Role In Epithelial Morphogenesis Research Articles

Sprouty genes regulate activated fibroblasts in mammary epithelial development and breast cancer

Stromal fibroblasts are a major stem cell niche component essential for organ formation and cancer development. Fibroblast heterogeneity, as revealed by recent advances in single-cell techniques, has raised important questions about the origin, differentiation, and function of fibroblast subtypes. In this study, we show in mammary stromal fibroblasts that loss of the receptor tyrosine kinase (RTK) negative feedback regulators encoded by Spry1, Spry2, and Spry4 causes upregulation of signaling in multiple RTK pathways and increased extracellular matrix remodeling, resulting in accelerated epithelial branching. Single-cell transcriptomic analysis demonstrated that increased production of FGF10 due to Sprouty (Spry) loss results from expansion of a functionally distinct subgroup of fibroblasts with the most potent branching-promoting ability. Compared to their three independent lineage precursors, fibroblasts in this subgroup are “activated,” as they are located immediately adjacent to the epithelium that is actively undergoing branching and invasion. Spry genes are downregulated, and activated fibroblasts are expanded, in all three of the major human breast cancer subtypes. Together, our data highlight the regulation of a functional subtype of mammary fibroblasts by Spry genes and their essential role in epithelial morphogenesis and cancer development.

SCRIB controls apical contractility during epithelial differentiation.

Although mutations in the SCRIB gene lead to multiple morphological organ defects in vertebrates, the molecular pathway linking SCRIB to organ shape anomalies remains elusive. Here, we study the impact of SCRIB-targeted gene mutations during the formation of the gut epithelium in an organ-on-chip model. We show that SCRIB KO gut-like epithelia are flatter with reduced exposed surface area. Cell differentiation on filters further shows that SCRIB plays a critical role in the control of apical cell shape, as well as in the basoapical polarization of myosin light chain localization and activity. Finally, we show that SCRIB serves as a molecular scaffold for SHROOM2/4 and ROCK1 and identify an evolutionary conserved SHROOM binding site in the SCRIB carboxy-terminal that is required for SCRIB function in the control of apical cell shape. Our results demonstrate that SCRIB plays a key role in epithelial morphogenesis by controlling the epithelial apical contractility during cell differentiation.

Imaginal disk growth factors are Drosophila chitinase-like proteins with roles in morphogenesis and CO2 response.

Chitinase-like proteins (CLPs) are members of the family 18 glycosyl hydrolases, which include chitinases and the enzymatically inactive CLPs. A mutation in the enzyme's catalytic site, conserved in vertebrates and invertebrates, allowed CLPs to evolve independently with functions that do not require chitinase activity. CLPs normally function during inflammatory responses, wound healing, and host defense, but when they persist at excessive levels at sites of chronic inflammation and in tissue-remodeling disorders, they correlate positively with disease progression and poor prognosis. Little is known, however, about their physiological function. Drosophila melanogaster has 6 CLPs, termed Imaginal disk growth factors (Idgfs), encoded by Idgf1, Idgf2, Idgf3, Idgf4, Idgf5, and Idgf6. In this study, we developed tools to facilitate characterization of the physiological roles of the Idgfs by deleting each of the Idgf genes using the CRISPR/Cas9 system and assessing loss-of-function phenotypes. Using null lines, we showed that loss of function for all 6 Idgf proteins significantly lowers viability and fertility. We also showed that Idgfs play roles in epithelial morphogenesis, maintaining proper epithelial architecture and cell shape, regulating E-cadherin and cortical actin, and remarkably, protecting these tissues against CO2 exposure. Defining the normal molecular mechanisms of CLPs is a key to understanding how deviations tip the balance from a physiological to a pathological state.

Epithelial morphogenesis in the Drosophila egg chamber requires Parvin and ILK.

Integrins are the major family of transmembrane proteins that mediate cell-matrix adhesion and have a critical role in epithelial morphogenesis. Integrin function largely depends on the indirect connection of the integrin cytoplasmic tail to the actin cytoskeleton through an intracellular protein network, the integrin adhesome. What is currently unknown is the role of individual integrin adhesome components in epithelia dynamic reorganization. Drosophila egg chamber consists of the oocyte encircled by a monolayer of somatic follicle epithelial cells that undergo specific cell shape changes. Egg chamber morphogenesis depends on a developmental array of cell-cell and cell-matrix signalling events. Recent elegant work on the role of integrins in the Drosophila egg chamber has indicated their essential role in the early stages of oogenesis when the pre-follicle cells assemble into the follicle epithelium. Here, we have focused on the functional requirement of two key integrin adhesome components, Parvin and Integrin-Linked Kinase (ILK). Both proteins are expressed in the developing ovary from pupae to the adult stage and display enriched expression in terminal filament and stalk cells, while their genetic removal from early germaria results in severe disruption of the subsequent oogenesis, leading to female sterility. Combining genetic mosaic analysis of available null alleles for both Parvin and Ilk with conditional rescue utilizing the UAS/Gal4 system, we found that Parvin and ILK are required in pre-follicle cells for germline cyst encapsulation and stalk cell morphogenesis. Collectively, we have uncovered novel developmental functions for both Parvin and ILK, which closely synergize with integrins in epithelia.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.

The Mammalian Crumbs Complex Defines a Distinct Polarity Domain Apical of Epithelial Tight Junctions

Epithelial apico-basal polarity is established through the asymmetric cortical distribution of the Par, Crumbs and Scribble polarity modules. Apical (Par and Crumbs) and basolateral (Scribble) polarity modules overlap at the apical-lateral border, which, in mammals, is defined by the apical junctional complex (AJC). The AJC is composed of tight junctions (TJ) and adherens junctions (AJ) and plays fundamental roles in epithelial morphogenesis and plasticity. However, the molecular composition and precise sub-junctional organization of the AJC and its associated polarity regulators are not well defined. Here, we used the peroxidase APEX2 for quantitative proximity proteomics (QPP) and electron microscopy (EM) imaging to dissect the architecture of the AJC in fully polarized MDCK-II cells. We present a high-confidence proteome of the apical-lateral border in which TJ and AJ components and apical and lateral compartment markers are spatially resolved. We further demonstrate that the Crumbs complex (Pals1, PatJ, Lin7c, and Crumbs3) defines a hitherto unidentified membrane compartment apical of TJ, which we coin the vertebrate marginal zone (VMZ). QPP, imaging, and immunoprecipitation assays showed that the HOMER scaffolding proteins, PKN2 and PTPN13, and the membrane-proximal HIPPO pathway proteins ARHGAP29 and STXBP4 are recruited to the VMZ via the PDZ domains of PatJ. Taken together, our work defines the spatial and molecular organization of the apical-lateral border in mammalian epithelial cells, reveals an intriguing molecular and spatial conservation of invertebrate and vertebrate cell polarity protein domains, and identifies a VMZ-associated protein network implicated in HIPPO signaling and the control of the cortical actin cytoskeleton.

A variational method for image-based inference of internal stress in epithelial tissues.

Cellular mechanics drives epithelial morphogenesis, the process wherein cells collectively rearrange to produce tissue-scale deformations that determine organismal shape. However, quantitative understanding of tissue mechanics is impaired by the difficulty of direct measurement of stress in-vivo. This difficulty has spurred the development of image-based inference algorithms that estimate stress from snapshots of epithelial geometry. Such methods are challenged by sensitivity to measurement error and thus require accurate geometric segmentation for practical use. We overcome this difficulty by introducing a novel approach - the Variational Method of Stress Inference (VMSI) - which exploits the fundamental duality between stress and geometry at equilibrium of discrete mechanical networks that model confluent cellular layers. We approximate the apical geometry of an epithelial tissue by a 2D tiling with Circular Arc Polygons (CAP) in which arcs represent intercellular interfaces defined by the balance of local line tension and pressure differentials between adjacent cells. The mechanical equilibrium of such networks imposes extensive local constraints on CAP geometry. These constraints provide the foundation of VMSI which, starting with images of epithelial monolayers, simultaneously approximates both tissue geometry and internal forces, subject to the constraint of equilibrium. We find VMSI to be more robust than previous methods. Specifically, the VMSI performance is validated by the comparison of the predicted cellular and mesoscopic scale stress with the measured myosin II patterns during early Drosophila embryogenesis. VMSI prediction of mesoscopic stress tensor correlates at the 80% level with the measured myosin distribution and reveals that most of the myosin activity in that case is involved in a static internal force balance within the epithelial layer. In addition to insight into cell mechanics, this study provides a practical method for non-destructive estimation of stress in live epithelial tissue.

Fog signaling has diverse roles in epithelial morphogenesis in insects.

The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, here we show in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog's role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway.

Active Tension Network model suggests an exotic mechanical state realized in epithelial tissues.

Mechanical interactions play a crucial role in epithelial morphogenesis, yet understanding the complex mechanisms through which stress and deformation affect cell behavior remains an open problem. Here we formulate and analyze the Active Tension Network (ATN) model, which assumes that the mechanical balance of cells within a tissue is dominated by cortical tension and introduces tension-dependent active remodeling of the cortex. We find that ATNs exhibit unusual mechanical properties. Specifically, an ATN behaves as a fluid at short times, but at long times supports external tension like a solid. Furthermore, an ATN has an extensively degenerate equilibrium mechanical state associated with a discrete conformal - “isogonal” - deformation of cells. The ATN model predicts a constraint on equilibrium cell geometries, which we demonstrate to approximately hold in certain epithelial tissues. We further show that isogonal modes are observed in the fruit y embryo, accounting for the striking variability of apical areas of ventral cells and helping understand the early phase of gastrulation. Living matter realizes new and exotic mechanical states, the study of which helps to understand biological phenomena.

Diverse expression patterns of the EMT suppressor grainyhead-like 2 (GRHL2) in normal and tumour tissues.

The transcription factor grainyhead-like 2 (GRHL2) plays a crucial role in various developmental processes. Although GRHL2 recently has attracted considerable interest in that it could be identified as a novel suppressor of the epithelial-to-mesenchymal transition, evidence is emerging that GRHL2 also exhibits tumour-promoting activities. Aim of the present study therefore was to help defining the relevance of GRHL2 for human cancers by performing a comprehensive immunohistochemical analysis of GRHL2 expression in normal (n = 608) and (n = 3,143) tumour tissues using tissue microarrays. Consistent with its accepted role in epithelial morphogenesis, GRHL2 expression preferentially but not exclusively was observed in epithelial cells. Regenerative and proliferating epithelial cells with stem cell features showed a strong GRHL2 expression. Highly complex GRHL2 expression patterns indicative of both reduced and elevated GRHL2 expression in tumours, possibly reflecting potential tumour-suppressing as well as oncogenic functions of GRHL2 in distinct human tumours, were observed. A dysregulation of GRHL2 expression for the first time was found in tumours of non-epithelial origin (e.g., astrocytomas, melanomas). We also report GRHL2 copy number gains which, however, did not necessarily translate into increased GRHL2 expression levels in cancer cells. Results obtained by meta-analysis of gene expression microarray data in conjunction with functional assays demonstrating a direct regulation of HER3 expression further point to a potential therapeutic relevance of GRHL2 in ovarian cancer. Hopefully, the results presented in this study may pave the way for a better understanding of the yet largely unknown function of GRHL2 in the initiation, progression and also therapy of cancers.

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, we examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb is necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. We found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis.

The transmembrane protein Crumbs displays complex dynamics during follicular morphogenesis and is regulated competitively by Moesin and aPKC.

The transmembrane protein Crumbs (Crb) functions in apical polarity and epithelial integrity. To better understand its role in epithelial morphogenesis, we examined Crb localization and dynamics in the late follicular epithelium of Drosophila. Crb was unexpectedly dynamic during middle-to-late stages of egg chamber development, being lost from the marginal zone (MZ) in stage 9 before abruptly returning at the end of stage 10b, then undergoing a pulse of endocytosis in stage 12. The reappearance of MZ Crb is necessary to maintain an intact adherens junction and MZ. Although Crb has been proposed to interact through its juxtamembrane domain with Moesin (Moe), a FERM domain protein that regulates the cortical actin cytoskeleton, the functional significance of this interaction is poorly understood. We found that whereas the Crb juxtamembrane domain was not required for adherens junction integrity, it was necessary for MZ localization of Moe, aPKC and F-actin. Furthermore, Moe and aPKC functioned antagonistically, suggesting that Moe limits Crb levels by reducing its interactions with the apical Par network. Additionally, Moe mutant cells lost Crb from the apical membrane and accumulated excess Crb at the MZ, suggesting that Moe regulates Crb distribution at the membrane. Together, these studies reveal reciprocal interactions between Crb, Moe and aPKC during cellular morphogenesis.

Regulation of a serine protease homolog by the JNK pathway during thoracic development of Drosophila melanogaster

The importance of the Jun N-terminal Kinase (JNK) pathway during normal development and tumor invasion has been well documented in Drosophila. Here, this pathway plays important roles in epithelial morphogenesis, wound healing, apoptosis, immunity and regulation of lifespan. However, which downstream molecules facilitate these effects is not very well elucidated. In this study, data are presented on a serine protease homolog (SPH), scarface. These data show that scarface is under regulatory control of the JNK pathway and that this pathway is both necessary and sufficient for its expression within the context of thoracic development. Consequently, down-regulation of scarface results in a thoracic-cleft phenotype that phenocopies the JNK pathway defect. A possible role of scarface during thoracic development in Drosophila is discussed.

Epimorphin expression in a rat model of pulmonary hypoplasia associated with congenital diaphragmatic hernia.

The pathogenesis of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains unclear. Interactions between the epithelium and surrounding mesenchyme play an important role in normal lung morphogenesis. Epimorphin, a stromal protein, plays a role in epithelial morphogenesis and lung branching, both of which are involved in pulmonary hypoplasia. In this study, we aimed to examine the relationship between epimorphin and pulmonary hypoplasia associated with CDH in an animal model. Time-pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on D16 and D20, and were divided into control, hypoplastic lungs with CDH (CDH+), and hypoplastic lungs without CDH (CDH-). Both lungs of each fetus were removed and subjected to morphometric and molecular biologic analyses. Lung-to-body weight ratios were calculated. Pulmonary RNA was extracted, and relative mRNA level of epimorphin was determined by quantitative real-time PCR(qRT-PCR). Protein expression of epimorphin was investigated by Western blotting. In groups D16 and D20, lung-to-body weight ratios in subgroups CDH+ were significantly lower than those of controls and CDH-. The relative mRNA expression levels of epimorphin were significantly increased in both lungs in subgroup CDH+ compared with controls and CDH- on D16. Pulmonary epimorphin gene expression levels were significantly decreased in CDH+ group on D20 compared to controls. Western blotting confirmed the qRT-PCR results showing decreased pulmonary epimorphin protein expression in CDH+ hypoplastic lungs compared to controls on D20. Our study shows that there is an association between the epimorphin expression and pulmonary hypoplasia associated with CDH. Although the cause-effect relationship is far from being established, epimorphin-related mechanisms have a more critical role in early (D16) developmental stage.

Spatial regulation of Dia and Myosin-II by RhoGEF2 controls initiation of E-cadherin endocytosis during epithelial morphogenesis

E-cadherin plays a pivotal role in epithelial morphogenesis. It controls the intercellular adhesion required for tissue cohesion and anchors the actomyosin-driven tension needed to change cell shape. In the early Drosophila embryo, Myosin-II (Myo-II) controls the planar polarized remodelling of cell junctions and tissue extension. The E-cadherin distribution is also planar polarized and complementary to the Myosin-II distribution. Here we show that E-cadherin polarity is controlled by the polarized regulation of clathrin- and dynamin-mediated endocytosis. Blocking E-cadherin endocytosis resulted in cell intercalation defects. We delineate a pathway that controls the initiation of E-cadherin endocytosis through the regulation of AP2 and clathrin coat recruitment by E-cadherin. This requires the concerted action of the formin Diaphanous (Dia) and Myosin-II. Their activity is controlled by the guanine exchange factor RhoGEF2, which is planar polarized and absent in non-intercalating regions. Finally, we provide evidence that Dia and Myo-II control the initiation of E-cadherin endocytosis by regulating the lateral clustering of E-cadherin.

Distinct roles of cadherin-6 and E-cadherin in tubulogenesis and lumen formation

Classic cadherins are important regulators of tissue morphogenesis. The predominant cadherin in epithelial cells, E-cadherin, has been extensively studied because of its critical role in normal epithelial development and carcinogenesis. Epithelial cells may also coexpress other cadherins, but their roles are less clear. The Madin Darby canine kidney (MDCK) cell line has been a popular mammalian model to investigate the role of E-cadherin in epithelial polarization and tubulogenesis. However, MDCK cells also express relatively high levels of cadherin-6, and it is unclear whether the functions of this cadherin are redundant to those of E-cadherin. We investigate the specific roles of both cadherins using a knockdown approach. Although we find that both cadherins are able to form adherens junctions at the basolateral surface, we show that they have specific and mutually exclusive roles in epithelial morphogenesis. Specifically, we find that cadherin-6 functions as an inhibitor of tubulogenesis, whereas E-cadherin is required for lumen formation. Ablation of cadherin-6 leads to the spontaneous formation of tubules, which depends on increased phosphoinositide 3-kinase (PI3K) activity. In contrast, loss of E-cadherin inhibits lumen formation by a mechanism independent of PI3K.

Myosin II regulation duringC. elegansembryonic elongation:LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. Here we analyze the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. We developed novel GFP probes to visualize the dynamics of actomyosin remodeling, and found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5, which we identified herein). To determine how myosin II regulates embryonic elongation, we rescued mlc-4 mutants with various constructs and found that MLC-4 is essential in a subset of epidermal cells. We show that phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. Finally, in an RNAi screen for potential myosin regulatory light chain kinases, we found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, we suggest that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4.

Regulation of VDR by ΔNp63α is associated with inhibition of cell invasion

The p63 transcription factor has a pivotal role in epithelial morphogenesis. Multiple transcripts of the TP63 gene are generated because of alternative promoter usage and splicing. DeltaNp63alpha is the predominant isoform of p63 observed during epithelial morphogenesis and in human cancers. Loss of DeltaNp63alpha expression has been shown to promote invasiveness in a subset of human cancer cell lines. Here, we studied whether the regulation of VDR by DeltaNp63alpha controls the invasiveness of an epidermoid cancer cell line. We demonstrate that VDR expression is induced by all p63 isoforms, including DeltaNp63alpha. Endogenous DeltaNp63alpha protein was observed to bind to the VDR promoter, and silencing of endogenous DeltaNp63alpha resulted in diminished VDR expression. Although silencing of p63 inhibits VDR expression leading to an increase in cell migration, overexpression of p63 or VDR results in reduced cell migration as a result of increased VDR expression. Therefore, it is conceivable that p63 inhibits cell invasion by regulating VDR expression. Finally, we observed that expression of p63 and VDR overlaps in the wild-type mouse skin, but a reduced or complete absence of VDR expression was observed in skin from p63-null mice and in p63-null mouse embryonic fibroblasts. In conclusion, we demonstrate a direct transcriptional regulation of VDR by DeltaNp63alpha. Our results highlight a crucial role for VDR in p63-mediated biological functions.

Epithelial phenotype and the RPE: Is the answer blowing in the Wnt?

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell-cell adhesion and melanization are linked by a common signaling pathway: the Wnt/beta-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of photoreceptors, theoretically contributing to aging retinal disease such as age-related macular degeneration (AMD). Analyzing sub-lethal stress that produces declines in RPE functional efficiency rather than overt cell death is suggested as a useful future direction for understanding the effects of age on RPE organization and physiology. As for phenotype and pigmentation, a role for the Wnt/beta-catenin pathway is also suggested in regulating the RPE response to oxidative stress. Exploration of this pathway in the RPE therefore may provide a unifying strategy for advancing our understanding of both RPE phenotype and the consequences of mild oxidative stress on RPE structure and function.