Regulated and polarized PtdIns(3,4,5)P3 accumulation is essential for apical membrane morphogenesis in photoreceptor epithelial cells

BACKGROUND: In a specialized epithelial cell such as the Drosophila photoreceptor, a conserved set of proteins is essential for the establishment of polarity, its maintenance, or both--in Drosophila, these proteins include the apical factors Bazooka, D-atypical protein kinase C, and D-Par6 together with D-Ecadherin. However, little is known about the mechanisms by which such apical factors might regulate the differentiation of the apical membrane into functional domains such as an apical-most stack of microvilli or more lateral sub-apical membrane. RESULTS: We show that in photoreceptors Bazooka (D-Par3) recruits the tumor suppressor lipid phosphatase PTEN to developing cell-cell junctions (Zonula Adherens, za). za-localized PTEN controls the spatially restricted accumulation of optimum levels of the lipid PtdIns(3,4,5)P3 within the apical membrane domain. This in turn finely tunes activation of Akt1, a process essential for proper morphogenesis of the light-gathering organelle, consisting of a stack of F-actin rich microvilli within the apical membrane. CONCLUSIONS: Spatially localized PtdIns(3,4,5)P3 mediates directional sensing during neutrophil and Dictyostelium chemotaxis. We conclude that a conserved mechanism also operates during photoreceptor epithelial cell morphogenesis in order to achieve normal differentiation of the apical membrane.     

Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine

Ezrin, Radixin, and Moesin (the ERM proteins) supply regulated linkage between membrane proteins and the actin cytoskeleton. The study of mammalian ERM proteins has been hampered by presumed functional overlap. We have found that Ezrin, the only ERM detected in epithelial cells of the developing intestine, provides an essential role in configuring the mouse intestinal epithelium. Surprisingly, Ezrin is not absolutely required for the formation of brush border microvilli or for the establishment or maintenance of epithelial polarity. Instead, Ezrin organizes the apical terminal web region, which is critical for the poorly understood process of de novo lumen formation and expansion during villus morphogenesis. Our data also suggest that Ezrin controls the localization and/or function of certain apical membrane proteins that support normal intestinal function. These in vivo studies highlight the critical function of Ezrin in the formation of a multicellular epithelium rather than an individual epithelial cell.     

Perp is a p63-regulated gene essential for epithelial integrity

p63 is a master regulator of stratified epithelial development that is both necessary and sufficient for specifying this multifaceted program. We show here that Perp, a tetraspan membrane protein originally identified as an apoptosis-associated target of the p53 tumor suppressor, is the first direct target of p63 clearly involved in mediating this developmental program in vivo. During embryogenesis, Perp is expressed in an epithelial pattern, and its expression depends on p63. Perp-/- mice die postnatally, with dramatic blistering in stratified epithelia symptomatic of compromised adhesion. Perp localizes specifically to desmosomes, adhesion junctions important for tissue integrity, and numerous structural defects in desmosomes are observed in Perp-deficient skin, suggesting a role for Perp in promoting the stable assembly of desmosomal adhesive complexes. These findings demonstrate that Perp is a key effector in the p63 developmental program, playing an essential role in an adhesion subprogram central to epithelial integrity and homeostasis.     

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Here, we show that Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. We find that mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic programme to promote aECM formation.     

Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.     

Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.

Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical beta(Heavy)-spectrin (beta(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of beta(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of beta(H) prevents the stable recruitment of alpha-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (alphabeta(H))(2)-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.     

TGF-beta signaling is essential for joint morphogenesis

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor beta (TGF-beta) signaling in mice lacking the TGF-beta type II receptor gene (Tgfbr2) in their limbs (Tgfbr2(PRX-1KO)). In Tgfbr2(PRX-1KO) mice, the loss of TGF-beta responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2(Prx1KO) joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2-green fluorescent protein-beta-GEO-bacterial artificial chromosome beta-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2(PRX-1KO) mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-beta receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2(PRX-1KO) growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-beta signaling represents a means of entry to initiate the process.     

Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.     

Aminopeptidase N is a marker for the apical pole of porcine thyroid epithelial cells in vivo and in culture

Summary An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization, lose their polarity and exhibit a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized in the apical cell pole. In monolayer cells, on the other hand, the enzyme is distributed over the whole surface facing the medium. In both types of cultures fluorescence is also observed in intracytoplasmic organelles. In vivo, aminopeptidase is a marker of the apical part of the thyroid plasma membrane, but its in vitro localization depends upon cell differentiation related to the culture conditions.     

Coordinating morphogenesis: epithelial integrity during heart tube assembly

Formation of the embryonic heart tube requires the medial migration and merger of bilateral precursor populations. A new study of zebrafish cardiogenesis published in this issue of Developmental Cell indicates that precursor migration involves formation of a coherent epithelium and that fibronectin plays an important role in maintaining cardiac epithelial integrity.     

Microtubule dynamics in neuronal morphogenesis

Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development.     

Epimorphin: a mesenchymal protein essential for epithelial morphogenesis.

A novel 150 kd protein expressed on the surface of mesenchymal cells of mouse embryonic tissues was identified. A monoclonal antibody to this molecule inhibited various processes of epithelial morphogenesis, such as hair follicle growth and lung epithelial tubular formation, in organ cultures of these tissues. Sequence analysis of cDNA encoding this protein revealed that it had 289 amino acids with a hydrophobic stretch at the C-terminus. NIH 3T3 cells transfected with the cDNA of this protein expressed the exogenous 150 kd protein on their surface. When lung epithelial cells were cocultured with these transfected cells, they showed normal tubular morphogenesis, but not with untransfected NIH 3T3 cells. These results indicate that this protein, termed epimorphin, plays a central role in epithelial-mesenchymal interactions.     

Shh signaling is essential for rugae morphogenesis in mice

Palatal ridges, or rugae palatinae, are corrugated structures observed in the hard palate region. They are found in most mammalian species, but their number and arrangement are species-specific. Nine palatal rugae are found in the mouse secondary palate. Previous studies have shown that epithelial Shh signaling in the palatal ridge plays an important role during rugae development. Moreover, Wnt family members, including LEF1, play a functional role in orofacial morphogenesis. To explore the function of Shh during rugae development, we utilized the maternal transfer of 5E1 (anti-Shh antibody) to mouse embryos. 5E1 induced abnormal rugae patterning characterized by a spotted shape of palatal ridge rather than a stripe. The expression patterns of Shh and Shh-related genes, Sostdc1, Lef1 and Ptch1, were disrupted following 5E1 injection. Moreover, rugae-specific cell proliferation and inter-rugae-specific apoptosis were affected by inhibition of Shh signaling. We hypothesize that the altered gene expression patterns and the change in molecular events caused by the inhibition of Shh signaling may have induced abnormal rugae patterning. Furthermore, we propose a reaction–diffusion model generated by Wnt, Shh and Sostdc1 signaling. In this study, we show that Sostdc1, a secreted inhibitor of the Wnt pathway, is a downstream target of Shh and hypothesize that the interaction of Wnt, Shh and Sostdc1 is a pivotal mechanism controlling the spatial patterning of palatal rugae.     

PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.     

Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis

Actin microfilaments form a three-dimensional cytoskeletal network throughout the cell and constitute an essential throughway for organelle and vesicle transport. Development of Arabidopsis trichomes, unicellular structures derived from the epidermis, is being used as a genetic system in which to study actin-dependent growth in plant cells. The present study indicates that filamentous actin (F-actin) plays an important role during Arabidopsis trichome morphogenesis. For example, immunolocalization of actin filaments during trichome morphogenesis identified rearrangements of the cytoskeletal structure during the development of the mature cell. Moreover, pharmacological experiments indicate that there are distinct requirements for actin- and microtubule-dependent function during trichome morphogenesis. The F-actin-disrupting drug cytochalasin D does not affect the establishment of polarity during trichome development; however, maintenance and coordination of the normal pattern of cell growth are very sensitive to this drug. In contrast, oryzalin, an agent that depolymerizes microtubules, severely inhibits cell polarization. Furthermore, cytochalasin D treatment phenocopies a known class of mutations that cause distorted trichome morphology. Results of an analysis of cell shape and microfilament structure in wild-type, mutant, and drug-treated trichomes are consistent with a role for actin in the maintenance and coordination of an established growth pattern.     

Protocadherin-19 is essential for early steps in brain morphogenesis

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.