Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis

Many organs consist of tubes of epithelial cells enclosing a central lumen. How the space of this lumen is generated is a key question in morphogenesis. Two predominant mechanisms of de novo lumen formation have been observed: hollowing and cavitation. In hollowing, the lumen is formed by exocytosis and membrane separation, whereas, in cavitation, the lumen is generated by apoptosis of cells in the middle of the structure [1, 2]. Using MDCK cells in three-dimensional cultures, we found an inverse correlation between polarization efficiency and apoptosis. When cells were grown in collagen, where cells polarized slowly, apoptosis was needed for lumen formation. However, in the presence of Matrigel, which allowed rapid polarization, lumens formed without apoptosis. If polarization in Matrigel was perturbed by blocking formation of the apical surface by RNAi of Cdc42, lumens formed by apoptosis. In a complementary approach, we plated cells at high density so that aggregates formed with little polarity. These aggregates required apoptosis to form lumens, whereas cells plated at low density formed cysts with rapidly polarizing cells and did not need apoptosis to form lumens. The mechanism of lumen formation in the 3D-MDCK model can shift between hollowing and cavitation, depending on cell polarization.     

斑马鱼管腔器官空腔形态发生及分子机制

后生动物复杂的体内结构和器官结构多以网络状的管道系统出现。中空的管腔作为这个系统的重要结构单元承担了运输物质、区分器官不同部位功能、分隔机体和外环境等诸多重要的生理功能。管腔的发育障碍将致使相关器官形态发生畸形、功能紊乱。管腔型器官形态发生易被直接观察以及各种相关突变鱼和荧光转基因鱼的出现, 使得斑马鱼(Danio rerio)成为管道器官研究的优秀模式动物。斑马鱼血管、神经管、小肠、胰腺外分泌腺、前肾管等几种重要的器官的形态发生都伴随着典型的腔道发育过程, 是研究管腔形成的重要器官模型。管腔形成由胞外信号诱导、细胞极性化、胞内物质定向运输、腔内液体形成和胞内细胞骨架重构等相关管腔细胞内外发生的结构功能变化过程所构成, 而这些结构与功能的变化过程是通过精确而复杂的分子调控网络来实现, 最终形成管道器官。文章对斑马鱼4种典型管腔型器官的空腔形态发生过程进行了综述, 并总结了此过程中的分子机制, 为今后的相关研究提供了参考。     

A molecular network for de novo generation of the apical surface and lumen

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization.     

Apical protein transport and lumen morphogenesis in polarized epithelial cells

Segregation of the apical and basolateral plasma membrane domains is the key distinguishing feature of epithelial cells. A series of interrelated cues and processes follow this primary polarization event, resulting in the morphogenesis of the mammalian epithelium. This review focuses on the role of the interactions between the extracellular matrix and neighbouring cells during the initiation and establishment of epithelial polarity, and the role that membrane transport and polarity complexes play in this process. An overview of the formation of the apical junctional complexes is given in relation to the generation of distinct membrane domains characterized by the asymmetric distribution of phosphoinositides and proteins. The mechanisms and machinery utilized by the trafficking pathways involved in the generation and maintenance of this apical-basolateral polarization are expounded, highlighting processes of apical-directed transport. Furthermore, the current proposed mechanisms for the organization of entire networks of cells into a structured, polarized three-dimensional structure are described, with an emphasis on the proposed mechanisms for the formation and expansion of the apical lumen.     

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.     

Mammalian PAR-1 determines epithelial lumen polarity by organizing the microtubule cytoskeleton

Epithelial differentiation involves the generation of luminal surfaces and of a noncentrosomal microtubule (MT) network aligned along the polarity axis. Columnar epithelia (e.g., kidney, intestine, and Madin-Darby canine kidney [MDCK] cells) generate apical lumina and orient MT vertically, whereas liver epithelial cells (hepatocytes and WIFB9 cells) generate lumina at cell-cell contact sites (bile canaliculi) and orient MTs horizontally. We report that knockdown or inhibition of the mammalian orthologue of Caenorhabditis elegans Par-1 (EMK1 and MARK2) during polarization of cultured MDCK and WIFB9 cells prevented development of their characteristic lumen and nonradial MT networks. Conversely, EMK1 overexpression induced the appearance of intercellular lumina and horizontal MT arrays in MDCK cells, making EMK1 the first known candidate to regulate the developmental branching decision between hepatic and columnar epithelial cells. Our experiments suggest that EMK1 primarily promotes reorganization of the MT network, consistent with the MT-regulating role of this gene product in other systems, which in turn controls lumen formation and position.     

Epithelial polarity and tubulogenesis in vitro

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.     

The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini

We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.     

Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways

The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.     

Proteomic characterization of biogenesis and functions of matrix vesicles released from mineralizing human osteoblast-like cells

Matrix vesicles (MVs), released by budding from apical microvilli of osteoblasts during bone formation and development, are involved in the initiation of mineralization by promoting the formation of hydroxyapatite in their lumen. To gain additional insights into MV biogenesis and functions, MVs and apical microvilli were co-isolated from mineralizing osteoblast-like Saos-2 cells and their proteomes were characterized using LC-ESI-MS/MS and compared. In total, 282 MV and 451 microvillar proteins were identified. Of those, 262 were common in both preparations, confirming that MVs originate from apical microvilli. The occurrence of vesicular trafficking molecules (e.g. Rab proteins) and of the on-site protein synthetic machinery suggests that cell polarization and apical targeting are required for the incorporation of specific lipids and proteins at the site of MV formation. MV release from microvilli may be driven by actions of actin-severing proteins (gelsolin, cofilin 1) and contractile motor proteins (myosins). In addition to the already known proteins involved in MV-mediated mineralization, new MV residents were detected, such as inorganic pyrophosphatase 1, SLC4A7 sodium bicarbonate cotransporter or sphingomyelin phosphodiesterase 3, providing additional insights into MV functions.     

Genetic control of single lumen formation in the zebrafish gut

Most organs consist of networks of interconnected tubes that serve as conduits to transport fluid and cells and act as physiological barriers between compartments. Biological tubes are assembled through very diverse developmental processes that generate structures of different shapes and sizes. Nevertheless, all biological tubes invariably possess one single lumen. The mechanisms responsible for single lumen specification are not known. Here we show that zebrafish mutants for the MODY5 and familial GCKD gene tcf2 (also known as vhnf1) fail to specify a single lumen in their gut tube and instead develop multiple lumens. We show that Tcf2 controls single lumen formation by regulating claudin15 and Na+/K+-ATPase expression. Our in vivo and in vitro results indicate that Claudin15 functions in paracellular ion transport to specify single lumen formation. This work shows that single lumen formation is genetically controlled and appears to be driven by the accumulation of fluid.     

Time-lapse imaging of Arabidopsis leaf development shows dynamic patterns of procambium formation

The principles underlying the formation of leaf veins have long intrigued developmental biologists. In leaves, networks of vascular precursor procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Understanding dynamics of procambium formation has been hampered by the difficulty of observing the process in vivo. Here we present a live-imaging technique that allows visual access to complex events occurring in developing leaves. We combined this method with stage-specific fluorescent markers in Arabidopsis (Arabidopsis thaliana) to visualize preprocambial strand formation and procambium differentiation during the undisturbed course of development and upon defined perturbations of vein ontogeny. Under all experimental conditions, we observed extension, termination and fusion of preprocambial strands and simultaneous initiation of procambium differentiation along entire individual veins. Our findings strongly suggest that progressiveness of preprocambial strand formation and simultaneity of procambium differentiation represent inherent properties of the mechanism underlying vein formation.     

Formation of axon-dendrite polarity in situ: initiation of axons from polarized and non-polarized cells

Neurons are polarized cells that extend a single axon and several dendrites. Historically, how neurons establish their axon-dendrite polarity has been extensively studied using dissociated hippocampal cells in culture. Although such studies have identified the cellular and molecular mechanisms underlying axon-dendrite polarization, the conclusions have been limited to in vitro conditions. Recent progress using live imaging has enabled us to directly observe axon formation in situ, revealing distinct cellular mechanisms that regulate axon-dendrite polarization in vivo. In this review, we compare the cellular events during axon formation studied in various systems both in vivo and in vitro and discuss possible common mechanisms underlying the axon-dendrite polarization.     

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. In this study we show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. Our findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation.     

Hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC in conjunction with Lgl

Hepatocytes differ from columnar epithelial cells by their multipolar organization, which follows the initial formation of central lumen-sharing clusters of polarized cells as observed during liver development and regeneration. The molecular mechanism for hepatocyte polarity establishment, however, has been comparatively less studied than those for other epithelial cell types. Here, we show that the tight junction protein Par3 organizes hepatocyte polarization via cooperating with the small GTPase Cdc42 to target atypical protein kinase C (aPKC) to a cortical site near the center of cell–cell contacts. In 3D Matrigel culture of human hepatocytic HepG2 cells, which mimics a process of liver development and regeneration, depletion of Par3, Cdc42, or aPKC results in an impaired establishment of apicobasolateral polarity and a loss of subsequent apical lumen formation. The aPKC activity is also required for bile canalicular (apical) elongation in mouse primary hepatocytes. The lateral membrane-associated proteins Lgl1 and Lgl2, major substrates of aPKC, seem to be dispensable for hepatocyte polarity establishment because Lgl-depleted HepG2 cells are able to form a single apical lumen in 3D culture. On the other hand, Lgl depletion leads to lateral invasion of aPKC, and overexpression of Lgl1 or Lgl2 prevents apical lumen formation, indicating that they maintain proper lateral integrity. Thus, hepatocyte polarity establishment and apical lumen formation are organized by Par3, Cdc42, and aPKC; Par3 cooperates with Cdc42 to recruit aPKC, which plays a crucial role in apical membrane development and regulation of the lateral maintainer Lgl.     

Drosophila Zpr1 (Zinc Finger Protein 1) Is Required Downstream of Both EGFR And FGFR Signaling in Tracheal Subcellular Lumen Formation

The cellular and molecular cues involved in creating branched tubular networks that transport liquids or gases throughout an organism are not well understood. To identify factors required in branching and lumen formation of Drosophila tracheal terminal cells, a model for branched tubular networks, we performed a forward genetic-mosaic screen to isolate mutations affecting these processes. From this screen, we have identified the first Drosophila mutation in the gene Zpr1 (Zinc finger protein 1) by the inability of Zpr1-mutant terminal cells to form functional, gas-filled lumens. We show that Zpr1 defective cells initiate lumen formation, but are blocked from completing the maturation required for gas filling. Zpr1 is an evolutionarily conserved protein first identified in mammalian cells as a factor that binds the intracellular domain of the unactivated epidermal growth factor receptor (EGFR). We show that down-regulation of EGFR in terminal cells phenocopies Zpr1 mutations and that Zpr1 is epistatic to ectopic lumen formation driven by EGFR overexpression. However, while Zpr1 mutants are fully penetrant, defects observed when reducing EGFR activity are only partially penetrant. These results suggest that a distinct pathway operating in parallel to the EGFR pathway contributes to lumen formation, and this pathway is also dependent on Zpr1. We provide evidence that this alternative pathway may involve fibroblast growth factor receptor (FGFR) signaling. We suggest a model in which Zpr1 mediates both EGFR and FGFR signal transduction cascades required for lumen formation in terminal cells. To our knowledge, this is the first genetic evidence placing Zpr1 downstream of EGFR signaling, and the first time Zpr1 has been implicated in FGFR signaling. Finally, we show that down-regulation of Smn, a protein known to interact with Zpr1 in mammalian cells, shows defects similar to Zpr1 mutants.     

Crucial polarity regulators in axon specification

Cell polarization is critical for the correct functioning of many cell types, creating functional and morphological asymmetry in response to intrinsic and extrinsic cues. Neurons are a classical example of polarized cells, as they usually extend one long axon and short branched dendrites. The formation of such distinct cellular compartments (also known as neuronal polarization) ensures the proper development and physiology of the nervous system and is controlled by a complex set of signalling pathways able to integrate multiple polarity cues. Because polarization is at the basis of neuronal development, investigating the mechanisms responsible for this process is fundamental not only to understand how the nervous system develops, but also to devise therapeutic strategies for neuroregeneration. The last two decades have seen remarkable progress in understanding the molecular mechanisms responsible for mammalian neuronal polarization, primarily using cultures of rodent hippocampal neurons. More recent efforts have started to explore the role of such mechanisms in vivo. It has become clear that neuronal polarization relies on signalling networks and feedback mechanisms co-ordinating the actin and microtubule cytoskeleton and membrane traffic. The present chapter will highlight the role of key molecules involved in neuronal polarization, such as regulators of the actin/microtubule cytoskeleton and membrane traffic, polarity complexes and small GTPases.     

Synthetic matrices reveal contributions of ECM biophysical and biochemical properties to epithelial morphogenesis

Epithelial cells cultured within collagen and laminin gels proliferate to form hollow and polarized spherical structures, recapitulating the formation of a rudimentary epithelial organ. However, the contributions of extracellular matrix (ECM) biochemical and biophysical properties to morphogenesis are poorly understood because of uncontrolled presentation of multiple adhesive ligands, limited control over mechanical properties, and lot-to-lot compositional variability in these natural ECMs. We engineered synthetic ECM-mimetic hydrogels with independent control over adhesive ligand density, mechanical properties, and proteolytic degradation to study the impact of ECM properties on epithelial morphogenesis. Normal cyst growth, polarization, and lumen formation were restricted to a narrow range of ECM elasticity, whereas abnormal morphogenesis was observed at lower and higher elastic moduli. Adhesive ligand density dramatically regulated apicobasal polarity and lumenogenesis independently of cell proliferation. Finally, a threshold level of ECM protease degradability was required for apicobasal polarity and lumen formation. This synthetic ECM technology provides new insights into how cells transduce ECM properties into complex morphogenetic behaviors.     

Evolutionary imprint of activation: The design principles of VSDs

Voltage-sensor domains (VSDs) are modular biomolecular machines that transduce electrical signals in cells through a highly conserved activation mechanism. Here, we investigate sequence–function relationships in VSDs with approaches from information theory and probabilistic modeling. Specifically, we collect over 6,600 unique VSD sequences from diverse, long-diverged phylogenetic lineages and relate the statistical properties of this ensemble to functional constraints imposed by evolution. The VSD is a helical bundle with helices labeled S1–S4. Surrounding conserved VSD residues such as the countercharges and the S2 phenylalanine, we discover sparse networks of coevolving residues. Additional networks are found lining the VSD lumen, tuning the local hydrophilicity. Notably, state-dependent contacts and the absence of coevolution between S4 and the rest of the bundle are imprints of the activation mechanism on the VSD sequence ensemble. These design principles rationalize existing experimental results and generate testable hypotheses.