In the Drosophila pupal notum, an increase in mechanical tension due to tissue compaction drives single-cell extrusion of less fit, caspase-activated cells (pink polygon). Caspase effectors promote disassembly of the medio-apical microtubule meshwork (brown lines) in the extruding cell before apical constriction. Junctional Myo-II (red lines) is enriched in the extruding cell, forming an actomyosin ring, and AJ-mediated mechanotransduction promotes the formation of a supracellular actomyosin cable (green lines) in the neighboring cells (grey polygons) that contracts and pushes the dying cell out of the epithelium. The extruded cell is then engulfed and eliminated via phagocytosis.
Invagination of the mesoderm primordium
During gastrulation in Drosophila, the presumptive mesoderm, a subset of cells in the ventral region, invaginates and undergoes posterior detachment from the ectoderm in a process known as epithelial-to-mesenchymal transition (EMT; see Glossary, Box 1). During this process, the cells of the presumptive mesoderm progressively lose their epithelial identity, ultimately acquiring a new mesenchymal one (Fig. 5A-D) (Font-Noguera et al., 2021; Ko and Martin, 2020). EMT is a multistep process conserved across species that requires remodeling of cell-cell junctions, loss of apicobasal polarity and acquisition of cell motility (Yang et al., 2020). EMT starts with the nuclear transport of the maternal morphogen Dorsal after fertilization, which triggers the expression of two transcription factors, Snail and Twist, which in turn activate the G protein-coupled receptor (GPCR)-Rho signaling cascade (Manning and Rogers, 2014). Briefly, the Fog ligand, a Twist transcriptional target, activates the GPCR Mist (Mthl1), a Snail transcriptional target (Costa et al., 1994), which in turn activates the Gα subunit Concertina (Parks and Wieschaus, 1991). Concertina and T48, another transcriptional target of Twist, bind to and activate RhoGEF2, resulting in the activation of Rho1 in the plane of AJs (Kölsch et al., 2007; Martin et al., 2009). Rho1-GTP activates Rok, which phosphorylates Myosin regulatory light chain, activating Myo-II (Fox and Peifer, 2007). Rho1 also activates Diaphanous (Dia), a formin protein that promotes the assembly of F-actin, resulting in a stable connection between the medial actomyosin meshwork and AJs (Homem and Peifer, 2008; Mason et al., 2013). These Rho-dependent events generate anisotropic contractile pulses in the plane of AJs, leading to a ratcheted apical constriction in the presumptive mesoderm (Mason et al., 2013).
Snail also activates the expression of the E3 ligase Neuralized (Neur) and represses Bearded (Brd), an inhibitor of Neur activity (Bardin and Schweisguth, 2006). In the mesoderm, Neur regulates changes in polarity by acting on the Sdt/Patj complex (Chanet and Schweisguth, 2012; Perez-Mockus et al., 2017). Brd inhibits Myo-II by an unknown mechanism, and thus brd repression in the mesoderm also contributes to increased apical contractility (Perez-Mockus et al., 2017). Snail and Twist additionally regulate the expression of cell-cell adhesion molecules such as E-cad (Cano et al., 2000), resulting in the deviation of cells from the epithelial sheet.
Ultimately, the ectoderm epithelium must be sealed, implying assembly of cell-cell adhesions, a step that remains poorly characterized. In vertebrates, ECM remodeling is required for EMT (Mogi and Toyoizumi, 2010; Park and Gumbiner, 2012). By contrast, in Drosophila, a condensed ECM and mature SJs are not yet present at this developmental stage (Tepass and Hartenstein, 1994). However, ECM proteins and SJ components are already expressed and their depletion results in developmental defects and embryonic lethality, suggesting that they may be important during these early gastrulation processes (Baumgartner et al., 1996; Hall and Ward, 2016; Lamb et al., 1998; Wang et al., 2008).
Single-cell ingression of neuroblasts
During neurogenesis, neuroblasts undergo a type of single-cell ingression in which they deviate from the prospective ectoderm in a snail- and twist-independent manner (An et al., 2017; Simões et al., 2017) (Fig. 5E). Early in the ingression process, neuroblasts generate cell-autonomous contractile medial Myo-II pulses and induce Myo-II flow towards one of the vertical junctions of the neuroblast, parallel to the anterior-posterior body axis, resulting in a local increase in mechanical tension (An et al., 2017; Simões et al., 2017). Upstream regulators of cell ingression include the polarity complex Crb, the RhoGEF Cysts and Rho1, which control Myo-II activity to promote apical domain loss in ingressing neuroblasts (Simões et al., 2017, 2022). The Crb polarity complex is polarized along vertical junctions, where it recruits the RhoGEF Cysts (Simões et al., 2022). Cysts activates Rho1, promoting junctional Myo-II contractility and generating periodic pulses of junctional Myo-II at the cell cortex (An et al., 2017; Simões et al., 2017). Crb turnover kinetics rely on the ESCRT/retromer dual recycling/degradation system and play a key role in controlling the speed at which neuroblasts ingress (Simões et al., 2022). Coupling between actomyosin and the cell cortex is ensured by adaptor proteins Smug and Canoe, with the latter localizing at TCJs in a tension-dependent manner (An et al., 2017; Simões et al., 2017; Yu and Zallen, 2020). The amplitude and frequency of the periodic pulses of junctional Myo-II increase over time, enhancing the anisotropic enrichment of Myo-II along the neuroblast's vertical junction. As a result, the vertical junction shrinks, two TCJs meet and are remodeled into a single TCJ. This process takes place in a progressive manner, with the shrinkage of one vertical BCJ at a time (Fig. 5E). Eventually, the neuroblast ingresses at the one remaining BCJ, between two neighboring non-ingressing cells (Simões et al., 2017) (Fig. 5E). Ultimately, these ingressed neuroblasts will go on to form the central nervous system (Ramon-Cañellas et al., 2019).
Epithelial cell extrusion
Cell extrusion serves to balance the cell division rate, accommodate new cell geometries and dissipate tissue tension induced by overcrowding (Etournay et al., 2015). Live-cell extrusion has been proposed to occur upon tissue compaction in the midline of pupal notum epithelium (Marinari et al., 2012). However, using more potent genetic tools, this notion has since been revisited. It appears that most, if not all, cells that extrude from this tissue are committed to die, with caspase activation preceding extrusion (Levayer et al., 2016). Caspase activation has also been observed across other tissues such, as the pupal abdomen (Teng et al., 2017; Toyama et al., 2008).
In the Drosophila pupal notum, caspase activation promotes disassembly of the medio-apical microtubule meshwork in the extruding cell, with no changes in junctional or medial Myo-II, before apical constriction (Fig. 5F) (Villars et al., 2022). Then, junctional Myo-II is enriched in the extruding cell, forming an actomyosin ring (Villars et al., 2022). Next, AJ-mediated mechanotransduction results in the formation of a supracellular actomyosin cable in the neighboring cells (Villars et al., 2022). Collectively, the resulting forces promote apical constriction and extrusion of the dying cell out of the epithelium, to be eliminated by macrophages (Villars et al., 2022). A similar mechanism has been observed in the Drosophila epidermis. During pupal abdomen metamorphosis, polyploid larval epidermal cells (LECs) undergo extrusion and are progressively replaced by proliferating histoblasts. Abdominal development and tissue growth are driven by LECs secreting matrix metalloproteases to promote ECM remodeling and histoblast proliferation before LEC extrusion (Davis et al., 2022). Caspase-3 activation in LECs precedes apical constriction (Teng et al., 2017). During single-cell extrusion of LECs, AJ remodeling relies primarily on the mechanical coupling between E-cad complexes and the contractile actomyosin network. Junctional Myo-II enrichment in the extruding cell promotes cell-autonomous apical constriction (Teng et al., 2017). AJ-mediated mechanotransduction promotes recruitment and activation of junctional Myo-II in neighboring histoblasts, resulting in the formation of a supracellular actomyosin cable (Teng et al., 2017). Coordination between the constriction of the supracellular cable and AJ disassembly ultimately results in extrusion of the LEC (Teng et al., 2017). During apical constriction in the LEC, SJ core complex components Neuroglian and ATPα are still present at the plasma membrane, suggesting that tissue sealing properties are preserved during extrusion and SJ integrity may be required to cohesively elicit this process (Prat-Rojo et al., 2020; Teng et al., 2017). Extrusion is not limited to LECs sharing an interface with histoblasts. Later in development, tissue-wide caspase activation results in an overall increase in tissue mechanical tension and LECs in the dorsal margin, surrounded by neighboring LECs, undergo extrusion (Michel and Dahmann, 2020). Strikingly, blocking E-cad endocytosis suffices to prevent extrusion, reinforcing the notion that AJ remodeling is an upstream regulator at the onset of extrusion processes (Michel and Dahmann, 2020). Endocytosis-mediated junctional remodeling may therefore represent a common strategy for removing single cells, both alive and caspase-activated, from an epithelium, as this is observed during neuroblast ingression and LEC extrusion (Michel and Dahmann, 2020; Simões et al., 2017).
Overall, it appears that the contribution of caspase signaling during extrusion is not yet fully understood. In the larval wing disc, caspase-activated cells continue to extrude even when activation of Myo-II is prevented, ultimately undergoing apoptosis (Franke et al., 2010; Monier et al., 2015). However, not all caspase-activated cells that extrude from the epithelium ultimately undergo cell death (Fujisawa et al., 2020). In the pupal notum, apical stabilization of microtubules is sufficient to prevent apical surface reduction and extrusion in caspase-activated cells, whereas destabilization of the apical microtubule meshwork in caspase-inhibited cells induces apical surface reduction and cell extrusion (Villars et al., 2022). Therefore, microtubule stability may act to limit the rate of extrusion. Although caspase activity is required for epithelia morphogenesis (Ambrosini et al., 2017), their contribution to extrusion may be context-specific. A closer look at ECM adhesion components also revealed that Integrins are required for cell survival in the tissue and reducing their expression activates caspase signaling, promoting basal extrusion and cell death (Valencia-Expósito et al., 2022). These observations raise new questions as to what the main regulators of cell extrusion could be.
The role of epithelial apoptosis is not restricted to the elimination of less fit cells. Indeed, over the past decades, apoptosis-mediated extrusion of epithelial cells has been shown to contribute to morphogenesis by mechanically modifying the surrounding epithelial cells (Ambrosini et al., 2017). This has been particularly well-characterized in the folding of the Drosophila leg imaginal disc epithelium. In this tissue, epithelial cells committed to death assemble an actomyosin cable that anchors the nuclear envelope to AJs (Monier et al., 2015) and an F-actin mesh linking the basal side of the nucleus to the basal cortex (Ambrosini et al., 2017). The actomyosin cable is contractile and produces forces along the apical-basal axis, inducing transient local deformation of the apical surface of neighboring cells (Monier et al., 2015). When several epithelial cells undergo apoptosis within a certain distance and time interval, they collectively contribute to the increase in tissue tension, ultimately leading to the folding of the epithelium (Ambrosini et al., 2017; Monier et al., 2015).
Control of apical versus basal cell extrusion
The direction in which cells leave the epithelium is another context-dependent feature of extrusion that involves distinct sequential remodeling of junctional complexes. In the larval wing disc, polarity-deficient clones undergo basal or apical extrusion depending on where they originate in the epithelium (Tamori et al., 2016). Basal extrusion and subsequent apoptosis in the larval wing disc occurs in specific tissue regions with weak basal ECM organization, a dense apical microtubule meshwork and actin-filled basal filipodia (Tamori et al., 2016). Conversely, apical extrusion and cell survival is observed in tissue regions with tight ECM laminae, presence of cortical microtubules basally, instead of apically, and intertwined basal protrusions (Tamori et al., 2016). These radically different tissue microenvironments are directly impacted by RhoGEF2, an activator of Rho-family small GTPases (Tamori et al., 2016). Loss of RhoGEF2 results in the depletion of apical F-actin, loss of basal integrin and basal translocation of cortical microtubules (Tamori et al., 2016). As a result, sites that favor basal extrusion and apoptosis are transformed into permissive sites of apical extrusion in which extruding cells escape cell-death (Tamori et al., 2016).
The impact of Ras overactivation on cell extrusion is a major concern in cancer biology. The establishment of Drosophila cancer models over recent decades has brought to light how oncogenes and mutations in tumor-suppressor genes synergize to enhance tumor fitness (Dillard et al., 2021). During normal development of the notum, the pro-survival EGFR/ERK signaling pathway becomes transiently activated in neighboring cells in contact with an extruding cell, preventing collective extrusion and tissue rupture (Valon and Levayer, 2019). This strategy is hijacked by Ras-overactivated cells, in a process known as ‘super-competition’. RasV12 cells overproliferate, resulting in tissue compaction (Levayer et al., 2016), and downregulate ERK signaling in neighboring wild-type cells to promote their extrusion (Moreno et al., 2019). Ras overactivation in combination with M6 depletion can promote both basal and apical extrusion (Dunn et al., 2018), suggesting mutations in TCJ components, and not just in tumor-suppressor genes, may contribute to enhancing tumor fitness. As basal and apical extrusion have been linked to cell death and survival, understanding the molecular regulators of these processes, as well as the junctional remodeling involved, would help decipher the mechanisms of these developmental events that are often observed in pathological contexts.
Conclusions
In this Review, we have assembled the molecular, multicellular and tissue-scale mechanisms that converge at cellular interfaces to elicit collective cell movements during development, predominantly drawing on findings from Drosophila. Junctional remodeling has emerged as a cornerstone of epithelial integrity, raising exciting new questions and presenting unmet challenges. How does spatiotemporal interplay of bi- and tricellular junctions regulate non-cell autonomous behaviors to render epithelia plastic yet robust? What are the minimal, permissive requirements and underlying hierarchy of junctional remodeling in coordinated, three-dimensional rearrangements? Functional and spatiotemporal multidisciplinary studies, such as 3D electron microscopy, together with mechanical or optogenetic perturbations, will deepen our understanding of junctional remodeling hierarchy, apicobasal interplay, dynamics and ultrastructure, unveiling how junctional remodeling contributes to homeostasis in living organisms.
Acknowledgements
We thank members of the R.L.B. team for discussions and Jacek Kubiak for critical reading and constructive comments on the manuscript. We thank the reviewers for their time, valuable remarks and insightful suggestions. We apologize to the many colleagues whose relevant work was not cited here owing to space limitations and manuscript scope.
Footnotes
Funding
This work was supported in part by La Ligue Contre le Cancer (M.M.-O.), Fondation ARC pour la Recherche sur le Cancer (PJA 20191209388 to R.L.B.) and the Association Nationale de la Recherche et de la Technologie programme PRC ACTriCE (ANR-20-CE13-0015).
References
Competing interests
The authors declare no competing or financial interests.