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From cells to organs: building polarized tissue

David M. Bryant* and Keith E. Mostov*‡ Abstract | How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.

Basement membrane The defining feature of metazoa is that their cells ultimately, underlying blood vessels. The basal and A thin layer are organized into multicellular tissues and organs. lateral surfaces are fairly similar in composition and that specifically lines the basal Although almost every eukaryotic cell is spatially organization and are often referred to together as side of epithelial sheets, and asymmetric or polarized, polarity must be coordi- the basolateral surface. The apical and basolateral certain other tissues, to which cells are attached. Also nated in space and time for individual cells to form surfaces, however, have very different compositions. 1 referred to as the basal lamina. a tissue . Cell polarity involves the asymmetric In vertebrates, tight junctions (TJs) are found at the organization of most of the physical aspects of the apical-most portion of the lateral surfaces, where Extracellular matrix cell, including the cell surface, intracellular organelles the TJs form barriers both between the apical and baso- An extracellular scaffolding gel and the cytoskeleton2,3. Analysis of the polarization lateral surfaces and between adjacent cells, limiting that consists of fibrous 7 structural proteins, complex of unicellular eukaryotes, such as yeast, has yielded paracellular permeability (FIG. 1a). sugars, fluid and signalling enormous insights into the mechanisms that underlie Many epithelial organs make use of interconnected molecules. the polarity of individual cells3. Formation of a tis- tubular networks, although the basic design principles sue, however, requires an ensemble cast; the emergent (as defined by Rafelski and Marshall8) are the same: a A diffusion barrier-forming properties of the tissue result from the combined roles series of tubes terminates in a spherical ending or cap, junction at the apical-most of the individual cells that are involved. Accordingly, which is referred to as an acinus, end bud, alveolus or region of the lateral membrane several biological processes, including cell division, cyst in different tissues. Tubular networks can either of vertebrate epithelial cells. cell death, shape changes, and dif- arise independently and then become interconnected, ferentiation, must be coordinated with the polarity or can be branching trees that form via new sprouts Design principle 4 A simple rule that increases the requirements of a tissue to form an organ . from existing tubes. Many conserved morphogenetic likelihood of the proper Evolutionarily, epithelia are the most archetypal processes give rise to these structures, including mech- assembly and function of a polarized tissues in metazoa, with ~60% of mamma- anisms of lumen formation and expansion, tubulogen- system. lian cell types being of epithelial or epithelial-derived esis, branching morphogenesis, mesenchymal–epithelial origin5. Accordingly, the best studied polarized tissue is transitions (MET) and epithelial–mesenchymal transitions the simple epithelium of the mammalian intestine and (EMT). kidney, the cells of which are columnar in shape (that is, Cellular specialization through polarization occurs they are taller than they are wide). The apical surfaces in almost all cell types. Neural have specialized 9 Departments of *Anatomy of these cells provide the luminal interface and are sites for neurotransmitter release and uptake (FIG. 1b). and ‡Biochemistry and specialized to regulate the exchange of materials, such The apical membranes of photoreceptor epithelium Biophysics, University of as nutrients from the intestine. The lateral surfaces of undergo light-sensing activity, whereas the basal sur- California San Francisco, these cells contact adjacent cells and have specialized faces connect to underlying (FIG. 1c). Migrating California 94143‑2140, USA. 3,6 (FIG. 1a) e‑mails: david.bryant@ucsf. junctions and cell–cell adhesion structures . cells, such as or Dictyostelium discoideum edu; [email protected] The basal surfaces of these cells contact the underly- amoebae, exhibit asymmetric front–back polarity as doi:10.1038/nrm2523 ing basement membrane, extracellular matrix (ECM) and, they move towards attractive cues10 (BOX 1). With a core

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a Tubular epithelium requirement for cellular asymmetry in biological func- Tight junction Lumen tion, understanding how cells polarize and coordinate this process to form a tissue is a central question. Although many biological processes contribute to the formation of an organ, we will focus on how cell polar- Golgi ity is controlled and contributes to morphogenesis in the context of whole tissues. We discuss the molecular control of tissue polarization in in vivo organs and in Adherens Nucleus Lumen junction in vitro organotypic models, including the establish- ment, transcriptional control and molecular regulation ECM of tissue polarization, control of polarity orientation, and regulation of polarity by ECM and Rho GTPase signal- Basement Integrins membrane ling. We emphasize the role of epithelial lumen and tube formation and expansion, as epithelial tissues have b Neural provided many fundamental insights into how polarity is Nucleus shaft coordinated at the cellular, tissue and organ level.

Soma Forming polarized tissue Axon terminal The organization of cells into tissues involves the Pre-synaptic concerted integration of polarizing cues from various Post-synaptic zone Synapse zone interdependent biological processes. First, cells must Dendrite sense their environment, including where they are in relation to their neighbours. This can be mediated by direct interaction of cells with the ECM through Neurotransmitter various receptors, such as integrin, dystroglycan receptor and proteoglycan molecules11,12. Cells can sense and modify the chemical composition, assembly, stiff- ness and other mechanical properties of the ECM13,14. Cells can also communicate with other cells through an array of adhesion molecules, such as cadherins15, Synaptic and through the sensing of diffusible factors, such vesicle as morphogens, chemoattractants and chemorepel- lants16. These combined cues provide instructions Adhesion molecules that enable cells to orientate their polarity and begin to assemble into groups. second, cells in a forming c Drosophila melanogaster tissue must coordinate the asymmetrical distribution ommatidium Rhabdomere terminal web of polarity complexes17 to establish and enforce the gen- eration of an axis of asymmetric organization (BOX 1). Rhabdomere Lumen Concurrently with this second step, the (IRS) and membrane-trafficking systems organize asym- metrically 2. These basic steps allow individual cells Stalk Zonula membrane adherens to become asymmetrically polarized (see the reviews by bornens and by Nelson and Mellman in this issue). Examples of polarized epithelial and migratory cell Nucleus polarity and of the formation of polarity complexes Figure 1 | cell polarization in diverse tissue types. a | Epithelial tubes are comprised are presented in BOX 1. of tightly adhering cells that display strong apico–basal polarity. Lateral membranes An important design principle is that polarization possess desmosomes, adherens junctions and tight junctionsNature Re (TJs),views providing | Molecular cell–cell Cell Biolog y must be coordinated between all cells in a tissue. For adhesion and diffusion barriers. Basal membranes interact with underlying basement example, although the organization of polarity com- membrane and extracellular matrix (ECM). Apical membranes are specialized for plexes, the cytoskeleton, membrane-trafficking events absorption and secretion, such as for electrolytes, milk or O2. b | Neurons polarize to form and adhesive junctions must be asymmetric in a single a soma (cell body), an axon shaft, an axon terminal and dendrites. Neural synapses cell, the orientation and organization of this asymmetry contain adhesion molecules for stabilization of the interaction between cells. The must be coordinated between neighbouring cells. In synapse provides a specialized region for neurotransmission to occur, through polarized addition to apico–basal polarity, some behaviours, such targeting and uptake of neurotransmitters. c | The Drosophila melanogaster retina contains ommatidia made up of tubular neuroepithelia surrounding a central lumen (or as cell division and migration, can also be polarized in interrhabdomeral space (IRS)). Cells are not radially symmetric in the tube but an orthogonal axis; that is, in the plane of the tissue. nevertheless follow a defined, polarized pattern. They have typical epithelial junctions Although this planar cell polarity (PCP) is extremely (zonula adherens at the most apico–lateral region in D. melanogaster) and a subapical important in tissue formation — for example, it regulates -dense network (the rhabdomere terminal web), but their apical surfaces are the expansion of a tissue along a particular axis — we specialized into two domains; the stalk membrane and the light-sensing rhabdomeres, will only briefly touch on it here as it has been reviewed which are specialized microvilli. excellently elsewhere18,19.

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Box 1 | Polarity complexes: conserved regulators of great plasticity Although the signalling mechanisms to induce and regulate morphogenetic movement during tissue for- Conserved, core protein complexes are involved in the generation and maintenance mation are well characterized, how these processes are of all types of polarization. Three major polarity complexes, the PAR (CDC42–PAR3– regulated at the cellular level has only recently started PAR6–aPKC), Crumbs (Crb–PALS–PATJ) and Scribble (Scrib–Dlg–Lgl) complexes to become clear. The identification of conserved, core function in such diverse contexts as asymmetric cell division, epithelial and neuronal polarity-regulating complexes that operate in various polarization, chemotactic migration and cell proliferation2,17 (see figure). Although these complexes perform seemingly different functions in different cell types and contexts has given many insights into these processes contexts, some overarching themes can be drawn. (BOX 1). Recent progress in understanding the orientation Polarity complexes distribute asymmetrically in cells, promoting the expansion of of tissue polarity, the molecular regulation of epithelial the membrane domain they associate with. In epithelial cells (panel a), the PAR and lumen generation and the moulding of biological tubes is Crumbs complexes promote apical polarity, whereas the Scribble complex operates discussed below. In addition, key roles for phosphatidyl- at the basolateral surface. The PAR complex can also be divided into two inositol-phosphates (PtdInsPs; BOX 2) and Rho-family subcomplexes: apical CDC42–PAR6–aPKC and tight junction (TJ)-localized GTPases in many of the aforementioned processes have PAR3–aPKC, which recruits the lipid phosphatase PTEN (BOX 2). Polarity complexes recently become clear, and their contributions to cell and 2,107 can be mutually antagonistic (panel b) , a design principle that allows the tissue polarity are discussed below. establishment of axes of asymmetry8. Inappropriate movement of the Scribble complex into the apical domain is antagonized by the phosphorylation of Lgl by Tissue morphogenesis by MET–EMT aPKC: phosphorylation of Lgl dissociates the protein from the cell cortex108. Similar reciprocal exclusion mechanisms between apical and basolateral complexes one design principle underlying morphogenesis is maintain this asymmetry17, allowing apical and basolateral regions to become that cells can switch between different types of polar- discrete, non-overlapping domains — a possible example of zero-order ity. In EMT, epithelial cells switch their polarity to that ultrasensitivity109. Several signalling receptors that disrupt or promote the formation of mesenchymal cells (FIG. 2a). EMT occurs in many of polarized adhesion appear to do so by modulating this balance68,110,111. Asymmetric developmental processes, such as during gastrulation polarity complex distribution also polarizes other cell types2 (for example, migratory and during formation of the neural crest and primi- cells; panel c), although the Crumbs complex is apparently specific to epithelia and tive streak, whereby well-polarized epithelial sheets epithelial-derived cell types, such as neurons. convert to motile mesenchymal cells and give rise to Despite the integral involvement of polarity complexes in morphogenesis, the another tissue type20. loosely defined, EMT involves mechanisms through which they promote asymmetry are still largely unclear. Recent evidence reveals that these complexes are central organizing platforms that modulate the disruption of polarized adhesion, such as epithelial the microtubule cytoskeleton, membrane traffic and phosphatidylinositol-phosphate (E)-cadherin-based junctions, disruption of apico– regulation2 (BOX 2), in part by controlling Rho GTPase activation through guanine basal polarity, reorganization of the cyto skeleton, nucleotide-exchange factors and GTPase-activating proteins 2,39,60. Disruption of altered basement-membrane composition and organiz- polarity complexes also has marked effects on cellular proliferation, revealing that ation, and adoption of motile behaviours and invasion these complexes have key roles in tumour suppression67,112. into surrounding tissue. As many aspects of EMT are reminiscent of tumour formation, inappropriate reca- pitulation of EMT has received much attention as a a Polarity complexes PAR6 in epithelia aPKC mechanism for metastasis of epithelial cells (this has Crb 20,21 CDC42 been excellently reviewed elsewhere ). PATJ PALS Conversely, MET occurs through condensation of mes- enchymal cells into tightly adhesive groups, generation of apico–basal polarity, adoption of epithelial characteristics TJ and transition to a polarized epithelial tissue21 (FIG. 2a). Scrib Lumen PAR3 MET contributes, for example, to certain sections of the Lgl aPKC developing kidney22. Dlg PTEN The exact definition of EMT is not clear-cut, perhaps Nucleus because different forms of morphogenesis may use some, but not all, aspects of complete EMT. It is also unclear whether EMT involves only changes in motility, adhe- sion and polarity, or whether EMT involves changes in b Interaction of complexes to c Polarity complexes cell fate and differentiation20,23. There are examples of par- balance polarity requirements in migratory cells tial EMT (pEMT) whereby epithelial cells become motile, PAR3 24–28 PAR3 PTEN PAR6 but do not completely lose adhesion and polarity and PAR6 aPKC do not seem to lose epithelial differentiation and fate. It aPKC CDC42 CDC42 is important, therefore, to emphasize that cells under- Crb going different forms of EMT do not lose all polarity; PATJ rather, they may simply substitute and PALS characteristics for mesenchymal polarity. Many migra- Scrib tory cells, such as neutrophils or D. discoideum, display Scrib Back Lgl Front strong front–back asymmetry and exhibit polarized Dlg migration towards attractive cues10. some of the core polarity complexes that modulate apico–basal polarity Apico–basal polarity Apical membrane (the PAR complex; see BOX 1) are also involved in the axis axis organization of front–back asymmetry2. Morphogenesis

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Box 2 | Phosphatidylinositol-phosphates specify membrane polarity of polarized tissues, which involves the contribution of both stable and motile phases during formation, can a Front therefore be seen as movement along a continuum of MET–EMT stages. such transitions between polarity PtdIns(4,5)P2 states are fundamental for shaping many metazoan tis- PtdIns(3,4,5)P 3 EBP50 sues, and alteration to polarity provides a mechanism PIP5KIβ G12, G13 Back pERM to change cell behaviour without necessarily changing Rac, CDC42 RhoA cell fate. Akt–PI3K c b Lumen Transcriptional control of polarity in EMT and MET. Asymmetric morphogen gradients can provide instruc- tive cues for a tissue to undergo morphogenesis, for example, by inducing transcription factors (TFs) to drive these processes16. The snail and ZEb families of Lumen TFs, for example, are potent inducers of, and are often required for, EMT events both in vivo and in vitro29. How these transcriptional modulators induce cellular outputs CDC42 to regulate tissue polarity has only recently started to Annexin-2 Akt–PI3K become clear. The traditional view is that the loss of E-cadherin and PtdIns(4,5)P 2 Rhabdomere PtdIns(3,4,5)P of other junction proteins induces altered cellular polar- 3 21 Stalk ity and EMT . Recent evidence, however, suggests that membrane some EMT-associated TFs also control morphogenesis TJ PTEN by directly repressing transcription of molecules that are involved in polarity complexes30,31, membrane- trafficking systems32, the cytoskeleton32 and the base- ment membrane33 (FIG. 2b). For example, snail and ZEb1 PtdIns(4,5)P2 ZA PtdIns(3,4,5)P repress key components of both the scribble and 3 Crumbs polarity complexes30,31, and re-expression of β1-integrin, RAC1, Akt–PI3K PTEN scribble and Crumb complex proteins partially rescues epithelial polarization. Although loss of apico–basal Phosphatidylinositol-phosphates (PtdInsPs) are phospholipids that are singly or multiply polarity is an inherent part of the definition of EMT, loss phosphorylated on the 3, 4 and/or 5 positions on an inositol head group113. PtdIns(3,4,5)P 3 of E-cadherin is not always induced by or correlated with can be generated from PtdIns(4,5)P by a family ofNa PtdIns3-kinasesture Reviews | Mol (PI3K),ecular and Ce ll Biology 2 expression of some of these TFs32. In these cases, altera- PtdIns(4,5)P can be generated from PtdIns(3,4,5)P by PTEN (a 3-phosphatase). The 2 3 tions to polarity complexes or to the ECM may suffice to balance between these two lipids is crucial to polarity homeostasis, and recent evidence reveals that PtdInsP and associated proteins have core roles (see figure, panels a–c) in allow for altered cellular morphogenesis. Indeed, during membrane identity and polarity generation. metastasis of colon carcinoma, invasive fronts of cells only occur at sites of lost basement-membrane integ- Asymmetric PtdIns(4,5)P2 and PtdIns(3,4,5)P3 distribution occurs in various cell types, including migrating neutrophils114, polarized kidney epithelia46 and Drosophila rity33, showing that ECM signalling is a key regulator of 115 melanogaster photoreceptors . In polarized MDCK cells (panel b), PtdIns(3,4,5)P3 EMT and thus of tissue polarity.

localizes exclusively at the basolateral membrane, whereas PtdIns(4,5)P2 is enriched stimulation of epithelial cells with various cytokines, apically46,116, like in D. melanogaster epithelia117,118. The PAR complex (PAR3–aPKC) such as hepatocyte growth factor (HGF) or transforming modulates asymmetric PtdIns distribution by recruiting the phosphatase PTEN to tight growth factor-β (TGFβ), can induce expression of snail- junctions (TJs)118–120, potentially restricting PtdIns(3,4,5)P from moving across the TJ into 3 or ZEb-family members21,29. snail expression can induce the apical membrane. Asymmetry of PtdIns(4,5)P :PtdIns(3,4,5)P can also occur without 2 3 co-expression of ZEb factors34, which further enables TJs, such as in migrating neutrophils (panel a), where the PtdInsP and associated proteins β (G12, G13, EBP50, phospho-ERM (pERM)) control ‘backness’ and ‘frontness’114. PtdInsP additional TGF expression, initiating a positive-feed- 21 distribution can also be asymmetric in a single membrane domain, such as in the apical back loop . Interestingly, this EMT-inducing module membrane of D. melanogaster ommatidia (panel c), where PTEN controls levels of is also under the control of a negative-feedback loop 115 that involves endogenous microRNAs (miRNAs)35,36 PtdIns(3,4,5)P3 . (FIG. 2b) Asymmetric PtdIns(4,5)P2:PtdIns(3,4,5)P3 distribution is fundamental to the . The miR-200 family of miRNAs promotes

maintenance of cell polarization. Addition of exogenous PtdIns(3,4,5)P3 to basal epithelial differentiation via downregulation of membranes results in their expansion into the surrounding matrix; apical addition ZEb1. loss of the miR-200 family is found in tumour induces rapid loss of apical identity, transcytosis of basolateral membrane to the apical samples that have lost epithelial polarity, and forced surface and projections from the apical surface121. Addition of PtdIns(4,5)P to the 2 re-expression of the miR-200 cluster restores epithelial basal surface results in rapid redistribution of apical proteins towards the basal polarization and differentiation. This downregulation, membrane46. The opportunistic pathogen Pseudomonas aeruginosa takes advantage of however, is also reciprocal; miR-200 members are this process by inducing ectopic PtdIns(3,4,5)P3 at the apical membrane, converting it into basolateral-like membrane and facilitating entry into the epithelium from the luminal targets for direct transcriptional repression by ZEb1. surface122. Actin-regulatory proteins and membrane-trafficking pathways that are Whether cells become polarized into an epithelial tissue influenced by PtdInsP, such as the vesicle-regulating exocyst complex123, may therefore (MET) or become motile (EMT) therefore depends

require PtdIns(4,5)P2:PtdIns(3,4,5)P3 asymmetry to ensure correct vesicle targeting, on a balance between mutually antagonistic miRNA cytoskeletal organization and maintenance of polarity. ZA, zonula adherens. and TF modules, which controls epithelial polarization,

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a Integrins Extracellular Mesenchymal–epithelial Basement matrix transition membrane Nucleus The de novo acquisition of epithelial characteristics, such as apico–basal polarity and Adherens epithelial-type junctions, by junction mesenchymal cells. Tight junction EMT Epithelial–mesenchymal Lumen transition The transition of epithelial cells MET to a mesenchymal state by Polarizing complete loss of apico–basal Back attractant polarity, epithelial-type Front junctions, basement Golgi membrane and the adoption of migratory behaviours.

Front–back polarity b Polarity A morphological characteristic, Crumbs-3 particularly in migratory cells, LGL2 wherein the front (leading edge) and the back () PATJ show morphological and functional asymmetry. Junctions E-cadherin Polarity complexes Claudin-4 TGFβ Conserved, multimeric protein Occludin miR-200 complexes that promote and Polarized epithelium family modulate the formation of Membrane traffic asymmetric cellular Snail ZEB architecture in diverse tissue RAB25 types and organisms. ECM

Planar cell polarity Laminin-5 (α3) (PCP). The polarization of epithelial cells along the plane Cytoskeleton of the epithelium, orthogonal to the apico–basal axis, Keratins directing the orientation of cell Figure 2 | emT and meT in tissue morphogenesis. a | Epithelial cells from tubes undergoing epithelial–mesenchymal shape, division, movement and Nature Reviews | Molecular Cell Biology differentiation. Non-epithelial transition (EMT) lose apico–basal polarity, downregulate cell–cell adhesion, change their cytoskeleton composition and 21 cells can also exhibit PCP. invade into the extracellular matrix (ECM) at areas where the basement membrane has broken down . As cells adopt a mesenchymal state, they may also be polarized, displaying front–back polarization and Golgi orientation towards the Zero-order ultrasensitivity leading edge (front) during migration112,116. Conversely, during mesenchymal–epithelial transition (MET), cells develop A reversible system, such as apico–basal polarity, express epithelial-specific proteins, form stable adhesions and generate luminal structures. phosphorylation, where b | Transforming growth factor-β (TGFβ) (and other morphogens) can induce EMT by inducing Snail and the ZEB family of modifying enzymes can transcription factors (which can also crosstalk)29. Snail and ZEB directly repress the expression of numerous proteins that become saturated with regard are involved in epithelial polarization, including polarity complexes30,31, cell–cell junctions29, the ECM33 and the to the protein being modified, cytoskeleton32. Snail-1 can also alter membrane trafficking, such as by direct repression of RAB25 (rEF. 32), a small GTPase resulting in a switch-like 130 movement of the substrate that is involved in apical membrane trafficking (although, paradoxically, RAB25 is overexpressed in some tumour 131 between modification states. types ). The miR-200 family of microRNAs promotes epithelial polarity and can induce MET by inhibiting translation of ZEB1, thereby blocking induction of EMT35,36. Condensation An event wherein non-adherent or loosely adherent cells can adhesion and the ECM. unravelling the factors that integrins, dystroglycan and proteoglycans, can ultimately move together and tightly adhere to one another. promote the expression of the miR-200 family will lead to changes in cell polarity and shape through various be key to understanding the control of epithelial mechanisms. Modulation of the cytoskeleton and signal- Partial EMT polarization, and may involve the local signalling ling through Rho-family GTPases appears to be key to The transient adoption of some microenvironment, such as the ECM. regulating ECM-directed polarity specification12,13,37,38. mesenchymal characteristics by epithelial cells without complete or permanent loss of Orientating polarity: ECM and GTPase signalling Orientation and maintenance of tissue polarity. A the epithelial phenotype. Cells in tissues are often surrounded by ECM. Recent fundamental design principle for forming a polarized studies have revealed another design principle: the tissue is that cells must interpret an initializing cue to Exocyst ECM, more than providing a structural scaffold, can polarize. When isolated cells, such as neutrophils or A highly conserved, octameric protein complex that regulates define positional information and differentiation cues D. discoideum, are stimulated with a chemoattractant, 13 vesicle docking and delivery to for tissues, ultimately influencing tissue polarity . they typically polarize and migrate towards the source the cell surface. Transduction of these cues via ECM receptors, such as of the molecule10. This orientation of polarity is set up

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Box 3 | In vitro and in silico modelling of epithelial morphogenesis inappropriate activation of the RhoA GTPase and its downstream effectors, RoCK1 and myosin II38. Despite Organotypic three-dimensional (3D) culture of epithelial cells in extracellular matrix this inversion, cell–cell junctions form and cells main- (ECM; matrigel, a basement membrane-like tumour product, or COLI gels) provides a tain some level of polarity; that is, proteins and lipids way to model epithelial morphogenesis in vitro. Many types of epithelial cells form are asymmetrically distributed. This suggests that the structures in 3D culture24,40,124–126, recapitulating varying levels of tissue polarity and architecture. For MDCK cells, at least, this is analogous to a mesenchymal– establishment and correct orientation of polarity are epithelial transition (MET). Motile cells that are embedded in matrix either migrate not obligate partners and can be molecularly uncou- together and adhere, or proliferate from an individual precursor to form stable pled. It further suggests that signalling at the cell–ECM adhesive complexes. Cells then undergo morphogenesis and polarization to eventually interface is a primary determinant of the axis along form simple epithelial cysts that surround a hollow, fluid-filled lumen. Stimulation with which epithelial cells orientate their polarity. growth factors, particularly hepatocyte growth factor (HGF), induces branching and Maintenance of polarization and migration of tubulogenesis of structures, modelling some aspects of embryonic kidney neutrophils towards a chemoattractant requires the 127,128 development . Notably, the tubules produced in this system closely resemble the continued sensing of the polarizing cue, such as by morphogenesis of the mouse embryonic nephric duct (C. Mendelsohn, personal G-protein-coupled receptor-mediated activation of communication). 3D culture has proved to be indispensable for examining aspects of PtdInsP signalling cascades10. Analogously, maintenance cell polarization, lumen formation, oncogene function and the influence of the ECM on tissue organization, which remain technically challenging to manipulate in vivo. of epithelial polarity in tissues may require the contin- Notably, mutants in many polarity regulators or oncogenes show vastly different ued sensing of polarizing cues, such as detecting the phenotypes in 3D culture than in traditional 2D culture1,24,37,46, even in instances where ECM through integrins and detecting cell neighbours no phenotype is observable in 2D culture. through cadherins. Notably, both of these receptors can 12,42–44 On the basis of 3D culture, researchers have established a simple set of rules that control the generation of PtdIns(3,4,5)P3 , which epithelial cells follow during polarization1,40. Each cell strives to have three types of has a fundamental role in generating and maintaining surface: a basal surface, which contacts the ECM, a lateral surface, which contacts basolateral membrane identity (BOX 2), at least in MDCK other cells, and an apical surface, which faces the lumen. A cell that does not contact cells. Cadherin and integrin receptors can signal through the ECM undergoes apoptosis, whereas a cell that lacks an apical surface will generate small GTPases, such as to the Rho and Arf families, a lumen at a region of contact with other cells, or even within itself. Notably, such which can function both upstream and downstream of parameters can be incorporated into an in silico model of epithelial morphogenesis and can yield remarkably life-like ‘cysts’ during simulations129. Computational and systems PtdInsP. Receptor–GTPase–PtdInsP signalling mod- biology approaches are likely to play an increasingly important role in the development ules (whether the receptor is integrins, cadherins or a and analysis of in vitro tissue systems, such as in regenerative medicine or G-protein-coupled receptor) may therefore be key to stem-cell-derived differentiation of epithelial tissue types, in which induction and both the generation and maintenance of tissue polarity maintenance of a polarized epithelium will be crucial. in diverse contexts.

Rho GTPase and PAR complex crosstalk. The three proto- Hepatocyte growth factor by an asymmetric gradient of the initiating cue, such as typical Rho GTPases, RhoA, RAC1 and CDC42, play (HGF). A multipotent ligand, a bacterium or chemokine. The PAR complex (BOX 1) is integral roles in cytoskeletal arrangement, membrane- also known as scatter factor, involved in such front–back polarity in migratory cell trafficking pathways and ECM interactions, and all of for the c-Met receptor. HGF 45 induces proliferation, types, in part by controlling asymmetric orientation of these roles are crucial for cell polarization . CDC42 39 46–48 scattering motility and the microtubule cytoskeleton . What governs the orien- function is crucial for epithelial lumen formation branching morphogenesis in tation of apical and basolateral polarity in epithelial tis- (see below and BOX 1), whereas RAC1 is associated with many epithelia. sues, on the other hand, is less clear, partially because of both integrin and cadherin signalling12,44 and controls the systems that are used to study this process. Polarized the orientation of polarity in epithelial and migrating Transforming growth 12,49 factor-β epithelial cells that are cultured on an artificial support, cell types . RhoA is associated with both apical and 50 Cytokine ligand that induces such as a dish or filter, receive strong, asymmetric initiat- basal membranes in epithelial cells and at the rear of strong epithelial–mesenchymal ing cues for polarization — the rigid substratum provides migrating cells51 and appears to regulate cell shape in transition in many epithelial a cue, while the ‘free’ medium provides another — and some systems45. cells and tissues. there is consequently an axis from which the cells can various connections between the PAR complex 1,40 MDCK collectively orientate their polarity . and Rho GTPases have recently emerged, emphasizing Madin–Darby canine kidney How is the orientation of polarity defined when their key roles in cell polarity. unique functions of Rho cells. A polarized epithelial cell cells are completely surrounded by ECM, such as GTPases occur at discrete subcellular locales, regulated line that is commonly used for in vivo? studies using epithelial cells grown in 3D by guanine nucleotide-exchange factor (GEF) and GTPase- studies of polarity, membrane (BOX 3) activating protein trafficking and cell adhesion. culture have provided some answers . Normally, (GAP) proteins. For instance, binding MDCK (Madin–Darby canine kidney) cyst structures of CDC42 to PAR6 is required for proper function of this Guanine form as a simple epithelium surrounding a central apical determinant52. In C. elegans embryos undergoing nucleotide-exchange factor luminal space40. Inhibition or loss of β1-integrin or radial polarity generation, CDC-42 targets the atypical A protein that catalyses the exchange of GDP for GTP on the downstream GTPase RAC1 results in inversion of protein kinase C (aPKC)–PAR-3–PAR-6 polarity com- 53 GTPase proteins, thereby this orientation such that the apical surface becomes plex to non-junctional membranes . This requires a GAP ‘activating’ the GTPase. orientated towards the matrix12,37. A similar inversion (PAC-1) to exclude CDC-42 from junctions and a (as of glandular epithelial polarity also occurs in a subset of yet unidentified) GEF protein to activate CDC-42 at GTPase-activating protein invasive ductal breast carcinomas41, demonstrating apical membranes. Deletion of this GAP causes ectopic Protein that catalyses hydrolysis of GTP to GDP on that inversion of polarity can also occur in vivo dur- mistargeting of polarity complexes and polarity defects. GTPase proteins, thereby ing tumor igenesis. by contrast, inversion of polarity PAR-3 binds the Rac GEF TIAM-1 to regulate cell–cell ‘inactivating’ the GTPase. orientation is promoted by, and dependent on, the junctions. TIAM-1, through aPKC, apparently controls

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Table 1 | Selected components involved in apical polarity and lumen formation component Function model system loss-of-function phenotype refs Polarity components PAR complex AP polarity Zebrafish gut; MDCK cyst Multiple lumens; accumulation of luminal 46,73,87 (PAR6–aPKC) apoptotic cells Crumbs complex AP polarity MDCK cyst; Dm salivary gland Multiple lumens, disrupted apico–basal polarity; 97,133,135 (Crumbs-3–PATJ) distended luminal size Scribble BL polarity MCF-10A cyst Lumen filling phenotype 68 Rho GTPases CDC42 (also ECT2) Rho GTPase; Embryoid body; MDCK cyst Disrupted polarity; multiple lumens 46,48,140 CDC42 GEF RAC1 Rho GTPase MDCK cyst Inverted apico–basal polarity 12,37 Apical transport Annexin-2 or -13 Apical transport MDCK cyst Multiple lumens 46,133 ARL3 Arl GTPase Dm trachea Inability to fuse lumens between branches 141 Eyes-closed (EYC) SNARE-complex Dm photoreceptor No lumen expansion 141 modulator Exocyst (SEC5–SEC6) Vesicle transport Dm photoreceptor Abnormal apical transport 139 FAPP2 Apical transport MDCK cyst Abnormal lumen expansion 142 RAB8 Apical transport Mouse intestine, Ce intestine Gross defect in apical organization 143 RAB11 complex Vesicle recycling Dm photoreceptor; MDCK cyst Abnormal apical transport, ectopic rhabdomere 98,144 (RAB11–RIP11–MyoV) formation; multiple lumens Syntaxin-2 or -3 t-SNARE(s) MDCK cyst Disrupted apico–basal polarity, small cysts 133,145 VIP17, galectin-3 Apical transport MDCK cyst Multiple lumens 133 Apical ECM/cytoskeleton Chitin synthetases and Luminal ECM Dm trachea Distended luminal size 94 deacetylases EPS-8 Actin regulation Ce intestine Distended luminal size 146 Ezrin Membrane–actin Ce intestine; mouse intestine Distended luminal size; multiple lumens 137,138 crosslinker Eyes shut (EYS) Proteoglycan Dm photoreceptor No lumen expansion 95 Piopio Luminal protein Dm trachea Inability to fuse lumens between branches 83 Slit–Robo– Repulsive Dm heart tube No lumen formed 74,75 dystroglycan complex Junction proteins E-cadherin Cell adhesion MDCK cyst Lumen filling, abnormal lumens 144,147 Na+/K+-ATPase Ion pump Dm trachea; zebrafish gut Distended luminal size; multiple lumens 87,90 TJs (APG2, claudins, TJ components Zebrafish gut; Dm trachea; Multiple lumens; disruption to lumen 87,132,148,149 JAM-A, ZO-1) MDCK cyst dimensions; disrupted apico–basal polarity Other β1-integrin ECM signalling MDCK cyst Inverted apico–basal polarity 12 BIM, BCL2 Apoptosis Mouse mammary gland, MCF-10A Accumulation of cells in lumen 47,65,66 cyst, MDCK cyst CFTR Chloride Mouse model of PKD, MDCK cyst Reduced lumen size 89 channel PTEN Lipid Dm photoreceptor; MDCK cyst Abnormal rhabdomere morphogenesis; multiple 46,115 phosphatase lumens Podocalyxin Sialomucin MDCK cyst No lumen expansion 76 AP, apical; BL, basolateral; Ce, Caenorhabditis elegans; CFTR, cystic fibrosis transmembrane conductance regulator; Dm, Drosophila melanogaster; ECM, extracellular matrix; GEF, guanine nucleotide-exchange factor; MDCK, Madin–Darby canine kidney; PKD, polycystic kidney disease; TJ, tight junction.

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a Cavitation

aPKC– BIM ECM PAR6 Scribble BCL2

Proliferation Lumen

ERBB2 Cyclin D1 Poorly polarized cells HPV16 E7 No lumen Cell death Expanded single lumen

RhoA b Hollowing PTEN ROCK1 CDC42 Annexin-2 Laminin aPKC `1-integrin PAR6 RAC1 Ezrin

Lumen

Newly polarizing cells

Vacuolar Small lumen/ exocytosis well polarized Expanded single lumen Claudin-15 Vacuolar apical Na+/K+-ATPase Nucleus compartments Crumbs-3 PATJ SEC10 Pinocytosis CFTR?

c D. melanogaster heart tube PAR3/Baz Midline DE-cadherin/Shg β-/Arm Dorsal Ena Junctions Repulsion

Robo

Nucleus Dg Ventral Slit Lumen Dorsal projections Ventral tube closure Enclosed tube Figure 3 | cavitation, hollowing and membrane repulsion as lumen-forming mechanisms. a | During cavitation, groups of cells proliferate to form a cell mass. Apoptosis of non-extracellular matrixNatur (ECM)-contactinge Reviews | Molecular inner Ce llcells Biolog y results in lumen and polarized tube or cyst formation. Correct lumen formation requires the PAR and Scribble complexes, which modulate cell proliferation68. Apoptosis requires BCL2-family members (BCL2 and BIM) and is inhibited by proliferation-inducing oncoproteins (ERBB2, cyclin D1 and HPV16 E7), causing luminal filling24,65,66. b | During hollowing, polarity establishment and orientation requires laminin–β1-integrin to signal RAC1 (rEFS 12,37), and is inhibited by RhoA–ROCK1 signalling38. Intracellular vesicles (varying in size in different systems71) containing apical membrane components and endocytosis- and/or trans-Golgi-derived material are delivered to regions between cells46,69,70. This delivery depends on PTEN-mediated segregation of phosphatidyl inositol-4,5-

bisphosphate (PtdIns(4,5)P2) to nascent apical regions, recruiting annexin-2 and, in turn, the CDC42–aPKC–PAR6 complex46,73. Once rudimentary lumens are formed (there may be multiple such lumens), tight junctions87,132,133, pump proteins87,134, the Crumbs complex133,135, the exocyst (Sec10 (rEF. 136)) and possibly ezrin137,138 promote formation of a single, expanded lumen. c | During Drosophila melanogaster cardiac tube formation, two rows of myoendothelial cells line up along the midline. Membrane processes extend and join between cells on either side of the midline, first at the ventral-most and then at the dorsal-most regions between cells. Resultant junctions containing PAR3 (Bazooka (Baz)), DE-cadherin (Shotgun (Shg)), β-catenin (Armadillo (Arm)) and Ena allow an enclosed lumen. Slit signalling to Robo at lumen surfaces, apparently regulated by dystroglycan (Dg), prevents extended adhesive contacts between cells (membrane repulsion), allowing lumens to form74,75.

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microtubule organization, providing a mechanism for been reviewed elsewhere50,63,64, we concentrate here the PAR complex to regulate asymmetric cytoskeletal on more recent developments in the different lumen- organization39. In certain Drosophila melanogaster and formation mechanisms of cavitation, hollowing and membrane chick embryonic tissues, GEF-mediated activation of repulsion. TABLE 1 lists the molecular machinery that is RhoA at the apical surface, coupled to GAP-mediated currently implicated in these processes. In these contexts, inactivation of RhoA at the basal surface, allows api- groups of poorly polarized cells can begin to tightly cal cell constriction and remodelling of the epithelium adhere to one another and generate lumens de novo while maintaining apico–basal polarity54–59. (see below; FIG. 3). For cavitation and hollowing, such The PAR6–aPKC subcomplex also regulates RhoA adherence is essentially a MET event and occurs, for signalling through direct interaction with and modu- example, as part of the condensation of the metanephric lation of p190A RhoGAP activity60. RhoA–RoCK during kidney development22. signalling can conversely disrupt aPKC–PAR6–PAR3 Cavitation occurs when a group of cells proliferate interaction and function by direct phosphorylation of in an adhesive, but initially only moderately polarized, PAR3 (rEF. 61). This emphasizes the emerging notion manner (FIG. 3a). The selective apoptosis of cells that are that PAR–Rho GTPase complexes act as discrete mul- not in contact with the ECM gives rise to an outer epithe- timeric signalling ‘hubs’ in different regions of the lial layer surrounding a now hollow lumen. This process cell, controlling various aspects of asymmetric cellular occurs, for example, in three-dimensional (3D) models organization and polarity. Identifying unique functions of mammary acini, and in in vivo mouse mammary end for Rho GEFs and GAPs will be crucial to understand buds24,65,66. In these situations, pro-apoptotic bCl2- Evagination Rho GTPases and PAR complex function at such dis- family factors have key roles in luminal cell apoptosis, The deformation of an crete locales. As PAR3–TIAM1 complexes can control although additional mechanisms, such as autophagy, epithelial sheet, without the microtubule organization39, and as the PAR6–CDC42 appear to contribute to luminal clearance in such loss of apico–basal polarity, 65,66 such that part of the sheet subcomplex controls recycling from endocytic com- tissues . The PAR, scribble and Crumbs complexes 62 extrudes into the extracellular partments , both the cytoskeleton and membrane have important roles in suppressing cell proliferation matrix. trafficking systems may be direct targets of such com- in D. melanogaster tissues67, and both the PAR (aPKC– plexes. similarly, given the key roles of Rac, CDC42 PAR6) and scribble complexes promote apoptosis Invagination 68 The deformation of an and Rho in cell polarity, it will be important to dissect of luminal cells during cavitation , thus contributing to epithelial sheet, without the overlapping or potential novel roles of the remaining the formation of a polarized tubular epithelium. loss of apico–basal polarity, Rho GTPase family members in tissue formation and During hollowing of rapidly polarizing groups of such that part of the sheet polarity. For a more extensive discussion of the role cells, intracellular vesicles (varying in size between sys- folds into the lumen of the of GTPase signalling in cell polarity, see the review by tems) are delivered to the cell surface at a coordinated tube. Iden and Collard in this issue. point between two closely apposed cells, creating a lumi- Radial tissue symmetry nal space de novo (FIG. 3b). These vesicles are thought The complimentary Putting in the plumbing: tube formation to contain fluid that is taken up by endocytosis, and to arrangement of cell polarity in During the morphogenesis of an epithelial tissue, cells contain apical proteins that are destined for delivery a symmetric manner around a central line, such as the apical often organize into biological tubes. such tubes provide to the lumen. Their movement to the cell surface results surfaces of cells in a biological the basic plumbing that is crucial for organ and organismal in the generation of space between two (or more) polar- tube. function, and their formation is therefore a fundamental ized cells, and concomitant cell-surface delivery of the event in the generation of diverse tissues during meta- apical, luminal membrane. The surrounding cells now Cavitation zoan development63. For example, vascular tubes allow exhibit apico–basal polarity and are orientated around The formation of a lumen between a group of cells by for transport of o2 and nutrients throughout the body, a lumen, and the whole tissue is subsequently expanded apoptosis of inner cells that are the digestive system lumen allows absorption of food and in a highly polarized manner. This mechanism has not in contact with the mammary tubes allow the secretion of milk64. Although been observed in 3D organotypic models of kidney extracellular matrix. classic embryology has shown that there is an enormous and vascular development, as well as in blood vessels 46,69,70 Hollowing diversity of mechanisms for tube formation, some common in vivo (although its observation in blood vessels is 71 The trafficking of vesicles themes and molecular regulators have emerged. controversial ). containing apical membrane to A molecular understanding of this process has a space between cells, or in a Making use of polarity: forming lumens. The paramount recently become clear46 and involves concerted inte- single cell, to form a lumen requirement for a biological tube is that a lumen must gration of PtdInsP, Rho-family GTPases and the PAR de novo. form, and the lumen must be enclosed and unobstructed. polarity complex. signalling from the ECM, through Membrane repulsion Many tubes form by rearrangement of existing epithe- integrin receptors, initially orientates apico–basal epi- Activation of a signalling lial sheets. such sheets can be deformed by evagination, thelial polarity in newly adhering groups of cells (see cascade that promotes invagination or similar folding to give rise to tubes that below). Enrichment of PtdIns(4,5)P2 at the apical plasma membranes between cells to 64 de-adhere or that inhibits any bud off , such as the branching of the ureteric bud dur- membrane by the lipid phosphatase PTEN results in attraction between membrane ing embryonic kidney development. A variant of this the apical recruitment of the small GTPase CDC42 regions. occurs when an epithelial sheet folds or rolls up along via the PtdInsP-binding protein annexin-2. Activated an axis that is parallel to the plane of the sheet, such as in CDC42 in turn binds the PAR6–aPKC polarity com- Mammary end bud the neuroepithelium of the developing chick. by merg- plex, thereby ensuring targeting to the apical membrane The spherical end of a (FIG. 3b; BOX 1) mammary tubule; referred to ing the epithelium only at specialized contact points, a . scaffolding of this complex to nascent as an acinus when fully tube with radial tissue symmetry and a central lumen can lumina is required to efficiently generate apico–basal enclosed in 3D culture. be formed63. As such morphogenetic movements have polarity and, consequently, a single polarized lumen.

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a D. melanogaster trachea

Chitin Chitin synthetases deacetylases

Lumen

Clathrin- Chitin fibrils dependent Narrow lumens endocytosis COPI/II complexes (CHC, DYN2, Tracheal (SAR1, SEC13, SEC23 RAB5, Wurst, cuticle α-COP, β-COP, δ-COP) HSC70-4)

Luminal Apical EE secretion Nucleus endocytosis LE Septate junction Secretory vesicle

b DPP c D. melanogaster retina

KNI Dorsal branch

Wg SAL RIP11– DE-cadherin Dorsal RAB11 recycling trunk ZA

Stalk Autocellular membrane junctions Rhabdomere Dorsal Lumen (IRS) terminal web Intercellular branch junctions Exocyst Nucleus RAB11 Dorsal RIP11 trunk Rhabdomere MyoV EYS

Figure 4 | membrane traffic and apical extracellular matrix secretion during lumen formation and expansion. a | In the embryonic Drosophila melanogaster trachea an initially narrow lumen is expandedNature Reduringviews a | rapidMolecular burst Ce ofll Biology secretion at the apical membrane, dependent on the COPI and COPII vesicle transport complexes92,93. Chitin fibrils in the lumen (made by chitin synthetases) signal to underlying cells to organize luminal diameter before being subsequently remodelled into tracheal cuticle (reviewed in rEF. 94). Clathrin-dependent endocytic activity at the apical surface directs luminal material to early endosomes (EE) and then to late endosomes (LE) for degradation, thereby clearing the lumen for gas entry93. Correct luminal secretion and expansion requires functional septate junctions94. b | Part of the D. melanogaster airway is divided into the dorsal trunk and dorsal branches, which sprout from the trunk. Wingless (Wg) signalling in the trunk induces the Spalt (SAL) transcription factor, which promotes DE-cadherin recycling through induction of the RIP11–RAB11 membrane-trafficking complex; lumens are formed by intercellular junctions between multiple cells. Decapentaplegic (Dpp) signalling in branches inhibits SAL expression, through the repressor Knirps (KNI), downregulating the RIP11–RAB11 complex and leading to formation of autocellular junctions and lumens in individual cells80. c | During ommatidium formation in the D. melanogaster retina, a tripartite complex of RAB11, RIP11 and the motor protein myosin V (MyoV) regulates transport of cargo to the rhabdomere, and is required for correct ommatidia organization98. RAB11 interacts with the exocyst complex, which also regulates apical transport to the rhabdomere139. Luminal secretion of the proteoglycan eyes shut (EYS) is required for expansion of the interrhabdomeral space (IRS), apparently occurring in an exocyst-independent manner95. ZA, zonula adherens.

Interestingly, when vacuolar exocytosis is inhibited, in 3D organotypic culture of mammary acini, activation lumens eventually form by cavitation47, which emphasizes of the ERbb2 oncoprotein, which promotes cell sur- the robust drive of epithelial cells to form a hollow lumen. vival and lumen filling in human cancers, also results The CDC42–PAR6–aPKC polarity complex appears to in a lumen-filling phenotype by inducing uncoupling be a master regulator of lumen formation; its disruption of PAR3 from the PAR6–aPKC complex68. The identi- results in either multiple small lumens or in the accumu- ties of the downstream effectors of this complex in lation of apoptotic cells in the lumen46,72,73. Interestingly, lumen formation are still unclear, although regulation

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of both glycogen synthase kinase-3β (GsK3β) and multilayered epithelia, or in lumina in single cells, organi- micro tubules have recently become good candidates39,73. zation of the tissue, such as location of cell–cell junctions Thus, the CDC42–PAR6–aPKC complex appears to and bio genesis of the apical membrane, is more complex, and regulate lumen polarity, formation and maintenance in how polarity is controlled in these situations remains diverse tissue contexts. unclear. For example, budding of the mammary gland During D. melanogaster heart-tube formation, mem- tubular tree has recently been shown to involve an inter- brane repulsion appears to regulate lumen formation. mediate step whereby the buds are filled with multilayered Here, two rows of myoendothelial cells line up along cells84; the polarization of multilayered cells is different the midline and extend membrane process towards from and much less well understood than that of simple the mirroring cell on the other side of the midline. In (monolayered) epithelia. When MDCK cysts are treated epithelial cells, this would normally result in adhesion with HGF to form tubules, cells undergo a pEMT, pass- between cells along the entire lateral contact. In these ing transiently through an intermediate stage of chains cells, however, junctions occur only at the dorsal- and and cords of cells, which have some properties of migrat- ventral-most regions, resulting in the formation of an ing mesenchymal cells, before returning to complete enclosed luminal tube between the rows of cells (FIG. 3c). epithelial polarity25,26. such chains and cords can also be COPI and COPII Interestingly, slit–Robo signalling, a ligand–receptor cou- viewed as small regions of multilayered cells, somewhat Coatomer protein complexes pling that governs repulsive signalling in other cellular analogous to the multilayering seen in mammary gland that regulate anterograde (COPII) and retrograde (COPI) contexts, occurs at the site where the future lumen will buds. The polarity requirements of different regions of 74,75 membrane transport between form , excluding cadherin complexes from the region a tissue, therefore, changes during morphogenesis; cells the endoplasmic reticulum and and promoting luminal development between cells. As can switch, even transiently, between polarized states. through cisternae of the Golgi cells must convert adhesive regions between cells into complex. nascent lumens during hollowing, it will be important Getting it just right: lumen length and diameter. once a Autocellular junctions to determine whether repulsive forces or anti-adhesive lumen is formed, how is it expanded to the appropriate 76 The formation of junctional mole cules (such as gp135 (also known as podocalyxin) ) physiological diameter? several diseases are caused by complexes in a single cell. They play analogous roles in other tissues. likewise, although tubes that are either too wide85 (for example, polycystic can be used to form a lumen in the fly homologue of PAR3 localizes to adhesive inter- kidney disease (PKD)) or too narrow80 (for example, vascu- a single cell. faces in developing lumens, whether any of the PAR, lar stenoses)86. In the cavitation model of lumen formation, Chain scribble or Crumbs polarity complexes play a role in this proliferation of the entire tissue to the required dimen- The extension of one or more mechanism of lumen formation is currently unclear. sion followed by regulated cell death of inner cells allows cells that have lost apico–basal for the generation of appropriate lumen diameter24,65. In polarity from an epithelial Tissue polarity during lumen and tubule formation. other circumstances, such as in lumens formed by hol- sheet into the extracellular matrix without losing cell–cell once rudimentary luminal structures are formed, tubes lowing, sheet wrapping or folding, various mechanisms adhesion or becoming must lengthen and become interconnected networks. In for lumen expansion exist. In the embryonic zebrafish multilayered. some systems, such as the mammalian lung and mam- gut, multiple small lumens are remodelled into a single mary ducts, this is achieved primarily through budding tube in a process that is dependent on the accumula- Cord 63 + Similar to the formation of a of new sprouts and branches . This design principle can tion of luminal fluid, regulated by certain pumps (Na / + chain, but comprised of make repeated use of such budding mechanisms, with K -ATPase) and modulated by the ion permeability of TJs multilayering of cells that may the branches following simple patterns77. Many rounds (claudin-15)87. similar regulation of lumen expansion is contain some disconnected of such iterative branching give rise to an extensive also observed in MDCK cysts and in thyroid 3D cultures luminal structures. Can build tree. In other networks, such as the developing D. mela- where chloride ion secretion is involved in determining on successful chain extension. nogaster trachea, different parts of the tubular network lumen diameter87,88. Notably, inhibition of the CFTR Polycystic kidney disease arise in an initially unconnected manner and sprouting chloride channel inhibits lumen overexpansion in both A group of diseases that cause branches extend towards each other, fusing and creating MDCK cyst and mouse models of PKD, suggesting that focal dilation of kidney tubules an anastomozing network78. Complex rearrangements of chloride transport may be a key regulator of lumen size89. resulting in the formation of + + large cysts and severely apical polarity and cell–cell adhesion must occur when Moreover, coordinated regulation of the Na /K -ATPase compromised renal function. two branches of a network eventually undergo fusion and septate junctions (the invertebrate equivalent of TJs) to maintain lumen integrity and generate an inter- also appears to be key for appropriate lumen expansion Vascular stenosis connected lumen79. In some cases, lumens form in single in the D. melanogaster trachea90,91. Interestingly, this func- A pathological vascular cells, although these often connect up to lumens between tion of the Na+/K+-ATPase appears to be independent of condition that involves the (FIG. 4) 90 narrowing of blood vessels and multiple cells . In D. melanogaster airways, for pump activity , suggesting that there are some differ- that results in hypoperfusion of example, this occurs via switching between intercellular ences in the mode of actions between species or tissues. tissues. (between two or more cells) and autocellular junctions80 (in Whether multiple small lumens occur as obligatory pre- a single cell). specialized adhesion molecules, luminal cursors to a single lumen and whether fluid accumulation Septate junction (SJ). An invertebrate cell–cell matrix proteins and modulation of endocytic recycling drives lumen expansion in other biological tubes remain 80–83 junction, localized to the pathways are involved in such rearrangements . to be determined. mid-lateral membrane region. Most, if not all, morphogenetic mechanisms involve At least two additional processes account for changes Like vertebrate tight junctions alterations in cell polarity at some level, although how in lumen diameter in the tracheal tube and salivary gland (TJs), SJs provide a paracellular polarity is regulated and remodelled during morpho- of D. melanogaster (FIG. 4a). Initially, the apical surfaces of diffusion barrier. Unlike TJs, SJs contain basolateral, rather genesis is poorly understood. In simple epithelia, the cells on opposite sides of the tracheal tube are close than apical, polarity PAR, scribble and Crumbs polarity complexes specify together, and the lumen is narrow. A rapid burst of mem- determinants. apico–basal polarity (BOX 1). In pseudo-stratified or brane traffic to the apical surface and secretion into the

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lumen occurs, concomitant with lumen expansion92,93. involved in surface delivery of D. melanogaster E (DE)- In the trachea, this corresponds to an increase in lumen cadherin complexes. RAb11 and RIP11 promote inter- diameter but not in length (which is apparently control- cellular adhesion and the formation of lumens between led by other mechanisms), whereas in the salivary gland two or more cells. Interestingly, repression of spalt by both the lumen diameter and length are increased92. At the repressor Knirps (induced by TGFβ–decapentaplegic roughly the same time an intra-luminal ECM, comprised signalling) in adjacent regions of the network results of fibrils of the polymer chitin, is assembled in the trachea. in downregulation of this cadherin-recycling pathway, The synthesis and subsequent modification of chitin promoting the formation of autocellular junctions and fibrils is thought to provide a mould over which the pre- lumens in single cells80. Notably, the RAb11–RIP11 cise dimensions of the tube can be modelled (reviewed complex is also involved in D. melanogaster retina devel- in rEF. 94). The burst of exocytic traffic around this time opment98 (FIG. 4c), and recycling from these endosomes controls some of the chitin-regulating enzymes, and thus is regulated by the PAR6–CDC42 complex62, suggest- likely controls some of the lumen-expansion mecha- ing that diverse lumen-formation contexts are a core nism. subsequently, and in the case of the trachea before requirement for this pathway. Thus, in addition to trans- gas enters into the lumen, the lumen is rapidly cleared criptional promotion or repression of junction proteins, via endocytosis at the apical surface of tracheal cells93. polarity complexes and basement-membrane proteins, Perturbation of exocytosis or endocytosis halts the proc- traditional ‘fate inducing’ signalling molecules, such as esses of lumen expansion and clearance, demonstrating Wnt and TGFβ, can induce tissue formation through that there is an integral role for membrane trafficking in the regulation of membrane-trafficking pathways. modulating lumen morphogenesis. It remains unknown Further examination of the transcriptional control of whether trafficking of chitin-modifying enzymes or of membrane traffic, of which little is known, should yield additional lumen-destined cargo is the most crucial enormous insights into the regulation of cell polarity and contributor to lumen expansion. However, these stud- morphogenesis during tissue formation. ies raise the possibility that rather than responding to a pre-existing apical ECM, tubular epithelial cells may Conclusions and future prospects transiently generate their own apical matrix, which may Although much has been learned about how the polarity act to regulate lumen expansion. of individual cells is established and maintained, we are As there is no homologue of chitin in vertebrates, the still in the early days of understanding how polarized extent to which these principles can be directly extended cells are put together to make tissues. Communication to mammalian tubular systems is unclear. Instead, secre- between cells, both through cell–cell contact and via the tion of other ECM molecules, such as proteoglycans into ECM and diffusible factors, and using both chemical and lumens, which is observed, for example, in the D. mela- mechanical signals, is at the heart of polarized tissue nogaster retina, may play an analogous role82,83,95 (FIG. 4b). and organ formation. The potential participation of an apical matrix in lumen over 85% of fatal malignancies in adults in the usA formation and expansion thus remains an attractive arise from epithelial tissues99, and loss of polarity is a concept. similarly, membrane-trafficking pathways to hallmark of increased malignancy. The mechanistic the apical surface will likely play a key role in lumen bases for this connection are rapidly being elucidated, as formation and tissue polarity. is described in several recent reviews20,67,100. Acute injury of major epithelial organ systems is collectively one of Transcriptional control of lumen formation the most important causes of death worldwide101–103. Although we have begun to understand how TFs can understanding polarization of epithelia, therefore, promote the loss of polarized epithelial characteristics, is important in analysing the response of a tissue to how do transcriptional regulators promote the expres- acute injury and in generating prospects for regenera- sion of genes that induce polarization of tissues? some tive medicine. Many organs, such as the kidney, lung insight has come from studying D. melanogaster tubular and liver, can recover from even severe or acute injury, epithelia, such as salivary glands, in which a network provided that the patient survives the initial insult. In of TFs, including hairy, huckebein and ribbon, control the case of the kidney, at least, this involves the local expression of genes involved in apical polarity and lumi- proliferation of epithelial cells, which replace their dead nal development, among other targets. These targets neighbours in denuded regions of the tubules through a include components of the Crumbs complex (BOX 1), process that appears to involve a pEMT104,105. Repeated as well as the apical transport machinery, such as the injury, however, leads to a permanent EMT, whereby molecular motor dynein and certain luminal ECM- epithelial cells become fibroblastic and contribute to modifying enzymes96,97, all of which have varied roles a fibrotic response, which ultimately destroys organ in creating the apical luminal structure. similarly, in function106. learning how to improve the response to the developing zebrafish gut, the TCF2 TF regulates the acute injury, as well as how to avoid fibrosis and EMT expression of certain TJ proteins and ion pumps87, which after chronic injury, such as by controlling the polarity Chitin regulate apical lumen expansion. state of cells, offers enormous possibilities to enhance A polysaccharide that consists During branching of the D. melanogaster airways, human health. As we begin to understand how polariza- of N-acetylglucosamine, the polymer of which is a primary the transcription factor spalt (induced by Wnt–Wingless tion occurs and is controlled at the tissue level, we move component of insect signalling) promotes expression of RAb11 and RIP11, closer to being able to translate such research potential . two members of a membrane-recycling pathway that is into medical reality.

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140. Liu, X. F., Ohno, S. & Miki, T. Nucleotide exchange 145. Sharma, N., Low, S. H., Misra, S., Pallavi, B. & Acknowledgements factor ECT2 regulates epithelial cell polarity. Weimbs, T. Apical targeting of syntaxin 3 is essential This work was supported by National Institutes of Health Cell Signal 18, 1604–1615 (2006). for epithelial cell polarity. J. Cell Biol. 173, 937–948 grants to K.E.M. and a Susan G. Komen Foundation 141. Jiang, L., Rogers, S. L. & Crews, S. T. (2006). Postdoctoral Fellowship to D.M.B. We thank R. Metzger, C. A. The Drosophila Dead end Arf-like3 GTPase 146. Croce, A. et al. A novel actin barbed-end-capping Hunt and A. J. Ewald for comments on the manuscript and controls vesicle trafficking during tracheal activity in EPS-8 regulates apical morphogenesis in members of our laboratory for discussions. This paper is fusion cell morphogenesis. Dev. Biol. 311, 487–499 intestinal cells of Caenorhabditis elegans. Nature Cell dedicated to the memory of our colleagues S. Ross and P. (2007). Biol. 6, 1173–1179 (2004). Kolodzeij. 142. Vieira, O. V., Verkade, P., Manninen, A. & Simons, K. 147. Troxell, M. L., Loftus, D. J., Nelson, W. J. & Marrs, FAPP2 is involved in the transport of apical cargo in J. A. Mutant cadherin affects epithelial morphogenesis DATABASES polarized MDCK cells. J. Cell Biol. 170, 521–526 and invasion, but not transformation. J. Cell Sci. 114, uniprotKB: http://ca.expasy.org/sprot (2005). 1237–1246 (2001). aPKC | CDC42 | claudin-15 | Crb | Dlg | ERBB2 | PAR-3 | PAR6 | 143. Sato, T. et al. The Rab8 GTPase regulates apical 148. Aijaz, S., Sanchez-Heras, E., Balda, M. S. & Matter, K. PATJ | RAC1 | RhoA | ROCK1 | Scrib | Snail | ZEB1 protein localization in intestinal cells. Nature 448, Regulation of tight junction assembly and epithelial 366–369 (2007). morphogenesis by the heat shock protein APG-2. BMC FURTHER INFORMATION 144. Desclozeaux, M. et al. Active Rab11 and functional Cell Biol. 8, 49 (2007). Mostov lab homepage: http://anatomy.ucsf.edu/ recycling endosome are required for E-cadherin 149. Wu, V. M. & Beitel, G. J. A junctional problem of apical mostovlabpage/Homepage.html trafficking and lumen formation during epithelial proportions: epithelial tube-size control by septate morphogenesis. Am. J. Physiol. Cell Physiol. 295, junctions in the Drosophila tracheal system. Curr. all links are acTive in The online pdF C545–C556 (2008). Opin. Cell Biol. 16, 493–499 (2004).

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