Role for mitogen-activated protein kinase p38 alpha in lung epithelial branching morphogenesis

In the early stages of lung development, the endoderm undergoes extensive and stereotypic branching morphogenesis. During this process, a simple epithelial bud develops into a complex tree-like system of tubes specialized for the transport and exchange of gas with blood. The endodermal cells in the distal tips of the developing lung express a special set of genes, have a higher proliferation rate than proximal part, undergo shape change and initiate branching morphogenesis. In this study, we found that of the four p38 genes, only p38α mRNA is localized specifically to the distal endoderm suggesting a role in the regulation of budding morphogenesis. Chemical inhibitors specific for the p38α and p38β isoforms suppress budding of embryonic mouse lung explants and isolated endoderm in vitro. Specific knockdown of p38α in cultured lung endoderm using shRNA also inhibited budding morphogenesis, consistent with the chemical inhibition of the p38 signaling pathway. Disruption of p38α did not affect proliferation or expression of the distal cell markers, Sox9 and Erm. However, the amount of E-cadherin protein increased significantly and ectopic expression of E-cadherin also impaired budding of endoderm in vitro. These results suggest that p38α modulates epithelial cell-cell interactions and possibly cell rearrangement during branching morphogenesis. This study provides the first evidence that p38α is involved in the morphogenesis of an epithelial organ.     

The Drosophila homologue of Arf-GAP GIT1, dGIT, is required for proper muscle morphogenesis and guidance during embryogenesis

GIT1-like proteins are GTPase-activating proteins (GAPs) for Arfs and interact with a variety of signaling molecules to function as integrators of pathways controlling cytoskeletal organization and cell motility. In this report, we describe the characterization of a Drosophila homologue of GIT1, dGIT, and show that it is required for proper muscle morphogenesis and myotube guidance in the fly embryo. The dGIT protein is concentrated at the termini of growing myotubes and localizes to muscle attachment sites in late stage embryos. dgit mutant embryos show muscle patterning defects and aberrant targeting in subsets of their muscles. dgit mutant muscles fail to localize the p21-activated kinase, dPak, to their termini. dPak and dGIT form a complex in the presence of dPIX and dpak mutant embryos show similar muscle morphogenesis and targeting phenotypes to that of dgit. We propose that dGIT and dPak are part of a complex that promotes proper muscle morphogenesis and myotube targeting during embryogenesis.     

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.     

In VITRO ASSEMBLY of the øX174 procapsid from external scaffolding protein oligomers and early pentameric assembly intermediates

Bacteriophage øX174 morphogenesis requires two scaffolding proteins: an internal species, similar to those employed in other viral systems, and an external species, which is more typically associated with satellite viruses. The current model of øX174 assembly is based on structural and in vivo data. During morphogenesis, 240 copies of the external scaffolding protein mediate the association of 12 pentameric particles into procapsids. The hypothesized pentameric intermediate, the 12S? particle, contains 16 proteins: 5 copies each of the coat, spike and internal scaffolding proteins and 1 copy of the DNA pilot protein. Assembly naïve 12S? particles and external scaffolding oligomers, most likely tetramers, formed procapsid-like particles in vitro, suggesting that the 12S? particle is a bona fide assembly intermediate and validating the current model of procapsid morphogenesis. The in vitro system required a crowding agent, was influenced by the ratio of the reactants and was most likely driven by hydrophobic forces. While the system reported here shared some characteristics with other in vitro internal scaffolding protein-mediated systems, it displayed unique features. These features most likely reflect external scaffolding protein-mediated morphogenesis and the øX174 procapsid structure, in which external scaffolding-scaffolding protein interactions, as opposed to coat-coat protein interactions between pentamers, constitute the primary lattice-forming contacts.     

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background

There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.

Principal Findings

We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.

Conclusions/Significance

These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.     

Plakophilin-1, a Novel Wnt Signaling Regulator,Is Critical for Tooth Development and Ameloblast Differentiation

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed Pkp1 knockdown experiments using ex vivo organ cultures and cell cultures. Loss of Pkp1 reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, Pkp1 knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex.     

Exocyst-mediated membrane trafficking is required for branch outgrowth in Drosophila tracheal terminal cells

Branching morphogenesis, the process by which cells or tissues generate tree-like networks that function to increase surface area or in contacting multiple targets, is a common developmental motif in multicellular organisms. We use Drosophila tracheal terminal cells, a component of the insect respiratory system, to investigate branching morphogenesis that occurs at the single cell level. Here, we show that the exocyst, a conserved protein complex that facilitates docking and tethering of vesicles at the plasma membrane, is required for terminal cell branch outgrowth. We find that exocyst-deficient terminal cells have highly truncated branches and show an accumulation of vesicles within their cytoplasm and are also defective in subcellular lumen formation. We also show that vesicle trafficking pathways mediated by the Rab GTPases Rab10 and Rab11 are redundantly required for branch outgrowth. In terminal cells, the PAR-polarity complex is required for branching, and we find that the PAR complex is required for proper membrane localization of the exocyst, thus identifying a molecular link between the branching and outgrowth programs. Together, our results suggest a model where exocyst mediated vesicle trafficking facilitates branch outgrowth, while de novo branching requires cooperation between the PAR and exocyst complexes.     

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.     

Polarity complex proteins

The formation of functional epithelial tissues involves the coordinated action of several protein complexes, which together produce a cell polarity axis and develop cell-cell junctions. During the last decade, the notion of polarity complexes emerged as the result of genetic studies in which a set of genes was discovered first in Caenorhabditis elegans and then in Drosophila melanogaster. In epithelial cells, these complexes are responsible for the development of the apico-basal axis and for the construction and maintenance of apical junctions. In this review, we focus on apical polarity complexes, namely the PAR3/PAR6/aPKC complex and the CRUMBS/PALS1/PATJ complex, which are conserved between species and along with a lateral complex, the SCRIBBLE/DLG/LGL complex, are crucial to the formation of apical junctions such as tight junctions in mammalian epithelial cells. The exact mechanisms underlying their tight junction construction and maintenance activities are poorly understood, and it is proposed to focus in this review on establishing how these apical polarity complexes might regulate epithelial cell morphogenesis and functions. In particular, we will present the latest findings on how these complexes regulate epithelial homeostasis.     

mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.     

A Non-sulfated Chondroitin Stabilizes Membrane Tubulation in Cnidarian Organelles

Membrane tubulation is generally associated with rearrangements of the cytoskeleton and other cytoplasmic factors. Little is known about the contribution of extracellular matrix components to this process. Here, we demonstrate an essential role of proteoglycans in the tubulation of the cnidarian nematocyst vesicle. The morphogenesis of this extrusive organelle takes place inside a giant post-Golgi vesicle, which topologically represents extracellular space. This process includes the formation of a complex collagenous capsule structure that elongates into a long tubule, which invaginates after its completion. We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella. It accompanies the tubulation of the vesicle membrane forming a provisional tubule structure, which after invagination matures by collagen incorporation. Inhibition of chondroitin synthesis by β-xylosides arrests nematocyst morphogenesis at different stages of tubule outgrowth resulting in retention of tubule material and a depletion of mature capsules in the tentacles of hydra. Our data suggest a conserved role of proteoglycans in the stabilization of a membrane protrusion as an essential step in organelle morphogenesis.     

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.     

Crumbs3 Is Essential for Proper Epithelial Development and Viability

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.     

RIP4 is a target of multiple signal transduction pathways in keratinocytes: Implications for epidermal differentiation and cutaneous wound repair

Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-κB) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury and might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications     

Cell division orientation and planar cell polarity pathways

The orientation of cell division has a crucial role in early embryo body plan specification, axis determination and cell fate diversity generation, as well as in the morphogenesis of tissues and organs. In many instances, cell division orientation is regulated by the planar cell polarity (PCP) pathways: the Wnt/Frizzled non-canonical pathway or the Fat/Dachsous/Four-jointed pathway. Firstly, using asymmetric cell division in both Drosophila and C. elegans, we describe the central role of the Wnt/Frizzled pathway in the regulation of asymmetric cell division orientation, focusing on its cooperation with either the Src kinase pathway or the heterotrimeric G protein pathway. Secondly, we describe our present understanding of the mechanisms by which the planar cell polarity pathways drive tissue morphogenesis by regulating the orientation of symmetric cell division within a field of cells. Finally, we will discuss the important avenues that need to be explored in the future to better understand how planar cell polarity pathways control embryo body plan determination, cell fate specification or tissue morphogenesis by mitotic spindle orientation.     

The shavenoid gene of Drosophila encodes a novel actin cytoskeleton interacting protein that promotes wing hair morphogenesis

The simple cellular composition and array of distally pointing hairs has made the Drosophila wing a favored system for studying planar polarity and the coordination of cellular- and tissue-level morphogenesis. The developing hairs are filled with F-actin and microtubules and the activity of these cytoskeletons is important for hair morphogenesis. On the basis of mutant phenotypes several genes have been identified as playing a key role in stimulating hair formation. Mutations in shavenoid (sha) (also known as kojak) result in a delay in hair morphogenesis and in some cells forming no hair and others several small hairs. We report here the molecular identification and characterization of the sha gene and protein. sha encodes a large novel protein that has homologs in other insects, but not in more distantly related organisms. The Sha protein accumulated in growing hairs and bristles in a pattern that suggested that it could directly interact with the actin cytoskeleton. Consistent with this mechanism of action we found that Sha and actin co-immunopreciptated from wing disc cells. The morphogenesis of the hair involves temporal control by sha and spatial control by the genes of the frizzled planar polarity pathway. We found a strong genetic interaction between mutations in these genes consistent with their having a close but parallel functional relationship.     

Study of possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in planarian regeneration processes using pharmacological inhibition analysis

Possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in the processes of regeneration and morphogenesis in freshwater planarian flatworms Schmidtea mediterranea was studied using a pharmacological inhibitor analysis. It was found that pharmacological inhibitors of these kinases significantly inhibit the regeneration of the head end of the animals and that this effect is realized due to inhibition of proliferative activity of neoblasts, planarian stem cells. It is shown that that the inhibition of the studied protein kinases in regenerating planarians markedly disturbs stem cell differentiation and morphogenesis.