A Polarizing Issue: Diversity in the Mechanisms Underlying Apico-Basolateral Polarization In Vivo.

Polarization along an apico-basolateral axis is a hallmark of epithelial cells and is essential for their selective barrier and transporter functions, as well as for their ability to provide mechanical resiliency to organs. Loss of polarity along this axis perturbs development and is associated with a wide number of diseases. We describe three steps involved in polarization: symmetry breaking, polarity establishment, and polarity maintenance. While the proteins involved in these processes are highly conserved among epithelial tissues and species, the execution of these steps varies widely and is context dependent. We review both theoretical principles underlying these steps and recent work demonstrating how apico-basolateral polarity is established in vivo in different tissues, highlighting how developmental and physiological contexts play major roles in the execution of the epithelial polarity program.

Development and Cell Biology of the Blood-Brain Barrier.

The vertebrate vasculature displays high organotypic specialization, with the structure and function of blood vessels catering to the specific needs of each tissue. A unique feature of the central nervous system (CNS) vasculature is the blood-brain barrier (BBB). The BBB regulates substance influx and efflux to maintain a homeostatic environment for proper brain function. Here, we review the development and cell biology of the BBB, focusing on the cellular and molecular regulation of barrier formation and the maintenance of the BBB through adulthood. We summarize unique features of CNS endothelial cells and highlight recent progress in and general principles of barrier regulation. Finally, we illustrate why a mechanistic understanding of the development and maintenance of the BBB could provide novel therapeutic opportunities for CNS drug delivery.

The basement membrane of hair follicle stem cells is a muscle cell niche.

The hair follicle bulge in the epidermis associates with the arrector pili muscle (APM) that is responsible for piloerection ("goosebumps"). We show that stem cells in the bulge deposit nephronectin into the underlying basement membrane, thus regulating the adhesion of mesenchymal cells expressing the nephronectin receptor, α8ß1 integrin, to the bulge. Nephronectin induces α8 integrin-positive mesenchymal cells to upregulate smooth muscle markers. In nephronectin knockout mice, fewer arrector pili muscles form in the skin, and they attach to the follicle above the bulge, where there is compensatory upregulation of the nephronectin family member EGFL6. Deletion of α8 integrin also abolishes selective APM anchorage to the bulge. Nephronectin is a Wnt target; epidermal ß-catenin activation upregulates epidermal nephronectin and dermal α8 integrin expression. Thus, bulge stem cells, via nephronectin expression, create a smooth muscle cell niche and act as tendon cells for the APM. Our results reveal a functional role for basement membrane heterogeneity in tissue patterning. PAPERCLIP:

Basement membrane remodelling regulates mouse embryogenesis.

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

Human blood vessel organoids as a model of diabetic vasculopathy.

The increasing prevalence of diabetes has resulted in a global epidemic1. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and amputation of lower limbs. These are often caused by changes in blood vessels, such as the expansion of the basement membrane and a loss of vascular cells2-4. Diabetes also impairs the functions of endothelial cells5 and disturbs the communication between endothelial cells and pericytes6. How dysfunction of endothelial cells and/or pericytes leads to diabetic vasculopathy remains largely unknown. Here we report the development of self-organizing three-dimensional human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks that are enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycaemia and inflammatory cytokines in vitro induces thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes found in patients with diabetes. DLL4 and NOTCH3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Therefore, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable systems for modelling and identifying the regulators of diabetic vasculopathy, a disease that affects hundreds of millions of patients worldwide.

Ontogenesis of the tear drainage system requires Prickle1-driven polarized basement membrane deposition.

In terrestrial animals, the lacrimal drainage apparatus evolved to serve as conduits for tear flow; however, little is known about the ontogenesis of this system. Here, we define the anatomy of the fully formed tear duct in mice, characterize crucial morphogenetic events for the development of tear duct components and identify the site for primordial tear duct (PTD) initiation. We report that the PTD originates from the orbital lacrimal lamina, a junction formed by the epithelia of the maxillary and lateral nasal processes. We demonstrate that Prickle1, a key component of planar cell polarity signaling, is expressed in progenitors of the PTD and throughout tear duct morphogenesis. Disruption of Prickle1 stalls tear duct elongation; in particular, the loss of basement membrane deposition and aberrant cytoplasmic accumulation of laminin are salient. Altered cell adhesion, cytoskeletal transport systems, vesicular transport systems and cell axis orientation in Prickle1 mutants support the role of Prickle1 in planar cell polarity. Taken together, our results highlight a crucial role of Prickle1-mediated polarized basement membrane secretion and deposition in PTD elongation.

Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation.

How extracellular matrix contributes to tissue morphogenesis is still an open question. In the Drosophila ovarian follicle, it has been proposed that after Fat2-dependent planar polarization of the follicle cell basal domain, oriented basement membrane (BM) fibrils and F-actin stress fibers constrain follicle growth, promoting its axial elongation. However, the relationship between BM fibrils and stress fibers and their respective impact on elongation are unclear. We found that Dystroglycan (Dg) and Dystrophin (Dys) are involved in BM fibril deposition. Moreover, they also orient stress fibers, by acting locally and in parallel to Fat2. Importantly, Dg-Dys complex-mediated cell-autonomous control of F-actin fiber orientation relies on the preceding BM fibril deposition, indicating two distinct but interdependent functions. Thus, the Dg-Dys complex works as a crucial organizer of the epithelial basal domain, regulating both F-actin and BM. Furthermore, BM fibrils act as a persistent cue for the orientation of stress fibers that are the main effector of elongation.

Endothelial k-RasV12 Expression Induces Capillary Deficiency Attributable to Marked Tube Network Expansion Coupled to Reduced Pericytes and Basement Membranes.

OBJECTIVE: We sought to determine how endothelial cell (EC) expression of the activating k-Ras (kirsten rat sarcoma 2 viral oncogene homolog) mutation, k-RasV12, affects their ability to form lumens and tubes and interact with pericytes during capillary assembly Approach and Results: Using defined bioassays where human ECs undergo observable tubulogenesis, sprouting behavior, pericyte recruitment to EC-lined tubes, and pericyte-induced EC basement membrane deposition, we assessed the impact of EC k-RasV12 expression on these critical processes that are necessary for proper capillary network formation. This mutation, which is frequently seen in human ECs within brain arteriovenous malformations, was found to markedly accentuate EC lumen formation mechanisms, with strongly accelerated intracellular vacuole formation, vacuole fusion, and lumen expansion and with reduced sprouting behavior, leading to excessively widened tube networks compared with control ECs. These abnormal tubes demonstrate strong reductions in pericyte recruitment and pericyte-induced EC basement membranes compared with controls, with deficiencies in fibronectin, collagen type IV, and perlecan deposition. Analyses of signaling during tube formation from these k-RasV12 ECs reveals strong enhancement of Src (Src proto-oncogene, non-receptor tyrosine kinase), Pak2 (P21 [RAC1 (Rac family small GTPase 1)] activated kinase 2), b-Raf (v-raf murine sarcoma viral oncogene homolog B1), Erk (extracellular signal-related kinase), and Akt (AK strain transforming) activation and increased expression of PKCε (protein kinase C epsilon), MT1-MMP (membrane-type 1 matrix metalloproteinase), acetylated tubulin and CDCP1 (CUB domain-containing protein 1; most are known EC lumen regulators). Pharmacological blockade of MT1-MMP, Src, Pak, Raf, Mek (mitogen-activated protein kinase) kinases, Cdc42 (cell division cycle 42)/Rac1, and Notch markedly interferes with lumen and tube formation from these ECs. CONCLUSIONS: Overall, this novel work demonstrates that EC expression of k-RasV12 disrupts capillary assembly due to markedly excessive lumen formation coupled with strongly reduced pericyte recruitment and basement membrane deposition, which are critical pathogenic features predisposing the vasculature to develop arteriovenous malformations.

The role of integrins in Drosophila egg chamber morphogenesis.

The Drosophila egg chamber comprises a germline cyst surrounded by a tightly organised epithelial monolayer, the follicular epithelium (FE). Loss of integrin function from the FE disrupts epithelial organisation at egg chamber termini, but the cause of this phenotype remains unclear. Here, we show that the ß-integrin Myospheroid (Mys) is only required during early oogenesis when the pre-follicle cells form the FE. Mutation of mys disrupts both the formation of a monolayered epithelium at egg chamber termini and the morphogenesis of the stalk between adjacent egg chambers, which develops through the intercalation of two rows of cells into a single-cell-wide stalk. Secondary epithelia, like the FE, have been proposed to require adhesion to the basement membrane to polarise. However, Mys is not required for pre-follicle cell polarisation, as both follicle and stalk cells localise polarity factors correctly, despite being mispositioned. Instead, loss of integrins causes pre-follicle cells to constrict basally, detach from the basement membrane and become internalised. Thus, integrin function is dispensable for pre-follicle cell polarity but is required to maintain cellular organisation and cell shape during morphogenesis.

Breaching multiple barriers: leukocyte motility through venular walls and the interstitium.

The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.

The dynamics of Hippo signaling during Drosophila wing development.

Tissue growth needs to be properly controlled for organs to reach their correct size and shape, but the mechanisms that control growth during normal development are not fully understood. We report here that the activity of the Hippo signaling transcriptional activator Yorkie gradually decreases in the central region of the developing Drosophila wing disc. Spatial and temporal changes in Yorkie activity can be explained by changes in cytoskeletal tension and biomechanical regulators of Hippo signaling. These changes in cellular biomechanics correlate with changes in cell density, and experimental manipulations of cell density are sufficient to alter biomechanical Hippo signaling and Yorkie activity. We also relate the pattern of Yorkie activity in older discs to patterns of cell proliferation. Our results establish that spatial and temporal patterns of Hippo signaling occur during wing development, that these patterns depend upon cell-density modulated tissue mechanics and that they contribute to the regulation of wing cell proliferation.

Forces drive basement membrane invasion in Caenorhabditis elegans.

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.

Fusion of airways during avian lung development constitutes a novel mechanism for the formation of continuous lumena in multicellular epithelia.

BACKGROUND: During development of the avian lung, the initially terminally branched epithelial tree later forms a continuous network of airways. This occurs via a large-scale epithelial fusion event, wherein airways that originate proximally collide with those that originate distally to form one continuous lumen. RESULTS: Here, we found that prior to fusion, the epithelium of the embryonic chicken lung undergoes a shape change to permit the initiation and extension of new branches which contain the cells that initiate contact. These changes in epithelial shape coincide with the differentiation of smooth muscle cells that wrap the airways. From these nascent epithelial branches, individual cells form cytoskeletal protrusions that extend toward and form a bridge with their target airway. Additional cells then join the fusion site, forming a bilayered epithelium. During this process, the basement membrane around the prefusion epithelium degrades and then reforms after fusion. The epithelial bilayer then undergoes apoptosis, clearing the path between the two lumens. CONCLUSIONS: The process of airway epithelial fusion in the developing chicken lung constitutes a novel mechanism for the generation of complex multicellular tubes and suggests a conserved role for smooth muscle in the shaping of airway epithelia.

Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta.

Mural cells (vascular smooth muscle cells and pericytes) play an essential role in the development of the vasculature, promoting vascular quiescence and long-term vessel stabilization through their interactions with endothelial cells. However, the mechanistic details of how mural cells stabilize vessels are not fully understood. We have examined the emergence and functional role of mural cells investing the dorsal aorta during early development using the zebrafish. Consistent with previous literature, our data suggest that cells ensheathing the dorsal aorta emerge from a sub-population of cells in the adjacent sclerotome. Inhibition of mural cell recruitment to the dorsal aorta through disruption of pdgfr signaling leads to a reduced vascular basement membrane, which in turn results in enhanced dorsal aorta vessel elasticity and failure to restrict aortic diameter. Our results provide direct in vivo evidence for a functional role for mural cells in patterning and stabilization of the early vasculature through production and maintenance of the vascular basement membrane to prevent abnormal aortic expansion and elasticity.

Bowman's layer in the cornea- structure and function and regeneration.

Bowman's layer lies immediately posterior to the epithelial basement membrane (EBM) and anterior to the stroma proper in humans, chickens, quail, zebra fish, deer, giraffe, antelope, California sea lions, guinea pig and several other species. It is not found in dog, wolf, cat, tiger, lions, rabbit, pigs, cows, goats, or horses. Developmental anomalies of Bowman's layer are rare, but acquired damage to Bowman's layer, or even complete destruction, is frequently seen in advanced bullous keratopathy or Fuchs' endothelial dystrophy. No detrimental effects of removal of Bowman's layer over the central 6-7 mm of central cornea have been noted in millions of patients who've had photorefractive keratectomy (PRK). Recent studies have suggested the randomly-oriented collagen fibrils that make up Bowman's layer do not have a significant barrier function in modulating the passage of moderate- to large-sized proteins. It is hypothesized that Bowman's layer develops in the corneas of those species that have one because of cytokine-mediated interactions occurring between corneal epithelial cells and underlying keratocytes, including negative chemotactic and apoptotic effects on the keratocytes by low levels of cytokines such as interleukin-1α that are gradually released as epithelial cells die and slough during their normal development. A "Bowman's like layer" can generate around stromal epithelial plugs after radial keratotomy, and possibly beneath the central corneal epithelial basement membrane many years after PRK.

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Integrin: Basement membrane adhesion by corneal epithelial and endothelial cells.

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet's membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFß1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

Subendothelial matrix components influence endothelial cell apoptosis in vitro.

The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. Dysregulation of apoptosis has been linked with embryonal death and is involved in the pathophysiology of various diseases. Specifically, endothelial apoptosis plays pivotal roles in atherosclerosis whereas prevention of endothelial apoptosis is a prerequisite for neovascularization in tumors and metastasis. Endothelial biology is intertwined with the composition of subendothelial basement membrane proteins. Apoptosis was induced by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells. Cells were either grown on polystyrene culture plates or on plates precoated with healthy basement membrane proteins (collagen IV, fibronectin, or laminin) or collagen I. Our results reveal that proteins of healthy basement membrane alleviate cytokine-induced apoptosis whereas precoating with collagen type I had no significant effect on apoptosis by addition of tumor necrosis factor-α to cycloheximide-sensitized endothelial cells compared with cells cultured on uncoated plates. Yet, treatment with transforming growth factor-ß1 significantly reduced the rate of apoptosis endothelial cells grown on collagen I. Detailed analysis reveals differences in intracellular signaling pathways for each of the basement membrane proteins studied. We provide additional insights into the importance of basement membrane proteins and the respective cytokine milieu on endothelial biology. Exploring outside-in signaling by basement membrane proteins may constitute an interesting target to restore vascular function and prevent complications in the atherosclerotic cascade.

Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Involvement of PUMA in pericyte migration induced by methamphetamine.

Mounting evidence indicates that methamphetamine causes blood-brain barrier damage, with emphasis on endothelial cells. The role of pericytes in methamphetamine-induced BBB damage remains unknown. Our study demonstrated that methamphetamine increased the migration of pericytes from the endothelial basement membrane. However, the detailed mechanisms underlying this process remain poorly understood. Thus, we examined the molecular mechanisms involved in methamphetamine-induced pericyte migration. The results showed that exposure of C3H/10T1/2 cells and HBVPs to methamphetamine increased PUMA expression via activation of the sigma-1 receptor, MAPK and Akt/PI3K pathways. Moreover, methamphetamine treatment resulted in the increased migration of C3H/10T1/2 cells and HBVPs. Knockdown of PUMA in pericytes transduced with PUMA siRNA attenuated the methamphetamine-induced increase in cell migration through attenuation of integrin and tyrosine kinase mechanisms, implicating a role of PUMA in the migration of C3H/10T1/2 cells and HBVPs. This study has demonstrated that methamphetamine-mediated pericytes migration involves PUMA up-regulation. Thus, targeted studies of PUMA could provide insights to facilitate the development of a potential therapeutic approach for alleviation of methamphetamine-induced pericyte migration.