SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

Molecular mechanisms of cell shape changes that contribute to vertebrate neural tube closure

During early development of the central nervous system, the neuroepithelial cells undergo dynamic changes in shape, cumulative action of which cause the neural plate to bend mediolaterally to form the neural tube. The apicobasal elongation changes the cuboidal cells into columnar ones, whereas apical constriction minimizes the cell apices, causing them to adopt wedge-like shapes. To achieve the morphological changes required for the formation of a hollow structure, these cellular changes must be controlled in time and space. To date, it is widely accepted that spatial and temporal changes of the cytoskeletal organization are fundamental to epithelial cell shape changes, and that noncetrosomal microtubules assembled along apicobasal axis and actin filaments and non-muscle myosin II at the apical side are central machineries of cell elongation and apical constriction, respectively. Hence, especially in the last decade, intracellular mechanisms regulating these cytoskeletons have been extensively investigated at the molecular level. As a result, several actin-binding proteins, Rho/ROCK pathway, and cell-cell adhesion molecules have been proven to be the central regulators of apical constriction, while the regulatory mechanisms of cell elongation remain obscure. In this review, we first describe the distribution and role of cytoskeleton in cell shape changes during neural tube closure, and then summarize the current knowledge about the intracellular proteins that directly modulate the cytoskeletal organization and thus the neural tube closure.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

The Genes Raw and Ribbon Are Required for Proper Shape of Tubular Epithelial Tissues in Drosophila

The products of two genes, raw and ribbon (rib), are required for the proper morphogenesis of a variety of tissues. Malpighian tubules mutant for raw or rib are wider and shorter than normal tubules, which are only two cells in circumference when they are fully formed. The mutations alter the shape of the tubules beginning early in their formation and block cell rearrangement late in development, which normally lengthens and narrows the tubes. Mutations of both genes affect a number of other tissues as well. Both genes are required for dorsal closure and retraction of the CNS during embryonic development. In addition, rib mutations block head involution, and broaden and shorten other tubular epithelia (salivary glands, tracheae, and hindgut) in much same manner as they alter the shape of the Malpighian tubules. In tissues in which the shape of cells can be observed readily, rib mutations alter cell shape, which probably causes the change in shape of the organs that are affected. In double mutants raw enhances the phenotypes of all the tissues that are affected by rib but unaffected by raw alone, indicating that raw is also active in these tissues.     

PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6beta and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 and alpha4/beta1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.     

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.     

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions

Epithelial cells assemble specialized actomyosin structures at E-Cadherin–based cell–cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.     

Apical constriction: A cell shape change that can drive morphogenesis

Biologists have long recognized that dramatic bending of a cell sheet may be driven by even modest shrinking of the apical sides of cells. Cell shape changes and tissue movements like these are at the core of many of the morphogenetic movements that shape animal form during development, driving processes such as gastrulation, tube formation, and neurulation. The mechanisms of such cell shape changes must integrate developmental patterning information in order to spatially and temporally control force production—issues that touch on fundamental aspects of both cell and developmental biology and on birth defects research. How does developmental patterning regulate force-producing mechanisms, and what roles do such mechanisms play in development? Work on apical constriction from multiple systems including Drosophila, Caenorhabditis elegans, sea urchin, Xenopus, chick, and mouse has begun to illuminate these issues. Here, we review this effort to explore the diversity of mechanisms of apical constriction, the diversity of roles that apical constriction plays in development, and the common themes that emerge from comparing systems.     

Transposon tagging of the Defective embryo and meristems gene of tomato.

The shoot and root apical meristems (SAMs and RAMs, respectively) of higher plants are mechanistically and structurally similar. This has led previously to the suggestion that the SAM and RAM represent modifications of a fundamentally homologous plan of organization. Despite recent interest in plant development, especially in the areas of meristem regulation, genes specifically required for the function of both the SAM and RAM have not yet been identified. Here, we report on a novel gene, Defective embryo and meristems (Dem), of tomato. This gene is required for the correct organization of shoot apical tissues of developing embryos, SAM development, and correct cell division patterns and meristem maintenance in roots. Dem was cloned using transposon tagging and shown to encode a novel protein of 72 kD with significant homology to YNV2, a protein of unknown function of Saccharomyces cerevisiae. Dem is expressed in root and shoot meristems and organ primordia but not in callus. The expression pattern of Dem mRNA in combination with the dem mutant phenotype suggests that Dem plays an important role within apical meristems.     

α-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium

Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of α- and β-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in?a simple model organism.     

Apical junctions and growth control in Drosophila

Recent studies have revealed unexpected links between cell polarity and proliferation, suggesting that the polarized organization of cells is necessary to regulate growth. Drosophila melanogaster is a genetically simple model that is especially suited for the study of polarity and growth control, as polarized tissues undergo a well-defined pattern of proliferation and differentiation during the development. In addition, genetic studies have identified a number of tumor suppressor genes, which later studies have shown to be associated with junctions, or in the regulation of junctional proteins. We will explore in this review the links between growth and apical junction proteins in the regulation of growth control in Drosophila.     

Rho GTPase controls invagination and cohesive migration of the Drosophila salivary gland through Crumbs and Rho-kinase

Coordinated cell movements shape simple epithelia into functional tissues and organs during embryogenesis. Regulators and effectors of the small GTPase Rho have been shown to be essential for epithelial morphogenesis in cell culture; however, the mechanism by which Rho GTPase and its downstream effectors control coordinated movement of epithelia in a developing tissue or organ is largely unknown. Here, we show that Rho1 GTPase activity is required for the invagination of Drosophila embryonic salivary gland epithelia and for directed migration of the internalized gland. We demonstrate that the absence of zygotic function of Rho1 results in the selective loss of the apical proteins, Crumbs (Crb), Drosophila atypical PKC and Stardust during gland invagination and that this is partially due to reduced crb RNA levels and apical localization. In parallel to regulation of crb RNA and protein, Rho1 activity also signals through Rho-kinase (Rok) to induce apical constriction and cell shape change during invagination. After invagination, Rho-Rok signaling is required again for the coordinated contraction and dorsal migration of the proximal half of the gland. We also show that Rho1 activity is required for proper development of the circular visceral mesoderm upon which the gland migrates. Our genetic and live-imaging analyses provide novel evidence that the proximal gland cells play an essential and active role in salivary gland migration that propels the entire gland to turn and migrate posteriorly.     

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.     

A quantitative and dynamic model for plant stem cell regulation

Plants maintain pools of totipotent stem cells throughout their entire life. These stem cells are embedded within specialized tissues called meristems, which form the growing points of the organism. The shoot apical meristem of the reference plant Arabidopsis thaliana is subdivided into several distinct domains, which execute diverse biological functions, such as tissue organization, cell-proliferation and differentiation. The number of cells required for growth and organ formation changes over the course of a plants life, while the structure of the meristem remains remarkably constant. Thus, regulatory systems must be in place, which allow for an adaptation of cell proliferation within the shoot apical meristem, while maintaining the organization at the tissue level. To advance our understanding of this dynamic tissue behavior, we measured domain sizes as well as cell division rates of the shoot apical meristem under various environmental conditions, which cause adaptations in meristem size. Based on our results we developed a mathematical model to explain the observed changes by a cell pool size dependent regulation of cell proliferation and differentiation, which is able to correctly predict CLV3 and WUS over-expression phenotypes. While the model shows stem cell homeostasis under constant growth conditions, it predicts a variation in stem cell number under changing conditions. Consistent with our experimental data this behavior is correlated with variations in cell proliferation. Therefore, we investigate different signaling mechanisms, which could stabilize stem cell number despite variations in cell proliferation. Our results shed light onto the dynamic constraints of stem cell pool maintenance in the shoot apical meristem of Arabidopsis in different environmental conditions and developmental states.     

The Arabidopsis SPIKE1 gene is required for normal cell shape control and tissue development

Regulated growth and cell shape control are fundamentally important to the function of plant cells, tissues, and organs. The signal transduction cascades that control localized growth and cell shape, however, are not known. To better understand the relationship between cytoskeletal organization, organelle positioning, and regulated vesicle transport, we conducted a forward genetic screen to identify genes that regulate cytoskeletal organization in plants. Because of the distinct requirements for microtubules and actin filaments during leaf trichome development, a trichome-based morphology screen is an efficient approach to identify genes that affect cytoplasmic organization. The seedling lethal spike1 mutant was identified based on trichome, cotyledon, and leaf-shape defects. The predicted SPIKE1 protein shares amino acid identity with a large family of adapter proteins present in humans, flies, and worms that integrate extracellular signals with cytoskeletal reorganization. Both the trichome phenotype and immunolocalization data suggest that SPIKE1 also is involved in cytoskeletal reorganization. The assembly of laterally clustered foci of microtubules and polarized growth are early events in cotyledon development, and both processes are misregulated in spike1 epidermal cells.     

Epithelial tube morphology is determined by the polarized growth and delivery of apical membrane

Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. We identified a new allele of hairy in a mutagenesis screen and showed that hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). HKB controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube.     

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.     

Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension.