Progenitor cell integration into a barrier epithelium during adult organ turnover

Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. These replacement cells--the progeny of basal stem cells--are born without apical, barrier-forming structures such as a protective, lumen-facing membrane and occluding junctions. How stem cell progeny acquire these structures to become part of the barrier is unknown. Here we use Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), Correlative Light-Electron Microscopy (CLEM), and volumetric imaging of live and fixed organs to investigate progenitor integration in the intestinal epithelium of adult Drosophila. We find that stem cell daughters gestate their future lumenal-apical membrane beneath a transient, basal niche formed by an umbrella-shaped occluding junction that shelters the growing cell and adheres it to mature neighbor cells. The umbrella junction both targets formation of a deep, microvilli-lined, apical invagination and closes it off from the contents of the gut lumen. When the growing cell is sufficiently mature, the umbrella junction retracts to expose this Pre-Assembled Apical Compartment (PAAC) to the gut lumen, thus incorporating the new cell into the intestinal barrier. When we block umbrella junctions, stem cell daughters grow and attempt to differentiate but fail to integrate; when we block cell growth, no umbrella junctions form, and daughters arrest in early differentiation. Thus, stem cell progeny build new barrier structures in the shelter of a transient niche, where they are protected from lumenal insults until they are prepared to withstand them. By coordinating this dynamic junctional niche with progenitor cell differentiation, a physiologically active epithelial organ incorporates new cells while upholding integrity of its barrier.

Neuronal immunoglobulin superfamily cell adhesion molecules in epithelial morphogenesis: insights from Drosophila

In this review, we address the function of immunoglobulin superfamily cell adhesion molecules (IgCAMs) in epithelia. Work in the Drosophila model system in particular has revealed novel roles for calcium-independent adhesion molecules in the morphogenesis of epithelial tissues. We review the molecular composition of lateral junctions with a focus on their IgCAM components and reconsider the functional roles of epithelial lateral junctions. The epithelial IgCAMs discussed in this review have well-defined roles in the nervous system, particularly in the process of axon guidance, suggesting functional overlap and conservation in mechanism between that process and epithelial remodelling. We expand on the hypothesis that epithelial occluding junctions and synaptic junctions are compositionally equivalent and present a novel hypothesis that the mechanism of epithelial cell (re)integration and synaptic junction formation are shared. We highlight the importance of considering non-cadherin-based adhesion in our understanding of the mechanics of epithelial tissues and raise questions to direct future work. This article is part of the discussion meeting issue ‘Contemporary morphogenesis’.

The transmembrane proteins M6 and Anakonda cooperate to initiate tricellular junction assembly in epithelia of Drosophila

SummaryCell vertices in epithelia comprise specialized tricellular junctions (TCJs) that seal the paracellular space between three adjoining cells [1, 2]. Although TCJs play fundamental roles in tissue homeostasis, pathogen defense, and in sensing tension and cell shape [3-5], how they are assembled, maintained and remodeled is poorly understood. In Drosophila the transmembrane proteins Anakonda (Aka [6]) and Gliotactin (Gli [7]) are TCJ components essential for epithelial barrier formation. Additionally, the conserved four-transmembrane-domain protein M6, the only myelin proteolipid protein (PLP) family member in Drosophila, localizes to TCJs [8, 9]. PLPs associate with cholesterol-rich membrane domains and induce filopodia formation [10, 11] and membrane curvature [12], and Drosophila M6 acts as a tumor suppressor [8], but its role in TCJ formation remained unknown. Here we show that M6 is essential for the assembly of tricellular, but not bicellular occluding junctions, and for barrier function in embryonic epithelia. M6 and Aka localize to TCJs in a mutually dependent manner and are jointly required for TCJ localization of Gli, whereas Aka and M6 localize to TCJs independently of Gli. Aka acts instructively and is sufficient to direct M6 to cell vertices in the absence of septate junctions, while M6 is required permissively to maintain Aka at TCJs. Furthermore, M6 and Aka are mutually dependent for their accumulation in a low-mobility pool at TCJs. These findings suggest a hierarchical model for TCJ assembly, where Aka and M6 promote TCJ formation through synergistic interactions that demarcate a distinct plasma membrane microdomain at cell vertices.

Transient opening of tricellular vertices controls paracellular transport through the follicle epithelium during Drosophila oogenesis

Paracellular permeability is regulated to allow solute transport or migration of cells across epithelial or endothelial barriers. However, how occluding junction dynamics controls paracellular permeability is poorly understood. Here we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicle epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-Cadherin, N-Cadherin, NCAM/Fasciclin-2 and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions terminates patency and seals the paracellular barrier. E-Cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin-2 expression, impeded endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport, and demonstrate that the dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.

MSCs ARE LOCATED IN THE PERINEURIUM OF THE RECIPIENT RAT AFTER ALLOTRANSPLANTATION INTO DAMAGED NERVE

The gene and cell therapy, stimulating the regeneration of damaged nerves is currently under development. In these experiments mesenchymal stem cells (MSCs) are often used. The purpose of this study is to describe the localization and morphological features of mesenchymal stem cells derived from bone marrow after their allografting into the damaged rat nerve. MSCs of the bone marrow of Wistar-Kyoto rats were obtained from Transtechnology LLC (Head G.Polyntsev, Ph.D.). MSCs were cultured, identified and labeled by 5-bromo-2’-deoxyuridine (BrdU) in vitro. The sciatic nerve of adult Wistar-Kyoto rats (n = 12) was damaged (ligature, 40 sec), and the suspension of BrdU+ MSCs (5 · 104 cells in 5 μl per animal) was immediately transplanted into the damaged sciatic nerve. In a previous study, we have showed that some transplanted cells are located in the epineurium of the recipient’s nerve. The perineurium of the recipient rats was studied in the present work. Perineurial cells have polygonal form, thin and flat cell nucleus and form several layers, the basement membranes being placed between them. Perineurium is characterized by the presence of occluding junctions that can be identified using anti-claudine antibodies. The use of antibodies to claudine allowed us to identify perineurium. Some BrdU+ MSCs were found to survive 5-7 d following surgery and according to their localization and morphology became perineurium cells. Such extracellular matrix proteins as laminin, fibronectin, collagen are present in the perineurium. Apparently, the presence of these proteins creates a favorable biological environment for MSCs survival and for their differentiation towards perineurium cells. The results of the study confirm the mesenchymal origin of perineurium cells.

Occluding junctions as novel regulators of tissue mechanics during wound repair

In epithelial tissues, cells tightly connect to each other through cell–cell junctions, but they also present the remarkable capacity of reorganizing themselves without compromising tissue integrity. Upon injury, simple epithelia efficiently resolve small lesions through the action of actin cytoskeleton contractile structures at the wound edge and cellular rearrangements. However, the underlying mechanisms and how they cooperate are still poorly understood. In this study, we combine live imaging and theoretical modeling to reveal a novel and indispensable role for occluding junctions (OJs) in this process. We demonstrate that OJ loss of function leads to defects in wound-closure dynamics: instead of contracting, wounds dramatically increase their area. OJ mutants exhibit phenotypes in cell shape, cellular rearrangements, and mechanical properties as well as in actin cytoskeleton dynamics at the wound edge. We propose that OJs are essential for wound closure by impacting on epithelial mechanics at the tissue level, which in turn is crucial for correct regulation of the cellular events occurring at the wound edge.

An alternative mode of epithelial polarity in the Drosophila midgut

Apical-basal polarity is essential for the formation and function of epithelial tissues, whereas loss of polarity is a hallmark of tumours. Studies in Drosophila have identified conserved polarity factors that define the apical (Crumbs, Stardust, Par-6, aPKC), junctional (Baz/Par-3) and basolateral (Scribbled, Discs large, Lgl) domains of epithelial cells (1, 2). Because these conserved factors mark equivalent domains in diverse vertebrate and invertebrate epithelial types, it is generally assumed that this system organises polarity in all epithelia. Here we show that this is not the case, as none of these canonical factors are required for the polarisation of the endodermal epithelium of the Drosophila adult midgut. Furthermore, unlike other Drosophila epithelia, the midgut forms occluding junctions above adherens junctions, as in vertebrates, and requires the integrin adhesion complex for polarity (3, 4). Thus, Drosophila contains two types of epithelia that polarise by different mechanisms. Since knock-outs of canonical polarity factors often have little effect on the polarity of vertebrate epithelia, this diversity of polarity mechanisms is likely to be conserved in other animals (5-8).

Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation

Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection.